Preamble

Monday, October 20, 2008

1:15 PM

Hey, everybody. These are my compiled learning objectives for Molecules to Medicine
when I took it in the fall of 2007. I hope you find them useful. A few notes:

1. These aren't to be taken as everything-you-need-to-know

material, or anything close to it. They can be, however, extremely
useful, if only to look at the material a second time in a different
format.
2. Learning objectives change. Granted, in our vaunted institution,
they often don't change a lot. But it's worth figuring out where these
overlap with what you're studying and where they don't to avoid any
unnecessary learning (God forbid).
3. They can be incorrect. I hope this is infrequent, but I'm sure
there are things in here that aren't accurate. I've tried to curate them
reasonably well; I hope one of your classmates will do likewise. If you
find an error, kindly let him or her know.
4. They are nothing more or less than my personal take on what
we happened to be learning on a given day. Sometimes they're very
detailed, sometimes they're uncomprehending, frequently they're
irreverent. I occasionally call babies vampires and things like that
(dude, they are). Internet lesson: free trumps tasteful. In any case
you are free to disagree with me.
5. To anyone who's wondering: I honored this block and all the
rest my first year. That's not supposed to impress you, but it is
supposed to give you some kind of confidence that I have a reasonably
good handle on what's going on.
6. To the many of you who are thinking, "How can I repay this
wonderful, wonderful man?" I would reply that I never turn down free
beer if I can help it. The problem with that is that I suspect I will never
meet most of your class, and beer-buying in absentia is a cold and
heartless thing. So if you find these useful and would like to do
something for me, I would prefer it if you donated $5 to the charity of
your choice; if you're stumped, I suggest browsing
www.charitynavigator.com for some good options. Kindly do not
donate money to armed insurrectionist groups.
i. Addendum on donating to charity: always always
ALWAYS have an email account that you set aside purely to
sign up for or donate to things (thus ducking all the spam
associated therewith). I think I have 1,200 emails in mine,
mostly from a donation I made to the SPCA a couple of years
ago. Gmail or Hotmail works well. I also recommend using a
false street address to avoid direct-mail campaigns.
ii. "That seems like a lot of trouble to go through to donate
five bucks"-- yeah, well, welcome to the world, sonny Jim.
Doing things for other people frequently is a pain in the ass.
Doesn't make it less worth doing.
Bioenergetics: 10/16/07
Wednesday, October 17, 2007
3:42 PM

LO's for Bioenergetics:

• Define: 1) Entropy, 2) Enthalpy, 3) Free energy, 4) High energy compounds,

5) Oxidation-reduction reaction:
o Entropy: randomness associated with a given system.
o Enthalpy: measure of heat content (thermodynamic potential) of a
system.
o Free energy: the amount of thermodynamic energy in a system that
can be converted into work at a given temperature and pressure.
o High energy compounds: compounds whose products, when put
through a given reaction, have a lower energy state than their
reactants. (ie. ATP -> ADP + Pi, or hydrolysis of thioesters S-C).
Notice that this is really a meaningless term without a given reaction
in mind, since "high-energy" refers to a relationship within a reaction
of the reactants to the products.
o Oxidation-reduction reaction: a chemical reaction which involves the
transfer of electrons away from one or more compounds (the oxidized
compounds) to one or more other compounds (the reduced
compounds).
• Review the first and second laws of thermodynamics:
o 1st law: Energy cannot be created or destroyed, only converted. Does
beg the question of where the energy came from.
o 2nd law: The entropy of the universe is constantly increasing. This
means that in any given set of reactions that occur simultaneously in a
system, the overall entropy of the system must increase. This usually
takes the form of some energy in the system being converted to heat.
Does beg the question of where we got so much order to begin with,
since the universe is currently progressing towards a state of complete
thermodynamic equilibrium (flat, completely uniform and sterile
space).
• Describe the different forms of energy:
o Relative to current discussion, there's chemical potential energy,
usually stored as bonds between atoms, and kinetic energy, which
usually involves transfer of energy from one atom to another.
• Memorize the following basic thermodynamic equations that involve free
energy,equilibrium constant, enthalpy and entropy. Know how to calculate
one unknown variable in an equation when all other variables are given.
o a) ∆G = ∆G0 + (RT * ln [PRODUCTS]/[REACTANTS])
o b) ∆G0 = -RT * ln (Keq)
o c) ∆G = ∆H – (T * ∆S )
• Know the relationship between the sign of the standard free energy and the
direction of a reaction under standard conditions.
o If ∆G is negative, the reaction will occur spontaneously. If ∆G is
positive, it won't go until forced.
 • Describe the effect of positive/negative entropy or enthalpy on the
thermodynamic forces driving a reaction based on the equation: ∆G = ∆H – T
∆S.
o Positive entropy of a reaction decreases the ∆G of that reaction
(makes it more spontaneous). Positive enthalpy change in a reaction
increases the ∆G of that reaction (makes it less spontaneous). And
vice versa.
• Know how to calculate the numerical conversion between free energy (∆G)
and Redox potential (∆E) in biological systems.
o Can calculate ∆E for a reaction by subtracting the Eo of the electron
donor from the Eo of the electron acceptor.
o Eo of a molecule is high when the molecule is more electronegative.
It's high negative when the molecule is more electropositive.
o ∆G = -nF∆E. Notice that when ∆E is positive (so that the electrons are
flowing away from an electropositive atom to an electronegative one),
∆G is negative and thus the reaction is more spontaneous.
• Recognize the fact that series of electron transfer in biological system can
generate energy (a common way of converting the solar thermal energy into
chemical bond energy in plant, or converting the chemical bond energy into
redox energy and other forms of energy).
o (ie. see mitochondrial electron transport chain and oxidative
phosphorylation.)
• Recognize the fact that the standard free energy changes for a set of
reactions are additive, or reactions with positive or negative free energy
changes can be coupled.
o Essentially, if you've got two reactions going on at the same time
(which is usually what we're talking about when we talk about a "set of
reactions"), the ∆G (free energy change) that determines whether or
not both of them go forward or not is just the sum of the ∆G of each
individual reaction. If the total ∆G is negative, all the reactions will be
spontaneous. This is why you can couple a favorable reaction (like
ATP->ADP + Pi) to an unfavorable one to make the unfavorable one
spontaneous.
• Know the major high-energy compounds used in biological systems and the
principle of energy storage in high-energy compound.
o Main types: thioester bonds (C-S, as in acetyl Co-A) and high-energy
phosphate bonds (like the two outer phosphate-to-phosphate bonds of
ATP)
o The "principle" that I can think of is that you have an interchangeable,
rechargable currency in high-energy bonds. You "spend" a phosphate
group off of ATP to drive a reaction, which leaves you with a "spent"
ADP molecule. You then "recharge" that ADP to ATP with another
phosphate group during mitochondrial respiration, at which point
you've got your original "whole" currency back to spend again.

DNA/RNA: 10/17/07
Wednesday, October 17, 2007
3:42 PM

LO's for DNA/RNA:

• Distinguish purines and pyrimidine bases, ribose and deoxyribose, ribo- and
deoxyribo nucleosides, nucleotides, nucleoside di and triphosphates
o Purines: adenine, guanine.
o Pyrimidines: cytosine, thymine, uracil.
o Ribose is a five-carbon sugar that's the primary building block of
ribonucleic acids. Deoxyribose has been de-hydroxylated at the 2'
position.
o Nucleosides are the central ribose sugar and a base attached to it at
the 1' position. A nucleoside with one phosphate group attached to the
5' position of the ribose is called either a nucleotide or a nucleoside
monophosphate. If there's a chain of two phosphates tagged on at the
5' position, it's a nucleoside diphospate; if the chain is three
phosphates long, it's a nucleoside triphosphate.
o Notice that one nucleoside triphosphate, ATP, is the universal energy
currency in the body.
• Evaluate the relative solubility of the different bases and the diseases related
to their insolubility.
o So phosphates are fairly hydrophillic. The bases attached to the ribose
are generally more hydrophobic. Among bases, purines are the least
soluble of the bunch.
o Diseases can result from the accumulation of excess purine derivatives
in tissues:
• Gout
• Lesch-Nyhan disease
• Identify the chemical basis for the 5’ –3’ polarity of DNA and RNA
polynucleotide strands DNA and the phosphodiester linkage.
o Each nucleotide has an open hydroxyl group at the 3' ribose position
and a phosphate at the 5' position (as well as a base stuck onto the 1'
carbon). It's the binding of one nucleotide's phosphate group to
another's OH group that makes the "chain" of DNA/RNA. Thus, at the
end of each DNA or RNA chain, there's either a 'spare,' unlinked
phosphate group (what's called the 5' end) or a 'spare,' unlinked
hydroxyl group (called the 3' end). The phosphodiester linkage is the
link between phosphate and hydroxyl.
• Review the important experiments that helped to establish DNA as the genetic
material.
o Avery, McCloud and McCarty: established DNA as the genetic material
with their Pneumococcus experiments (transformation of nonlethal
P.coccus to lethal P.coccus by addition of heat-killed lethal P.coccus).
o Chargaff's (see below)
o Franklin and Wilkins: x-ray diffraction suggesting a helical structure.
o Watson and Crick discover definitive double-helical structure.
• List Chargaff’s rules.
o The molar ratios of total purines and total pyrimidines are roughly
equal.
o The molar ratios of adenine to thymine, and guanine to cytosine, are
roughly equal.
• Describe the Watson-Crick model for DNA structure, recognize the major and
minor grooves the phosphodiester backbone and the base pairs.
o Briefly: phosphate groups on the outside of the helix (makes sense,
they're hydrophilic), base pairs on the inside (also makes sense,
they're hydrophobic). Major groove is the larger of the two grooves
running down the helix (due to the geometry of the molecules in either
side of the helix); minor groove is the smaller. There are about 10
base pairs per turn of the helix. The horizontal distance covered by A-T
is almost identical to that covered by G-C.
• Describe the chemical basis for the stability of the double helix DNA in
solution.
o You'd think all the phosphate groups right next to each other would
generate some electrostatic repulsion and destabilize the molecule.
But (a) they're mostly neutralized by positively charged species in the
cell; (b) the base pair linkages give the helix a lot of stability; and (c)
adjacent base pairs "stack" on top of each other, providing additional
delocalization options for the electrons and giving, often, more stability
than comes from the base pairing itself.
o Notice that increases in salt concentrations and extremes of pH will
decrease the stability of the DNA molecule (thus reduce its Tm)
respectively due to deneutralized PO4 groups or different ionizations of
bases, disrupting H-bonds.
o Notice also that both the length of the DNA molecule and its relative
GC content will positively affect its stability (more H-
bonding/delocalization potential).
• Describe the chemical modifications of bases in DNA including different forms
of DNA damage (methylation, deamination, depurination, UV cross linking)
and their significance to disease.
o Methylation of bases:
• Methylation tends to occur at the (5') cytosine ends of CpG
sequences.
• [CpG sequences are CG sequences. Not sure why somebody felt
it necessary to add the phosphate in between, as it kind of goes
without saying.]
• This usually deactivates the gene unless repaired (see next
section).
o Deaminination of bases:
• A couple of problems with this.
• One is that if you deaminate cytosine, you get uracil, which
shouldn't be in DNA at all- this is usually repaired by base
excision repair (see next section).
• The other, more serious problem is that if the cytosine has
already been methylated and it's deaminated, you get
thymine-- which is a much subtler change, since you expect to
find thymine in DNA anyway. If this isn't corrected before the
next replication of the DNA, the mutation can cause one of the
replicated helices to contain AT instead of CG at this point.
o Depurination of bases:
• Effectively, this is a hydrolysis reaction, breaking off the purine
base from the ribose and leaving a hydroxyl group.
• It's usually caught by DNA repair enzymes.
 • The problem is that it significantly weakens the phosphodiester
backbone at the depurination site-- so if you have a couple of
these nearby, can result in a double-stranded break.
o UV-caused cross-linking of bases:
• Usually occurs between thymines to create cyclobutane
thymine dimers.
• Distort the DNA helix and can block replication enzymes.
• Generally repaired by nucleotide excision repair (see next
section)
• Explain the chemistry of DNA polymerization and how nucleoside analogues
are used as drugs.
o I think this question relates more to the second than the first part, as
DNA polymerization is covered in the next section. Essentially, you can
use molecules that are very similar to nucleosides to block replication
of virally infected cells by having the replicating DNA (which recruits
free-floating nucleosides as it replicates) incorporate the analogues
into the growing chain. The analogues are different enough from actual
nucleosides to ensure that the resulting DNA chains are nonfunctional.
o Notice you can also use differences in the viral reverse transcriptase
pathways to design nucleoside analogues preferentially incorporated
by reverse transcriptase pathways.
o More-specific nucleoside analogues usually used against retroviruses.
Less specific nucleoside analogues usually used as chemotherapy
against cancer.
• Define the major similarities and differences between DNA and RNA:
o DNA: has no hydroxyl group at the 2' position of the ribose, which
makes it stabler and less prone to breakdown. DNA binds cytosine,
guanine, adenine, and thymine as its bases. RNA: hydroxylated at its
2' ribose position; binds uracil instead of thymine. RNA is usually
single-stranded, although it can form double-stranded loops (often
called 'hairpin loops') with itself.
• Define 5 classes of RNA in a human cell.
o 3 main types: structural, regulatory, and information-containing.
• Structural: rRNA for ribosome assembly, tRNA for protein
assembly, and small nuclear and small nucleolar RNA for a
variety of in-cell modifications.
• Regulatory: miRNA and siRNA to downregulate gene
expression.
• Information-containing: mRNA to be translated into proteins.
• Describe the chemical basis for nucleic acid melting and annealing and the
how it can be used to detect one specific DNA sequence in total cellular DNA.
o Essentially, you're breaking and re-making the hydrogen bonds
between base pairs. A given strand of DNA will re-anneal itself to its
complementary pair more or less by itself. This means you can take a
bunch of someone's DNA, melt it, throw a tagged DNA strand from
something you want to test for in the mix, and see if it anneals to
anything. If it does, there's a match in the person's DNA.
• Distinguish between linear and circular and relaxed and supercoiled forms of
DNA
o linear DNA: well, it's linear (ends not attached to each other). You can
guess about circular DNA. Relaxed DNA: DNA with normal amount of
coiling. Supercoiled DNA: DNA with more coils than normal (for
 example, can result from helicases partially unwinding a segment of
DNA and thus pushing the "coil" from that segment ahead of and
behind the helicases).

DNA/RNA Replication and Repair: 10/18/07

Thursday, October 18, 2007
8:04 AM

LO's for DNA replication and repair and RNA synthesis and splicing:
(DNA replication and repair)
• Describe the meaning of "semi-conservative", "bidirectional", "Okazaki
fragments", "origin", and "fork" as it relates to DNA replication.
o Semi-conservative: Each DNA strand is preserved as one half of a new
double DNA strand; thus each new DNA strand has one-half original
material and one-half newly synthesized material.
o Bidirectional: Means that when the replication machinery attaches to
the DNA double helix, replication proceeds in both directions along that
helix at once.
o Okazaki fragments: Small stretches of DNA synthesized during
replication in the 5' to 3' direction. Since the synthesized strand is
running in the 3' to 5' direction, and since new dNTPs can only be
added at the 3' hydroxyl group, the DNA synthesis process takes a
kind of "leapfrogging" approach whereby small segments on that
strand are copied 5' to 3' and then melded together later. Class notes
have some illustrations of this.
o Origin of replication, aka replication origin: Specific sequences for
recognition by binding proteins. Usually contain multiple short repeats,
as well as an A-T rich streak.
o Replication fork: where the DNA helicases have unwound the double
helix. Effectively the H-bonds of the base pairs have been split apart
and the two strands are peeled away from each other, thus forming a
"fork" in which the replication machinery sits and synthesizes
complementary strands.
• Know the functions of the following proteins during DNA replication: origin
binding proteins, helicase, single-strand binding proteins, primase, Pol I, Pol
III, processivity clamp, DNA ligase, telomerase, topoisomerase/gyrase, and
reverse transcriptase
o Origin binding proteins: Not sure about this, but presumably it's the
protein complex that forms to bind to the origin. Possibly these are the
helicase proteins.
o Helicases: enzymes that catalyze the breaking of H-bonds between
base pairs and the subsequent 'unwinding' of the helix (though this
isn't really unwinding as much as pushing the winding part further
back; see under topoisomerases).
o Single-strand binding proteins: bind to the melted strands of original
DNA to prevent them from re-annealing. I would imagine this has two
functions: one, making sure the strands don't re-anneal to each other,
 and two, making sure the strand matched to the Okazaki fragments
doesn't attach itself to the fragments before they're combined.
o primase: Enzyme that catalyzes the addition of the RNA primer to
begin replication.
o Pol I: DNA polymerase I, "distributive." Synthesizes a new DNA strand
from its complementary strand. Low processivity (speed and extent of
synthesis) compared to Pol III, due to a lack of Pol III's 'sliding clamp'
mechanism. Used when removing RNA primers and replacing them
with DNA segments during replication, since it has 5' to 3' exonuclease
activity.
• Common to both Pol III and Pol I: "proofreading" activity, or 3'
to 5' exonuclease activity. If the wrong dNTP is added during
DNA synthesis, the synthesis stops and 'backs up' slightly (in
the 3' to 5' direction) and chops off the last nucleotide added.
o Pol III: DNA polymerase III, "processive." Also synthesizes DNA strand
from its complement. High processivity due to a sliding clamp
mechanism that holds the polymerase tightly to the DNA. No 5' to 3'
exonuclease activity (thus can't be used to remove RNA primers).
Synthesizes both the leading strand and the Okazaki fragments of the
lagging strand; thus the main synthesizer of DNA.
o Processivity clamp: the aforementioned 'sliding clamp' mechanism.
Present in Pol III.
o DNA ligase: enzyme responsible for sealing Okazaki fragments
together once the RNA primers have been replaced by Pol I.
o (Telomere:) sequence at the ends of chromosomes, consisting of a
large number of repeating segments. Gets consistently shorter every
time the chromosome is replicated, since the RNA primer on the very
last O. fragment can't be replaced by Pol I (Pol I needs to have a
nearby 3' OH from the next fragment to bind and replace the RNA
primer). After a certain point, the telomeres get short enough that the
cell becomes unstable and is destroyed.
o Telomerase: Enzyme responsible for ensuring that the telomeres of
chromosomes in certain immortal structures, such as germ cells, never
shorten. Effectively, they act as reverse transcriptases, binding to the
ends of DNA sequences and adding on some extra dNTPs. The reason
this works is that the telomeres have more or less a uniform
repetitious sequence (so it's not hard to predict what the replacement
sequence will have to be).
o Topoisomerase/gyrase: Enzyme responsible for relieving torsional
strain in the DNA helix in the region ahead of the replication fork. It
does this by clipping the phosphodiester backbone in selected places
(the helix stays intact, as it's a single-strand break, but the tension is
relieved).
o Reverse transcriptase: Enzyme responsible for copying a base
sequence INTO DNA (as opposed to out from it), usually from RNA.
This can be endogenous (ie. telomerases) or exogenous (ie.
retroviruses).
• Understand how DNA polymerase creates the phosphodiester bond during
addition of dNTPs:
o It breaks off a diphosphate group from the dNTP and uses the energy
liberated from that reaction to bind the remaining phosphate group to
the hydroxyl group of the previous nucleotide on the chain.
• Know that DNA polymerase requires an RNA primer.
• Know that DNA synthesis only occurs in the 5' to 3' direction.
• Know that errors are corrected by proof reading (3' to 5' exonuclease
activity).
• Understand the order of events that occur during, the differences between,
and coordination of, DNA synthesis on the lagging and leading strands
o Leading strand: pretty simple, relatively speaking:
• origin binding proteins bind to origin.
• DNA melted apart locally by helicases.
• Topoisomerases relieve tension ahead of the replication fork.
• Pol III elongates DNA complementary to leading strand.
• The two strands, one new, one old, are annealed.
o Lagging strand: similar but a little more complicated since DNA
synthesis can only occur in the 5' to 3' direction. Share first 3 steps
with leading strand, then:
4. Primase attaches RNA primer to lagging strand segment
5. Pol III elongates DNA from RNA primer back a short ways,
forming an Okazaki fragment.
6. RNA primer is removed and replaced with DNA by Pol I.
7. Fragments are sealed together with DNA ligase.
8. The two strands, one new and one old, are annealed.
• Describe the relationship between mutation and cancer, both at the
mutational level and at the level of heritable defective human DNA repair
pathways, giving examples of human DNA repair diseases
o Three things seem to need to happen to wind up with cancer. One is
that a mutation or mismatching event has to occur in DNA to begin
with. Another is that the repair mechanisms have to either miss it or
be overwhelmed by too many such events (ie. exposure to lots and
lots of UV radiation). The third is that the self-destruction pathways in
the cell need to misfire. If all three conditions are met, can result in
cancer.
o Couple of examples of diseases resulting from mutations in DNA repair
mechanisms: Cockayne's syndrome, Xeroderma pigmentosum
(generally involved with light sensitivity, neurodegeneration,
premature aging, and cancer).
• Explain the basic steps of mismatch repair, describing the type of damage
repaired by this pathway, and understand the marking of the old strand of
DNA by methylation in E. coli
o Mismatched base recognized almost immediately after synthesis on
new strand (not old strand; old strand recognized by methylation in E.
coli, mechanism of recognition unknown in humans). A stretch of DNA
behind and in front of the mismatch is clipped by endonucleases,
excised by helicase and exonucleases, and replaced with the correct
sequence by DNA Pol I (and sealed with liagases).
• Describe the basic mechanism of base-excision repair, nucleotide-excision
repair, recombinational repair, NHEJ and the types of lesions corrected by
these pathways:
o Nucleotide-excision repair (NER): tends to repair more overt
modifications that alter the helical pattern of the affected DNA.
Process: recognition, clipping the backbone by endonucleases, excision
of the affected part, replacing by Pol I, resealing by DNA ligase.
 • Notice that the recognition pathways here need a transcription
factor, TFIIH, to work properly.
• Notice also that there's two kinds of NER:
 transcribed: the distortion is within a gene being actively
transcribed
 global: the distortion isn't within a gene being actively
transcribed
o Base-excision repair (BER): tends to repair subtler modifications, like a
mismatched base pair not caught by either proofreading or mismatch
repair. Process: recognition, clipping off the inappropriate base by
glycosylases, clipping the backbone by endonucleases, chewing off by
exonucleases of the affected part, replacing by Pol I, resealing by DNA
ligase.
o Recombinatorial repair: also called homologous recombination. Repairs
double-stranded breaks in DNA. Process: partially degrades both sides
of the break to create primers for DNA synthesis, attracts intact,
homologous sequence from other chromosome, each strand aligns
itself with a strand on homologue and fills in its gap from that strand.
o NHEJ [Non-Homologous-End-Joining]: A form of double-stranded
break repair that doesn't involve the homologous chromosomes.
Essentially you unwind the two ends with helicases, pair up a few
matching bases, and reseal the phosphodiester backbone. Note that
this can be inaccurate, as you often lose a few bases off the unpaired
strands during the resealing. (handout p.13 has a good picture of
this.)
• Describe the sources and nature of lesions to DNA, the type of repair pathway
used to repair the lesion and the molecular consequences of failure to repair
the lesions, for:
o thymine dimers: Good candidate for nucleotide excision repair. Usually
caused by UV radiation causing linkage between adjacent thymine
residues, causing a bulging deformation of the helical structure. If
unrepaired, can cause problems with normal processing due to
malformed helix. (also see next bullet point)
o uracils in DNA: Good candidate for base excision repair. Usually caused
by the deamination of a cytosine residue to produce uracil in the DNA.
If uncorrected, can cause problems with both the process of
replicating/transcribing this portion of the DNA and also with
recognition sites of transcription enzymes.
• Specifically, if this portion of the DNA is transcribed or
replicated as is, it will pair with an adenine residue, not a
guanine; thus will have effectively swapped a C for a T.
o bulky chemical adducts: like thymine dimers, except usually caused by
chemotoxic binding of large molecules to bases in a DNA helix.
o double-strand breaks: Good candidate for either homologous
recombination repair or non-homologous end joining (good to know:
non-homologous end joining is the major form of double-strand
breaks). Caused by a double break of the phosphodiester backbone
(not sure what underlying causes are). If unrepaired, since a
chromosome is one long DNA sequence, can lose up to half of the
chromosome (very bad).
 • Describe the mechanism that enables replication to continue in the face of
DNA lesions, and know the unfortunate consequence of this process for the
cell.
o The mechanism is called lesion bypass polymerization and usually
occurs when the cell doesn't have enough resources to fix all the
thymine dimers occasioned by UV exposure. Effectively it allows
replication to proceed, despite the fact that the dimer interferes with
normal processing, by eliminating the polymerase's ability to do base
proofreading (ie. 3' to 5' exonuclease activity).
o The error rate is 2 to 4 orders of magnitude higher than normal
replication, thus frequently results in cancers, etc.

(RNA synthesis and splicing)

• Describe the chemical reaction catalyzed by RNA polymerase and why it is
unidirectional.
o Notice that a single RNA polymerase enzyme is responsible for
catalyzing the entire elongation of transcription, even for genes that
take 12 hours to transcribe.
• Distinguish five steps in the transcription cycle common to bacterial and
eukaryotic RNA polymerases.
o 1: Polymerase binds to promoter sequence on the helical DNA in a
"closed complex."
o 2: Polymerase melts DNA strands apart near transcription start site,
forming an "open complex." They seem to insist on calling this (the
unwound region) the "transcription bubble."
o 3: Polymerase catalyzes phosphodiester linkage of two initial rNTPs.
o 4: Polymerase advances 3' to 5' down template strand, melting DNA
and linking rNTPs.
o 5: At transcription stop site, polymerase releases completed RNA and
dissociates from DNA.
o Note that steps 1-3 are called initiation, step 4 is elongation, and
step 5 is called termination.
• Name the four cellular RNA polymerases and their main functions.
o E. coli RNA polymerase: transcribes in E. coli.
o [human] RNA polymerase I: makes ribosomal RNA or rRNA.
o [human] RNA polymerase II: makes messenger RNA (mRNA), small
nuclear RNA (snRNA), and microRNA (miRNA). Note that RPol II has a
C-terminal domain.
o [human] RNA polymerase III: makes primarily tRNA.
• Define a promoter and name sequence elements characteristic of promoters
in human genes.
o A promoter is a sequence of DNA upstream of the transcription start
site that positively affects the expression of the gene.
• The TATA box is a frequently conserved TATA sequence about
30 bases upstream from the start site. Mutations in the TATA
box often result in reduced expression of the gene (ie beta-
thalassemia with B-hemoglobin), probably because the TATA
box binding protein effects assembly of the pre-initiation
complex of general transcription factors at the start site.
• Promoter proximal elements are promoter DNA sequences
between 30-1000 bp upstream of the start site.
 • Enhancer elements are promoter DNA sequences much farther
upstream (10,000-50,000 bp).
• Describe how alpha-amanitin and rifampicin block transcription.
o alpha-amanitin: extremely toxic substance found in death cap
mushrooms. Acts by inhibiting the movement of RNA Pol II, binding its
bridge substructure so that translocation of the polymerase down the
DNA chain can't happen.
o rifampicin: broad-spectrum antibiotic. Acts by binding the beta subunit
of bacterial RNA polymerase, plugging up the exit chamber where
assembled RNA exits the transcriptional complex. Thus elongation is
prevented from going farther than a few base pairs due to having
nowhere to go.
• Name 4 components of the RNA polymerase II pre-initiation complex.
o TFII [transcription factor II] A, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.
o Of these, TFIID and TFIIH are of particular interest:
• TFIID binds the TATA box on the DNA sequence.
• TFIIH facilitates nucleotide excision repair, adds PO4 to C-
terminal domain of Pol II, and act as helicases to open DNA
strands.
• Describe the clinical syndromes caused by mutations in TFIIH subunits.
o Problems with nucleotide excision repair: Xeroderma pigmentosum,
Cockayne's syndrome, Trichothiodystrophy (light sensitivity, abnormal
pigmentation, cancer susceptibility, neuorological abnormalities, etc).
• Describe the three major ways in which most pre-mRNA's are processed.
o Capping: replacement of the 5' triphosphate (from the first rNTP to be
added) with a backwards, 5' to 5' 7-methylguanosine (no phosphate
groups).
o Splicing: excision of introns and desegmentation of exons.
o Cleavage/Polyadenylation: cleavage of RNA at 3' end past the
consensus sequence and polyadenylation (> 200 A's) of cleaved
site.
o Note that these processing steps take place while the RNA is still being
made, not after it's finished and released. For example, the
cleavage/polyadenylation step is partly responsible for Pol II being
released from the DNA.
• Compare and contrast a pre-mRNA with a mature mRNA.
o Pre-mRNA: considerably longer (introns are usually much longer than
exons and there are a lot of them), tri-phopshate group at 5' end, no
poly-A tail at 3' end.
• List the functions of the 5' cap of the mRNA.
o It makes the 5' end resistant to exonucleases (which, if you recall,
target the "lone ends" of single D/RNA strands).
o It helps with splicing and processing through a cap-binding complex
that recognizes the cap.
o Translation factor eIF4E (eukaryotic initiation factor 4E) recognizes the
cap for transport to the ribosomes.
o When the cap is eventually removed, it signals for the mRNA to be
degraded.
• List the three reactions required to add a 5' cap to pre-mRNA.
o (1) Cut off the last PO4 from the triphosphate group at the 5' end of
the mRNA.
 o (2) Add guanosine triphosphate (GTP). It loses two PO4 groups of its
own (making GMP) and forms a 5'-to-5' triphosphate bond (2 PO4
from mRNA, 1 PO4 from GMP) to the end of the mRNA.
o (3) Methylate the 7-position of the guanosine cap with S-adenosyl
methionine (SAM; nearly universal methyl donor in cell; donates
methyl to form homocysteine) to form 7-methyl-guanosine at cap.
• Memorize the conserved sequences at the 5' and 3' ends of most introns and
the consensus sequence at the polyA site.
o Splice site at 5' end of intron: GU.
o Splice site at 3' end of intron: AG.
o Consensus sequence at poly-A site: AAUAAA.
• Describe how alternative splicing permits multiple proteins to be produced by
splicing defects.
o Since the 5' splice site (the one identified by snRNA that recruits the
spliceosome) at any given intron is part of the DNA sequence, it's
vulnerable to being corrupted by mutation and being unrecognizable
as an intron, in which case the finished mRNA has an extra sequence
in it and the final protein winds up significantly different.
o If this question was meant to be how ordinary splicing produces
multiple proteins from one pre-mRNA sequence, essentially various
splicing sites can be turned on or off, allowing mixing and matching
from the various portions of the mRNA as either exons or introns.
• Provide examples of genetic disorders caused by splicing defects.
o Marfan's syndrome: caused by mutations that disrupt splicing of the
fibrillin gene transcript (fibrillin is a connective tissue protein that is
important for the integrity of the walls of the heart and blood vessels).
o Abnormal splicing of CD44 (cell-surface glycoprotein) is a predictor of
tumor metastasis.
• Describe the function of U1 and U2 snRNA's in splicing.
o U1snRNA (also called U1snRNP, or "U1 small nuclear ribonuclear
protein") binds to the GU 5' splice site.
o U2snRNA/U2snRNP binds to the branch point (point on the pre-mRNA
sequence between the 5' and 3' splice sites).
o Essentially the U1snRNP brings the 5' splice site into proximity to the
branch point. The U2snRNP activates the 2' hydroxyl group at the
branch point, which attacks the phosphodiester bond just past the GU
5' splice site.
o Now there's a free 3' OH on the end of the 5' exon, which attacks the
phosphodiester bond at the AG 3' splice site-- linking the two exons
and excising the snRNP/intron complex to be degraded.
• Identify on a diagram of a gene the following:
o a) transcription start site: Look for either the +1 position or the little
bent arrow coming out of the sequence at this point.
o b) introns: Should be between the GU (5') and AG (3') splice sites.
o c) 5' splice sites: Look for GU.
o d) 3' splice sites: Look for AG.
o e) branch points: should be in the introns, between the GU (5') and AG
(3').
o f) exons: should be between the introns. Mark the introns with GU at
5' and AG at 3', then look between them for the exons.
 o g) 5' UTR: look for a region between position +1 (start of transcript)
and the start codon (see below) on the processed mRNA strand.
o h) 3' UTR: look for a region after the stop codon (see below) until the
end of the transcript, including the consensus sequence and the poly-A
tail.
o i) initiation codon: also called start codons. Encodes methionine. Look
for 5' AUG.
o j) termination codon: also called stop codons. Look for 3' UAG, UAA, or
UGA.
o k) poly A site: look for consensus sequence AAUAAA
• Describe the two reactions that make the mature 3' end of mRNA's.
o (1) recognition of consensus sequence at pre-mRNA's 3' end (AAUAAA)
and cleavage of the mRNA soon after this sequence.
o (2) polyadenylation of free hydroxyl at 3' end.
o Notice that the poly-A tail is NOT coded in the DNA of which the mRNA
is a transcript.
• Describe the relationship between 3' end processing of the pre-mRNA and
termination of transcription at the end of a gene.
o The poly-A tail seems to be necessary for the RNA polymerase
complex to detach from the DNA (termination).
• Describe the two major functions of the mRNA's poly A tail.
o Actually, I found four:
o (1) protection from exonucleases
o (2) termination of transcription
o (3) export of mRNA from nucleus
o (4) facilitates translation
• Provide an example of how alternative poly A sites can be used to make more
than one protein from a single gene.
o two different forms of immunoglobulin M (IgM), membrane-bound and
secreted, are formed by alternative poly-A sites in their common gene.

Gene expression: 10/19-22/07

Friday, October 19, 2007
8:07 AM

LO's for gene expression:

• List the different eukaryotic DNA control elements:
o Notice that "DNA control elements" mean transcription-influencing
segments of DNA on or associated with the gene being transcribed.
o 1) TATA box/initiator sequence: usually 25-35 bp upstream from start
site. Determines site of transcription initiation and directs binding of
RNA Pol II. Also the site at which GTPs (general transcription factors,
like TFIID and TFIIH) bind.
o 2) promoter proximal element: usually within 200 bp upstream of start
site, about 20 bp long. Bound by transcription factors to regulate
transcription.
o 3) enhancers: usually much farther upstream, or downstream, than
promoters, although still fairly short in themselves (8-20 bp). Can be
 upstream of the start site, downstream of the last exon, or within
introns in the gene itself. Similar function to promoters.
• Describe a disease that arises from a mutation in a DNA control element, and
how the mutation leads to the disease state.
o Beta-thalassemia (from thalassa, Greek for sea): mild inherited
anemia (low hemoglobin count). Caused, here, by a mutation in the
promoter of the b-globin gene, resulting in lowered rate of production
of b-globin protein.
o Gamma-delta-beta thalassemia: more serious anemia caused by a
deletion in the locus control region for the transcription of all globin
genes, resulting in the loss of globin transcription.
o Hemophilia B Leyden: X-linked disease (usually males) that affects
clotting. Again, a problem in the promoter region of a clotting protein
gene. Tends to get partially better at puberty.
o Fragile-X syndrome: Again, usually a disease of men. Results in
mental retardation and atypical development of the face with enlarged
testicles (macroorchidism). Caused by an expansion in the CGG count
upstream of a particular gene (the FMR1 gene), which results in an
abnormally high rate of methylation in that region and transcriptional
silencing of the gene.
• Describe the role of transcriptional activators and repressors.
o They bind to either the DNA control elements (ie, to the DNA itself) or
to other factors bound to control elements (ie, to other proteins that
are bound to the DNA).
o They increase or decrease the rate of transcription of the gene's
protein(s).
• List the two classes of activators and repressors.
o Those that bind to the control elements in the DNA are called
sequence-specific DNA binding proteins or SSDBPs. They usually
bind to short (6-8 bp) sequences by inserting their alpha-helices into
the major groove of the sequence in question.
o Those that bind to SSDBPs are called co-factors.
• Describe the domains of a sequence specific DNA binding protein.
o Generally two domains. Notice that these domains are modular-- that
is, if you pull one domain off of one protein and attach it to another,
the second protein will have the function of the domain you just
attached.
o First domain: DNA binding domain. Very highly structured
(obviously, since they need to attach to very specific sequences) and
evolutionarily conserved.
o Second domain: activation (or repression) domain. Fairly
unstructured and less conserved; they recruit other proteins (either
co-factors or general transcription factors) to bind and affect
transcription one way or the other.
o Note the difference between co-factors and general transcription
factors: co-factors influence the rate of transcription, while GTFs
provide the pre-initiation complex needed to begin transcription.
• List the four major families of sequence specific DNA binding proteins and
describe the means for categorizing the proteins into these families.
o These are categorized based on tertiary structure differences:
o Homeodomain proteins have a helix-turn-helix structure. They tend
to be regulators of development and affect many genes at once.
 o Zinc-finger proteins have a "finger" made up of two antiparallel beta
sheets and a helix, held together by a zinc ion. This finger is what
binds with the DNA. The largest family of SSDBPs. Include androgen
and estrogen receptors.
• Most common classes of zinc-finger proteins: C2H2, which bind
the zinc ion with two cysteine and two histidine residues, and
C4, which bind it with four cysteine residues.
o Basic leucine zipper proteins (bZIP): (the "basic" here refers to
the fact that they have a high-pH region that binds to the DNA, and
not that they're uncomplicated-- just in case anyone else made that
mistake.) Every 7 amino residues, there's a hydrophobic residue (like
leucine). Often form homodimers to bind DNA.
o Basic helix-loop-helix proteins (bHLH): also has basic region for
DNA binding.
• Describe a particular human disorder that arises from a mutation in a
sequence specific DNA binding protein, explaining how the mutation leads to
the disorder.
o Craniosynostosis: premature closure of the skull sutures in infants.
Arises from a mutation in the homeodomain protein that causes the
protein to bind more strongly, creating a "hypermorphic allele" that
activates genes more strongly than it should.
o Androgen insensitivity syndrome: indifference of androgen receptors to
androgen. Caused by a mutation in the zinc-finger androgen receptor
lessening binding potential of androgen.
o Waardenburg syndrome: deafness, pigmentation defects. Associated
with mutations in the MITF gene (which codes for a bHLH binding
protein responsible for melanocyte development).
• Describe combinatorial control as a mechanism for controlling gene
expression.
o Combinatorial control refers to the fact that SSDBPs can dimerize,
making possible a wide variety of possible DNA binding sequences.
o For example:
• Say you've got two SSDBPs, one that binds to AAAA and
another that binds to GGGG. If they can't dimerize, you can
bind to two sequences: AAAA and GGGG.
• But if they can dimerize, you can bind to four additional
sequences: AAAA--AAAA, AAAA-GGGG, GGGG-AAAA, and
GGGG-GGGG.
• Effectively, given n dimerizing SSDBPs, you can bind to (n2 +
n) combined or single sequences instead of just n sequences.
Allows a much higher degree of specificity in DNA binding
(which, given how many DNA sequences there are in the
genome, is a good thing).
• List the 2 classes of chromatin remodeling factors and briefly describe how
they work.
o DNA-dependent ATPases: these disrupt the histone octamers, opening
up the chromatin and exposing DNA for binding. (cited example is the
SWI/SNF complex)
o Factors that reversibly modify histones through acetylation:
• Can either be acetylators (histone acetyltransferases, or
HATs) or de-acetylators (histone deacetylators, or HDACs).
 • Acetylation is associated with increased transcription of the
DNA on the affected histone; deacetylation is associated with
the reverse.
• Current theory is that HATs' pattern of histone acetylation
recruits co-factors to effect increased transcription rather than
directly affecting it themselves.
• Define HATs and HDACs and describe how their activity influences
transcription.
o See above.
• Give an example of a disease in which histone acetylation is altered, and
describe the defect that leads to altered histone acetylation.
o Leukemia: haematopoietic (formation of blood cell components)
disease involving chromosomal translocations over-activating fusion
proteins that alter the activity of HATs or HDACs.
o Rubinstein-Taybi syndrome: growth and mental retardation, broad
thumbs and toes, craniofacial dysmorphism. Results from mutations in
one copy of CREB binding protein gene; CBP is a widespread HAT
important in development and its insufficiency has particularly drastic
effects.
• Describe how activators/repressors modulate transcription via their
interaction with general transcriptional machinery vs. with chromatin.
o Activators/Repressors can either (1) bind to GTFs or the RNA Pol II
complex to influence initiation or elongation of the primary transcript,
or (2) interact with chromatin to regulate the accessibility of the DNA
to the Pol II transcription apparatus.
• Discuss the basic principles of transcriptional regulation including how
specificity is achieved and how protein-DNA interactions contribute to
transcriptional control.
o (1) Specificity depends on binding of transcriptional
activators/repressors to DNA control elements.
o (2) Regulation depends on DNA-protein and protein-protein
interactions
o (3) The interactions affect the conformation of DNA, modification of
chromatin structure, and formation of the transcription initiation
complex.
o (4) Control elements are combinatorial, which allows for thousands of
transcriptional activators/repressors to alter the expression of various
genes in response to varying stimuli.

(10/22/07)
• List at least 4 mechanisms by which sequence specific DNA binding proteins
are regulated.
o (1) Alter the conformation of the protein with ligand binding
o (2) Regulate entry into the nucleus (regulate access to DNA)
o (3) Regulate amount of protein in the cell
o (4) Regulate DNA binding action of protein
o (5) Post-translationally modify protein (ie PO4ation)
• Describe how the activity of nuclear hormone receptors is controlled, and how
tamoxifen acts in breast cancer therapy.
o Hormone receptors (many of which, remember, have zinc-finger DNA
binding domains) are activated by (big surprise) hormones, which act
as ligands to induce a conformational change that allows the receptors
 to dimerize (which is required for them to bind to the DNA control
elements).
o Tamoxifen acts as an antagonist to estrogen, binding to the estrogen
receptors as a ligand without providing dimerization-- thus effectively
preventing estrogen from binding to its site of action and preventing
the transcriptional effect of estrogen receptors.
• Give an example of a sequence specific DNA binding protein regulated by
nuclear entry and describe the mechanism by which its entry is controlled.
o NF-κB: normally bound to IκB, which holds it in the cytoplasm (and
away from genetic material). Under certain conditions, IκB is
phosphorylated, which targets it for degradation. Degrading IκB allows
NF-κB to migrate into the nucleus and affect transcription. (This is one
of the pathways by which aspirin acts as an anti-inflammative and one
of the reasons low-dose aspirin is given to prevent buildup of
atherosclerotic plaque).
• Describe how the amount of an activator/repressor can be regulated within
the cell.
o Specific genes can target activators or repressors for degradation (like
APC targets beta-catenin, a cell proliferation activator protein). By
regulating the amount of the targeting genes (like APC), the amount of
activator/repressor (B-catenin) can be regulated.
o Clinical fact: ~50% of colon polyps observed in the clinic are caused
by mutations in the APC gene, resulting in insufficient degradation of
(and thus proliferation of) B-catenin.
• Describe a mechanism by which the DNA binding activity of a sequence
specific DNA binding protein can be inhibited.
o Id proteins have a helix-loop-helix (HLH) domain, but no basic
domain-- recall that the basic domain is what allows HLH proteins to
bind to DNA, while the HLH domain allows dimerization of these
proteins. Recall also that bHLH proteins usually dimerize to be able to
bind to DNA. By keeping a store of non-genetically active half-dimers
(ie. Id proteins, HLH) in the cell, a bunch of otherwise active half-
dimers (regular ol' bHLH proteins) are kept in a ready but inactive
state.
o My point here is that this is a better solution than just degrading the
SSDBPs because it allows the cell to keep around a store of SSDBPs all
the time without actually having to have them active. The fact that
they need dimerization is kind of an "on/off switch" that allows the cell
to keep them bound up and inactive when not needed and to remove
the Id proteins and turn them all "on" at once for quick response to
cellular stimuli (rather than having to slowly build them all from
scratch).
• List a protein modification that can alter the activity of a sequence specific
DNA binding protein, and explain the mechanism by which the activity is
altered.
o PO4ation (phosphorylation) can be used to activate CREB (cyclic AMP-
responsive element binding protein), a SSDBP that recruits HATs to
affect histone acetylation and gene transcription. When not PO4'd, it
can't recruit.
• Aside from transcriptional regulation, list at least 3 additional mechanisms to
control levels of gene expression.
o Control of mRNA export from nucleus
 o Control of mRNA degradation (ie small interfering RNA action)
o Control of efficiency of translation (ie next lecture IRE/IRP at 5' UTR)
o Control of protein degradation (ie ubiquitination)

Translation: 10/22/07
Monday, October 22, 2007
2:10 PM

LO's for Translation:

(be warned that these LO's were written poorly and cover a lot of ground. The
following, then, is kind of spotty depending on how much effort I felt like putting into
it.)

• Understand the steps in the initiation of protein synthesis in prokaryotes and

eukaryotes and the differences between prokaryotic and eukaryotic
translation initiation:
o Prokaryotic initiation of translation: dependent on a specific RNA
sequence called the Shine-Dalgarno sequence (8-13 bp). S-D
sequence binds to 30S subunit of prokaryotic ribosome.
o Notice that the S-D sequence can be at multiple sites on the gene,
allowing one transcript to code for multiple proteins (ie
polycistronic).
o In contrast, eukaryotic mRNA usually only encodes one protein.
o Eukaryotic initiation is complicated and irritating and relies on multiple
initiation factors assembling a translation complex that includes the
ribosome:
• eIF-4E (cap-binding protein) binds to 5' cap.
• eIF-4G (scaffolding protein) is recruited by eIF-4E.
• eIF-3, which binds to the 40S ribosome.
• eIF-2, which binds to a tRNA charged with methionine.
• eIF-4A, a helicase to melt any RNA structure encountered.
o After the initiation complex and ribosome are assembled, it "scans"
down the mRNA until it finds the start codon.
o Notice that the 5' UTR can influence the rate of this process or can
stop it entirely, depending on how convoluted it is (remember that the
ribosome complex has to scan through the 5' UTR to get to the AUG
start codon).
o Notice that the start codon, AUG, means that the first AA on the
translation is Met.
• Understand the mechanism by which eukaryotic cap-dependent translation is
inhibited by certain viruses and how these viruses initiate translation:
o Certain viruses (rhinovirus, poliovirus, etc) synthesize a protease that
cleaves eIF-4E so that it can't bind to eIF-4G and the rest of the
complex.
o Instead of using the eukaryotic initiation machinery, these viruses
recruit the ribosome to areas of the 5' UTR called Internal Ribosomal
Entry Sites (IRESes).
• Understand the general characteristics of the genetic code, including the start
and stop codons:
o mRNA structure: 5' cap, 5' UTR, start codon (AUG), open reading
frames, stop codon (UAG, UAA, or UGA), 3' UTR, poly-A tail.
• Understand the role of tRNA, the process of charging tRNA and the concept of
"humanizing" a protein at the level of translation:
o tRNA: brings in amino acid residues to be added to the polypeptide
chain.
o Amino acid residues are "activated" by adenylation prior to being
attached to a tRNA. Once a tRNA has a residue attached to it, it's
"charged" and can be used to transfer that residue onto the
polypeptide chain.
o "Humanizing" a protein: essentially, various codon-AA tRNAs are found
in varying concentrations in various organisms. If a given organism
has more XXX tRNAs coding for tyrosine (say) than XXY tRNAs coding
for tyrosine, and if a given protein needs a tyrosine at a particular
place, you may want to change the mRNA code to use a different
codon for the same amino acid residue. If you don't, you may rate-
limit the translation due to the low concentration of the specific codon-
AA tRNA in the organism you're translating in.
o (this last part is generally important when you're trying to make one
species' protein in another species-- like using bacteria to make
human proteins. Bacteria may normally use XXX to get tyrosine and
humans may normally use XXY. If you want to use a human mRNA (or
gene) to make protein in bacteria, you probably want to change it to
read XXX instead of XXY so as not to slow down your translation.)
• Understand the mechanism of peptide bond formation and the processes of
elongation and termination:
o Elongation:
• 3 sites on ribosomes: A, P, E.
• Methionine begins attached to the tRNA at the P site.
• The next tRNA comes in and attaches at the A site.
• The Met residue is transferred to the residue on the A site.
• The "uncharged" tRNA moves to E site where it can be
dislodged.
• The polypeptide-bound tRNA moves to the P site and another
charged tRNA comes in at the A site.
• Repeat.
o Peptide bonds are formed by the nucleophilic attack of the A site AA's
amino nitrogen on the end carbonyl carbon of the P site polypeptide.
o Termination: ribosome reads a codon for which there is no tRNA
match; it then binds a releasing factor that hydrolyzes the peptide-
tRNA bond and dissociates the ribosome complex.
• Know the various kinds of mutations that affect translation:
o Point mutations, or changes in a single base pair:
• nonsense mutations: Result in a stop codon where there
should be an AA-coding codon. This creates truncated proteins,
either nonfunctional or dysfunctional, that are usually quickly
degraded by the cell.
• missense mutations: Result in a different AA from the
original being encoded by mRNA. Can create a protein with
different folding and binding characteristics.
 • silent mutations: Result in no change at the protein level. Eg.
a mutation from one codon coding for tyrosine to another codon
coding for tyrosine.
o Deletion mutations, or deletions of one or more base pairs:
• Major problem here is a frame shift. If you're reading codons
three at a time, and one base pair gets deleted, it throws off
the correct "decoding" of the entire gene after that point. This
pretty much FUBARs your protein.
• Note that if you deleted three base pairs at once, you would
have no frame shift but would lose an amino acid residue from
the final protein.
• Understand the mechanisms by which antibiotics inhibit protein synthesis:
o Often they target the ribosome assemblage itself. If the two subunits
of the ribosome (30S and 50S in bacteria) can't bind together, or if the
smaller subunit can't bind to the Shine-Dalgarno sequence, bacterial
translation can't occur. They can also target the 3 sites (A, P, E) of the
assembled ribosome complex.
o Notice that you can inhibit translation in eukaryotes by sequestering
eIF-4E using eIF-4E binding proteins. Or, given the convoluted
inhibition logic of cells, you can inhibit the inhibitor of the binding
proteins (thus inhibiting the inhibitor of the sequesterer of eIF-4E,
resulting in sequestration). This is how rapamycin works.
• Understand how fluctuating intracellular iron concentrations can alter
translation of specific mRNAs:
o This was a long, meandering aside about a class of mRNA domains
called iron response elements (IREs) that are found on mRNAs
encoding proteins involved in iron metabolism. The proteins that bind
to these IREs are called IRPs (iron-response binding proteins).
o IREs can be found in either the 5' UTR or the 3' UTR of mRNAs and
give the intracellular concentration of iron a way to affect the
translation of these mRNAs.
o In the 3' UTR, IPEs affect stability. When there's a IRP bound to the
mRNA, it's stable. When not, it tends to be degraded. When the iron
concentration in the cell increases, the iron preferentially binds the IRP
instead, and the mRNAs degrade before translation.
o In the 5' UTR, IPEs affect translation rate. Recall that the ribosome
complex has to travel through the 5' UTR before it finds the start
(AUG) codon and begins translation. If there's a IRP bound to the 5'
UTR, the translation machinery can't move through it (or moves much
more slowly through it). If iron levels are high, the IRPs are bound to
iron and mRNA translation goes forward. If not, the IRPs are bound to
the 5' UTR and translation slows or stops.
o To sum: for IRPs at the 5' end, high iron results in no translation. For
IRPs at the 3' end, low iron results in no translation.
• Understand how interferon inhibits viral protein synthesis:
o Interferon synthesizes two proteins in cells near the one under viral
attack:
• One synthesizes an enzyme that activates a protease which
cleaves viral mRNA, preventing translation.
• The other phosphorylates eIF-2, inactivating it. This means that
no initial methionine tRNA can be attracted to begin translation,
which means no proteins can be translated.
Primary and Secondary Protein Structure: 10/22-23/07
Monday, October 22, 2007
2:20 PM

LO's for Primary and Secondary Protein Structure, 10/22-23/07:

• Identify alpha carbon, the NH2, COOH, and side chains (R groups) of an
amino acid.
o alpha carbon sits between the others. Shouldn't be too hard to identify
the rest.
• Distinguish between amino acids with hydrophobic, polar, acidic and basic
side chains.
o Groups discussed in class:
• Aliphatic R groups: ALIGVM (alanine, leucine, isoleucine,
glycine, valine, methionine)
• Aromatic R groups: FWY (phenylalanine, tyrosine, tryptophan)
• Polar uncharged groups: SCTPQN (serine, cysteine, threonine,
proline, asparagine, glutamine)
• Polar positively charged groups: HRK (histidine, arginine,
lysine)
• Polar negatively charged groups: DE (aspartate, glutamate)
o Presumably one could make the argument that aliphatic and aromatic
R groups are "hydrophobic," the other three are "polar," and that the
last two are the basic and acidic side chains.
• Distinguish three covalent bonds including the peptide bonds that make the
backbone of a polypeptide chain.
o Peptide bond (C1-N): formed from dehydration reaction of COO- and
NH3+. Stabilized by partial double bond character from proximal
carboxyl group, also rigidified there so that no rotation around that
bond is possible.
o Bond from carbonyl-side alpha carbon to carbon of peptide bond (C2-
C1). Free rotation.
o Bond from nitrogen of peptide bond to amine-side alpha carbon (N-
C3). Free rotation.
• Understand the function of disulphide bonds within proteins.
o S-S bonds (formed from two proximal cysteine residues-- recall that
cysteine's the only side chain with a free sulfhydryl group) stabilize
both intra-protein (tertiary) and inter-protein (quaternary) structure.
o Examples are insulin (A and B) and the keratin in hair.
• Describe the major post-translation covalent modifications of amino acid side
chains in proteins.
o Hydroxylation
• Hydroxylation of proline residues in collagen, mediated by
vitamin C, is required to prevent scurvy.
o Carboxylation
• Carboxylation of glutamate residues, mediated by vitamin K, is
required for effective blood clotting.
 • Notice that warfarin targets the carboxyl group of glutamate
residues and acts as an anticoagulant.
o Glycosylation
o Acetylation
• Acetylation and de-acetylation of histones in gene regulation
o Methylation
o Phosphorylation
o Ubiquitination
• Used for targeting proteins for degradation by the proteasome.
• Describe hydrogen bonds and their role in secondary structure formation.
o Weak, but numerous, bonds between a hydrogen atom and an
electronegative atom such as oxygen. The former is the "donor" of
electron density; the latter is the "acceptor" of electron density.
o "Driving force to form 2o structures." -- on alpha-helices, the H-bonds
between every
o Here, concentrating mainly on the hydrogen bonds between the
backbone carbonyl oxygen atom (acceptor) and the backbone amine
hydrogen (donor).
• Describe two major types of protein secondary structures.
o alpha-helix: ~30% of all protein structures
• Note that all biological alpha-helices are right-handed.
• All side chains point towards the outside of helix.
• Every five residues there's a H-bond between the carbonyl O
and amine H.
o beta-sheet: ~30% of all protein structures
• The parallel or anti-parallel conformations are held together by
H-bonds (anti-parallel more common biologically).
o Notice that the primary structure of the protein determines its
secondary structure-- different side groups influence the formation of
different patterns of H-bonding.
o There's also turns and loops to compact proteins (mainly glycine,
because it's small and flexible, and proline, because it's slightly
kinked).
o Notice there's a triple helix structure in collagen: different
conformation from either alpha-helices or beta-sheets. Has a lot of
glycine and proline, like turns or loops, but the proline residues are
often hydroxylated (via vitamin C), resulting in massive H-bonding
between strands to form the characteristic triple helix.
• Every three residues there's a glycine residue (in order to
compact the helix).
• Describe the major approaches to purifying a protein.
o According to size by gel filtration chromatography:
• Have proteins run through a filtration column; large proteins
flows through quickly, small proteins more slowly.
o According to charge by ion exchange chromatography:
• Have proteins run through a cation exchange column filled with
negatively charged beads; positively charged proteins stick and
can be eluted off with salts (more slowly), negatively charged
proteins flow directly through.
o According to ligand binding properties by affinity chromatography:
 • Attach a given ligand to beads of column (ie glucose)-- run
protein solution through column and only the ligand-binding
proteins should stick. Then elute the proteins off the beads into
a separate container
• Ligands: enzyme substrates, DNA, metal ions, carbohydrates,
peptides, etc.
• [Not required but handy:]
o Electrophoresis: determine size of protein.
• Use a polymerized acrylamide gel to separate based on size.
Electric field causes denatured, negatively charged proteins to
run down gel at a rate dependent on size.
• Also need to run a "marker" of known molecular weight to give
a "size ladder" to determine the approximate size of the protein
under investigation.
o Mass spectrometry: determine sequence of (unknown) protein by
molecular mass.
o Edman Degradation: Label and remove the N-terminal amino acids one
at a time and identify.
o Western Blots: use immunology to identify proteins on an acrylamide
gel.
• Transfer proteins onto a membrane, react with primary
antibody for a particular protein, wash off anything unbound,
react with fluorescent secondary antibody to detect.
• One use is to identify HIV infection: use patient's serum as
primary antibody (checking for HIV antibodies in serum)
against HIV proteins.
• Describe the basis for how protein structure determines function and how
genetic mutation can affect protein function.
o Depending on the specific assortment and order of side chain from the
peptide backbone, the pattern of hydrogen- and sulfur-bonds in a
protein will be very different. Collagen is a good example, as without
the proline residues to be hydroxylated, the strong H-bonds holding it
together couldn't happen. Keratin is another example-- without being
cysteine-rich, the multiple disulfide bonds that give it its tensile
strength would be absent.
o Re mutations: if the ratio of glycine residues is messed with in collagen
(thus deforming the tight helix structure), it results in a fatal condition
in infants.

Tertiary and Quaternary Protein Structure: 10/23/07

Tuesday, October 23, 2007
9:00 AM

LO's for tertiary and quaternary protein structure, 10/23/07:

• Know the role of protein loops in the structure and function of proteins
o (1) Fold the protein back on itself (compaction)
o (2) Hold the protein in the correct structure and primary AA sequence
for binding. Ie: variation in the six loops of antibodies is what allows
variability in binding antigens.
• Know the reasons why drugs are more often used to inhibit the interactions of
small molecules with proteins rather than protein interactions with other
proteins
o Protein-protein interactions tend to occur over large areas of the
protein(s). Thus it's tough to inhibit efficiently-- but protein-small
molecule interaction occurs with a very punctiliar character and can be
targeted efficiently.
o Also, the primary AA sequence of small ligand-binding domains can be
studied and drugs designed to interact specifically with that area and
no others (the larger the surface area of affect, the larger the chance
of unintended interactions).
o Usually antibodies are used to deal with protein-protein interactions.
• Know the five different ways in which proteins are modified after synthesis
o Disulfide bonds can be formed and modified with protein disulfide
isomerases.
o Prolyl isomerase catalyze rotation of proline residues from cis to trans
or vice versa.
o Restricted cleaving by proteases can "activate" various proteins
(cleave off the portion that keeps it inactive).
o Glycosylation of secreted and cell surface proteins
• N-linked: added to asparagine (-X-N-X-S/T-)
• O-linked: added to serine or threonine
o Various other non-genetically coded factors: metal ions, vitamins, etc.
• [Not required but helpful:]
o "Induced fit": change in tertiary structure (shape) of protein when
substrate is bound.
o "Molten globule form": unstable tertiary/secondary structure passed
through in a protein refolding to a different form.
o Often multiple configurations are at relative equilibrium in a cell; given
substrates can bind to one form, but not the other. Certain substrates
bind to one form and make it favorable for that form to change to the
other form.
• allostery: a change in conformation of one domain or subunit
triggers a change in conformation of other subunits in the same
protein. (ex. hemoglobin, a tetramer in which one subunit
shifting from T to R (O2-receptive) forms (or vice versa)
facilitates the conversion of the other three.)
• Know the features which control the tertiary structure of proteins:
o N-terminus of protein synthesized first, meaning that the AA's at that
end have an opportunity to fold first (affects overall folding and
refolding after denaturation).
o Interactions between adjacent AA's in secondary structures
o Hydrophobic AA's tend to cluster in the cores of proteins (vs.
hydrophilic AA's).
o Various proteins assist with "chaperone" activity in folding. (about
30% of all eukaryotic proteins are folded by chaperones.)
• Know the names and general functions of 4 of the chaperone proteins
involved in protein refolding:
o Heat shock proteins: induced by brief exposure to high temperature.
Bind to hydrophobic regions in non-native or denatured prots to
prevent "irreversible aggregation." Use ATP.
 o Chaperonin complex: 7 distinct subunits. Bind hydrophobic regions of
unfolded proteins, use ATP to encapsulate and correctly fold the
protein.
o Disulfide exchange proteins: form/reform disulfide bonds in proteins.
o Prolyl isomerases: alter configuration of proline residues in proteins.

Enzyme Kinetics: 10/24-25/07

Thursday, October 25, 2007
8:04 AM

LO's for Enzyme kinetics, 10/24-25/07:

• Be able to describe what an enzyme is, the characteristics of enzymes, and
thermodynamically how they increase the rate of a reaction. Be sure to
understand the terms: catalyst, activation energy, free energy of the
reaction.
o Activation energy: dictates the rate of the reaction (no change in
equilibrium of rxn).
o Free energy: dictates the spontaneity of the reaction
• Be able to describe how enzymes work, using the terms: binding of the
transition state, induced fit, covalent chemistry, metal ion chemistry, general
acid-base chemistry.
o What you're essentially talking about is the following equation: E + S
-> ES -> EP -> E + P, where E = enzyme, S = substrate, and P =
product.
o So you want the enzyme, when it binds to the substrate, to change
something about that bond that makes it favorable for the substrate to
change conformation to the product. The thing that gets changed is
generally the conformation of the enzyme. The enzyme bends in a
particular way that forms a lot of weak covalent bonds (covalent
chemistry) with the substrate (thus energetically favorable). But that
bending also puts pressure on the substrate to change to the product,
made easier by the favorable energy of all those weak covalent bonds.
This, I think, is what's meant by "binding of the transition state"-- the
fact that multiple bonds are formed from the enzyme to the substrate
that makes the transition state (the energetic "bump" between
substrate and product) easier to get through.
o Induced fit refers to the fact that the enzyme usually has to change
conformation to bind the substrate to its active site-- this is good,
since you don't want the ES complex to be a stable one. Basically the
point is that the "lock and key" enzyme model isn't accurate- the
enzyme doesn't have a big substrate-shaped hole in it.
o Where metal ion chemistry comes in is in the fact that often enzymes
need metal ions (also called cofactors, see below) to induce their
conformations into a shape to be able to accept substrate.
o Acid-base chemistry: this refers to the fact that the enzyme can
sometimes create a local environment with a higher or lower pH than
the cellular environment by either temporarily donating or receiving
protons. Although the enzyme doesn't wind up permanently altered
(its gives or takes the proton back), its ability to temporarily "lend" a
 different pH to the substrate helps ease the changeover to the
product.
• Define the terms cofactor and coenzyme.
o cofactor: Metal ions required for enzymatic activity.
o coenzyme: Another enzyme that binds to the first enzyme and allows
it to act on the substrate.
o Both of these are often derived from nutritional (ingested) materials.
o Some terms associated with this:
• A group that's covalently linked to the enzyme is a prosthetic
group.
• The complex of a cofactor or coenzyme with an enzyme is a
holoenzyme. (ie "whole enzyme")
• The enzyme dissociated from its cofactors or coenzymes is an
apoenzyme.
• Understand the significance of the terms Km and Kcat (know what it means if
one enzyme has a lower or high Km than another, etc.). Be able to estimate
the value of Km from a graph of reaction velocity versus [S].
o Km:
• Km is the substrate concentration at which the 'enzyme
velocity' of the reaction is 1/2 the maximum velocity of the
enzyme.
• Generally, enzymes in biological systems tend to operate
around Vm concentrations.
• If an enzyme has a lower or higher Km than another, that
means it probably needs a higher or lower concentration,
respectively, of substrate to operate in the cell.
• Lineweaver-Burke plot: a way to empirically figure out Km and
Vmax.
o kcat:
• kcat : "turnover number", a rate constant for a given enzyme of
how many substrate molecules are converted to product in a
given unit of time by a molecule of that enzyme under
saturated conditions. (ie when there's a surplus of substrate).
• Effectively this is a kind of measure of efficiency of the enzyme.
o So Km vs kcat: Km tells you at what concentration of substrate the
enzyme starts to work, and kcat tells you how quickly the enzyme
turns substrate into product when it does start to get going.
o Smaller Km: less substrate needed to begin. Bigger kcat: faster action
of enzyme once started. Thus a large kcat/Km is indicative of a highly
active, highly efficient molecule.
• Describe 4 different types of inhibitors and, in general, how each works
(competitive, uncompetitive, mixed, irreversible).
o Reversible inhibitors: Binds to enzyme and decreases enzyme function
until released. Sort of like a ball and chain around the enzyme's leg-
slows it down until taken off.
• Competitive inhibitor: inhibitor goes into the substrate binding
site. Means it has to compete for the binding site with the
normal substrates.
• Uncompetitive inhibitor: inhibitor binds somewhere other than
the binding site. Means it doesn't have to compete with normal
substrates for binding.
• Mixed inhibitor: inhibitor does a combination of both.
 • Basically, you get different kinds of Lineweaver-Burke plots for
these different types of reversible inhibitors. So you can figure
out experimentally what kind of inhibitor you're looking at by
using these plots.
o Irreversible inhibitors: Binds to enzyme and changes something
fundamental about the enzyme that prevents it from working again,
even if the inhibitor is removed. More like a bullet to the enzyme's
head-- even if you take the bullet out, the outcome is generally the
same.
• Ex.: Penicillin irreversibly inhibits enzyme responsible for
polymerizing peptidoglycans in bacterial cell walls. Goes into
active site and forms a covalent, irreversible complex with
enzyme.
• Describe 4 types of enzyme regulation mechanisms and, in general, how each
functions (allosteric, covalent modification, binding of another protein,
proteolytic cleavage).
o Basic principles: generally the first enzyme in a long regulatory
pathway is the one most highly regulated (since that one controls all
the rest of them).
o Allosteric regulation: Some positive or negative modulating molecule
binds to the enzyme and causes a conformational change to allow or
disallow the action of the enzyme on the substrate.
• Sometimes this is involved in biofeedback loops where the end
product of the pathway is the allosteric inhibitor of the first
enzyme in the pathway.
o Covalent modification of the enzyme: Enzymes can be modified by
other enzymes to be turned on or off.
• Many enzymes have phosphorylation on/off switches.
• The enzymes that modify enzymes to turn them on or off are
usually called kinases.
• Also a variety of other modifications: adenylations,
methylations, etc, etc.
o Regulatory protein binding: Some enzymes are bound to by proteins
or peptides to activate or inactivate them.
o Activation of enzyme by proteolytically cleaving it: Some enzymes are
inactive until cleaved by a particular enzyme (ie trypsinogen doesn't
become active until it gets to the duodenum and is cleaved to trypsin).

Mitosis and Cell Cycle: 10/25/07

Thursday, October 25, 2007
8:05 AM

LO's for Mitosis and cell cycle, 10/25/07:

• Understand how cells regulate their size by coordinating growth with division
at the restriction point ("R") in G1 phase.
o In somatic cells, there's a "R point," or restriction point, at which the
cell has to make a decision - based on whether or not various
hormones or growth factors are there, as well as whether the cell is
large enough - whether or not to undergo replication and mitosis.
 o "R" point = first (thus most highly regulated) step in cell cycle
pathway.
o Not much to understand, at least from lecture. If the cell is too small,
it can "divide itself out of existence"- so it needs to be big enough to
divide sustainably, thus the need for a size check.
• Know that the main goal of the somatic cell cycle is to ensure exact
duplication of the genome in S phase followed by exact of division of the
genome in M phase to produce identical daughter cells.
o Ok.
• Know how cells prevent re-replication of their genomes by keeping the
assembly and activation of replication complexes in separate cell cycle
phases.
o Orc proteins [Origin replication complexes] initiate replication by
binding to the DNA origin and binding to it; other proteins complex
with Orcs to begin the process (DNA helicases, etc).
o The assembled Orc-protein-helicase complex = the pre-replication
complex (pre-RC). Required for replication.
o However, an assembled pre-RC is not the same as an activated pre-
RC.
o The pre-RCs are assembled in the G1 phase all over the genome.
o They're only activated in the S phase by high levels of CDKs. If the cell
doesn't make it to the S phase, no activation takes place.
o Notice (pertinent to the LO) that those high levels of CDK restrict the
assembly of more pre-RCs. So the same switch turns on the
assembled replication complexes but turns off the assembly process
that makes more of those complexes.
o This ensures no re-replication takes place-- the high levels of CDK are
what allow the cell to move into the activation phase (S), but it's also
what ensures no additional replication will take place beyond what's
already been put in place in the G1 phase.
o In short: pre-RCs assembled (but not activated) during G1, and
activated (but not assembled) during S, driven respectively by the low
and high concentrations of CDK in the cell.
• Know that genomic instability either by chromosome re-replication in S phase
or mis-segregation during mitosis produces human disease such as cancer
and birth defects (Trisomy 21).
o Essentially your "quality control" breaks down, creating a lot of
proliferated cells with serious problems or infidelities in them.
• Compare the cell cycle of somatic cells (mitosis) with that of germ line cells
(meiosis), which produces haploid gametes.
o Point of mitosis: 2 identical diploid cells from 1 diploid cell.
o Point of meiosis: 4 different haploid cells from 1 diploid cell.
• Start: chromosome A and chromosome a.
• 1st: replication of chromosomes (AA, aa)
• 2nd: homologous recombination of chromosomes to provide
genetic variation
• 3rd: separate homologues (diploid to diploid, or meiosis I) (one
cell: AA; another cell: aa).
• 4th: separate chromosomes of daughter cells (diploid to
haploid, or meiosis II) (two cells: A, two cells: a).
• Know that differentiated, post-mitotic cells such as neurons are stuck at the
"R" point in that they continue to grow without cycling.
 o Ok.
• Know how alterations in many cell cycle regulators that are found in cancer
cells are being used for patient diagnosis and prognosis.
o CDKs [cyclin-dependant kinases] : 9 enzymes central to regulating the
cell cycle.
o Active CDKs are produced by growth factor hormones (and cancer
factor proteins) and result in cell replication and duplication.
o Particular CDKs tend to be found at particular levels in various types of
cells. So if an elevated level of a given CDK is found in a tumor, that's
an indication of active proliferation.
o What CDKs do is to inhibit the inhibitors of the cell cycle (RB, or
retinoblastoma proteins).
o So can also look for abnormally low levels of RB, or highly
phosphorylated (inactivated) RB as a cancer marker.
o Notice that a couple of other kinase proteins are required to actually
begin replicating the DNA and also to initiate mitosis. These are
activated by separate pathways but still dependant on CDKs.
o Presumably, can look for levels of these other proteins in tumors as
well.
o There's also a family of proteins called CDIs [CDK inhibitors] that turn
off CDKs (thus repressing the cell cycle]. Mutations in these proteins
often turn off the ability to turn off a given CDK, thus result in
uncontrolled proliferation.
o Most frequently seen in cancer cells: mutation in Ink4 (inhibitor of
CDK4) CDI family that lets CDK4 push past the "R" point regardless of
other growth factors.
o So can look for mutations in CDI genes as a cancer marker as well.
o mitogen: signal protein that leads to activation of CDK4. Notice that
this leads to a positive feedback cycle which leads to the irreversible
"commitment" to completing the cell cycle.
• Understand the importance and the mechanism of cell cycle checkpoints in
maintaining genomic stability.
o Checkpoint at G1: Is the cell large enough?
o Checkpoint at S: Is the DNA replicating faithfully?
o Checkpoint at G2: Are there now two copies of every chromosome?
o Checkpoint at M: Did the spindles form and act as they should?
o Failing any of these checkpoints results in a cessation of the cell cycle
(cell stays in phase it's in when the checkpoint's failed) until the
damage is repaired or the checkpoint is passed. Notice that you can
repair DNA in any phase.
o If the damage can't be repaired, have apoptotic signaling.
o Mechanism: usually through sensor proteins transducing activity in
effector proteins (like inhibitors of CDK) that halt the cell cycle or
induce apoptosis.
o Essentially, if the genome becomes "unstable" (has infidelity of
information) in certain cells but is still able to replicate itself, there's a
whole world of genetic hurt that it can inflict (of which cancer is a
good, representative example).
o Notice that most cancer is not inherited unless the mutation occurs in
a germ cell.
Clinical vignettes:
Thursday, October 25, 2007
11:04 AM

LO's for clinical vignettes, such as they are:

Alzheimer's 10/23/07:
• Seems to be caused by incorrectly cleaved Alzheimer's
precursor proteins (APPs).
• APPs are membrane-bound proteins; the incorrect cleavage
comes while it's still in the membrane and is being excised out of it.
• Pathogenic forms of cleaved APP protein: AP40 and AP42.
• Pathway: beta-enzyme cleaves too small a portion from the
APP. Then the gamma- enzyme cleaves the membrane-binding portion
of the APP and it's released from the membrane.
• Primary pathophysiological cause of symptoms: amyloid
plaques (agglomerations of incorrectly cleaved AP proteins), which
lead secondarily to tangles inside nearby cells.

Prion disease 10/24/07:

• Examples: bovine spongiform encephalopathy (mad cow
disease), scrapies in sheep, Creuzfeld-Jacob disease in humans, some
in deer and elk as well.
• Caused by a misfolded (usually beta-sheet-rich) protein that
induces other, similar proteins nearby to adopt the same shape. These
misshapen proteins often agglomerate into clumps or plaques (due to
increased hydrophobicity of beta-sheet-rich content?).
• Can be cross-species, but only if the protein from the original
species is similar enough to the new species to induce the same
conformational change.

Li-Fraumeni Syndrome 10/25/07:

• LO's for LF:
• (1) describe criteria for classifying hereditary cancer as LF
• [proband: patient reported, diagnosed, or treated]
• sarcoma in proband before 45 years of age
• 1st degree relative with any cancer under 45 years of age
• 1st or 2nd degree relative with any cancer under 45 years of
age or a sarcoma at any age
• (2) describe Knudson "two-hit" hypothesis
• One p53 allele is mutated already at birth. The other needs to
get knocked out at some point along the way to cause the
cancer.
• Notice that mutation in another gene, instead of the p53 gene,
may be enough to cause cancer as well.
• (3) Describe the function of p53 in response to UV exposure.
• p53 protects from the damaging effects of radiation exposure;
thus a mutation in p53 can lead to susceptibility to radiation
damage (which is also why p53 mutation-caused cancers aren't
treated with cancer radiation therapy).
 • Notice that p53 arrests the cell cycle to allow DNA to repair
itself.
• Notice that p53 also causes apoptosis if excess DNA damage.
• LF:
• rare autosomally inherited cancer susceptibility
• large age range of presentation
• must have more than one family member with cancer to diagnose
• over 250 mutations in p53 gene, which is the predominant gene assoc.
with LF
• often test "hot spots" in p53 gene (in DNA binding domains) rather
than all 2300 bps
• can be sequestered in cell by mdm2 protein until needed

Tools of molecular biology: 10/26/07

Thursday, October 25, 2007
11:19 AM

LO's for Tools of molecular biology, 10/26/07:

(notice these are a little wooden, since I didn't actually attend the lectures in
question and answered the LO's from the printed notes.)
• Write out any three double stranded DNA sequences that are likely to be cut
by restriction endonucleases, i.e. palindromic (a known restriction enzyme
does not have to exist for your sequences).
o Palindromic usually means they read the same forwards and
backwards (ie "A man, a plan, a canal, Panama"). Here it seems to
mean that the same sequence, read 5' to 3' on the two
complementary strands (NOT on the same strand) will read the same.
o Here's our sequence: 5' TACGTA 3'. This implies its complementary
sequence: 3' ATGCAT 5'. If you read the 5' to 3' sequence on each, it's
the same. Note that TACGTA is not a classical palindrome (it doesn't,
by itself, read the same backwards) so don't get confused.
o Some other sequence examples:
• TTTAAA
• TCCGGA
• CTATAG
• Etc.
• Explain the principle of electrophoretic separation of DNA to a premed
student.
o Listen up. You want a method to separate proteins by size. You do it
with a polarized electrical field in a gel that will exert a uniform
electromotive force on all the proteins in that gel to move down its
gradient.
o The gel is thick but porous, meaning that small proteins will move
through it faster than large proteins. It's made of polymerized
acrylamide.
o This requires all proteins to have negative charges and to be
"unwound" from their tertiary and/or secondary configurations (so that
they move with equal speed; otherwise a globular protein would move
 slower than an identically sized linear protein and thus falsely appear
to be larger than it actually is). The way you do this is to denature
them in a sodium dodecasulfate (SDS) solution that unwinds the DNA
and coats it in negative charge.
o Once that's done, you stick your solution in the top of an acrylamide
gel and run an electric current down the gel. The proteins will migrate
down the electrical gradient and separate out by size: large proteins at
the top, small proteins at the bottom. You can use a "marker" of
known molecular weight proteins on the side to help you figure out
what relative sizes your proteins are.
• Give an example of a disease that can be diagnosed using a restriction
fragment length polymorphism (RFLP) and a use of DNA fingerprinting.
Describe at least three experimental stages required in each of these
procedures.
o RFLP: Example of looking for the HbS mutation of sickle cell anemia.
• Digest patients' DNA with diagnostic restriction enzymes
• Southern/PCR analysis
• Electrophoresis/detection of altered size of restriction fragments
o DNA fingerprinting: Example: paternity testing
• PCR/restriction digestion of region containing variable number
tandem repeats
• Southern blotting of digest/PCR
• Electrophoresis/detection of altered size of DNA fragment
patterns.
• List the names given to the transfer of DNA, RNA and protein respectively
from an electrophoresis gel to a membrane. Describe three characteristics of
a hybridization probe that you will use to detect a specific DNA sequence on a
membrane.
o Transfer of DNA: Southern blot (actually named after somebody)
o Transfer of RNA: Northern blot (to follow Southern)
o Transfer of proteins: Western blot (to follow Northern)
o Characteristics of probe:
• Specific length: you need to know what temperature to anneal
the probe to the DNA with, and that's dependent on the
primer's length.
• Specific sequence: so's it can bind to the DNA you're interested
in.
• Radioactivity: so you can detect it later.
• Quantity: add it in sufficient quantities to outcompete the other
strand of DNA in annealing to its target DNA.
• Recall the classes of enzymes that are used in recombinant technology to:
o a) copy a DNA sequence into a DNA sequence: DNA polymerases.
o b) copy an RNA sequence into a DNA sequence: reverse
transcriptases.
o c) join DNA fragments: DNA ligases.
• Describe the three main stages that are repeated multiple times during PCR
amplification. State the approximate temperature of each step, and relate this
temperature to the state of the DNA molecules in the PCR reaction.
o Step 0: add primers complementary to both strands of a specific
sequence.
o Step 1: add thermal-stable DNA polymerase (usually Taq polymerase)
and dNTPs (deoxyribonucleoside triphosphates).
 o Step 2: Heat to 95 degrees C (denaturing DNA).
o Step 3: Cool to 55 degrees C (allows primers to hybridize).
o Step 2.5: While cooling, the polymerase uses the primer to make
copies of the gene at around 72 degrees C.
• Describe at least 1 distinct use for PCR amplification in the diagnosis of a
genetic condition in your patients.
o Prepare a primer that hybridizes with a mutant copy of a gene but not
the normal copy. Then hybridize and replicate as per normal PCR with
your patient's chromosomes. PCR will generate a detectable
amplification of genetic material if the mutant gene is present. Thus
can use PCR as a diagnostic test for discovering mutant genes that are
markers for cystic fibrosis, beta-thalassemia, etc.
o Notice that since you're testing the entire genome at once anyway,
you can test for multiple mutant genes at once, as long as you can
distinguish between their amplified gene products (by size, for
example).
• Compare and contrast the molecular details of the processes of DNA
sequencing and PCR amplification in a short paragraph.
o DNA sequencing: uses a primer for a given start sequence and four
differently labeled dideoxynucleic acids, as well as normal unlabeled
dNTPs, in DNA synthesis. When the DNA synthesis incorporates a
ddNTP, it stops synthesis with a particular final "color" or label,
indicating which base it ended with. You run this a while and allow a
lot of different lengths of DNA to be made. Then you put all those
lengths next to each other and "read" the last color off each length.
o Maybe an example would help:
o You have a DNA stretch AAATCGA. You make a primer for AAA, then
throw in the ddNTPs (say T=blue, C=green, G=red, A=yellow) and the
dNTPs and let the reactions go. You'll get something like:
• AAAT (last residue blue)
• AAABC (last residue green, 1 residue longer than the one
before)
• AAABCG (last residue red, 1 residue longer than the one
before)
• AAABCDA (last residue yellow, 1 residue longer than the one
before)
o Then you just read off the colors -- primer, blue, green, red, yellow --
and you've got your sequence.
o PCR amplification: uses a primer for a given start sequence and DNA
polymerase to make more copies of a gene. Use varying temperatures
to melt DNA, reanneal to hybridized probe, and have polymerization
reaction happen.
o The point of PCR is to amplify (make lots of copies of) a given gene.
The point of sequencing is to determine the sequence of an unknown
DNA fragment.
o Similarities: Both use primer sequences to initiate replication of genes.
o Differences: PCR is used on double-stranded DNA, sequencing on a
single-stranded DNA fragment. Sequencing used ddNTPs, PCR doesn't.
• Be aware of the different types of cloning vectors and their general features.
o Plasmids: Vectors for amplifying DNA sequences in bacteria.
o Bacteriophage: Used to infect E. coli and use its replication machinery
to produce the recombinant vector. Can take up to 20kb insert.
 o Cosmids: hybrid of bacteriophage and plasmid: use plasmid replication
origin; can take up to 45 kb insert.
o [Retro]viral vectors: can carry very large inserts; introduce DNA into
eukaryotic tissues.

Unit II
Tuesday, October 30, 2007
6:50 AM

Pedigree and Mendelian Inheritance

Tuesday, October 30, 2007
6:50 AM

Pedigree and Mendelian Inheritance, 10/20/07:

• Understand and use standard pedigree symbols.

o males as squares, females as circles, deceased with lines through
them, black as phenotype positive, white as phenotype negative.
Diamonds = unspecified-gender kids/fetuses.
o I, II, III: generations from oldest to youngest). Three generations =
complete. Symbols go on the lines.
o See slide 10 for symbols.
• Be able to draw a pedigree (elicit family medical, use symbols correctly to
draw a pedigree).
• Be able to interpret a family history.
• Be able to discuss how the distribution of phenotypes in a pedigree is a
reflection of segregation of gene variants (genotypes)
o I think what he's saying is to be able to say "Hey, this guy with the
phenotype had parents who didn't have it. Must be some kind of
autosomal recessive disease," or "hey, every time this disease
phenotype comes up, at least half the children show it. Must be some
kind of autosomal dominant disease," etc. Just a guess though.
• Recognize the patterns of Mendelian inheritance.
o Genes come in pairs (sort of- note X-linked diseases in males [only
have one X chromosome] and genetic mitochondrial diseases, since
mitochondrial DNA is derived exclusively from maternal DNA).
o Genes come in different versions ('alleles'), which lead to observed
phenotypes.
o Law of Segregation: Alleles segregate at meiosis into the gametes.
o Law of Independent Assortment: The segregation of each pair of
alleles is independent. (exception: genes close together on
chromosome, aka linked genes)
o "Hemizygous": Means you only have one particular gene, not two. Ie:
males have a single copy of each X-chromosome gene. Also,
 presumably, anyone who has only one working copy of a given gene
through deletion (or, possibly, imprinting).
o "Horizontal" patterns of affected phenotype: tends to be autosomal
recessive (more likely affected in siblings and not in parents).
o The rarer the disease/alleles, the greater proportion of affected
persons will be due to consanguinity.
o Autosomal dominant: tends to appear in every generation;
phenotypically normal parents tend not to pass it on to their children.
Note new mutations (spontaneous generation of disease) can occur
with some frequency.
o X-linked recessive: incidence much higher in males (X-linked
dominant disease incidence is higher in females). Affected males pass
on mutations to all daughters and no sons. Carrier females' offspring
(male and female) have 50% chance of inheriting.
• Explain how genes ‘segregate’ and apply this knowledge to determining
probabilities of inheritance in simple pedigrees.
o Punnett square, simple Mendelian inheritance: AA x aa -> AA + 2Aa +
aa.
• Understand the factors that affect development of the phenotype in single-
gene disorders (roles of: environment, modifier genes, stochastic effects).
o Modifier genes: genetic factors outside a trait's genetic locus that
influence that trait's phenotype
o Stochastic (random) events: The "we have no idea" category; hand of
God reaching down to give someone cystic fibrosis, or the less
blasphemous metaphor of choice.
o Phenocopies: Diseases due to non-genetic (environmental) factors.
• Explain the terms: penetrance, expressivity, and pleiotropy.
o Penetrance: The fraction of individuals with a disease genotype who
show manifestations of the disease.
• High penetrance: most individuals with genotype get disease
• Low penetrance: most individuals with genotype don't get
disease
• Can be dependent on age, etc.
• "Light switch"-- have it (penetrant) or don't (non-penetrant).
o Expressivity: The degree to which a trait is expressed in an individual
(ie. severity).
• "Dimmer"-- how much does a person have the disease?
o Pleiotropy: When a mutation leads to multiple different phenotypes.
Notice that this isn't the same as different degrees of the same
phenotype (that's expressivity).
• Define population genetics and use the Hardy-Weinberg principle to estimate
carrier frequencies for autosomal recessive disorders.
o Population genetics: the study of allele frequencies and changes in
allele frequencies in populations-- where 'populations' is an arbitrarily
defined, but large, number of people.
o Note that nuclear DNA > 99% similar between human individuals.
o Notice "polymorphism" refers to any common genetic variant of an
allele- that is, it occurs in greater than or equal to 1% of the
population.
o Hardy-Weinberg principle: based on the fact that p + q = 1, where
p is the frequency of a common allele at a particular locus and q is the
frequency of a rare allele at that locus.
 o If you square the equation, you can get a nice equation for dealing
with frequency of homo- and heterozygous phenotypes in a
population: p2 + 2pq + q2 = 1.
o Notice that you calculate p or q by dividing the number of genes (each
individual has 2 genes, each of which may be affected or not) by the
total number of genes in the population.
o Notice that you calculate p2, 2pq, or q2 by dividing the number of
individuals who fit that genotype [usually assessed by their
phenotype] by the total number of individuals in the population.
o This makes it possible to calculate the autosomal recessive carrier
population (2pq) given the number of affected (q2) individuals.
o Example: You've got two individuals in a population of 10 that are
affected by an autosomal recessive disease. 2 individuals with
homozygous recessive (q2) out of a population size of 10: q2 = 2/10
= 0.2. Taking the square root of that, q = 0.45. Thus p = (1 - 0.45) =
0.55. So p2 = 0.3 and 2pq = 0.5. Thus you can say of our small
population that the number of homozygous dominant individuals (no
autosomal recessive genes) is (0.3 * 10 = ) 3, and the number of
heterozygous (carrier) individuals is (0.5 * 100 = ) 5.
o Notice that if a disease is very rare (1/10,000 or rarer), 2pq can
approach 2q, since p is pretty much equal to 1 (100% frequency).
• Use basic principles of population genetics (mutation rates, fitness, effects of
consanguinity, addition of new mutations to gene pool).
o Notice that Hardy-Weinberg doesn't usually work perfectly-- non-
random mating, mutation and selection pressures, small population
size, and medicinal palliative effects on affected individuals throws off
the H-W equilibrium.
o If a disease is recessive, it's less affected by fitness and selective
criteria.
• Notice this in the lecture notes: "By observing the number of cases of disease
in a population it is possible to calculate rates of mutation for different
conditions."
o What this means: for an autosomal dominant disease, the number of
new conditions with no previous family history of the condition can be
regarded as the number of new gene mutations causing that condition.
o Notice that the math here is about genes, not individuals:
o So if there's 5 new cases of X disease in 100 live births, that means 5
newborn genes out of 200 (100 newborn individuals with 2 genes
each) have the mutation.
o The symbol for the mutation rate for a given disease is mu (μ).
o 5 genes with the mutation divided by 200 total newborn genes = μ =
(5/200) = 0.025.
o Also can express μ as simply 5 mutations in 200 or 1 in 40.

Genome organization
Tuesday, October 30, 2007
6:52 AM

Genome organization, 10/30/07:

• Know what proteins human genomic DNA combines with, what they are likely
to do, and how they are arranged with human DNA
o They combine with histones/nonhistone proteins. DNA +
histones/nonhistones: chromatin.
o Four core histone subunits: H2A, H2B, H3, H4, each with two copies
(make up a histone octomer all together).
o Roughly 140 bp wrapped around a given histone; roughly 60 bp
connecting one histone to the next. Thus 200 bp = repeating unit of
DNA per histone.
• Know the terms associated with various DNA/protein structures mentioned in
the above outline, e.g. nucleosome, chromatin, etc.
o (nucleosome = histone + DNA complex)
• Know the various structural levels of organization of genomic DNA/protein
complexes as described in the above outline:
o Double helix: DNA
o "Beads on a string": DNA wrapped around histones (nucleosomes)
o "Solenoid": Nucleosomes wound into long cylinder
o Aggegrations of solenoids as 100 kbp [1 kilo-base pair = 1,000 base
pairs. Often notated as "kb" instead of "kbp."] loops attached to
nonhistone scaffolds
• Know that in addition to a nuclear genome, human cells contain the
mitochondrial genome
o Mitochondrial DNA inherits solely through the mother
• Know the features related to the mitochondrial genome described in the
following outline
o Mitochondria: ~ 16 kbp: a few dozen genes
o Uses slightly different codon code than nuclear DNA
o Notice that most proteins acting in mitochondria are made by nuclear
DNA instead.
• Know the fundamental principles regarding the evolution and organization of
the human genome mentioned in the outline
o Genome is both a trace of past evolution (shows various evolutionary
pressures) and a base for future evolution.
o Roughly 30 new mutations occur in each individual.
o Thus- there isn't "a" human genome, any more than there's "a" human
fingerprint.
• Know that genome variation is an essential fuel of evolution and adaptation,
but it also produces human disease.
o See next point.
• Know that random variation, when it has an effect, is almost always harmful
o Given the absurd degree of complexity in protein-protein interaction,
this makes sense. Also gives you a sense of the amount of time that
evolution [ie beneficial mutation trends] tends to require.
• Know the dynamic nature and non-random organization of the human
genome as described in the outline
o Gene-rich and gene-poor, stable vs unstable regions, GC-rich vs AT-
rich, euchromatic and heterochromatic regions (see below).
o Notice that, in general, the location of genes in a given gene family in
the chromosomes are not related to each other.
o Notice that chromosomes are numbered by size (#bp) but the number
of genes encoded by each chromosome doesn't correlate with its size.
• Know how frequently a SNP is likely to occur between two individuals
o SNP: Single nucleotide polymorphism (difference is a single base pair)
o Average: 1 SNP per 1000 bp
o This means 99.9% identical between individuals
o Also means roughly 3,000,000 differences between individuals (1/1000
of three billion bp in the human genome)
• Know the types of variations that occur between genomes
o Insertion-deletion polymorphisms:
• Minisatellites: tandemly repeated 10-100 bp blocks of DNA
(highly variable number).
 Here, labeled as VNTR (variable number of tandem
repeats), though notice the Wiki for this has both mini-
and microsatellites as subsets of VNTRs.
 Notice VNTR can be used as individual genetic
fingerprinting.
• Microsatellites: 2-,3-,4-bp repeats (> 50,000 per genome).
Also known as short tandem repeat polymorphisms (STRPs).
• The point, I think, is that you'd expect a lot of the variation
between individuals to come from variations in these large
blocks of small repeating segments, instead of variations
between important genes.
o Single Nucleotide Polymorphisms (SNPs):
• Frequency of ~ 1 per 103 bp.
• Can be detected by PCR and used for genetic fingerprinting.
o Copy number variation
• Variance in the number of copies in a particular gene in an
individual.
• Variation in size of segments of genome copied: ranges from
200 bp to 2 Mbp [1 mega-base pair = 1,000,000 base pairs.
Also abbreviated Mb.].
• Know the points listed in the outline related to the following: a) gene-rich, b)
gene-poor, c) stable, d) unstable, e) GC-rich, f) AT-rich, g) euchromatic, h)
heterochromatic
o There are "gene-rich" regions or chromosomes (ie Chr 19) and "gene-
poor" (ie Chr 13, 18-21), corresponding to how many genes
(expressed segments) each contains.
o Most of genome is classified as stable (ie doesn't change/mutate a
lot). Of the remainder that's unstable, a lot of its variation is related to
diseases.
o GC-rich (38% of genome) vs AT-rich (54% of genome): pattern of
GC/AT-rich areas is not random.
o Euchromatic regions: more relaxed, less repeats. Makes up most of
the genome.
o Heterochromatic regions: more condensed, more repeats. Tends to
be near centromeres and makes up less of the genome.
• Know that genome sequencing efforts are focused on euchromatic regions
o Right.
• Know that many sequencing gaps still remain even in euchromatic regions
o It's not a completely finished project yet.
• Know to what categories genomic DNA sequences can be assigned as
described in the outline; know the frequency of each class
 o 1.5% = translated (ie exons that code for protein)
o 20-25% = genes (introns, exons, UTRs, gene regulating factors)
• Notice the vast majority of "genes" are not translated.
o 50% = "single copy" sequences (one copy of gene)
o 40-50% = "repetitive DNA" (a sequence that's repeated 100-
1,000,000 times)
• As described in the outline, know the types, locations and frequency of
repetitive DNA sequences that exist in the human genome
o Tandem repeats: "Satellite DNA"
• Seems to be part of the basis for the differential dye binding
that's used in cytogenetic chromosome banding (ie G- or M-
binding, see "Cytogenetics").
• Some found in heterochromatic regions
• Alpha-satellite repeat: 171 bp repeat unit found near
centromeric region of all human chromosomes.
o Dispersed repeats
• These are retrotransposon elements in the DNA. Effectively
they can copy their own sequences into other locations in the
DNA.
 Having worked in a lab looking into these, there's some
thought that they're some kind of foreign DNA using our
DNA to propagate itself, like mitochondrial DNA but less
clearly symbiotic.
• Short Interspersed Nuclear Elements (SINEs) (aka Alu family):
 500,000+ copies in genome (Wiki says up to 13% of
human genome, which is considerably more than that),
~300 bp per copy.
• Long Interspersed Nuclear Elements (LINEs) (aka L1 family):
 100,000+ copies in genome (Wiki says more like
900,000), ~6 kbp per copy.
• Can be medically relevant: retrotransposition of copy in the
middle of another gene may inactivate that gene.
• Know the estimated number of human genes, and the different types of genes
as described in the outline
o The gene number = 25,000-30,000; comprised of:
• protein-encoding genes
• RNA-encoding genes
• "pseudogenes": non-functional but homologous copies of
existing genes.
 Split up into "intron-containing" (presumably a gene
duplication that's inactive for lack of a promoter region)
or "intronless" (presumably a segment of transcribed,
processed DNA that's been retrotransposed into the
genome).
• Know that genes can exist in families and gene families arise by gene
duplication
o Gene family: genes with high (>85%) sequence similarity. Perform
similar functions.
• Know the advantage of gene duplication as an evolutionary mechanism
o When a gene duplicates, it frees one copy to vary while the other copy
continues to perform its function.
Clinical vignettes
Tuesday, October 30, 2007
8:08 AM

Clinical vignettes: Down's Syndrome, Prader-Willi Syndrome, Thalassemia,

von Hippel-Landau Syndrome, Diabetic Ketoacidosis, Multiple Sclerosis:

Down's syndrome
The most common chromosomal abnormalities in newborns (others are often lethal)
Polyhydramnios: excess of amniotic fluid (baby not able to swallow fluid)
Points on handout:
• Advanced maternal age (no prenatal care – can discuss what tests might have
led to suspicion of DS prenatally had mother received prenatal care)
o Down's syndrome and trisomy incidence rises with the age of the
mother ("advanced" = 35 or older): risk of DS baby at 35 is 1:200. (at
40, 1:100- exponential increase in risk after 35)
o Karyotypic analyses:
• Amniocentesis to look at baby's chromosomes is routinely
offered to expecting mothers of advanced maternal age. Done
in early 2nd trimester. Needle into amniotic fluid, picking up
shed skin cells. Process takes about 1.5-2 weeks.
• Chorionic villous sampling: Take fetal tissue from where fetus
attaches to uterine wall. Results much quicker; done after 11
weeks (1st trimester).
o Fetal ultrasound: look for fat neck, short femurs
o Blood screens in mother: looking for fetal blood markers
o Risk of a person having a second trisomic baby after having a previous
one: about 1:100.
• Typical phenotype of DS, including hypotonia:
o Flattened back of head (occiput) : brachecephaly
o Midface hypoplasia (incomplete midface development)
o Epicanthal folds (folds at corners of eyes)
o Ears (pinnae) small and set low in head
o Bilateral transverse palmar creases
o Accentuated space between 1st and 2nd toes
o Decreased muscle tone (hypotonia)
o Abnormal tooth development
o GI tract problems (mainly esophageal/duodenal atresia: ~ 15%)
o Notice that the tongue is normal-sized in a too-small cavity.
• Congenital heart disease (can discuss incidence, and what are the most
common lesions)
o Diagnosed with ultrasound/echocardiogram
o Between 1/3 and 1/2 Down's syndrome babies are born with
congenital heart disease (septal defects, etc).
o Common heart problem: atrioventricular canal (hole between all four
heart chambers, requires surgical repair).
o Notice that heart conditions of babies with other trisomies are not
routinely repaired.
• Esophageal atresia – discuss GI tract atresias in DS, polyhydramnios, need
for immediate surgery
 o Esophageal atresia: esophagus ends in blind pouch (no ability to
swallow amniotic fluid, leading to polyhydramnios). Obviously needs to
be able to swallow, thus need for surgery.
• Genetic testing – timing of results for karyotype and FISH (more impt for
other trisomies)
o FISH usually used looking for trisomy; results within hours. (note
trisomy isn't strongly correlated with Down's syndrome)
o Karyotyping: see above (amniocentesis, chorionic villous sampling)
• Trisomy characteristics:
o Classically, trisomy on chromosome 21 is associated with Down's
syndrome. Sometimes, chromosome 21 gets stuck on other
chromosomes as well-
o Some DS patients have 21 trisomy in all of their cells, but some only
have it in some of their cells. This latter condition is called mosaic
trisomy; people with it tend to be on the higher-functioning end of
the curve that those with trisomy in all their cells.

Prader-Willi syndrome

• Presentation in newborn period with hypotonia and dysmorphic features,

undescended testicles.
• Diagnosis made with FISH – will further discuss importance of particular
region of 15q during presentation
o Notice that Prader-Willi and Angelman Syndromes both occur due to
irregularities with the long arm of chromosome 15.
• Early course of failure to thrive and feeding difficulties which reverses during
preschool age when children develop hyperphagia and gain weight
o Driven by hypothalamic change?
o Treated with growth hormone: prevents development of obesity,
promotes height
• Mild-moderate developmental delay leading to mental retardation as adults
• Ophthalmologic problems common – especially strabismus and nystagmus
o strabismus: "lazy eye" condition
o nystagmus: "jiggly eye" condition

Thalassemia

• Notice HbS, HbC, and HbE: mutant forms of hemoglobin (caused by beta
globin mutations).
• S form: common in east Mediterranean and Africa. [sickle-cell]
• C form: common in Africa
• E form: common in SE Asia and west Pacific
• Thalassemia common (most to least): SE Asia, Africa, west Pacific, east
Mediterranean

• see handout for case history

• Swollen stomach can be due to hemoglobin production in liver and spleen
after birth
• "SS" disease: sickle cell disease.
 • "Hemoglobin H": HbH disease, compound heterozygosity; one allele has no
alpha globin production, the other has impaired alpha globin production.
Usually found SE Asia. Doesn't usually result in hepatosplenomegaly.
• MCV = "Mean corpuscular volume": size of red blood cells.
• "Reticulocytes": young red blood cells (sign of increased RBC production).
• Erythroblasts: red blood cell precursor. Usually found in marrow- not normally
found in blood.
• Notice an excess of alpha cells can bind to and rupture red blood cell
membranes. This is why, clinically, you see bilirubin buildup and result in a
jaundiced appearance.
• Nice summary of beta-thalassemia syndromes on the handouts, p. 8.

Von Hippel-Lindau

• Identify the typical manifestations of von Hippel-Lindau (VHL) disease and

recognize their vascular nature.
o A familial cancer syndrome, manifesting mainly in vascular tumors.
o Constellation of CNS/retinal hemangioblastomas, renal cancer,
pheochromocytoma (tumors on adrenal cells)-- although some families
have the adrenal tumors and some don't.
o Uncommon: 1/36,000 births. Inherited in an autosomal-dominant
fashion, though it's a heterozygous germ-line mutation.
o Common causes of death: CNS hemangioblastoma (formerly), renal
carcinoma (currently).
• Understand the role pVHL plays in the regulation of Hif 1-alpha and the
consequences of mutations in VHL.
o Hif 1: hypoxia-induced factor 1. Controlled transcription of genes
related to hypoxia.
o [Gene:] VHL disease shows abnormality on short arm of chromosome
3: common deleted region, which codes for pVHL (VHL protein).
o Proteins that surround pVHL target other proteins from degradation
(ubiquitinylation).
o Normally, pVHL, in the presence of oxygen, targets Hif 1α for
degradation. At low O2 levels, the Hif 1α protein changes conformation
and is no longer recognized by pVHL complex (thus turns on anti-
hypoxia genes).
o In VHL disease, when you lose pVHL, the Hif 1α protein isn't degraded
by pVHL and is thus constantly transcribing anti-hypoxia genes even
when O2 supply is adequate.
o One of the things this results in is an uncontrolled transcription of
vascular endothelial growth factor (VEGF), which bind to its receptor
(VEGF-R) to cause carcinogenesis in the vascular cells.
o Timing of tumor development in familial VHL disease seems to support
LOH (see "Molecular basis of carcinogenesis") and Knudson's 2-hit
hypothesis.
• Recognize that VHL is sporadically mutated in many cases of renal cell
carcinoma that are not associated with the inherited von Hippel-Lindau
disease.
 o 60-70% of sporadic (not familial VHL) clear-cell kidney cancers have
inactivation of both VHL alleles-- common cause (which means it's an
important gene in vascular kidney tumors).
• Recognize that recently approved biologically targeted agents to treat renal
cell carcinoma are based on the pathophysiology of VHL mutation.
o Little success in treating Hif 1α levels.
o Successful treatments are based on VEGF - vascular endothelial
growth factor.
• Monoclonal antibody against VEGF (not particularly effective in
kidney cancer).
• Also tyrosine kinase inhibitors against VEGF-R: these seem to
be more effective.
 Notice that VEGF-R is crucial to developing new blood
vessels-- thus this treatment is not necessarily going to
do anything about existing vascular tumors. May
become a game of managing existing tumors rather
than eradicating them.

Diabetic Ketoacidosis (DKA):

• Summary:
o Basically, during uncontrolled diabetes, the cells can't get enough
glucose inside them to make sufficient energy to survive-- effectively,
the cells are starving because there's no insulin (or no effective
insulin) getting the glucose into cells and out of the bloodstream. Thus
diabetic ketoacidosis.
o Because the cells need energy and they can't get it from glucose, they
begin to break down other substances, like muscle proteins and stored
fat, for energy. One of the byproducts of non-glycolytic energy
production (lipolysis) is ketone bodies (thus ketoacidosis), which
accumulate more rapidly than they're able to be disposed of.
• Notice that the 'fruity' odor on the breath of DKA patients
results from the high levels of ketone bodies in the blood.
o The accumulation of circulating ketone bodies lowers the pH of the
blood (thus ketoacidosis). Lowered blood pH can break down the
proteins of tissues throughout the body, leading to multiple organ
failure.
• More to the clinical diagnostic point, when the blood pH goes
down, the breath patterns get very different (deeper) in an
effort to clear CO2 from the blood (remember CO2 makes blood
more acidic).
o Dehydration frequently results, even with good fluid intake, because
the kidneys aren't able to keep up with all the excess glucose in the
blood, and some glucose leaks out into the urine. This draws a lot of
water with it and the patient becomes dehydrated.
o Generally treat DKA with insulin-- corrects acidosis, which allows the
true K+ levels to be seen in the ECF-- so can treat the low K+ levels
accurately at that point. Also treat with fluids to correct dehydration.
• Review insulin release:
o DKA usually hits type I diabetics, less frequently type II diabetics.
o Insulin is released during food consumption.
 o Promotes uptake of glucose into cells, inhibits formation of glucose in
the liver, begins production of glycogen as a storage mechanism.
• Review pathophysiology of DKA:
o Notice that diabetes often occurs along with other autoimmune
diseases (like hypothyroidism) in family histories.
o Rapid breathing, nausea, vomiting, thirsty abnormally high urine
output.
o Elevated glucose, low venous pH, high potassium levels in blood.
• Understand the evolution of potassium during the development of initial
treatment of DKA.
o In DKA, as the [H+] in the ECF goes up, the H+/K+ exchangers take
H+ into cells and pump K+ out of them; this results in total body K+
depletion even though extracellular levels of K+ are high.
• Describe the risk of cerebral edema during treatment of DKA:
o Cerebral edema: shows as sluggish, combative behavior and
headaches. Major source of mortality in DKA, also can result in
significant neurological damage.
o Cerebral edema results in part from the fact that, after the
administration of insulin, the cells in the brain uptake glucose much
faster than the rest of the body. This means that the brain cells have a
high concentration of glucose relative to the blood-- and osmotically,
water travels down that gradient out of the blood into the cells, which
expand and cause edema in the brain.
o This is treated by mannitol-- this raises the concentration of the ECF in
all of the body aside from the brain (doesn't cross the blood-brain
barrier), which helps equalize the solute concentrations so that the
osmotic gradient doesn't flow into the brain cells.

Multiple Sclerosis:

• MS: disease of CNS: spares peripheral NS.

• progresses either as attacks of symptoms that come and go (85%) or a time-
progressive course (15%).
o See lots of different progression charts in powerpoint. Very variable
course.
o Notice that acute attacks locally break down the blood-brain barrier.
• Autoimmune disorder: immune system attacks CNS and demyelinates axons.
o Hallmark: slowing of axon conductivity. However, does also cause
axon loss.
• 75% of new cases present between 15-45; 5% are under 18; mostly women
(2/3) and whites. Most common inflammatory CNS disease. Gets more
common away from equator.
• Characteristic lesions on axons: round or ovoid, next to ventricles, optic
nerves, spinal cord, etc. These remain but become less inflamed over time.
• Possible causes: related to DR2 and IL-7 genes; increased risk in mono- vs.
dizygotic twins; Apo E e4 homozygosity; chlamydia and various viruses
(epstein-barr), smoking, sunlight/vit D.
• Diagnosis: CNS lesions disseminated in space (two distinct loci), time,
objective abnormalities on neuro exam, no other cause can be identified.
 • Symptoms: numbness, tingling, loss of or abnormal vision, weakness,
balance, urinary urgency, constipation; later on, fatigue (main symptom), sex
dysfxn, gait dysfxn, depression, pain, dysphagia.
• Tests: MRI, CSF, biopsies, evoked potentials (testing conduction velocity with
visual/brainstem/auditory/somatosensory stimulation)
o CSF: abnormal immunoglobulins (oligoclonal bands on gel), but
rarely high WBC.
• Treatment of acute attacks: IV methylprednisolone, 1g/day x 3-5d, or plasma
exchange once a day for about 2 weeks if steroids don't work.
• Can also suppress or modulate immune system:
o Modulate with interferons (reduce attack rate by 1/3, slow
progression).
o Can also use natalizumab (reduce attacks and new lesions
substantially but is assoc. with fatal brain infections).
• Demyelination of axons produces proliferation of Na+ channels down axon-
slows nerve conduction, can lead to calcium influx. Can use Na+ channel
blockers to treat.
• Note that demyelination symptoms get worse under high heat.

Chromosomal Abnormalities
Wednesday, October 31, 2007
8:03 AM

Chromosomal Abnormalities I, 10/31/07:

• Describe the events in meiosis that produce genetic variability among

offspring.
o ["Mosaicism": abberant process in which cells with two different
genetic complements are produced during mitosis after the zygote is
already formed. (Doesn't occur during meiosis, but often cells will
reproduce mitotically before meiosis, thus this affects meiosis as
well.)]
o Meiosis:
• Chromosomes replicate into sister chromatids (identical
chromosomes).
• Meiotic recombination: homologous crossing-over occurs
between non-sister chromatids.
• Recall that only one of the four haploid products of female
meiosis actually becomes an egg; the rest atrophy into polar
bodies.
o About meiotic recombination:
• Each cross-over is called a chiasmata.
• These cross-overs have a structural purpose: they steady the
pairs of homologous chromosomes next to each other so that
division can occur smoothly between them. This implies that
successful meiosis can't occur without crossing-over
 recombination (which in turn makes recombination more
frequent).
• Cross-overs also occur between X and Y chromosomes in male
meiosis.
• This results in a reshuffling of genes between chromosomes in
meiosis
• Compare and contrast mitotic and meiotic cell divisions.
o Mitosis in somatic cells and primordial germ cells; meiosis exclusively
in germ cells
o Mitosis: two diploid products; meiosis: four haploid products
o Mitosis: two identical genetic products; meiosis: shuffled or
randomized genetic products
o Mitosis: centromeres divide at anaphase; meiosis: centromeres divide
not at the first but the second anaphase (in splitting the sister
chromatids)
o Mitosis: one S (DNA replication) phase per division; meiosis: one S
phase for both divisions.
• Describe the relationship between meiotic recombination (cross-overs) and
chromosome nondisjunction and differentiate between the reproductive
consequences of nondisjunction events in meiosis I versus meiosis II.
o Recombination is a normal swapping of genetic material between
homologous chromosomes in meiosis I before separation.
Nondisjunction events are abberant and result in an uneven
distribution of genetic material when the cell divides.
o Nondisjunction event in meiosis I: both pairs of sister chromatids go to
one daughter cell after first division. This results in one quadroid (4
chromosomes) daughter cell and one daughter cell with no genetic
material at all. When meiosis II occurs, you wind up with two diploid
cells and two nongenetic cells, none of which are correctly functional
as products of meiosis.
• Nondisjunction in meiosis I: All daughter cells dysfunctional.
o Nondisjunction in meiosis II: meiosis I progresses normally, with the
homologues separating as they should. However, in meiosis II, one
pair of sister chromatids fails to separate, resulting in two normal
haploid meiotic cells, one abnormal diploid cell, and one cell with no
genetic material.
• Nondisjunction in meiosis II: Half of daughter cells
dysfunctional.
• Understand and interpret the karyotypic designations used in clinical genetics
reports to describe both numerical and structural chromosomal abnormalities.
o (A lot of stuff he talked about can fit under this rubric:)
o Giemsa: dye used to create banding patterns in chromosome based on
selective binding. (ie. "G-banding," see "Cytogenetics.")
o Ideogram: depiction of the banding pattern of chromosome, with the
bands numbered outwards from the centromere. Notice bands 11 and
12 are called "one-one" and "one-two" respectively (as opposed to
normal-people talk "eleven" and "twelve").
o There are three types of chromosome structures based on the position
of the centromere relative to the two arms (centromere also called
"primary constriction"):
• Metacentric: The centromere is in the center of the
chromosome.
 • Submetacentric: The centromere is offset from center,
producing a longer and a smaller arm.
• Acrocentric: The centromere is near one end of chromosome;
there's a small nubbin of chromosome above it (contains
"stalk" with ribosomal rRNA-producing DNA, and "satellite"
region above this) and a very long arm below.
o "p" = short arm of chromosome (petite)
o "q" = long arm of the chromosome
o Notice that in metacentric chromosomes, this designation is
conventional, but arbitrary.
o "Proximal" or "distal" designations are relative to the centromere.
o Ways to notate chromosomal abnormality [he spent a lot of time
hyping this, so pay close attention to it]:
• General format: (number of total chromosomes), <gender>
<type of mutation>(<location of mutation>)
• types of mutation notations:
 Addition of chromosome (+)
 Deletion (del)
 Inversion (inv)
 Duplication (dup)
 Insertion (ins)
 Ring (r)
 More in Thompson + Thompson p. 66
• examples:
 46, XX del(5p) = normal number chromosomes, female,
deletion in chromosome 5, petite (short) arm.
 47, XX +21 = one additional chromosome, female,
additional copy of chromosome 21 (ie Down's
Syndrome).
 46, XY, del(4)(p16.3) = normal number chromosomes,
male, deletion in chromosome 4, on band 16.3 on the
petite arm.
o Aneuploidy: loss or gain of selected chromosomes (usually fatal but
note below about clinical trisomies).
• Often due to selective problems with meiotic disjunction.
• Specifically, due mostly to maternal meiosis I disjunction.
o Polyploidy: extra copies of all chromosomes (triploidy, tetraploidy);
almost always fatal.
• Often due to complete (all chromosome) meiotic disjunction:
can be result of two sperm fertilizing an egg; can also result
from sperm fertilizing a diploid egg or a diploid sperm fertilizing
an egg (due to nondisjunction event during egg or sperm
meiosis).
o Mosaicism: When the zygote contains two cell lines differing in
chromosome number.
• Post-zygotic mitotic event results in chromosomal abnormality
• Affects various tissues depending on nature of abnormality
• Can be polyploid mosaic or aneuploid mosaic
• Generally less severe than complete aneu/polyploidy
• Describe the clinical features of common human trisomies: 21, 18, and 13.
o 21: Down's syndrome: most common survivable trisomy
• Congenital heart disease
 • Hypotonia
• GI abnormalities
• Early onset Alzheimer's disease
• Note mosaicism (incomplete 21 trisomy)
o 18: Edwards syndrome
• Congenital heart disease
• Hypertonicity (clenched hands, narrow hips)
• Severe CNS abnormalities/retardation
o 13: Patau syndrome
• Most clinically severe of the 3
• CNS abnormalities
• Omphalocele (herniation of GI organs outside abdomen)
• Renal dysplasia
• Congenital heart disease
• Other notes:
o Abnormalities in sex chromosomes tend to be less severe than in
autosomal chromosomes.
• Klinefelter syndrome (47, XXY) : tall, hypogonadism, breast
tissue, frequently infertile, language/reading retardation.
 Often due to a defect in paternal, as opposed to
maternal, meiosis I.
 about 15% due to mosaicism (dysfunction in a post-
zygotic mitotic division)-- zygote okay, problem occurs
afterwards.
• 47, XYY:
 Often due to a defect in paternal meiosis II
 delayed speech and language skills, behavioral problems
• Turner syndrome (45, X) :
 Webbed neck, swelling of hands and feet, renal,
cardiovascular abnormalities
 Mainly spontaneously aborted
 about 25% due to mosaicism

Biochemical Basis of Genetic Disease

Wednesday, October 31, 2007
9:00 AM

Biochemical Basis of Genetic Disease, 10/31/07:

• Understand at a molecular/gene level those sites where mutation can occur,

and how these specific mutations can affect the normal gene product.
o Missense/nonsense mutations within exons can affect structure of
proteins
o Mutations in promoter/enhancer elements can affect rate of
transcription
o Mutations at cap site or poly-A consensus site can affect stability of
transcript and translation
 o Mutations can occur pretty much everywhere, seems to be the take-
home message, and can result in pretty much every bad thing you can
think of.
o Specifically, lots of mutations have been identified in hemoglobin and
glucose-6-phosphate dehydrogenase.
• Understand why many biochemical variants are not deleterious. Appreciate
the number of mutant lethal genes we all carry in our genomes.
o (1) Degeneracy of the genetic code: change in base pair doesn't
necessarily give rise to a different amino acid.
o (2) If one amino acid is changed for a similar one (basic polar to basic
polar, aromatic for aromatic, etc), often there's not a noticeable
change in protein function.
o (3) Amino acid replacement at a non-structural or non-catalytic site
may not affect protein functions.
o (4) Diploid nature of genes means that we automatically have a
backup.
• This also means that most of us are carrying around lethal
mutant genes that don't kill us because we rely on the working,
'spare' copy of the gene.
• Compare dominantly and recessively inherited diseases in terms of their
mutant gene products. Understand when a particular mutation will be
expressed in a dominantly inherited fashion.
o Recessively inherited diseases: need both genes to be bad before a
disease phenotype crops up.
o Dominantly inherited diseases: only need one bad copy of a gene to
produce disease or death.
o Dominant inherited disease mutations tend to be in genes coding for:
• Structural proteins
 Disease: Osteogenesis imperfecta (improper collagen
assembly, often fatal)
• Regulatory proteins
 Disease: Waardenburg syndrome (mutation in trans.
factor PAX3)
• Proteins which induce growth or development
 Disease: Holoprosencephaly (mutation in Shh [sonic
hedgehog] gene)
• Enzymes whose concentration determines the rate of reaction
 (normally enzymes are at saturating levels in cells)
 Disease: Acute intermittent porphyria (insufficient
levels of urophorphobilogen synthase, which enables
formation of heme groups)
o Recessive inherited disease mutations tend to be in genes coding for
proteins that are normally present at a surplus level in cells.
• Understand the concept of "compound heterozygote", and how it is related to
the variable phenotype of many autosomal recessive disorders.
o Compound heterozygotes: inherit two different mutant alleles;
display variable severity.
• Ie: I get one copy of XYZ gene from Mom with a nonsense
mutation in its middle (thus that gene produces half-length XYZ
proteins). I get another copy of that gene from Dad with a
nonsense mutation right at the beginning (thus that gene
produces no XYZ proteins). I don't have the exact same
 mutation in both genes (thus not exactly "homozygous" at that
allele) but since I have reduced protein capacity from both
genes, I probably have the disease, even if it's recessively
inherited. Notice that if my brother had similar mutations in
both genes, but near their ends, he would probably have more
functional copies of the protein than I would (and thus have a
less severe manifestation of the disease), even though we're
both lumped in as "compound heterozygous."
• Understand how genetic defects in regulatory proteins, transport proteins,
receptor proteins, and structural proteins can give rise to disease.
o Transport:
• Cystinuria: Defect in cysteine transport protein results in
crystallization of cysteine in bladder
• Cystic fibrosis
o Receptor:
• Hyperlipoprotein II: defect in LDL (low-density lipoprotein)
receptor which picks up cholesterol and transports it into cells.
Results in hypercholesterolemia-- cholesterol accumulates in
the plasma (thus leads to cardiovascular disease).
• Androgen Insensitivity syndrome: we've seen this before
(mutation in androgen receptor zinc finger DNA binding region).
o Structural:
• Hereditary spherocytosis: mutations in structural elements
of red blood cells. This forces the cell to lose its tight disc shape
and assume a fragile spheroid form. This sticks inside the
spleen, gets lysed, and leads to anemia.
o Cofactor interaction with apoenzyme ("regulatory" protein?):
• Cystathionuria: Km defect (see below) in which the
cystathionase enzyme is altered and cystathionine is excreted
in the urine. Can be treated with an excess of vitamin B6.
• Homocystinuria: Similar; leads to accumulation of
homocysteine in the urine due to a mutation in the appropriate
enzyme. Treated the same way.
o Catalytic defect in biosynthetic pathway:
• Orotic aciduria: Anemia and a buildup of orotic acid due to a
mutation in pyrophosphorylase and decarboxylase enzymes.
Effectively, can't make new nucleic acids. Treated with uridine.
• Understand the concepts: “dominant negative mutation”, “gain of function”
mutation, “novel function” mutation, and “Km defect” mutation.
o Km defect: mutation where an enzyme's affinity for substrate or for
its cofactor are altered (thus raises Km, amount of substrate needed
for cellular function of the enzyme).
o Dominant-negative mutations: occur when the mutated gene
product adversely affects the non-mutant gene product.
o Gain of function: interchangeable term for "novel function
mutation". They both mean effectively that the gene product gets a
new, abnormal function.

Cytogenetics
Thursday, November 01, 2007
8:03 AM

Cytogenetics, 10/1/07:

• Recognize the landmarks of human metaphase chromosomes and know which

specimens yield different levels of band resolution
o (good to know that metaphase is when you're generally looking for
chromosomes.)
o short arm, centromere, long arm, telomere at ends. In acrocentric
chromosomes, also have stalks (containing ribosomal genes) and
satellites
o Types of chromosomes: metacentric, submetacentric, acrocentric (see
"Chromosomal Abnormalities").
o "G-banding" = current normal process for dye-banding chromosomes.
Essentially you use a dye that binds to various regions of DNA and not
to others, creating a characteristic "banding pattern" on each
chromosome. Good for diagnosing gross problems (ie loss or
translocation or chromosomes) but not good at detecting small
deletions (since the deletion makes up a tiny fraction of the
chromosome and its loss can't be readily observed). You need FISH to
detect microdeletions.
o G-light bands: GC-rich (chromatin more compact), contain a lot of
short interspersed repeat elements, replicated early, rich in transcribed
and housekeeping genes.
o G-dark bands: AT-rich (chromatin more open), contain a lot of long
interspersed repeat elements, replicated late, have sparse genes and
simpler sequence, tend towards tissue specific genes (heart, liver, etc)
as opposed to tissue-universal genes.
o Number of bands/lengths of chromosomes differ between tissue
samples:
• Very short to short chromosomes [fewer bands] found in:
 -bone marrows
 -solid tumors
 Have 300-400 bands/haploid chromosome
• Short to medium chromosomes [more bands] found in:
 -chorionic villous sampling
 -amniotic fluid
 -products of conception (don't ask me what this means)
 -some newborns
 Have 400-550 bands/haploid chromosome
• Medium to long chromosomes [most bands] found in:
 -peripheral blood
 -mitogen (DNA replicating agents)-stimulated tissue
 Have 450-650 bands/haploid chromosome
• Delineate types of FISH probes and how they compliment standard
cytogenetic analysis
o Note she only mentions the first three in the handout.
o Centromere FISH probes: "cen" probes
• Used for enumeration of chromosomes (prenatal diagnosis of
trisomy, etc)
o Locus-specific probes : "LSI" probes
• Used for detecting deletion/duplication of genes (loci)
 o Dual fusion/fusion: "DF/F" probes
• Used for detecting translocation
o Break apart: "BAP" probes
• Used for detecting rearranged + translocated chromosomes
o Whole chromosome paint: "WCP" probes
• Used for identifying markers or translocations
o FISH [Fluorescent in situ hybridization]: Method to examine subtle
deletions or changes in chromosomes that may not be picked up by
banding patterns alone:
• Small deletions
• Test host vs donor marrow cells after a transplant
• Can use FISH to look at a large number of cells at once
• Usually done after preliminary chromosome dye banding
• Identify which chromosomes can be carried by patients in a mosaic state and
what is the implication for the clinical phenotype
o Recall: mosaicism is two distinct cell lines in an individual (one normal,
one abnormal).
o Chromosomes for which mosaicism is viable: 8, 13, 18, 21 (last 3
most common)
o Often mosaicism results in a partial or incomplete pathophenotype.
• Characterize the laboratory test algorithm for children who present with
learning disorders, developmental delays, dysmorphic features, and/or failure
to thrive
o First, run a standard high-res chromosome banding analysis.
• If abnormal, check parents' chromosome banding.
• If normal, run aCGH (see below) to check for gene
deletions/duplications.
 If one's detected, look into database of genetic variants
to match it up with a disease syndrome. Can check
parental chromosomes with FISH if there's ambiguity.
o Clinical note: trisomy 21 can be 'free trisomy' (extra free-floating chr.
21), translocation (extra chr. 21 stuck on another chromosome), or
mosaic. Of the three, free trisomy is by far the most common.
• Appreciate that the field of clinical cytogenetics is dynamic, with evolving
molecular tests, such as array comparative genomic hybridization (aCGH),
that challenge genetic diagnosis and medical care
o aCGH: slide or silicon matrix in which gene-specific probes are
arrayed, covering the entire genome. Label patient DNA one color,
control DNA another color; look for color variations showing deletion or
duplication of genes.
• Essentially looking at copy number of different genes.
• Notice it can't detect balanced translocations of genes and it's
not good at detecting mosaicism.
• Notice that array studies are always confirmed with FISH.

Chromosomal Abnormalities II + Imprinting

Thursday, November 01, 2007
8:03 AM

Chromosomal Abnormalities II + III (with imprinting): 11/1/07

• Describe the mechanism of common chromosome structural rearrangements.
• Common mechanism: double-strand breakage in DNA and subsequent
repair by non-homologous end joining (which, recall, is kind of like
having two blindfolded monkeys assemble a radio with glue: bits are
always lost, put in backwards, eaten, etc).
• Another common mechanism: crossing-over between repetitive DNA
sequences. This can delete segments of a repetitive stretch, can delete
on one stand and duplicate on the other, invert, reciprocally
translocate, etc.
• Compare and contrast various forms of balanced and unbalanced
chromosomal structural rearrangements and understand their reproductive
consequences.
ο Balanced: normal, but rearranged, complement of chromosomal
material. Often phenotypically neutral.
• Inversion (double-stranded segment flipped around, 5'-3' to 3'-
5'):
 Paracentric inversions exclude the centromere
 Pericentric inversions include the centromere (inverted
segment goes from p to q)
 During meiosis, the chromosome now has to loop
around to pair effectively with its homologue, which
introduces a whole new set of problems with crossing-
over. See PP slides if you're interested.
• Reciprocal translocation (breakage/reforming create
recombination of two non-homologous chromosomes:
effectively chromosome A gets a chunk of chromosome B and
vice versa)
 Notice this is different from just straight up
translocation, which is when one chromosome sticks
itself whole cloth right onto another one.
 Observed in ~ 1:500.
 This really messes things up during recombination in
meiosis: creates "quadravalance" in which four
homologous chromosomes align instead of two.
 This can lead to disease states: chronic myelogenous
leukemia from translocation of chromosomes 9-22.
 Risk of lethality with reciprocal translocation ~ 5-10%.
• Robertsonian translocations (fusion event between long arms of
two acrocentric chromosomes- means you lose stalks/satellites
of those chromosomes)
 Notice that the chromosome count goes down by one.
 Notice that even so, this is considered a "balanced"
rearrangement.
 Notice that Robertsonian translocations can result in DS
(trisomy 21) without an abnormal chromosome number
(two normal 21s, one fused on another chromosome).
 Frequent occurrence of this: chr.14 - chr. 21 fusion.
Notice this often results in trisomy 21 (gamete has the
copy of chr. 14 with 21 stuck onto it as well as the
normal copy of 21).
 ο Unbalanced: abnormal chromosomal context. Often phenotypically
abnormal.
• Deletion:
 Flavor one: the deleted segment on one chromosome
arm: produces a deleted fragment (not stably
transmissible) and the transmissible rest of the
chromosome. This is called a "terminal deletion."
 Flavor two: the deleted segment contains the
centromere (ie goes from one arm to the other). This is
called an "interstitial deletion." Notice that the deleted
segment can form into a ring chromosome (see below).
• Duplication:
 Generally less harmful than deletion
• Isochromosomes (one in which one arm is missing and the
other has mirrored itself, replacing [or displacing] the missing
arm):
 Most commonly found on the X chromosome
 Sometimes found on chromosome 21:
• 100% of the viable offspring of a carrier of
isochromosome 21 are abnormal. --since the
progeny either have three 21 chromosomes or
one (thus not viable).
• Marker (ring) chromosomes:
 Occurs when an interstitial deletion fragment becomes
circular and (since it contains the centromere) is stably
transmissible to offspring.
 Recall that the majority of Down's Syndrome patients have trisomy 21 due to
a maternal meiosis I nondisjunction event.
 Assess the recurrence risks of various chromosomal rearrangements for the
progeny of carriers of these rearrangements.
ο Most chromosomal abnormalities are unlikely to recur (caused by
random event).
ο Some stable, balanced rearrangements in the parents (like some
translocations of chromosomes) are transmissible to offspring.
 Define and give examples of contiguous gene syndromes.
ο A disorder due to overexpression or deletion of multiple gene loci that
are adjacent to one another.
ο Ex: Velocardiofacial syndrome (del 22q11) and DiGeorge syndrome
(del 22q11)
 Define the term "epigenetics" and understand how DNA and chromatin
modifications may affect gene expression.
ο Epigenetics: Heritable changes in gene expression that occur without
a change in DNA sequence.
ο Examples are patterns of reversible post-translational modifications of
histones and pattern of DNA methylation (alters configuration of DNA,
not DNA sequence itself).
 Describe genetic imprinting and its molecular basis.
ο Small subset of genes that are inherited in a transcriptionally active
state from one parent and transcriptionally inactive state from the
other parent: this is called genetic imprinting.
ο Clinical interpretation: we're normally hemizygous (only one working
copy of a gene) for all of our imprinted genes. This is synonymous with
 saying we're particularly vulnerable at all of those genes, since there's
no backup.
ο Activation or inactivation seems to depend on the methylation or
nonmethylation of cytosine residues in CpG islands in promoter regions
of particular genes.
ο Methylation binding proteins attract other proteins that act as HDACs
to silence transcription and compact chromatin at the site. (Point:
generally, DNA methylation leads to transcriptional inactivation.)
 Describe the different fates of DNA methylation imprinting patterns in germ
line and somatic cells.
ο 3 rules for epigenetic DNA methylation:
• Modification must be established during gamete genesis (all
paternal gametes must be imprinted the same way, all
maternal gametes must be imprinted the other way).
• Modification must be stably maintained in somatic cells (which
will contain half paternal and half maternal modifications) after
fertilization.
• Modifications must be reversible so that they can be reset
during gametogenesis to transmit the appropriate sex-specific
imprint to progeny.
ο Somatic cells: DNA methylation patterns (silencing/expression of
imprints) are set and don't change (some genes are maternally
imprinted, some are paternally imprinted).
ο In primordial germ cells, all alleles undergo universal demethylation
(erasing the inherited imprinting pattern). Then they're methylated
again in a sex-specific manner (if they're in a female, maternally
methylated; if in a male, paternally methylated)
ο This happens to ensure that all genes coming from the father to
offspring have ONLY paternally imprinted gene patterning, and that all
genes coming from the mother have ONLY maternally imprinted gene
patterning.
 Describe the relevance of genetic imprinting in Prader-Willi and Angelman
syndromes.
ο Both PW and Angelman syndromes have to do with a deletion in a
region of chromosome 15.
ο This region codes for two imprinted regions (one per disease)- one is
maternally activated (Angelman syndrome), the other is paternally
activated (P-W syndrome).
ο So you have two copies of chr. 15, one from your mother, one from
your father. The one from your dad has active Prader-Willi genes and
inactive Angelman genes. The one from your mom has active
Angelman genes and inactive Prader-Willi genes.
ο If the chromosome region with the PWS-active genes [paternal
chromosome] is deleted, results in Prader-Willi syndrome.
• Notice that this also results in the deletion of a Angelman gene
region, but since it's an inactive region, no Angelman
phenotype results.
• This happens because the PW region on the other, maternal
chromosome is inactivated due to imprinting-- thus no working
copies of this region remain once the paternal genes have been
deleted.
 ο If the chromosome region with the Angelman-active genes [maternal
chromosome] is deleted, results in Angelman Syndrome.
• Notice that this also results in the deletion of a PW gene region,
but since it's an inactive region, no PW phenotype results.
• This happens because the Angelman region on the other,
paternal chromosome is inactivated due to imprinting-- thus no
working copies of this region remain once the maternal genes
have been deleted.
ο One can think of PW Syndrome as "paternal insufficiency" and
Angelman Syndrome as "maternal insufficiency."
ο Notice that both of these can occur in both male and female offspring.
(imprinting pattern is dependent on your parents, not your gender.)
 Describe the origin and clinical effects of uniparental disomies with regard to
Prader-Willi and Angelman syndromes.
ο Here, disomy is when one gamete has two copies of a chromosome.
ο If that gamete fuses with another, normal gamete, the zygote will
have trisomy for that chromosome.
ο If the zygote has a nondisjunction mitotic event early in life, may
continue on with normal chromosome number, but has both
chromosomes from one parent.
ο This means that whatever genes are meant to be activated from the
other parent aren't activated.
ο This means if it's maternal disomy for chromosome 15 (no copies of
chr. 15 exist from the father), it results in Prader-Willi syndrome.
ο Or if it's paternal disomy for chromosome 15 (no copies of chr. 15
exist from the mother), it results in Angelman syndrome.
ο Notice the frequencies:
• PW: 70% occurs from a deletion in the paternal gene, 25%
from uniparental disomy from the mother.
• Angelman: 70% occurs from a deletion in the maternal gene,
less than 5% from uniparental disomy from the father.
• Uniparental disomy in the father is rarer since nondisjunction
meiotic events in male gametes are rarer than in the female.

Sex Determination
Friday, November 02, 2007
10:34 AM

Sexual Determination, 11/2/07:

• Understand the biological advantages of sexual reproduction

o Diploidy: protects against effects of mutation (still one working copy)
o Recombination: creates new combinations of haploid genes in
individual's germ line
o Sex: allows random chromosomal assortment by combination of
haploid cells
o Sex also makes possible gender-dependent epigenetic imprinting.
 o This permits rapid evolution and increases survival of species through
increasing genetic variability
o Sexual dimorphism allows for division of labor and cooperation (not
bloody likely).
• Describe X chromosomal inactivation and its implications
o All diploid somatic cells have a single active copy of the X
chromosome, whether it belongs to a male or a female.
o In normal females, this means one X chromosome is inactivated in any
given cell. The other remains as a "Barr body."
• Note that this inactivation is a mosaic: that is, there's a 50-50
chance of either X chromosome in a female cell being active or
inactive.
• How inactivation works: DNA methylation and modifications of
histones.
• How that works: XIST region on inactive X transcribes a special
type of RNA that spreads across that X chromosome to coat it,
modifying the genomic structure to attract DNA methylators
and histone deacetylases (HDACs).
• Note that not all genes on an "inactive" X chromosome are
actually silenced; some of the genes (10-15%) in Barr bodies
are still transcribed.
o "Nonrandom X inactivation" occurs when an X chromosome is
abnormal. This results in the abnormal X chromosome being
preferentially inactivated. Instead of a mosaic pattern where a 50-50
random pattern of inactivation, you get almost all cells with an inactive
abnormal X due to nonviability of cells with active, abnormal X
chromosomes.
• Can still have abnormal phenotypes due to translocations, etc,
but rare.
• Describe the genetic regulation of sexual differentiation
o Before you get to that point, you need the WT gene, which directs the
differentiation of the embryological genital ridge (which you need to
have any gonads at all).
o Gonadal differentiation is dependent largely on whether or not genes
promoting the development of testes are present.
o Specifically, it depends on the SRY gene on the Y chromosome, which
drives differentiation of gonadal tissue into testes (without SRY,
ovaries develop).
o Other important genes for proper sexual differentiation: SOX9
(interacts with SRY), SF1, DAX1
o Mullerian inhibiting factor (MIF) produced by testicular cells: allows
paramesonephric ducts to atrophy away and the mesonephric ducts to
grow.
• Describe the basic embryology of dimorphic human reproductive organs
o Testosterone (derived from testicular non-germ cells) drives the
differentiation of external genitalia.
o However, testosterone production largely driven by pituitary gland-- so
problems with the pituitary gland can result in external sexual
ambiguity.
o Mullerian inhibiting factor, as mentioned, allows the formation of
ductus deferens, etc, from mesonephric ducts. Notice that this is not
dependent on the pituitary gland.
 • Describe the clinical characteristics of disorders of sex chromosomes
o True hermaphroditism: 46 XX/46 XY: show ovaries, testes, partial
formation of uterus
o Sex reversals:
• XX males in which the Y chromosome has translocated
autosomally.
• XY females in which regions of the Y chromosome have been
deleted or certain sex-developing genes on the X chromosome
have been duplicated.
o 45 X : called Turner syndrome:
• Normal early female gonadal development in utero, but
degeneration of developing ovaries later in fetal life.
• Clinical features: short height, perceptual disorders, coarctation
of the aorta (narrowing of aorta between upper-body and
lower-body supply), fused kidneys.
o 47 XXY : called Klinefelter syndrome. Develop as anatomic males,
but have degeneration of gonads. Infertile, low levels of testosterone
development.
• Clinical features: tall stature, gynecomastia (development of
breasts).
o Turner and Klinefelter are mostly driven by meiotic nondisjunction
events.
o Androgen insensitivity: presents as non-menstruating females.
• Notice that androgen insensitivity does not result in a uterus
(testes still produce working mullerian inhibiting factor even if
the testosterone isn't able to affect development).
o "Pseudohermaphroditism": Individuals with ambiguous external
genitalia but normal ovaries or testes (not both).
• Describe the clinical approach to disorders of sexual differentiation
o **[Not much discussed in lecture or in the notes. I've emailed Dr.
Manchester, but no response as yet. About all I know is that he said
that you always start off with a chromosome analysis of the subject
(and parents if warranted).]

Multifactorial Inheritance
Friday, November 02, 2007
10:34 AM

Multifactorial inheritance, 11/2/07

• Identify and describe the characteristics of diseases and other traits that
demonstrate multifactorial inheritance
o Spectrum of disease: simple Mendelian to extremely complex
multifactorial
o Many diseases have characteristics that aren't explained by the
genotype at the causative locus. In addition, different alleles at the
same gene can result in different levels of severity (as in cystic
fibrosis).
o Multifactorial: combination of genetic variants and nongenetic factors.
 o Tend to aggregate in families but don't follow simple modes of
inheritance.
o Specific characteristics of complex traits:
• Incomplete penetrance: not everyone with predisposing
genetic variant develops the disease.
 Ie: Type I diabetes (20% population have predisposing
genotype, incidence is 0.4%)
• Variable expressivity: No people with the same genetic variant
have the exact same disease characteristics.
 Ie: Maturity Onset Diabetes
• Heterogeneity: "Same" or similar diseases can be caused by
(1) different alleles at one location in the gene or by (2) alleles
at different locations (or loci) in one gene or among many
genes.
 (1) = allelic heterogeneity
 (2) = locus heterogeneity
 Ie: cystic fibrosis (allelic: lots of alleles lead to CF,
variable severity)
 Ie: Alzheimer's (locus: mutations in chr. 1, 14, 21 all
lead to AD)
• Phenocopies: People who have the disease (same clinical
presentation) for reasons that aren't primarily genetic.
 Ie: thalidomide-induced limb malformation v.
genetically-induced
• Give specific examples of diseases and other traits that demonstrate
multifactorial inheritance
o Some cancers
o Diabetes, I and II
o Alzheimer's
o Inflammatory bowel disease
o Asthma
o Schizophrenia
o Hypertension
o Cleft lip/palate
o Rheumatoid arthritis
• Describe the strategies used to determine the relative importance of genetic
vs. non-genetic factors in contributing to the variation in a complex trait
o Epidemiologic twin, adoption, and immigration studies
• Twins: compare monozygotic to dizygotic twins
• Adoptive: compare biological siblings raised apart and adoptive
siblings
o Examine disease frequency and risk pattern in relatives
o Notice that if you have one major gene associated with high
penetrance of a given disease trait, that's about as simple-Mendelian
as you get.
• Understand the potential difficulties associated with quantifying the role of
genetic factors in contributing to risk of disease at both the population level
and the individual level
o Study of heritability (heritability: proportion of total variance in a
trait that's due to genetic variation).
 o High heritability: differences among individuals with trait can be
largely attributed to differences in genetic makeup.
o Low heritability: differences among individuals with trait can be largely
attributed to differences in environmental factors.
o Notice that high or low heritability can still mean that the other
variability source is significant-- it's just not as significant.
o Re this bullet point: the emphasis here is that the roles of genetic and
non-genetic factors vary from trait to trait and individual to
individual-- thus hard to lay down strict guidelines for what markers
mean what about disease predispositions.

Autosomal Recessive Disorders

Tuesday, November 06, 2007
7:51 AM

Autosomal Recessive Disorders, 11/6/07:

• locus: physical position of a gene on a chromosome.

• alleles: different isoforms of same gene
• 1) Overview of Autosomal Recessive Disorders
o (a) Know the common characteristics of disorders that are of
autosomal recessive inheritance
• Phenotype expressed only in homozygotes.
• Males and females usually equally affected.
• Horizontal inheritance pattern (sib-sib more than parent-child).
• Parents of an affected child are obligate carriers (or affected
themselves).
• The recurrence risk for each potential child of two carriers is
1/4.
• The chance of unaffected children of two carriers being carriers
is 2/3 (unaffected means it's not the 1/4 that's homozygous
recessive, thus 3 possibilities left, 2 of which are carriers).
• Predisposition in certain ethnic groups
o (b) Calculate allele frequency and carrier frequency of a given
autosomal recessive disease when provided with the disease
frequency, and vice versa
• Frequency of disease = q2. Can take the square root of that to
get the allele frequency q, then subtract q from 1 to get the
frequency of the wild-type p allele. Once you have p and q, you
can calculate the carrier frequency by 2pq.
• If given q, can get p by subtracting q from 1; then you have
enough information to get q2 and 2pq (thus calculate the
carrier frequency and allele frequencies.
• Notice that if all you're given is 2pq, you don't have enough
information to calculate anything else.
o (c) Understand the following concepts:
• 1) Allelic heterogeneity: Existence of multiple alleles of a
single gene
 • 2) Compound heterozygote: A person who carries different
mutant alleles at the same genetic locus (see "Biochemical
Basis of Genetic Disease").
• 3) Parental consanguinity: Parents sharing one or more
common ancestors. (Obviously, we're all related if you go back
far enough-- Wiki says consanguinity is usually defined as
second-cousins or more closely related.
• 4) High-risk group: An ethnic group in which an autosomal
recessive disease occurs with higher frequency. This is high-risk
both because of the increased frequency of the allele and also
because people who intermarry within that ethnic group are at
a much higher risk for being homozygous recessive for that
allele.
• 2) Phenylketonuria (PKU)
o (a) Discuss the biochemical deficiencies in PKU patients and the
appropriate treatments:
• PKU: defect of phenylalanine (F) metabolism (can't break down
F). Results in high F levels in the blood and high levels of F
metabolites in the urine. Shows as hyperactivity, epilepsy, and
mental retardation.
• This is usually a result of a defect in the gene coding for
phenylalanine hydroxylase (PAH), which converts F into
tyrosine (Y).
• Occasionally, it's instead a result of coenzyme genes, so that
the reaction doesn't go forward despite having intact PAH
enzymes.
• Has a large variety of mutant alleles-- thus incidence of
compound heterozygosity is fairly high.
• Preventable: treatment with low-F diet prevents mental
retardation. Notice that F is an essential AA and can't be
eliminated from the diet completely. The diet can be
discontinued after the school years, unless the patient gets, or
plans to get, pregnant (see next).
o (b) Explain maternal PKU and its treatment:
• Pregnant women who have PKU and are not on F-restricted
diets have a high incidence of miscarriage and developmental
abnormalities in their children.
• These abnormalities in children are not caused by the genotype
of the child but the high levels of F in the maternal circulation
when the child is in utero.
• The developmental abnormalities are called maternal PKU
since they aren't a result of the child's genes.
o (c) Know newborn screening procedures for PKU and importance of the
timing of the test:
• Can be tested with mass spectrometry to look for abnormally
high levels of F (as well as low levels of Y, its metabolite) in the
blood.
• Timing of the test is important: before ingestion of food, the
new baby won't have elevated F/Y ratios. So take a baseline
blood sample at birth, then wait for a couple of days and test
again to compare.
• 3) Alpha1-Antitrypsin Deficiency (ATD)
 o (a) Know the clinical features of alpha1-antitrypsin deficiency and the
influence of environmental factors on the expression and severity of
the disease (ecogenetics):
• Disorder is late-onset, more common among Northern
Europeans.
• 20-fold increased risk of developing emphysema; also much
higher risk of developing liver cirrhosis.
• Notice that smoking aggravates both problems ("environmental
factors").
• What's missing: the alpha1-antitrypsin enzyme.
o (b) Which enzyme is the primary target of alpha1-antitrypsin?
• Mainly targets and inhibits elastase, a serum protease, by
irreversible binding.
• Elastase is released by neutrophils in the lung to break down
elastin (structural protein in the lung alveoli). Basically,
elastase breaks down lung tissue to be remodeled.
• If elastase isn't inhibited by alpha1-antitrypsin, lung tissue
breaks down more quickly, causing inflammatory responses
that cause the lung tissue to break down even faster--
eventually causing emphysema.
o (c) Know the two most common mutant alleles that cause ATD and the
severity of different allelic combinations. Why do some ATD patients
have liver failure?
• Not a lot of different mutant alleles:
 Z allele: most severe and common form (ZZ genotype =
15% normal alpha1-antitrypsin function). Makes an
improperly folded protein which gets stuck in the liver
cells, which is why elevated rates of liver cirrhosis are
observed in ATD patients.
 S allele: less severe; makes unstable ATD proteins as
opposed to stable but misfolded proteins (thus no liver
disease).
 Notice that ZZ smokers have an average life span of
about 40 years. (ZZ non-smokers live an average of 60
years.)
• 4) Tay-Sach Disease (T-S)
o (a) Explain the biochemical defects in Tay-Sachs disease and why the
brain is the major target:
• Early-onset, fatal disease targeting the CNS. Born normal,
symptoms show about 9-12 months, usually death occurs 2-4
years.
• The problem in patients is a defect in their ability to get rid of a
ganglioside (lipid) that makes up about 5% of brain mass.
Instead of being able to break it down and turn it over, the
ganglioside gets stuffed into lysosomes until the lysosomes are
massively engorged.
o (b) Compare the biochemical and genetic differences between Tay-
Sachs disease and Sandhoff disease:
• T-S: mutant alpha subunit of the enzyme required to break
down ganglioside
 • Sandhoff: mutant beta subunit of the enzyme required to break
down ganglioside (also results in inability to break down other
lipids).
o (c) Know the high-risk group for Tay-Sachs disease and the available
methods for carrier screening and prenatal screening in the high-risk
population
• High-risk: Ashkenazi Jews for the most part.
• Notice that three mutant alleles account for > 95% of the
mutations in that population; DNA testing for these alleles are
offered for carrier and prenatal screening.

Molecular Genetics of Hemoglobinopathies

Tuesday, November 06, 2007
7:52 AM

Molecular Genetics of Hemoglobinopathies, 11/6/07:

• Describe the layout of the alpha- and beta-globin gene clusters and the switch
between different forms of hemoglobin (Hb) during development. Explain the
function of the locus control region (LCR).
o All of the alpha genes are in the alpha-cluster of chromosome 16; all
of the beta genes are in the beta-cluster of chromosome 11. Recall
that hemoglobin is a tetramer, composed of two alpha and two beta
subunits. (Also can consider it two alpha-beta dimers.)
o Notice alpha and beta subunits have very similar sequences,
structures, and heme-binding groups. That said, 2 alphas and 2 betas
are vastly more efficient at binding oxygen than 4 alphas or 4 betas.
o Alpha-cluster has this sequence: zeta-alpha1-alpha2.
o Beta-cluster has this sequence: epsilon-gammaG-gammaA-delta-beta
o (simplified) Normal development of types of globins: first zeta-
epsilon (early embryo), then alpha-gammas (fetus), and finally alpha-
beta (post-birth).
o Cool thing: genes in cluster are arranged 5' to 3' in the order in which
they will be expressed during development. This is called "globin-
switching."
o Major form of hemoglobin: A2B2. Minor form: A2D2. Delta globin is a
minor auxiliary of beta globin.
o Alpha replaces zeta and remains on throughout life.
o Major form of fetal hemoglobin: A2G2.
o Gamma is dominant beta cluster hemoglobin until birth, at which point
beta form predominates.
o The location of erythrogenesis changes as well: yolk sac to liver to
spleen in utero, then bone marrow after birth.
o Locus control region (LCR): 10-20 kbp sequence far upstream of the
cluster.
• In the globin clusters, each genes has its own promoter; LCR
controls which of these promoters are turned on or off.
• The LCR controls both the timing and the level of expression.
• [Most hemoglobinopathies are either structural (altered globin properties),
thalassemias (low levels of synthesis of one globin), or defective globin
switching (hereditary persistence of fetal hemoglobin-- gamma hemoglobin
continues at elevated levels through life).]
• Describe the mutations that cause sickle cell anemia and hemoglobin C
disease and their consequences. Know the DNA diagnosis method of the sickle
cell disease mutant allele.
o Sickle-cell anemia: Affects beta-globin gene; caused by mutation in
exon 1: A-to-T mutation at codon 6 in that exon causes glutamate
residue to be replaced with valine. This makes it less soluble and more
likely to polymerize, causing sickle-shaped agglomerations of globins
to result, which then block up capillary vessels.
o Usually heterozygotes for sickle-cell are phenotypically normal unless
put through heavy exertion. These heterozygotes are said to have
"sickle cell trait."
o Hemoglobin C: Also an A-to-T mutation in codon 6 in exon 1 of beta-
globin gene at a slightly different position. Changes the same
glutamate residue to lysine, making the protein less soluble (though
not as much as in sickle-cell). Less severe than sickle cell.
o DNA diagnosis: use a restriction enzyme that cleaves at the normal
site sequence in exon 6. With the mutation for sickle cell, can't cleave
there and have a larger fragment in that position. Can use a DNA
probe to detect these particular fragments and run PCR to detect
larger or smaller amounts of cleavage at this site.
• Know the six possible genotypes of alpha-globin locus, their clinical
phenotypes, and the geographical distributions of alpha-thal-1 (--) and alpha-
thal-2 (alpha -) alleles.
o α-thal-1 (--): caused by deletion of both copies of alpha globin gene in
alpha cluster on the same chromosome.
• (1) Homozygotes (--/--): results in stillbirth.
• (2) Heterozygotes (αα/--): Mild anemia ("alpha-thalassemia-1
trait").
• Mainly found in East Asian areas.
o α-thal-2 (α-): deletion of one copy of alpha globin genes. 50%
decrease in α-globin synthesis.
• (3) Homozygotes (α-/α-): Mild anemia in ("alpha-thalassemia-2
trait").
• (4) Heterozygotes (αα/α-): no disease phenotype.
• Mainly found in African and Mediterranean regions.
o α-thal-1/α-thal-2 (α-/--):
• (5) Compound heterozygotes, who have only 25% of normal
alpha globin levels. Severe anemia, called "HbH disease".
• Mainly found in people who have at least part SE Asian
ancestry.
o (6) Sixth genotype is normal people, who are fine.
• Understand the following concepts about beta-thalassemias:
o [Something to keep in mind: since there's two alpha globin genes on
each chromosome, you effectively get four chances to have at least
one of them work. With beta globin genes, you only get one per
chromosome, which means the odds are less in your favor.]
o (a) thalassemia major & thalassemia minor:
 • A clinical way of characterizing beta-thalassemias:
• Major is characterized by severe anemia, in which most red
blood cells are destroyed before being released into circulation.
• Minor means a clinically normal carrier of one beta-thalassemia
allele.
o (b) beta0-thalassemia & beta+-thalassemia:
• A molecular-biology/genetics way of characterizing beta-
thalassemias:
• beta0: Zero beta-globin synthesis. Caused by deletion of gene,
nonsense mutation, frameshift mutation in early beta coding
region. Generally this leads to death after birth.
• beta+: Most common beta-thalassemia. Some beta hemoglobin
still made. Often caused by mutations affecting transcription,
protein stability, etc.
o (c) beta0-thal allele & beta+-thal allele:
• I really don't know what this is. I presume it has something to
do with the beta0 and beta+ thalassemia characterizations,
above-- maybe beta0 means that the allele will produce no
function beta globin and beta+ means that it will produce a
limited amount.
o (d) simple beta-thalassemias & complex thalassemias :
• A biochemical way of characterizing beta-thalassemias:
• Simple: Caused by mutations or deletions that only target the
beta globin gene.
• Complex: Mutations or deletions that target not only the beta
globin gene, but other genes in the beta cluster, or the LCR.
Notice this can cause HPFH (below).
• Explain hereditary persistence of fetal hemoglobin (HPFH) and its significance.
Give examples of two types of mutations that are known to cause HPFH.
o HPFH is a condition that results from not switching over to beta globin
from gamma globin after birth (beta and delta globin genes deleted).
As such, it has genetic components that are heritable. Two common
genetic causes:
• Large deletion that brings an enhancer closer to gamma gene.
Leads to persistent expression of gamma hemoglobin in adult
(enhancers overcome the repressors).
• Point mutations in gamma gene destroy the targets of
transcriptional repressors, which means the genes can't be
turned off in adults.
o Note that gamma hemoglobin can substitute for defective beta
hemoglobins in part.

Pharmacogenetics
Tuesday, November 06, 2007
7:52 AM

Pharmacogenetics, 11/6/07:

• Know the difference between pharmacogenetics and pharmacogenomics.

 o Pharmacogenetics: Study of how variance in a single gene influences
variability in drug response, usually based on prior knowledge of drug
action pathways.
o Pharmacogenomics: Study of how variance across multiple genes
influences variability in drug response, usually not based on prior
knowledge of drug action pathways.
• As described in the notes, have a historical perspective on some of the key
events that led to the establishment of pharmacogenetics as a discipline.
o There were events. Some of them were key.
• Know the cost, in terms of mortality, morbidity and health care dollars, of
adverse drug responses in the human population.
o Fourth to sixth leading cause of death in US hospitals.
o Morbiditity/mortality estimated at $30 billion/year in US.
• Understand the cost/benefit considerations related to widespread
pharmacogenetic testing.
o Benefit: reduce adverse drug reactions.
o Cost: extremely expensive to genotype every person.
• Know that drug metabolism has Phase I and Phase II steps and understand
the characteristics of each as described in the notes.
o Phase I: "first pass" metabolism: hydroxylates drug, usually by
cytochrome P450 enzymes in the liver.
o Phase II: Conjugation reactions: glycosylation or acetylation to
deactivate drug, make it more soluble, and excrete it faster.
• Know the list of known single gene differences and their known pharmacologic
effects described in the notes.
o Malignant hyperthermia:
• Adverse response to inhaled anesthetics
• Caused by defect in RYR1 and other calcium channels
o Reaction to succinylcholine:
• Succinylcholine is a paralytic agent for surgeries.
• Reaction is caused by defect in pseudocholinesterase enzyme.
• Results in prolonged apnea (suspension of breathing)
o Glucuronyl conjugation:
• Results in bilirubin buildup; caused by problems in
glucuronyltransferases
• Results in Crigler-Najaar syndrome
o Isoniazide acetylation:
• Isoniazides are used as a treatment for tuberculosis.
• Defect in N-acetyltransferase-2 (can't acetylate isoniazides).
o Flushing response to alcohol
• Caused by deficiency in aldehyde dehydrogenase.
o Primaquine sensitivity
• Primaquine: anti-malarial treatment.
• Caused by defect in glucose 6-phosphate dehydrogenase
enzyme.
o Cytochrome P-450 polymorphisms
• Cytochrome P-450s are involved in metabolizing many different
drugs.
• Know that differences in drug response may be due to any of a number of
different types of genomic variations.
• Understand why it now appears that there may be more differences between
human genomes than previously appreciated: know the types of variations
(SPNs, structural variations, etc.) as described in the notes.
o SNPs
o Structural variations (duplications, insertions/deletions, inversions,
etc)
• Know why human genomic variation has been underestimated in the recent
past.
o Method used to map the human genome (shotgun sequencing) misses
a lot of variation in highly similar sequences within the genome.
• Know why pharmacogenomics is of great interest to pharmaceutical
companies.
o Reduce adverse drug effects
o Target drugs to particular populations
o MAKE A FUCKLOAD OF MONEY on account of they're the devil.
• Understand what is known about variations in the CYP2D6 gene, e.g. what
types of variations exist, what the range of responses are, and what types of
drugs are involved, as described in the notes. Also know about the interethnic
variation in CYP2D6 gene copy number and its consequences.
o CYP2D6: enzyme responsible for ~20% of drug metabolism in the
liver, across a wide variety of drug classes.
o Over 75 variant alleles of this gene: SNPs, gene deletions, gene
duplications.
o Classification of patients based on phenotypes:
• Ultrarapid metabolizers (UMs)
• Extensive metabolizers (EMs)
• Intermediate metabolizers (IMs)
• Poor metabolizers (PMs)
o Copy number variation: from 0 to 13 copies.
o High copy number: Saudi Arabians and Ethiopians among others.
• High copy number seems to correlate with rapid drug
metabolism (less drug action, tapering out more quickly).
• Know about TPMT variation and thiopurines and about CYP2C9 variation and
warfarin.
o TPMT: thiopurine-S-methyltransferase.
o Involved in metabolism of thiopurines (used for cancer treatment).
o Variation here seems to mainly be SNP-inactivation of the TPMT gene,
which results in slower metabolism of thiopurine drugs (need smaller
dose to be effective).
o Zygosity of TPMT-inactivating mutations in patient indicates how much
of normal thiopurine dose should be given.
o CYP2C9:
• Related to warfarin metabolism, as CRP2D6 is to thiopurine
metabolism.
• Inactivations in CYP2C9 are associated with lower rates of
metabolism of warfarin (thus dosing requirements go down).
• Understand how response to drugs can involve many different genes and
pathways (not just drug metabolizing enzymes), as described in the notes on
pharmacokinetics and pharmacodynamics.
o (transporters, signal transduction downstream, etc.)
 o (and I thought he said several times in lecture that we weren't
responsible for the table in the notes.)
• Understand the clinical relevance of drug target pharmacogenomics and the
targets that are listed in the table in the notes.
o Effectively not much different from "why drug companies want to use
it," above.
• Understand the "double-edged sword" nature of human genome variation.
o Produces greater "insurance" against species- or community-wide
catastrophes, and drives adaptive change, but also results in high
disease incidence.

Genetic Testing
Wednesday, November 07, 2007
8:02 AM

Genetic Testing, 11/7/07:

• Be able to define what constitutes a ‘genetic test’:

o (1) Analyzing an individual's genetic material to determine
predisposition to a particular health condition or to confirm a diagnosis
of genetic disease.
o (2) Examining a sample of blood or other body tissue for biochemical,
chromosomal, or genetic markers that indicate the presence of
absence of genetic disease.
o Notice this involves DNA and non-DNA factors, and looking at risk as
well as disease.
• Identify methods of ‘genetic testing’ that do NOT involve the direct analysis of
DNA sequence
o Biochemical tests (for amino acids, organic acids, etc), enzyme activity
assays, protein electrophoresis, cholesterol testing, X-rays, medical
history and family history, blood pressure, etc.
• Understand the basic approaches, advantages and limitations of the following
types of genetic tests:
o (a) Chromosome analysis:
• Used when chromosomal abnormality is suspected. Often done
with amniocentesis on pregnant women, or from peripheral
blood.
• Method: stain, look at chromosomes under microscope, look for
abnormal chromosome number and large (3-5 Mbp) changes
(large deletions, duplications, insertions, rearrangements).
• Note that it can't detect small chromosomal changes.
o (b) Fluorescent in situ hybridization (FISH):
• Used to diagnose deletions, some translocations, abnormality of
copy number (smaller scale than chromosome abnormalities).
Often used in prenatal settings and to detect certain cancer
genes.
• Method: use fluorescently labeled DNA probes to pick up copies
of complementary DNA in patient's denatured genetic material.
• Note that FISH needs a known genetic sequence to work-- it
can't detect unknown genes (it needs to be able to bind to a
 complementary sequence, which means that you need to know
that sequence when you're making it). Ironically, you can't go
'fishing' for an unknown deletion with this method.
o (c) Restriction digests:
• Used when a known mutation alters the pattern of an
endonuclease digest.
• Method: use a restriction endonuclease and look at the sizes of
the fragments that come out of it. If the mutation changes the
normal fragmentation pattern, should show up on a gel or
however else you want to detect it.
• Note that this is pretty much useless unless a specific mutation
occurs right at the restriction site of a given endonuclease.
o (d) Linkage analysis:
• Linkage: the tendency for alleles physically close to each other
on a chromosomal segment to be transmitted together, as an
intact unit, through meiosis (not following Mendel's Law of
Independent Assortment).
• Can be used to determine the probability of an offspring
inheriting a DNA locus containing a mutation, based on looking
at some nearby or 'linked' allele that presumably is inherited
along with the disease allele.
 You look for a pattern of alleles that indicates disease by
tracking the disease through the family, then test for
that pattern in patient.
• Notice that this analysis doesn't identify the particular
mutation, just that one's occurred.
o (e) Genetic sequencing:
• Amplify given sequence of DNA with PCR; purify. Use
radiolabeled oligonucleotides to replicate DNA (see 10/26/07,
"Tools of Molecular Biology") to determine the sequence of a
given gene in a given individual.
• Notice this can miss mutations in promoters, some introns, etc.
• No detailed questions on exam.
o (f) Detection of tri-nucleotide repeats:
• Can use Southern blotting: depending on how many repeats
are present in the DNA, it's run slower on the gel. Generally,
the slower the DNA runs on the gel, the worse the phenotype
is.
o (g) Microarrays:
• Can test the presence or the activity of genes (which are being
transcribed, which aren't). Can study transcription of 1000's of
genes at once using microarrays.
• Effectively you pick up cellular material (containing DNA/mRNA)
from the patient, purify it, and "dot" it across a glass slide. On
that glass slide are lots of different DNA probes, one for each
gene you want to look at. Once you "dot" the same amount of
cellular mRNA onto each such DNA sequence and allow the
probe to hybridize, you can detect whether mRNA for that gene
is being expressed, and at what level, in the patient's cells.
• No detailed questions on exam.
• Interpret genetic testing results and distinguish between ‘informative’ and
‘non-informative’ results.
 o Informative: The genetic test definitively proves or disproves presence
of disease.
o Non-informative: It's possible to have false negative results and false
positive results.
• Explain how allelic heterogeneity and genetic heterogeneity can affect the
sensitivity of genetic tests.
o Allelic heterogenuity: multiple mutations in a given gene can cause
disease (thus have to test for all of them to be sure).
o Genetic heterogenuity: mutations in multiple genes can cause
disease (again, have to test them all to be sure).
o So with sickle-cell, it's one gene, one mutation: if you don't have the
mutation, you don't have sickle-cell. On the other hand, cystic fibrosis
shows up on one gene but 1000's of mutations-- so testing for one or
two is non-informative.

Treatment of Genetic Diseases

Wednesday, November 07, 2007
8:05 AM

Treatment of Genetic Diseases, 11/7/07:

• Understand the concept that while curing genetic diseases remains

challenging, many genetic diseases are amenable to some level of
treatment/management.
o Can be either medication-based or behavioral (diet, etc).
• Recognize some of the genetic conditions that currently can be treated and
those for which treatment may soon be available.
o Disease
Intervention
Substance/Technique
G6PD deficiency
Avoidance
Antimalarials, barbituates
PKU
Diet Restriction
Phenylalanine-depleted diet
Galactosemia
Diet Restriction
Galactose-depleted diet
Hypothyroidism
Replacement
Thyroxine
Biotinase deficiency
Replacement
Biotin
Urea cycle defic.
Diversion
Sodium benzoate
Hyperchol.emia
 Diversion
Oral resins
Hyperchol.emia
Inhibition
Statins
Hyperchol.emia
Depletion
LDL apheresis

o Notice variety of interventions above: avoidance, diet

restriction, substance replacement, diversion to a different chemical
pathway, inhibition of pathways, depletion of overexpressed
substances.

o Strategy
Example
Status
Cofactor admin.
Biotinase defic.
50% patients responsive

Replace extracellular protein

Factor VIII in hemophiilia, α1-antitrypsin
Effective

Replace intracellular protein

ADA deficiency
Effective

Targeted replacement of intracellular protein

Gaucher disease
Effective

• Identify examples of pharmacogenetic management of patients (e.g.

medication selection and dosing).
o Mentions cytochrome P450 in the notes (recall, is responsible for most
of first-pass metabolism in the liver) as well as N-acetyltransferase
(recall, is responsible for metabolizing isoniazides). Ideally, should
figure out if there's a defect in either of those enzymes before you go
giving standard doses of the drugs that they metabolize.
o Also mentioned in lecture:
• Copy number of CYP2D6 gene: determines fast or slow
metabolism of certain drugs.
• TMPT mutations inactivate body's ability to metabolize
thiopurines (thiopurines used to combat common cancers in
children).
• Be able to discuss examples of genetic disorders that are treated on the basis
of protein replacement therapy.
 o Alpha-1 antitrypsin deficiency can be addressed by giving the patient
recombinant AT1; if caught early, can prevent accumulation of
elastase-mediated lung injury.
o Fabry disease (deficient in alpha-galactosidase A enzyme, leading to
buildup of galactosides in lysosomes): leads to kidney disease,
neuropathy, and cardiac complications. Can be treated with
recombinant alpha-galactosidase specifically targeted to the lysosome.
o Pompe disease: enzyme missing (alpha-glucosidase) that breaks down
glycogen, leading to muscular failure. Can be treated in a similar way
with IV drugs.
• Identify the principles that need to be addressed to ‘cure’ a genetic disease
through gene therapy.
o The idea is to introduce DNA/RNA into human cells to cure/slow
progression of the disease.
o Considerations:
• Easy production
• Targeting: specificity
• Sustained production
• Efficacy
• Integration
• Size capacity
• Toxicity
o Retroviral (RNA viruses), Adenoviral (DNA viruses), Non-viral (insert
DNA in lipids):
• Retroviral therapy:
 Advantages: integrate into host genome, minimal host
immune response
 Disadvantages: limited insert size, able to infect only
dividing cells
 Safety: risk of insertional mutagenesis/germline
integration
 Efficiency: Retroviral titers relatively low-- efficient at
infection, but only in dividing cells.
 Duration: The gene can be passed on to the daughter
cells of the host.
• Adenoviral therapy:
 Advantages: wide variety of cell types able to be
targeted, large insert size, stable
 Disadvantages: doesn't integrate into host genome,
transiently expressed, there's a risk of malignant
transformation, can be severe immune response.
 Safety: Lower risk of insertional mutagenesis, but note
potential for severe immune response.
 Efficiency: Can infect non-dividing cells; possibility of
higher titers.
 Duration: Typically short-lived (not passed to daughter
cells).
• Non-viral therapy:
 Advantages: very large insert size (can deliver mini-
chromosomes), minimal host immune response.
 Disadvantage: low efficiency, transiently expressed.
 Safety: Does not integrate into host genome.
  Efficiency: Low due to degradation by cellular
mechanisms.
 Duration: Typically short-lived (not passed to daughter
cells).
• Understand the theoretical risks of gene therapy.
o There are ethical risks to gene therapy (no kidding).
o There are physical risks to gene therapy (no kidding).

Finding Disease Genes

Wednesday, November 07, 2007
8:05 AM

Finding Disease Genes, 11/7/07:

[Note that these are kind of sketchy, partly because I'm not sure how well I
understand this and partly because the lecture and notes didn't always line up well
with the LO's. Take with a larger than usual grain of salt.]

• Understand the concept of individualized medicine and how this will change
the traditional medical model:
o Effectively can predict disease before it happens in the individual, as
well as treating according to individual genotype.
• Know what evidence is usually taken as evidence that a disease/trait involves
a genetic component and how this is measured:
o Honestly, I'm not sure what he means here. I would guess it probably
involves looking at heredity patterns as a first step. He may be
referring to this:
o Positional cloning: make use of linkage analysis to determine a disease
gene's position in genome, then identify gene(s) involved, using
human genome project data.
• Know the two principal types of polymorphic markers typically used in
genomic medicine and the differences between them:
o Notice that you can only track differences between people or through
families.
o Microsatellites: tandem repeat segments to fingerprint individuals and
establish framework against which to locate genes
o Single-nucleotide polymorphisms: single-nucleotide differences
between individuals.
• Notice that frequency of SNPs vary between ethnic groups
o Copy-number variations: not much known regarding their disease
significance.
• Understand haplotypes and haplotype blocks:
o Haplotype: As I understand it, a haplotype is a pattern of
polymorphisms over a region of the genome.
o Haplotypes are differentiated from each other by recombination
events.
o International haplotype mapping project: determined allelic
frequencies in various ethnic groups, how they fit together in genome
 o Haplotype blocks: patterns of polymorphisms (SNPs) that stay the
same over long periods of time. These are generally clusters of genes
on areas of the genome that don’t exhibit much recombination and
thus stay fairly stable.
• [How to find disease genes:]
o Hypothesis-driven: think a given gene is responsible, sequence it
(candidate gene sequencing), look for mutations in that gene. Most of
the time, the hypothesis is incorrect, especially in diseases partially
due to environmental factors or diseases due to a variety of genes.
• Candidate gene association studies: look for mutations in genes
nearby or associated with gene of interest.
• Notice that in hypothesis-driven research, can't discover new
genes or pathways, just features or patterns among known
genes.
• Most of these studies are case-control studies.
• Most of them are also completely wrong-- 96% false positive
rate.
• Other type of study: "family-based," compares allele
transmission from parents to patients.
 "transmission disequilibrium test": compares
transmission frequency of marker alleles from parents to
affected offspring (if gene causes disease, transmission
should be higher than chance [50%]).
o Discovery-driven: don't have a particular gene in mind before you
start looking.
• Genetic linkage studies: look for regions of genome that are
systematically co-inherited along with disease: can discover
new genes within regions of genome that you mark as relevant
to disease.
• Obviously this is easier with Mendelian alleles that have strong
phenotypic effects and are fairly uncommon.
 Genome-wide association studies: the handout's definition is wrong
here. NIH defines these as "any study of genetic variation across the
entire human genome that is designed to identify genetic associations
with observable traits (such as blood pressure or weight), or the
presence or absence of a disease or condition."
• I gather that you're effectively doing case-control studies
looking at not just a few genes of interest but thousands of
polymorphisms across the entire genome. With haplotyping, I
think you can define more precisely what genetic group a
certain person belongs to, so you can match them more
accurately with others from that genetic group as controls.
Presumably this means that your false-positive rate falls
dramatically and that you can accurately pinpoint the genetic
basis for disease much more quickly.
• Understand the differences between, appropriate applications, and limitations
of:
o A) Genetic linkage studies:
• (i) Know how recombination can define genetic intervals:
 Close-together genes on chromosome: less
recombination, more often inherited together.
  Far-apart genes: more recombination, less often
inherited together.
• (ii) Know LOD score criteria for linkage:
 LOD is a statistical measure of likelihood that loci are
linked together given the inheritance/disease pattern
• (iii) Know definition of centiMorgan (cM):
 One cM is 1% recombination between any two genetic
loci per meiosis.
 Used to measure "genetic distance" between two genes
(or, inversely, "linkage" between two genes).
o B) Genetic association studies:
• (i) Gene-specific: (see above)
 (a) Know strengths and weaknesses of case-control
studies:
• "controls" often are from a genetically distinct
group from the cases. Also, looking at one gene
may not give you a good picture of what's going
on unless you get crazy lucky.
 (b) Know strengths and weaknesses of family-based
studies:
• Strengths: can be fairly sure your controls are
from the same genetic group as your cases.
Weaknesses: limited external validity (thank you,
biostats) and difficulty tracking everyone down
and getting them in one place.
• (ii) Genome-wide:
 Test all parts of genomes simultaneously between
individuals for patterns of SNPs (look for SNP patterns
highly associated with disease); look for differences that
are statistically significant.
 Requires large number of cases and controls but not
limited to families.
 Works best for common alleles with strong or weak
effect
 Can locate mutations with high accuracy

Genetic Counseling
Wednesday, November 07, 2007
9:50 AM

Genetic Counseling, 11/7/07:

[Really, this is pretty much common sense-- of course, they could always whip a
ninja move and ask something crazy on the exam. But I'm betting not. These aren't
really done and, in my opinion, don't deserve to be. Essentially: Be cuddly and
understanding and nonjudgmental and everything will be okay. Oh, and people think
"1 in 10,000" is different from "9 times the risk." And the sky is blue and fish live
underwater. Next question.]

• Identify the goals of genetic counseling.

 o comprehend medical facts, understand condition's heredity,
understand options for dealing with recurrence risks, adjust to
condition
• Recognize the basic tenets and the ethical principals of genetic counseling.
o educational, nondirective, unconditional, supportive
o respect for patient autonomy, beneficence, nonmalfeasance, justice
• Recognize indications for genetic evaluation and counseling.
o Hereditary condition in family, fetus/child with birth defect, child with
mental retardation, exposure to carcinogen, consanguinity, advanced
maternal age, family history, ethnicity, etc.
• Recognize reproductive options currently available for families at increased
risks and apply appropriate options based on genetic condition and family.
• Recognize factors that may impact the client's perception of risk, selected
course of action, and utilization of services.
o Honestly, most of these are pretty obvious.
• Participants will gain an appreciation of emotional and ethical issues
associated with presymptomatic testing for adult onset disorders lacking
effective treatment therapies.

Autosomal Dominant Disorders

Thursday, November 08, 2007
8:00 AM

Autosomal Dominant Disorders, 11/08/07:

• Know the characteristic features of autosomal dominant inheritance.

o Disorder is expressed in the heterozygote.
o Affected individuals have at least one affected parent, unless it's a new
mutation.
o Offspring of an affected parent have a 1/2 chance of inheriting the
defective gene (and thus having the disorder).
o Tends towards a "vertical pedigree": disorder appears in each
generation, as opposed to the "horizontal pedigree" of autosomal
recessive.
o Autosomal (not sex-linked).
o Frequently have late-onset (ie Huntington's disease).
o Frequently involve structural protein defects (ie Osteogenesis
imperfecta).
• Understand why autosomal dominant disorders are in general less lethal than
other inherited disorders, and why they show such a variable phenotype even
within families.
o If they were particularly lethal as well as dominant, they would have
died out (not have had a chance to be transmitted).
o This implies that AD diseases aren't magically less lethal than AR
diseases-- just that the lethal AD mutations don't reproduce
successfully (thus have no pedigree).
• What factors may complicate the assessment of an autosomal dominant
pedigree? What is the difference between penetrance and expressivity, and
between genocopy and phenocopy when describing an inherited disorder.
 o AD diseases tend to have a wider range of clinical presentations than
AR.
o Penetrance tends to be more of an issue (whether or not a diseased
individual shows the disease phenotype).
o Expressivity as well (to what degree the affected individual shows the
phenotype).
o Genocopy: A mutation in a different gene causing the same syndrome.
o Phenocopy: A syndrome caused by environmental factors that mimics
a genetic disease.
• Understand the characteristic features and molecular basis of:
o Achondroplasia: Most common form of dwarfism. Results from a
mutation in FGF-R3 protein, which leads to its ligand-independent
activation, inhibiting chondrocyte synthesis (chondrocytes: cells
causing long bone growth). About 80% of achondroplasia cases are
new mutations.
o Marfan Syndrome: Connective tissue disorder; musculoskeletal and
cardiovascular (weakening of connective tissue in aorta) problems as
well. Caused by mutation in fibrillin gene on chromosome 15. Physical
exertion can cause dissection of the aorta.
o Neurofibromatosis-I: Frequency 1/3500, many new mutation cases.
Show café-au-lait spots and peripheral nerve tumors later in life.
Caused by a mutation in the NF-1 gene on chromosome 17, which may
be a tumor suppressor gene (turns off oncogene ras).
o Huntington's Disease: Frequency 1/10,000; usually late-onset in 3rd
or 4th decade. Causes neuronal atrophy in the caudate nucleus of the
basal ganglia (resulting in involuntary muscle twitching and jerking),
slowly progressing to death in 5-15 years. Caused by expansion of
CAG-repeat in HD gene on chromosome 4. Notice homozygosity for
this disease is no different from heterozygosity in terms of clinical
symptoms. Notice also that early-onset Huntington's tends to occur
when it's inherited from their fathers (expansion of CAG repeat occurs
during male genetic transmission).
o Familial Alzheimer Disease: Tends to show early-onset Alzheimer's.
Notice that the great majority of Alzheimer's is sporadic.
• What is a paternal age effect? What problems do late-onset disorders create
for genetic counseling?
o Paternal age effect: increased frequency of inheriting disease gene
when father is over 39 years old.
o Problems for counseling: since disease may not have shown by the
time the parents want to have children, may not be able to counsel
them accurately on the genetic risks posed to their offspring.

X-Linked Recessive Inheritance and Mitochondrial Diseases

Thursday, November 08, 2007
8:01 AM

X-Linked Recessive Inheritance and Mitochondrial Diseases, 11/8/07:

• Know the characteristic features of X-linked inheritance.
o Incidence is primarily in males.
o Trait can't be transmitted from father to son.
o Disease gene is transmitted from an affected father to all his
daughters, who will either be carriers or show the trait depending on
whether they also inherit a diseased X gene from their mother.
o Carrier females transmit the gene to 1/2 of their sons and daughters.
o Occasional clinical manifestation in carrier females.
o Disorders are generally severe, with early onset.
o If the disorder is lethal (or causes infertility) in males, 1/3 of
the male cases are new mutations (mother not a carrier).
• When do females show clinical manifestations of X-linked disorders? When do
females show “full blown” X-linked recessive disease?
o Lyonization (inactivation of one X chromosome in any given cell):
sometimes the abnormal X is activated in cells in a given tissue.
o Females can show full-blown X-linked recessives when both parents
have the genes, or have Turner's syndrome (one X chromosome), or
have a translocation of the affected portion of the X chromosome onto
an autosomal chromosome.
• Why are about 1/3rd of the individuals who inherit an X-linked recessive
disorder that is eventually lethal, considered to be new mutations?
o About 1/3 of inherited, diseased X chromosomes are in boys, who will
die before reproducing. If the disease rate stays the same, that means
that 1/3 of the disease states result from new mutations. (note this
isn't exact but a good approximation.)
• [Notice the following descriptions of intergenic and intragenic recombination:
intragenic recombination results in a swapping of chromosomal material in
the middle of a gene (thus makes a hybrid gene), intergenic recombination
results in a swapping of chromosomal material between genes (thus no hybrid
genes).
• What are the clinical, biochemical/cytogenetic and molecular features of:
o Red/green colorblindness: Affects ~8% of Causasian males; due to
defective red or green pigmentation (opsin) gene on the X
chromosome. Often results from intragenic (hybrid pigmentation) or
intergenic (non-hybrid pigmentation) recombination between red and
green genes-- frequently occurs due to misaligning at meiotic
recombination, since red and green genes are very similar. Notice that
a locus control region only allows the first two opsin genes to be
expressed (thus if you have a red gene and a hybrid, mostly-red gene,
even if you have a few more green genes after that, you're still green-
colorblind).
o Hemophilia A: Frequency is 1/10,000 male newborns in US, shows a
defect in anti-hemophilia globulin (Factor VIII-- effectively, can't clot),
often resulting from intragenic inversion on X chromosome. Notice that
recombinant factor VIII can now be made and used to treat this
disease.
• That intragenic inversion often results from the long arm of X
flipping back and recombining with itself at factor VIII site
(results in particularly severe hem A).
• Can diagnose affected or carrier status with restriction
fragment length polymorphism, PCR.
 • Genocopies:
 Hemophilia B: X-linked recessive disorder in Factor IX.
 Von Willebrand's disease: AD-inherited disorder affecting
Factor VIII.
o Duchenne muscular dystrophy: Fatal disease, frequency 1/3,500.
Involves progressive muscle weakness. Characterized by a defect in
dystrophin protein production (dystrophin anchors the actin filaments
to the muscle cell wall), usually caused by frameshift deletions in the
extremely large DMD gene. Notice that there are less severe forms of
the disease (Becker's MD) caused by in-frame deletions within the
gene (Becker's is allelic with Duchenne's).
• Notice that disease can sometimes be shown in heterozygote
carriers when the affected X chromosomes translocated to
autosomal chromosomes.
o Fragile-X syndrome: Characterized by a constriction near the distal
end at long arm of X chromosome. Results in moderate retardation
with dysmorphic facial features. There is reduced penetrance in men
(often don’t show symptoms).
• Caused by an expansion in the CGG repeat region of the FMR1
gene, enhancing methylation and silencing FMR1.
• Frequency is about 1/4000 male births (lecture says 1/1000-
1/1500).
• CGG triplet expansion occurs during female genetic
transmission.
o [Lesch-Nyhan disease: very rare. Involves self-mutilation.]
• X-linked dominant example: Vitamin-D resistant ricketts.
• What are the characteristic features of Maternal Inheritance pedigrees? What
genetic material is mutated? Clinical symptoms of these diseases are usually
restricted to which tissues?
o Diseases transmitted by a mother to all of her children, male and
female.
o Notice that affected males can't pass it on to any of their children.
o Due to mitochondrial defects.
o Diseases usually assoc. with oxidative phosphorylation and occur in
tissues with high energy requirements-- brain, eyes, muscle, heart,
kidneys, and liver.
o Example: Leber's hereditary optic neuropathy.

Research Ethics I
Thursday, November 08, 2007
8:01 AM

Research Ethics I, 11/8/07:

[Again: this is not really learning-objective friendly. Use your common sense here.
It's not okay for parents to say "Sure, test new random drugs on my kid without any
connection to therapy." It's not okay for researchers to promise potential subjects
that the drug will cause peace and happiness in the Middle East, or to say "it's
perfectly safe" while ducking behind lead-lined barriers (though my dentist still gets
away with this). Non-PC way of saying all this: IRBs are there to cover institutions'
collective asses by making sure nothing gets approved that could cause lawsuits to
be filed against the institution.]

• Identify the major responsibilities of Institutional Review Boards.

• Apply the ethical principles that underlie the responsible conduct of human
subjects research to three case studies.
• Understand the role of the informed consent process in human subjects
research.
• Explore the significance of coercion and decision-making capacity to the
informed consent process.
• Identify some of the risks that financial conflicts of interest pose to the ethical
conduct of human subjects research.

Molecular Analysis of Mendelian Disorders

Friday, November 09, 2007
8:58 AM

Molecular Analysis of Mendelian Disorders, 11/9/07:

• What are restriction fragment length polymorphisms (RFLPs), and do they

have to be within the gene in question to be informative? Are RFLPs ever
100% accurate?
o DNA fragments generated by restriction endonucleases.
• Use A: Since sometimes the genetic sequence at a restriction
site mutates, this can make the site either more or less
amenable to being cut. By comparing the restriction length
fragments from a subject to a control, "normal" DNA sequence,
you can tell if the sequence at that point has changed by
looking at the size of the fragments that are generated.
• Use B: Can use that principle, combined with observations
about who's sick and who's not, to establish and follow linkage
between a distant gene and the disease gene. The restriction
sites don't have to be within the gene itself to be used to
diagnose disease-- they just have to be within a gene that's
linked to it (low recombination rate). Can only use this within
families in which you can match the different restriction lengths
to a phenotype.
 Further note on this: effectively you're using a mutation
on the same chromosome as a disease gene to 'follow'
that portion of the chromosome through offspring. As
long as the portion of the chromosome that you're
following doesn't swap over to a different chromosome
during meiosis, you can use the fragment length of an
individual's DNA to predict whether or not they've
inherited the chromosome that you know (from
empirical evidence) causes the disease.
o Generally this has about 90% accuracy (10% chance of recombination
throwing off linkage).
 o Notice you don't need to know much of anything about the disease
gene itself to follow it with this technique.
• Know how synthetic oligonucleotide probes can provide specific information as
to the inheritance of a particular mutation.
o Once you know the sequence across a given possible mutation site,
can make two different oligonucleotides, one with the normal and one
with the disease sequence, and use hybridization to see which one
binds to the patient's DNA.
• Note can use this for alpha-1 antitrypsin deficiency (distinguish
between the point mutation variations in alpha-ATD) and
certain beta-thalassemias.
• Understand how PCR technology has improved the speed and specificity of
molecular diagnosis of specific genetic disorders.
o Recall that PCR, given a 5' "priming" sequence, can massively replicate
DNA very quickly.
o If you use primers that are designed to either bind to a 'normal' DNA
sequence at the disease site or to a specific disease DNA sequence,
then PCR will either amplify tremendously or not at all depending on
whether that sequence is present or not, and the difference in
expression can easily be shown on a gel.
o Specifically, can use to look for specific cystic fibrosis allele mutations.
• Appreciate how DNA microarrays will greatly improve diagnosis of
multifactorial genetic disorders.
o Basically it's more efficient. You can look at thousands of different
SNPs at once and establish a "constellation" or pattern with which to
predict or diagnose disease states.
• What is "genetic anticipation", and how does it relate to triplet repeat
expansion disorders?
o It's when the symptoms of a genetic disorder show up at an earlier
and earlier age as the disease is passed from generation to generation.
o Effectively, here's the thing. Say you have nine CAG repeats in front of
some gene or other. Say that's enough to cause disease symptoms at
age 60. Suppose, during meiosis in your germ cells, the chromosomes
line up slightly incorrectly at this point on account of all the repeats,
and suppose that a cross-over occurs in which, after the sixth CAG
repeat on one chromosome, everything after the third CAG repeat on
the other chromosome is transferred and vice versa. Now you have
two recombinant chromosomes, one with 12 CAG repeats and one with
6. Anticipation, in this context, is the process in which more and more
repeats are accumulated this way through generations.
• When do triplet repeat expansions cause disease? What is the current
thinking as to how trinucleotide repeats expand, and why their polyglutamine
containing gene products lead to neuronal cell death in HD?
o They cause disease in a variety of ways: they can cause a protein to
be nonfunctional (ie methylation of CGG-rich region in Fragile-X
syndrome), they can make an abnormal protein with novel function (ie
Huntington's Disease, in which the abnormal huntingtin protein now
interacts with a variety of transcriptional regulators), or they can make
novel RNA that futzes about with things it shouldn't (ie myotonic
dystrophy, in which the abnormal RNA binds to all kinds of receptors).
o As far as how they expand, it occurs during replication, repair, or
recombination:
 • During replication: may have something to do with hairpin loop
formation (DNA bonding with itself) on lagging strand during
Okazaki fragment synthesis.
• During repair: may be associated with mismatch repair proteins
putting in too many repeats, forming a DNA hairpin.
• During recombination: may involve recombination within the
repeating tract, particularly when the chromosomes aren't
correctly aligned.
o Polyglutamine-containing proteins: occur in Huntington's from CAG-
repeats. Cause trouble both because they aggregate with each other in
beta-sheets and also interact with a variety of transcriptional
regulators abnormally.
• Understand how to calculate recurrence risks for genetic diseases based on
pedigree information and molecular analyses.
o I believe this is the whole "mother's brother's dog's cousin has the
disease, what's the risk to the mother's child" thing. Need to be able to
distinguish autosomal recessive/dominant and X-linked recessive, also
probably X-linked dominant and maternally inherited patterns.
Remember the thing about 1/3 of all fatal X-linked recessive diseases
being new mutations.

Molecular Basis of Carcinogenesis I, II, III

Monday, November 12, 2007
7:58 AM

Molecular Basis of Carcinogenesis I, II, III, 11/12/07:

[These are a little scattered, partly because it's new to me and partly because Dr.
Sclafani is often more enthusiastic than he is organized.]

• Describe the properties of malignant cancer cells. You should be able to give
at least five different properties.
o Phenotype properties:
• Unresponsive to normal signals for proliferation control.
• De-differentiated (lack specialized structures of tissues in which
they grow).
• Invasive (can grow out into neighboring tissues).
• Metastatic (can shed into circulation and proliferate elsewhere
in the body).
 Generally it's the metastasis that's fatal in cancers.
• Clonal origin (derived from a single cell).
o Increased transport of glucose.
o Lack of contact inhibition (will grow over each other).
o Immortality (ability to grow indefinitely).
o Can grow without an attachment to a solid substrate.
• What is the multi-step process for carcinogenesis? You should be able to
discuss the relative importance of heredity and the environment and why
early events may include mutations in DNA repair genes.
o Not considered to be an inherited disease (not inherited as a single
Mendelian gene).
 o On the other hand, cancer susceptibility genes are certainly heritable
(see Knudson below).
o Carcinogenesis is characterized by the accumulation of many genetic
alterations or mutations, particularly over a long period of time-- thus
age is strongly associated with cancer, as are environmental factors
that produce high rates of mutations.
o If DNA repair genes are damaged early on, the rate at which you
accumulate DNA mutations - since you can't repair them as well - goes
up markedly.
o Multi-step process for cancer:
• (1) Normal cell
• (2) Increased proliferation: With a mutation or two, an
immortalized cell (see below for examples).
• (3 + 4) Early/progressive neoplasia: With a few more
mutations, get abnormal growth patterns.
• (5) Carcinoma: A full-on tumor.
• (6) Metastasis: A tumor that's spreading through the circulatory
system.
• I think the point he's trying to make here is that you need a
fairly wide assortment of mutations (though order isn't
important) to result in a cancer:
 Turn on oncogene or make oncogene protein much more
active
 Turn off tumor suppressor genes (both cell cycle
regulatory and DNA repair genes)
 Turn off apoptotic genes and turn on anti-apoptotic
genes.
[Notice that the notes have the following unelaborated steps: Tumor
initiation, promotion, conversion, progression.]
• What types of genes are usually mutated in tumor initiation? Describe the
effect on cellular proliferation that the product of these genes has.
o Either activated oncogenes (proliferation genes) or silenced tumor
suppressor genes.
o Oncogenes: accelerate proliferation. Tumor suppressors: slow down
proliferation.
• What type of cytogenetic abnormalities are associated with malignancy? You
should be able to give at least two different examples.
o Translocations of chromosomes, deletions on chromosomes:
• Can activate oncogenes (for example, by putting an extremely
active promoter upstream of one).
• Can inactivate tumor suppressor genes (for example, by
translocating another gene into the middle of the tumor
suppressor sequence)
o Notice that this is kind of a silly point to make, at least on its face.
Anything that can bring a promoter nearby an oncogene (like pretty
much any chromosomal rearrangement) or anything that can interrupt
transcription or promotion of a tumor suppressor gene (likewise) can
be associated with malignancy.
o Notice that aneuploidy (recall: loss or gain of a chromosome) is
associated with a poor outcome in many cancers.
• What events can produce LOH? Give at least two examples and state how
they support Knudson’s theory.
 o LOH = Loss Of Heterozygosity.
o Means you inherit a heterozygous state (say, for a working tumor
suppressor gene), but convert to (negative) homozygosity at some
point.
o Loss of this heterozygosity means you lose the one working copy of
the gene that you have, through:
• mutation
• mitotic recombination
• chromosome loss
• and/or environmental factors
o Knudson sez: If you're heterozygous, you have one "strike" against
you through your genes (one copy of suppressor gene knocked out in
your parents' passed-on DNA). If you have one more "strike" (ie,
exposure to UV light causes an unrepaired mutation in the other copy
of that gene), you're unable to produce that tumor suppressor gene
product at all-- which leads, potentially, to cancer.
o Example: Familial retinoblastoma vs acquired retinoblastoma (in
children vs. adults, bilateral vs. unilateral)-- former is easier to acquire
since it only requires one mutation event.
• Are cancers associated with both dominant and recessive syndromes? You
should be able to give a different example of each type.
o Familial retinoblastoma: A recessive disease but inherited in a
dominant way: need both tumor suppressor (RB) genes knocked out
to show a phenotype (thus recessive), but everyone who inherits
heterozygosity winds up with the disease (thus dominant) due to LOH
problems.
• Notice that this inheritance pattern shows a vertical pedigree
(looks like autosomal dominant).
o Sporadic retinoblastoma: Mutations in both tumor suppressor genes
needed to knock out function- thus not strongly inherited (not sure
how he's tying this to the LO, as somatic sporadic mutations shouldn't
be inherited at all).
• Describe how the RB (retinoblastoma) gene was first identified. You should be
able to describe the important cytogenetic and molecular evidence.
o Sorry, I was scrambling around trying to figure out the last thing he
said when he was going over this. It probably has something to do
with examining pedigrees of familial RB patients, noticing the
inheritance pattern, and using some kind of tagging technology to
figure out which gene was present in those with a greater tendency to
get RB and absent in those without that tendency.
• What are the properties of the protein product of the RB gene? List at least
three biochemical properties
o [Notice that the RB protein is a universal protein-- not just found in
the retina.]
o An inhibitor of the cell cycle that prevents proliferation.
o Normally hypophosphorylated (little PO4) to prevent proliferation.
o Hyperphosphorylated by CDKs (cyclin-dependent kinases) to be turned
off.
o When turned off, allows normal cell proliferation.
o When kept off or inhibited, allows unchecked cell proliferation and
carcinogenesis.
 o General note: RB protein works by binding to a variety of transcription
factors.
• Describe how the RB protein functions during the cell cycle and why it is
important in cancer. You should be able to give an explanation of how the loss
of RB may produce a malignancy.
o As mentioned, functions to block G1 moving to S (replication) phase.
Without RB, a cell has no 'brake' on its proliferation.
o Notice that there are certain tissues that are particularly susceptible to
the loss of RB-- that is, losing RB there has a particularly acute effect.
Example is, obviously, the retina.
• What is the hallmark of a tumor suppressor gene or anti-oncogene? You
should be able to use the RB gene as an example
o Prevents cells from proliferating by controlling cell cycle.
• How were oncogenes discovered? You should be able to describe the method
with at least three different examples
o Discovered in oncogenic retroviruses (specifically Rous Sarcoma Virus
in chickens). With one particular viral gene segment (v-onc), tumors
are rapidly induced in the infected cells after infection; without it,
integration into the host genome occurs without activation of
oncogenes.
o Examples: v-src, v-erb, v-myc, etc. Watch for the v- at the beginning
of it. (as opposed to c-myc, which is an endogenous oncogene in the
human genome.)
o Often oncogenes mimic growth factor receptors to achieve their
nefarious ends.
o Method: take cells, put them in agar, watch for proliferation. Normal
cells won't be able to proliferate (no anchorage to grow on)-- infected
cells will proliferate regardless of anchorage (see characteristics of
cancer cells, above).
• What functions do the protein products of viral oncogenes perform? You
should be able to give at least four examples of oncogenes of known function
o RB protein is a target of some tumor viruses (eg. human papilloma
virus), which produce proteins which inactivate RB (and/or p53) in the
cell in which the virus has taken up residence.
o This is a common theme: RB and/or p53 inactivation by viral proteins
allowing rapid, unchecked cell proliferation.
o Notice Karposi's sarcoma [HIV/AIDS] is also caused by RB/p53-
inactivating proteins.
o Notice also that retroviral-induced cancers (ie, resultant from viral
reverse-transcription of their oncogenes into human DNA) are very
rare in humans. Our viruses tend to just inactivate p53 and RB rather
than encode oncogenes themselves.
o Oncogenes:
• As mentioned, the viral src, erb, myc, etc, sequences. Notice
that viral copies of oncogenes tend to be more powerful effects
than their endogenous counterparts.
• v-src: phosphorylates various tyrosine residues in other
proteins (similar to ABL in humans).
• v-erb: mimics epidermal growth factor receptor (unregulated).
• v-sis: mimics platelet-derived growth factor (unregulated).
• Endogenous oncogenes are marked c-onc:
  Notice that c-onc genes are part of normal functioning of
human cells; therapy can't target all c-onc, just their
overexpression.
 c-onc genes need to undergo mutation before they
become carcinogenic.
• [Not on LOs but probably helpful:]
o APC gene product: keeps beta-catenin outside the nucleus; without
APC, beta-catenin goes to nucleus and begins uncontrolled
transcription of oncogenes (like c-myc).
• LOH in APC produces familial adenomatous polyposis (FAP),
which leads to colon polyps and, eventually, metastatic colon
cancer.
o BRCA1 (breast-cancer gene 1): Its gene product forms the scaffold for
protein assembly that "checks up on" the cell cycle to make sure that
the DNA has replicated faithfully. When it's knocked out, the check on
the cell cycle is removed.
• Can either be familial (LOH as above) or sporadic.
• Note that in sporadic cases, the mutation can be on other
(unspecified) genes that regulate or have an effect on BRCA's
expression.
o p53 you may recall from Li-Fraumeni syndrome-- mutant or
inactivated p53 is found in ~50% of all cancers.
• Protein involved in DNA mutation repair at "checkpoint" in cell
cycle.
• Notice that mutations in p53 can be "dominant-negative;" that
is, one bad copy of p53 can inactivate the other, good copy of
p53.
• Along with RB, one of the two major gene products knocked out
by oncoviruses to produce cancers.
• Why are oncogenes useful as molecular markers in prognosis? You should be
able to give at least two examples of oncogenes that are currently being used
and also include the evidence of why these are good markers.
o The level of expression of oncogenes tends to correlate with the
rapidity of the progress of the cancer.
o One example: The level of expression of the N-myc gene is used in
prognosis analysis for neuroblastoma.
o Another example: Increased expression of the HER2/neu gene
correlates with poor prognosis in breast cancer.
• What is the difference between oncogenes and tumor suppressor genes? You
should be able to describe the function of these two types of cancer genes
and how mutations in them may combine to produce cancers.
o Again: oncogenes promote cell proliferation, tumor suppressor genes
inhibit it. If you have an over-activation mutation in an oncogene and
an inhibiting mutation in a tumor suppressor gene, presumably you
can have a real problem.
• How can our knowledge of oncogenes and tumor suppressor genes be used
for targeted therapy? You should be able to give an example of each.
o Can design either small molecules or antibodies as therapy.
o Targeting oncogenes:
• Herceptin: drug antibody therapy against the HER2/erb2
oncogene product.
 • Small molecules: able to inhibit action of cancerous proteins by,
among other pathways, binding to their active sites. Example is
Gleevac, an ATP analogue, that inhibits ABL tyrosine kinase in
patients with BRC-ABL translocation on the Philadelphia
chromosome.
 (BRC-ABL: translocation causing leukemia; resistant to
radiation therapy. ABL is an ATP-dependent tyrosine
protein kinase that PO4s various other proteins.)
 Gleevac = specific to tyrosine kinase proteins: specific fit
to their ATP-binding pocket.
• Notice can use combined oncogene targeting therapy and
radiation therapy: former makes the cancer more susceptible to
the radiation.
o Targeting tumor repressor genes:
• You can inject RB directly into RB-negative tumors
• You can use drugs that only kill cells with p53 deficiencies
• You can use drugs which correct the mutant conformation of
dominant-negative p53 proteins (see above).

Composition of Cells
Tuesday, November 13, 2007
8:00 AM

Composition of Cells, 11/13/07:

• Know typical values for the volumes of plasma (3 liters), extracellular fluid
(ECF, about 13 liters + another 5 for the 'third space'), and intracellular
fluid (ICF, about 27 liters) compartments.
o Of the roughly 45 liters of fluid in the body:
• 99% is H2O
• 0.8% is Na+, K+, Cl-
• 0.2% is everything else.
o Notice that there's roughly a 2:1 ratio between ICF and ECF.
o 'Third space': fluid in GI tract, urine, CSF, sweat, etc. Effectively this
is any fluid outside of cells but separated from interstitial fluid by
epithelial cells. At the moment, more or less irrelevant.
• Know the major difference in composition between ECF and ICF.
o In ICF: mostly K+ cations (98% of K+ is inside cells) and a variety of
anions (A-n, where -n is the average charge on the anions).
o In ECF: mostly Na+ cations and Cl- anions.
• [Types of ECF: interstitial fluid, lymph, plasma]
• [Types of ICF: mitochondrial, nuclear, endoplasmic reticular, etc]
• [For purposes of discussion, we're lumping all these together into ICF and
ECF.]
• Normal ion values:

ICF (mM)
ECF (mM)
 Membrane Permeability (yes/no)
Na+
14
140
(no) (yes, but actively pumped out)
K+
145
5
yes
Cl-
5
145
yes
A-n (anions)*
126
~0
no
H2O
~55,000
~55,000 (same as ICF)
yes

* = proteins, inorganic ions, etc.

(notice this is close to the concentration of pure water)

• Know the two most important functional properties of membranes, one

conveyed by lipids, the other by channels and transporters.
o Lipids are resistant to charge diffusion: that is, they can hold a
differential charge on either side without having the charge leak across
and balance out. There are two components to this:
• One is that they are impermeable to charged substances (ie
water and ions).
• The other is that they are 'strong' vs. electric force: they can
withstand the large amount of electric force imposed by the
charge differentiation. (note that if you increase the membrane
differential by another 50 mV or so, this breaks down.)
o Channels and transporters (proteins in lipid membrane) are the ways
by which a cell gets around this impermeability. This allows a cell to be
selectively permeable and move ions back and forth in a controlled
fashion.
• Channels: allows passive diffusion out of or into cell. Notice:
 They are generally selective for a particular type of ion,
and
 They're usually 'gated'-- that is, there's a particular type
of stimulus that will cause it to open (particular
receptor-binding ligands, stretch receptors, voltage
changes, etc).
• Transporters: actively pump ions out of or into cell.
 Generally selectively bind large molecules for transport.
 Can also pump ions against a gradient (ie. Na+ pump)
using ATP.
• Understand the routes by which a given substance can traverse a membrane.
 o See last two points (channels and transporters).
o Notice, peripherally, that if the substance is lipid-soluble, it can just go
straight into (and potentially through) the membrane itself (how it
would have gotten to the membrane through the external aqueous
environment is another question).
• Identify physical forces that can determine the gating properties of ion
channels.
o Electric field (voltage-gated)
o Mechanical (stretch, hair cells in cochlea)
o Chemical (synaptic receptors)
o Temperature (cutaneous temp receptors)

Cell Volume Regulation

Tuesday, November 13, 2007
8:03 AM

Cell Volume Regulation, 11/13/07:

• Be able to determine which direction a given uncharged substance will move

across a membrane, given its concentrations on the two sides.
o Basic law of everything: substances tend to move from areas of high
concentration to areas of low concentration. This probably is connected
to the second law of thermodynamics (entropy, or randomness, or
diffusion, is constantly increasing).
o Notice that the diffusion of water down a gradient (ie. towards the
area of low concentration) is called osmosis. This usually is discussed
in the context of water flowing through a membrane that's
impermeable to other substances, to balance out uneven osmotic
concentrations of those concentrations on either side of the
membrane.
• Determine under a given set of conditions whether a cell will swell or shrink.
o Notice that, given the relative concentrations of water vs. everything
else inside of (and outside of) cells, water alone is effectively going to
determine changes in the volume of cells.
o If the cell is immersed in water that's at a higher concentration (that
is, it's more pure water and less solute) than the water inside the cell,
the cell will swell.
o If the cell is immersed in water that's at a lower concentration (that is,
it's less pure water and more solute) than the water inside the cell, the
cell will shrink.
o Notice there's another way of saying this:
• If the cell is immersed in water that has a lower concentration
of non-water stuff than the concentration of non-water stuff
inside the cell, the cell will swell (water flows into cell to
balance the concentrations).
• If the cell is immersed in water that has a higher concentration
of non-water stuff than the concentration of non-water stuff
inside the cell, the cell will shrink (water flows out of cell to
balance the concentrations).
 o Notice that in pure water, our cells will burst (too much swelling!)
because they're always going to have a higher concentration of solutes
than their surroundings.
• List the three mechanisms that different cells have evolved to keep from
swelling and bursting.
o (1) Make cell membranes impermeable to water.
• This is not a popular option, since during cell division you're
effectively going from 1 cell-full of water to 2 cell-fulls, and
you've got to get water in there somehow to make up the extra
volume. In other words: cells need to grow.
• However, this can work sometimes in specialized systems (ie
kidney cells when you need to retain water).
o (2) "Brute force": build a wall around the cell which resists the osmotic
pressure.
• This is by far the most popular option-- all plant matter and
pretty much everything other than animal cells.
• The wall is permeable to water but resists the osmotic swelling
force.
• One problem is that it really limits mobility and morphology of
the cell. The other problem is that it takes a lot of uptake and
energy to maintain that wall.
o (3) Balance the water concentrations osmotically.
• Ie: put solute in extracellular fluid to make the water outside
cells the same concentration as the water inside cells.
• Notice that it doesn't really matter what solute you put in the
ECF to dilute the water, as long as the solutes aren't able to go
into the ICF (which would render the whole point moot).
• Know which direction water will move across a semi-permeable membrane,
given the solute compositions of the fluids on either side of the membrane.
o Detailed answer: it depends on whether or not those solutes can cross
the membrane and how fast they do so. See below, under "reflection
coefficients," for details. Essentially if you have one solute that
permeates the membrane more quickly than the other, the water will
follow that solute first. If you have two permeable solutes, one on
each side of the membrane, that both diffuse at the same rate, then
the net water flow should be nada.
o Simple answer: if the solutes can't cross the membrane, water will
flow in the direction of greater solute concentration.
• Know the difference between diffusion and osmosis.
o Osmosis is the net inward movement of water across a semi-
permeable membrane. Notice more detail in the definition on the first
bullet point.
o Diffusion is the more general movement of any solute from an area of
high concentration of that solute to an area of low concentration of
that solute.
• Know which substances must move into or out of a cell in order for the cells
volume to change.
o Water alone is necessary and sufficient. To a first approximation.
• Know the effect of having a membrane with different, non-zero permeabilities
(ie, reflection coefficients less than 1) to different solutes.
o ['reflection coefficient' = how permeable the cell membrane is to a
substance: 0 = water, 1 = impermeable.]
 o Effectively, some things pass into cells more quickly than others, some
things can't come into cells at all.
o This has consequences: given the same concentrations of different
diffusable solutes on each side of the membrane, until they equilibrate
out, one of them will likely diffuse faster than the other one, which
means that the balance of solute will not be balanced throughout the
diffusion process (which means the cell is going to either swell or
shrink in the process as water follows the solute that diffuses faster).
• Know the definitions of molarity, osmolarity, equivalents, and tonicity, and
know how to convert between them.
o Molarity: a standard measure of concentration (grams of solute per
liter of water).
o Osmolarity: sum of molarity of all the solute particles. Effectively, you
want to (ie. 1 M NaCl = 2 OsM since you have 1 M of Na+ and 1 M of
Cl- when dissolved). Can calculate this for inside and outside the cell
and compare them.
• Notice that 1 M glucrose (C6H12O6) doesn't wind up being
something crazy like 24 OsM, even though there's 24 atoms in
each glucose molecule, how many atoms are in each , because
sucrose doesn't dissolve into constituent atoms in water like
ions in a salt do.
• This can be used to determine whether a cell will swell or
shrink, as noted above. Basically if you have a higher
osmolarity outside the cell than inside, the cell will shrink. If
you have a lower osmolarity outside the cell than inside, the
cell will swell. If the osmolarity is the same inside and outside,
the cell will stay the same size.
• Important point: in a simplified situation (not comparing
reflection coefficients or two opposing diffusing solutes), a
solute that can cross the lipid membrane does not change the
volume of a cell at all. It will go to equilibrium across that
membrane, which means that the osmolarity both inside and
outside the cell increases by the same amount, which means
the difference in osmolarity doesn't change at all-- thus no
volume change.
 But note caveat about relative permeabilities, above.
o Tonicity: a measure of how a cell reacts to a given ECF solution. Also a
way of describing the osmolarity of the impermeable solutes in the
ECF.
• Hypotonic: any solution that makes a cell swell. (total
impermeable osmolarity = lower outside the cell)
• Hypertonic: any solution that makes a cell shrink. (total
impermeable osmolarity = higher outside the cell).
• Isotonic: any solution that doesn't make a cell shrink or swell-
same impermeable osmolarity as what's in the cell.
o Equivalents: Take the ions that are the solute, break them down into
osmolarity as above, then multiply the osmolarity by the charge on the
ion. Basically gives you a feel for how much of a given osmolarity is
acidic or basic. I find it unlikely he'll ask a question about this, but just
in case.
Membrane Potential I, II
Wednesday, November 14, 2007
8:01 AM

Membrane Potential I, II, 11/14/07:

• Compare qualitatively the relative strengths of electric and osmotic forces:

o Electric forces are way crazy bigger than osmotic forces.
o This is why a small number of ions can overcome a large concentration
gradient.
• Describe the two forces acting on an ion moving across a membrane:
o Concentration gradient (higher concentrations diffuse to lower)
o Electrical gradient (like charges repulse, opposite charges attract)
• Define equilibrium potential:
o For a given ion, the equilibrium potential is the potential across the
membrane that that ion would 'like' to see exist in order to be in
equilibrium.
o If the membrane potential isn't at the equilibrium potential for the
given ion, either the ion isn't able to permeate the membrane or
there's some mechanism that keeps the ions from crossing the
membrane effectively in order to equilibrate (ie, an ion pump).
o Measured in millivolts.
• Understand the difference between an equilibrium potential and a recorded
membrane potential:
o Membrane potential = electrical differential across the cell membrane.
Empirical.
o Equilibrium potential = calculated from Nernst equation for a particular
ion. Notice that the equilibrium potential of all permeating, non-
pumped ions have to be equal to the membrane potential, at
equilibrium.
o On membrane potential: by convention, notated about the inside of
the cell with respect to the outside of the cell (so a negative
membrane potential means there's a potential for positive ions to flow
into the cell).
• Know that each and every ion species has its own, independent equilibrium
potential:
o The equilibrium potential is dependent on particular electrical
properties of the ion.
o However, as above, note that for a non-pumped, permeating ion
species (ie. Na+ or Cl-), the equilibrium potential is equal to the
membrane potential for that membrane.
• Understand that the number of excess anions in a typical cell is small
compared to the total number of anions:
o Doesn't need to be big to keep distinct ion concentration gradients in
place.
• Know that bulk solutions are always electrically neutral:
o This, I think, is effectively because electrostatic repulsion doesn't allow
charged solutions to stay together (see Betz's analogy about a one-
liter box of cations needing a quarter of the weight of the earth to
keep the lid on).
 o Technically this is not true across cell membranes - there's a small
electric differential of ~-60 millivolts - but the electrical difference is
small enough that we can get away with it.
o What this means: in any given bulk fluid (ie. ECF/ICF) the electric
charges must equal out.
o This is able to keep concentration gradients away from equilibrium-
that is, being electrically neutral takes precedence over being at a
state of concentration equilibrium.
• The cell is able to take advantage of this fact to maintain
different compositions of ions inside and outside the cell.
• Understand how an artificial cell can be in a state of equilibrium even though
the concentrations of ions are not the same inside and out:
o Okay. It's in a state of equilibrium because the electrostatic forces
keep the ions from sliding along their concentration gradients.
o Basically you've balanced the electrical equilibrium with the
concentration equilibrium, This means that the cell isn't electrically
neutral (though it's very close), and it also means that the cell isn't at
a concentration neutrality (ie. different species of ions have different
concentrations across the membrane).
• Not to wax philosophical, but this is what allows us to be alive.
Maintaining differences between things in the face of the 2nd
law of thermodynamics - which drives things to a universal
diffusion as the greatest expression of entropy - is what allows
us to control fluxes in those differences, which in turn is the
basis for pretty much all life as we know it, from arterial gas
exchange to action potentials to which football team you root
for.
o Notice this electrical-vs.-concentration gradient thing does not apply to
uncharged solute atoms (electrostatic forces don't have any grip on
uncharged atoms).
• Be able to apply osmotic balance, charge neutrality, and the Nernst equation
to calculate ion concentrations and the membrane potential of an artificial
cell:
o Nernst equation: Equilibrium potential is proportional to the natural log
of the concentration of a given ion outside the cell over its
concentration inside the cell.
• ie: E is proportional to ln ([Iono]/[Ioni]) divided by the valence
of the ion (+1 for Na+, -1 for Cl-, etc), where [Iono] is the
concentration of the ion outside.
• Approximation: E = 60/z * log ([Iono]/[Ioni]), where z is
the valence (+1 for Na+, -1 for Cl-, etc).
o (important) Donnan equation: [Ko][Clo] = [Ki][Cli]
o Notes on possible test questions:
• Dr. Betz seems to be fond of making charts where, given
known and unknown concentrations of various ions inside and
outside the cell, you fill in the blanks. There's a three-part
process to solving these. This is what you need, not in any
particular order:
 (1) Balance the electric charges in each compartment
(ECF + ICF). ECF and ICF are always electrically neutral,
more or less. So if inside a cell you've got 200 mosM of
Na+ (valence +1) and 100 mosM of K+ (valence +1)
 and 50 mosM of proteins with a valence of -1, the total
charge inside the cell right now is ((200*1) + (100*1) +
(50*-1) = 250). To make it neutral, then, you need 250
mosM of Cl- (250*-1). If the valence of the proteins was
-2, then the total charge inside the cell is ((200*1) +
(150*1) + (50*-2) = 200). In which case you'd only
need 200 mosM of Cl- to make it neutral. If this doesn't
make sense, go find someone who understands it and
threaten them until they verbally explain it.
 (2) Balance the osmolarity between compartments. If
you add all the osmolarity of the ions inside the cell, it
needs to be the same as the total osmolarity of the ions
outside the cell. So if you've got 200 mosM of Na+, 100
mosM of K+, 50 mosM of proteins, and 250 mosM of Cl-
in the ICF, total osmolarity = (200 + 100 + 50 + 250 =
600 mosM). This means that the total osmolarity in the
ECF also has to be 600 mosM. So if you're missing one
concentration in the ECF (in this example), just total the
rest of them and subtract them from 600; what's less is
what you have to have to balance the osmolarity. Again,
if this doesn't make sense to you, find someone what
can verbally explain it.
 (3) Use Donnan equation to get [K+] and [Cl-] for both
compartments. Because the product of [K+] and [Cl-] in
the ICF is the same as the product of the concentrations
of those ions outside the cell, if you know three of the
concentrations, you can find the fourth. Ie: if AB = CD,
and you know A, B, and C, D is pretty easy assuming
you passed 9th grade algebra (which, for the record, I
nearly didn't).
• Something else he spent some time on in class: given a
permeating ion's equilibrium potential (E) and the membrane
potential (Vm) of the membrane, can you tell if there's an ion
pump operating, and if so, in which direction it's operating?
 This first part is easy. If a permeating ion's E isn't the
same as the Vm, then there's a pump at work.
 To figure out which direction it's going in, compare E
and Vm.
• If E is larger (more positive) than Vm, the ion
wants to make the inside of the cell more
positive.
• This means if the ion's positively charged,
it wants to go into the cell. Conversely, if
the ion's negatively charged, it wants to
go out of the cell.
• If E is smaller (more negative) than Vm, the ion
wants to make the inside of the cell more
negative.
• This means if the ion's positively charged,
it wants to go out of the cell. Conversely,
if the ion's negatively charged, it wants to
go into the cell.
 • Try this on K and Na: K's E = -80, Na's E = +60.
An average membrane potential is about -60.
• Na's E is more positive than Vm, and it's a
positively charged ion, so it wants to go into the
cell.
• K's E is more negative than Vm, and it's a
positively charged ion, so it wants to go out of
the cell.
• Once you've figured out which direction the ion
wants to go, flip it. Then you know which
direction the pump has to be pumping it to stop
the leak in its preferred direction.
 This is kind of a cheat sheet. But it's good to work
through it on your own as well if you like mind pain.
 Notice that, given the concentrations of ions inside and
outside the cell, you can figure out E for those ions. So
really, all you need to solve these problems is Vm and
the concentrations of the ions you're looking at.

Other, random, notes: make membrane permeable to chloride but not sodium.
Chloride atoms come into the cell, bringing a negative charge- when enough Cl ions
are in there, electrostatic repulsion.
Then when any Cl ion comes in along its concentration gradient, one gets kicked out
by electrostatic repulsion.
For K: same idea. Electrical gradient wants to keep K inside cell (since cell is
negatively charged), even though concentration gradient wants to take K out of cell.

Na pump: transports Na out, K in.

*In a real cell: the equilibrium potential for sodium is about +60. The equilibrium
potential for potassium is about -80. The actual membrane potential is about -60.
This means, since both K and Na are permeating, that there's a constant leak of K
out of the cell (to make it more negative to approach equil potential) and a constant
leak of Na into the cell (to make it more positive to approach equil potential). Thus
rely on pump to pull back towards -60 as Vm. In other words, the current carried by
sodium into the cell must equal the current carried by potassium out of the cell at
equilibrium.

If you allow Na to come into cell (ie if you make the membrane more permeable to
Na), get depolarization- cell membrane potential rises towards 60 mV.
*Take-home here: any time you increase the membrane permeability of one ion, the
membrane potential of the cell moves toward the equilibrium potential of that ion.
And recall that the concentration gradient across the membrane determines the
equilibrium potential for that ion. "The ion with the highest permeability wins."

Put another way: the ratio of the permeability of sodium to the permeability of
potassium in a given cell is what determines the membrane potential of that cell.
Notice that the permeability of a given membrane to an ion is largely determined by
the number of open channels for that ion in that membrane. So if you have more
open K channels than Na channels, your membrane is effectively more permeable to
K than Na (and thus membrane potential will be closer to the equilibrium potential
for K, -80, than it will be to the E of Na, 60).
Acids, Bases, Buffers
Wednesday, November 14, 2007
8:02 AM

Acids, Bases, and Buffers, 11/4/07:

• Describe the law of mass action [le Chatelier's principle]:

o For any reversible reaction, the forward and reverse rates of the
reaction are directly dependent on the concentrations of the reactants
and the products respectively.
o Ie: in A <--> B, if you increase the concentration of A, the rate of A ->
B goes up. If you increase the concentration of B, the rate of B -> A
goes up instead.
o Equilibrium constant for a reaction Keq = [products] / [reactants].
o Keq of water = 1.8 x 10-16 = [H+][OH-] / [H2O].
o [H+][OH-] = 1 x 10-14.
o This is universally, always, everywhere, true. No exceptions.
Even for you.
o In a neutral solution, [H+] = [OH-] = 1 x 10-7.
• Define pH and pKa:
o pH is the negative log of the proton concentration in a solution.
o ie: pH = -log [H+].
o In neutral solutions, [H+] = 1 x 10-7; log [H+] = -7, negative log = 7.
• That is: in neutral solutions, pH = 7, which is what you already
know.
o Notice this means that small differences in pH mean large differences
in the concentration of protons-- which our bodies generally handle
very poorly.
o Ka measures the tendency of a weak acid to dissociate in water.
o pKa is the negative log of Ka for a given weak acid.
o The lower the pKa, the stronger the acid (more proton donation in
water).
o The higher the pKa, the weaker the acid (more proton acceptance in
water).
• Write the Henderson-Hasselbalch (H-H) equation for any given weak acid or
base:
o pH = pKa + log ([A-]/[HA]).
o This means: pH = pKa + log ([proton acceptor]/[protein donor]).
o Which means that, given the ratio of dissociated to undissociated acids
in solution, you can calculate the pH of the system given the pKa, or
can calculate pKa given the pH, or if you're given the pH and the pKa,
can calculate the ratio of undissociated to dissociated acid in a given
solution.
• (ie: given two elements of that equation can get the third.)
o Notice that when pH = pKa, the concentrations of deprotonated and
protonated acid are equal.
 • (ie: when pH = pKa, the acid is half deprotonated and half
protonated.)
• Define the H-H equation for the bicarbonate buffer system in extracellular
fluid and use it to evaluate clinical lab data:
o pH = 6.1 + log ([HCO3-]/(0.3*PCO2)).
o Essentially: the deprotonated bicarbonate (HCO3-) levels inversely vary
with the partial pressure [levels] of CO2 in the blood.
o What the hell that means: given the partial pressure of CO2 in the
blood and the pH of the blood, can calculate the concentration of
bicarbonate; or given the partial pressure of CO2 and the
concentration of bicarbonate, can calculate the pH of the blood; or
given the concentration of bicarbonate and the pH, can calculate the
partial pressure of CO2 in the blood.
• (ie: given two elements of that equation can get the third.)
o Important note: [HCO3-] needs to be measured in millimolar units;
PCO2 needs to be measured in millimeters mercury or mm Hg.
o Some background on all this mess: the buffering system (see below
for details on buffering) in the body is predominantly made up of
carbonic acid-to-bicarbonate:
H2CO3 <--> H+ + HCO3-
• This depends on the carbon dioxide levels in the blood--
dissolved CO2 reacts with water to form H2CO3.
• This means that the equilibrium of the dissociation of
H2CO3 is driven by the levels of CO2 in the blood (by le
Chatelier's principle).
• This works as a buffer, even though the nominal pKa of
H2CO3 is 3.8 (thus would buffer optimally at 2.8-4.8, way
outside physiological pH), because ion exchange in the kidney
and input of CO2 changes the effective, actual pKa.
• Define normal blood pH, [HCO3-] and pCO2 (please see your handout or
learning objective):
o Normal arterial blood pH: 7.34-7.44
o Normal venous blood ph: 7.28-7.42 (lower due to higher [CO2])
o Normal concentration of HCO3- in blood: 24 mM
o Normal PCO2 in blood: 40 mm Hg
o Normal concentration of CO2 in blood (he didn't ask, but it's in his
practice problems): 1.2 mM
• Describe how weak acids and bases work to buffer pH and define the pH
range of maximal buffering capacity:
o Effectively, you have a mix of weak bases and acids: the weak acids
neutralize added bases without changing the pH much, the weak bases
neutralize added acids without changing the pH much.
o Optimal buffering capacity is determined by the pKa of the buffering
acids/bases. There's a whole lot of titration chemistry behind this.
Effectively there's a pH at which your buffer system can neutralize
added acids or bases with very little change in overall pH, which is
what you want to avoid a lot of big jumps in your blood pH (which
would kill you): this optimal buffering pH is plus or minus 1 pH unit
from the pKa of the acid/base.
• Use the H-H equation to solve problems of how pH changes in defined buffers
i.e. you should be able to determine how many equivalents of acid or base
 are needed to titrate the ionizable groups(s) of a weak acid or base from a
starting pH to a final pH, given its concentration and pKa:
o Quick way to think about this: every factor of ten that you have of
deprotonated acid (A-) more than protonated acid (HA), you raise the
pH by 1 over the pKa. If you have 1 HA molecule and 10 A- molecules,
you just raised the pH by 1 from the pKa. If you have 1 HA and 100 A-,
now the pH will be 2 plus the pKa.
o Conversely, every factor of ten that you have of HA more than A-, you
lower the pH by 1 from the pKa. So if you've got 10 HA molecules and
1 A- molecule, the pH of the solution is going to be the pKa minus 1.
o So if you start out with a pH of 6 and the pKa is 6, to get to a pH of 7,
you'll need to add in ten times the current amount of A- (which will add
one to the pKa and result in a pH of 7). If you want to get to pH 5, you
need to add in ten times the current amount of HA. pH 8: 100 times
the current concentration of A-. pH 4: 100 times the current
concentration of HA. Etc.
o Again: find someone who gets this and beat or plead an explanation
out of them if this is Greek to you.

Side notes: histidine is the only amino acid side chain that protonates or
deprotonates at physiological pHs- thus important for acid-base balance in body.

Notice that the kidney controls a lot of pH by retaining or excreting HCO3- and NH4+.

Notice that the low pH in the stomach, which allows compounds to be easily
protonated, allows drugs that need to be protonated to be absorbed into the cell to
be so-

Remember: Higher levels of CO2 in the blood lower the blood's pH. This is because
you make more carbonic acid, which drives the deprotonization of carbonic acid to
form bicarbonate + a proton. These extra protons float around in the blood and
lower the pH.

Membrane Structure
Thursday, November 15, 2007
8:03 AM

Membrane Structure, 11/15/07:

• Describe the lipid components of biological membranes and what differences

exist between membranes of the different organelles
o Lipid bilayers: roughly 5 nm thick, spanned by proteins.
o Head groups: polar. Tail groups (inside the membrane): hydrophobic.
o Classes of lipids in membranes:
• Phosphoglycerides
 Phosphotidylethanolamine
 Phosphotidylcholine
 Phosphotidylserine
  Phosphotidylinositol
• Sphingolipids
• Cholesterol
• All are synthesized at least in part in the ER and have polar and
nonpolar ends.
o Phosphoglycerides: have a glycerol-3-phosphate backbone with 2 fatty
acyl chains (either saturated or mono- or polyunsaturated) at one end
and a polar head group at the other end. What type of head group
determines which phosphatidylglycerol it is (ethanolamine, inositol,
etc).
o Sphingolipids: have a 'sphingosine' (long acyl unit) with either of two
polar head groups (which one it is distinguishes which type of
sphingolipid it is) and an attached fatty acid chain.
• Sphingomyelin: phosphocholine head group.
• These are started to be made in the ER and finished in the
Golgi apparatus.
o Cholesterol: have a characteristic hydroxylated ring structure at one
end, and a fatty acid chain on the other.
o About 30% of all genomic proteins are membrane proteins
o There are some double-membrane (mitochondria) and some single-
membrane (nucleus) organelles.
o Notice that there's roughly ten times the surface area of the plasma
membrane contained as surface area of membranes within the cell;
notice that the cytoskeleton
has another order of magnitude of surface area over the intracellular
membrane area.
o Notice that under physiological pH, lipid bilayers spontaneously
reform compartments to minimize contact of their hydrophobic tails
with water.
• Bilayers are asymmetric. Notice that particular phospholipids don't flip from
one side of the membrane to another without enzyme activity, but lots of
lateral diffusion all the time.
o [Not on LO's so learn at own risk:]
o [Exoplasmic face: phosphotidylcholine, sphingomyelin, glycolipids
(glycosylated lipids).
o Cytoplasmic face: phosphoidylethanolamine, phosphatidylserine,
phosphatidylinositol.]
o Cholesterol is probably roughly equally distributed on both sides of the
membrane.
• Understand how concept of membrane fluidity and how membrane fluidity is
modified
o Membrane fluidity: the degree to which lateral motion is possible
among adjacent phospholipids on a given side of the bilayer. Some
lipids are anchored, some are free-floating within any given
membrane.
o Notice that different physical compartments of a membrane can have
different degrees of fluidity.
o Having acyl chains unsaturated makes the membrane more fluid (less
tightly packed).
o Cholesterol, when intercalated in membranes, stiffens the membrane
and makes it less fluid.
 o Membrane composition determines thickness and curvature:
• Membranes high in cholesterol also thicken the membrane by
straightening out the nonpolar, hydrophobic groups inside the
bilayer.
• Curvature is dependent on the size of the polar head groups on
both sides of the membranes.
• Understand the many different ways proteins interact with membranes
o [Cholesterol production: Proteins SCAP and SREBP, under high-
cholesterol conditions, stay inactive in the ER. Under low-cholesterol
conditions, SCAP and SREBP go on to the Golgi and wind up triggering
nuclear receptors to synthesize more cholesterol.]
o Different kinds of membrane proteins:
• Integral proteins (fully or partially embedded in membrane)--
can go multiple times back and forth through the membrane
(multiple transmembrane domains).
 Notice that transmembrane domains are usually alpha-
helices, but can also have beta-sheet barrel formations
(usually bacterial).
 Can have transmembrane proteins that are only bound
to one side of the bilayer.
 Can have proteins that are only attached at their ends to
the membranes.
• Peripheral membrane proteins: covalently interacting with
proteins bound in the membrane, but not themselves
embedded in the membrane.
• Describe the importance of sphingolipids and cholesterol in biological
membranes
o Cholesterol: see above (thickness and nonfluidity).
o Lipid rafts: formed primarily from sphingomyelin and cholesterol, thus
extremely nonfluid-- effectively a lipid platform for various proteins.
Used for signaling processes.
• Although the different carriers and channels that allow molecules to be
transported across membranes is discussed by Dr. Betz in his lectures,
understand how these molecules' function should be integrated into the
function of biological membranes.

Membrane Fusion
Thursday, November 15, 2007
8:59 AM

Membrane Fusion, 11/15/07:

• Understand the importance of sub-cellular protein targeting.

o A cell has a lot of compartments through which it needs to move big
proteins without compromising the membranes' integrity-- thus
transport vesicles (membrane package-wrap, made from membrane of
membrane-bound organelle of origin, containing cargo to be
transported to target membrane-bound organelles).
 o This (vesicular transport) is the basic principle of transporting
substances from one intracellular compartment to another.
o Incorrect transport frequently results in disease: cystic fibrosis is due
to a failure in chloride ion channel-protein transport.
o Myelin/tubulin cytoskeleton elements: act as "highways" to transport
vesicles throughout cell. "Motor" proteins use ATP to drive vesicles
along these highways (actin for myelin, kinesin/dynein for tubulin).
The particular motor proteins are specific for particular destinations--
one variety of kinesin may take a protein to one location, another to
another, etc.
• Know the basic principles of membrane and viral fusion:
o Membranes don't just automatically merge when they get close
together (which is good, otherwise things would not be well separated
in the cell).
o Two membranes: begin with them coated with water molecules. First
you have to remove the water before you can fuse the lipids; then you
need to overcome charge repulsion between lipids in order to allow a
greater efficiency of merging. Also need to make sure these specific
lipids should be merging in the first place (specificity of membrane
fusion).
o Early research: Botulin and tetanus toxins inhibit vesicle secretion by
neurotransmitters. They do this by cleaving particular proteins: the
SNARE proteins.
• Know the function and structure of SNARE proteins:
o Come in three shapes:
• VAMPs: transmembrane domain at one end, with a helical
domain in it.
• Syntaxin: transmembrane domain, still a helical domain.
• SNAP25: no transmembrane domain, two helical domains, fatty
acid binding region that more or less acts as a membrane-
binding domain.
 (notice that there are different flavors of each protein,
each of which can only bind to certain flavors of their
counterparts.)
• The helical domains of all three proteins serve to bind with each
other-- form an aligned bundle or tetramer.
• All vesicles contain VAMPs. All target membranes contain
syntaxin and SNAP25.
• When the vesicle nears the target membrane, the helical
domains on the vesicle bind to those on the target membrane:
these effectively press the two lipid layers together, squeeze
out water and overcome charge repulsion, and promote the
lipid fusion.
• To do this, the helical binding needs to be extremely strong,
and so it is.
 This means a problem after the lipids have fused: how
do you get apart the SNARE proteins once they've bound
together so strongly?
• Know regulation of SNARE-based fusion:
o Enzyme that regulates the dissociation of SNARE proteins: NSF
protein. Forms a kind of turning barrel around the SNARE complex and
twists it apart with the help of a lot of ATP.
 o After being unwound, syntaxin becomes unfolded; need another
protein, Sec1, to refold it properly.
o SNARE cycle: VAMP on vesicle, syntaxin and SNAP25 on target
membrane, helical binding and lipid fusion, NSF-mediated unwinding,
refolding of syntaxin with Sec1.
• Know the mechanism of viral fusion:
o ["Envelope viruses" (influenza, ebola, HIV, etc): unlike other viruses, it
has a membrane envelope around its capsid (capsid: protein structure
containing genetic material).]
o In order to infect a cell, the envelope needs to fuse with the target
membrane.
• Notice that the target isn't always the plasma membrane:
influenza virus gets endocytosed first, then when it's inside the
cell it goes after the endosome membrane.
o Notice that when a cell becomes infected by an envelope virus, the cell
makes viral fusogenic proteins that go to the cell's surface-- will put
these fusogenic proteins on emergent viral capsids.
o Mechanism is similar to SNARE-mediated fusion, but virus has only one
protein:
• Has a transmembrane domain as well as a non-transmembrane
hydrophobic domain hidden inside the folded protein.
• Binding to the cell surface causes the exposure of this hidden
hydrophobic domain, which inserts into the host membrane.
• The virus has a lot of these proteins on its envelope: effectively
they bind the envelope to the host membrane.
• The other feature of the protein is multiple helical domains: one
they're bound to the host membrane, these domains bind
strongly to each other and ratchet the envelope tightly onto the
host membrane, squeezing out water and overcoming charge
repulsion as per the SNARE mechanism.
• Know the regulation of viral fusion:
o Only thing I can think of that he mentioned was that influenza viruses
are dependent on a cellular drop in pH to release their genetic material
into the cell.

Membrane Potential III

Thursday, November 15, 2007
9:39 AM

Membrane Potential III, 11/15/07:

• Know the difference between equilibrium and steady state:

o Equilibrium: Ion concentrations are not changing, inside or out, over
time.
o Steady-state: Ion concentrations are not changing, inside or out, over
time because the cell is furiously working to keep them that way.
o Equilibrium is Zen (no ions have any striving ideas to go in or come
out).
o Steady-state is a contained prison riot occurring at the same time as
an attempted storming of the Bastille (lots of things want to go out,
 lots of things want to come in, the guards are beating everyone the
hell up to keep them put).
• In a cell at rest (steady state), given an ion's concentration inside and out,
the membrane potential, and knowledge that the membrane is permeable to
the ion in question, be able to determine whether or not a pump for that ion
must exist, and if so, which direction it pumps the ion:
o I went over this in detail at the end of the notes for "Membrane
Potential II".
• Understand that membrane potential depends on relative, not absolute,
permeabilities to ion:
o Example: a membrane is very permeable to sodium but not to
potassium. The membrane potential then approaches the equilibrium
potential of sodium (+60). If the membrane is very permeable to
potassium but not to sodium, the Vm approaches the E of potassium (-
80). If they permeabilities are equal, the Vm won't change - within a
first approximation - no matter how much absolute quantity there is
(note, however, that if the membrane gets really really permeable, the
Vm will indeed change and approach 0 as the changing concentrations
of the ions make their Es approach 0).
• Understand that the short term determinant of membrane potential is not the
Na/K pump, but relative membrane permeabilities:
o Depending on the size and the number of channels in the membranes
(larger cells with more channels are more vulnerable, quicker, to Na/K
pump failure than others).
• Define Driving Force of an ion:
o The ion "wants" to have the membrane potential approach its own
equilibrium potential. It will drive the membrane potential towards that
equilibrium, aided or counteracted by other ions, which are also driving
the membrane potential towards their own equilibrium potentials.
• Understand why, in neurons and other excitable cells, membrane potential is
sensitive to small changes in [K+]o, but not [Na+]o:
o (Understand that [Na+o] is the sodium concentration outside the cell.)
o (1) Because the membrane potential of the cell is nearly at the E of K+
anyway, the loss of extracellular Na+ (which brings the E of Na+ closer
to 0) will make the Vm slightly hyperpolarized (closer to -80 mV) but
have not much other effect.
o (2) [K+]o is more sensitive because its starting concentration outside
the cell are much smaller than Na+, and thus is much more sensitive to
small changes in concentration.
• Know the mechanisms by which calcium, glucose + insulin, Na/K ion
exchange resins, and renal dialysis counteract the signs of hyperkalemia:
o Hyperkalemia: Excess of potassium in blood (outside cells, in ECF).
This changes the membrane potential of the cell because it alters the
concentration gradient on which the electrical gradient of the
membrane is based.
o Effectively, this raises the equilibrium potential of K+, which in turn
makes the membrane potential rise substantially (by ~20 mV for a 2%
calcium efflux).
o This is almost universally fatal. The reason it's so dangerous is that
messing with membrane potentials is a good way to screw up action
potential propagation, particularly in the pacemakers in the heart--
thus can't get heart contraction signals.
 o Caused by a variety of things: massively lysed cells, kidney failure,
severe muscle damage.
o Treatment:
• Can stabilize cardiac conduction system by giving Ca2+
• Can get cells to take up the K+ by giving insulin and glucose to
stimulate Na-K pump. (Insulin gets glucose into the cell, where
it activates the pump to work harder).
• If you give alka-seltzer and alkylyze blood, can stimulate
proton pumps to get K+ into cells.
• Can use cation exchange resins: Effectively, Na+-lined
"flypaper" for K+ in the blood, swapping out Na+ for K+.
• Can use renal dialysis to pherese excess K+ out of blood.

Membrane Transport
Thursday, November 15, 2007
10:41 AM

Membrane Transport, 11/15/07:

• [Transporters: Proteins in the membrane that move specific substances, as in

moving particular large molecule or pumping ions against their gradients.
• Know the difference between primary and secondary active transport:
o Primary active transporters: use ATP for energy source.
• Ie: Na+-K+ pump, Ca2+ pump, proton pumps.
o Secondary active transporters: don't use ATP. Uses energy derived
indirectly from primary pumps.
• Amino acid transporters: intake AA into cells; derives energy
from leakage of Na+ into cell (slight amount of energy released
by Na+ leak).
• H+-to-Na+ exchange (protons out powered by energy of Na
coming in).
• Define cotransport and exchange transport:
o Cotransport: secondary active transporters that move different solute
species in the same direction.
o Exchange transport: secondary active transporters that move solutes
in opposite directions.
• Na-Ca pump: Na+ in cell, Ca2+ out of cell (or, sometimes, the
other direction)
 A note about calcium: [Ca2+] inside the cell is kept
extremely low (about 0.1 micromolar; about 10,000
times lower than outside the cell).
 Thus: calcium has very high equilibrium potential (~120
mV). This allows single-channel openings to allow Ca2+ in
can have large effects inside the cell.
 The Na-Ca exchanger effectively works by balancing the
favorable energy from 3Na+ entering the cell with the
unfavorable energy from Ca2+ exiting the cell.
 Notice that this can change direction depending on the
current state of the membrane potential and the
concentrations of Ca and K in the cell.
 • Be able to determine which direction a non-electrogenic secondary active
transporter will operate, given the concentrations of the participating ions
inside and out:
o Electrogenic: one cycle produces a net charge transfer across the
membrane. Thus non-electrogenic are transporters that don't change
the net charge across the membrane. In this case, my guess would be
that they tend to follow the direction of ion concentration (see the
theoretical H/K exchanger below).
• Know that electrogenic secondary active transporters can reverse direction,
depending on membrane potential.
o (see under Na-Ca pump above)
• Understand the non-existence, but conceptually useful idea, of the H/K
exchanger:
o If you drop the proton concentration in the blood, can promote cell
uptake of K in exchange for protons going out into the blood, and vice
versa. The H-K exchanger doesn't actually exist as an entity, but you
can think about it like that.
o Essentially: if you have excess K, you can give bicarbonate (HCO3-) to
decrease the total H+ concentration in the blood. When that happens,
the exchanger that switches K for H swaps out K in the blood for H in
the cell, thus making the proton concentration in the cell go down and
the potassium concentration in the cell go up. This effectively takes K
out of the blood, treating hyperkalemia.
• Know how cells can concentrate glucose inside, even though glucose
transporter cannot pump glucose against its concentration gradient
o Inside cell, glucose gets phosphorylated, which makes it unable to fit
into the glucose transporter-- thus glucose flow is more or less
unidirectional into the cell. Notice that glucose transporters are
normally sequestered within vesicles in the cell until insulin signals the
cell to make those vesicles merge with the cell surface and transport
glucose into the cell.
• Notice that the CHF drug Digitalis blocks the Na-K pump. It does this in order
to increase the contractile strength of the heart muscle; what it does is raise
the effective concentration of Na+ in the cell-- which then renders the Na-Ca
pump less effective, allowing more Ca2+ to stay in the cell, increasing the
strength of the muscle cell contraction.

Epithelial Transport
Thursday, November 15, 2007
8:02 AM

Epithelial Transport, 11/16/07:

• Epithelial cells: cells which sequester 'third space' fluids from extracellular
fluid.
o These cells are polarized: not electrically, but meaning that they
transport in only one direction (apical to basolateral).
o Side of epithelial cells facing the third-space fluid is called the 'apical'
or mucosal membrane; the side facing the ECF is the basolateral or
serosal membrane.
 o The basolateral membrane is generic across the entire surface.
o The apical membrane is specialized and compartmentalized (with tight
junctions) for special functions.
• Mainly, these involve transporters of various kinds.
• Tight junctions: here, protein-based 'glue' that holds nearby
cells together but blocks lipid exchange with the basolateral
membrane.
• Understand the generic transport mechanisms for NaCl and water into the
blood:
o NaCl: Na+ picked up by the apical membrane (high permeability of
apical membrane for Na+) and is transported into the cell, then is
pumped out by the Na/K pump out the basolateral membrane. Cl-
passively follows the Na+ to equalize the electrical potential.
o Water follows the influx of NaCl into cells to balance osmolarity.
o Notice all this is passive (no ATP usage) absorption.
• Know the basic transport mechanisms by which glucose and amino acids are
absorbed into the blood:
o Epithelium in GI tract: AA, sugars, and glucose are pumped
(secondary active transport) through the apical membrane and diffuse
out the basolateral side passively into the blood.
• AA and sugars are picked up by their target cells by secondary
active transport mechanisms.
• Glucose, as mentioned before, diffuses into cell along its
gradient (but is phosphorylated in the cell to prevent efflux).
• Know the major differences between 'tight' and 'leaky' epithelia:
o Tight: more junction proteins, tighter seal between cells. Used
particularly in the distal tubules of the kidney.
o Loose: less junction proteins, looser seal between cells.
o Tight: "fine tuning"-- strictly controlled substance transport at lower
levels, don't want backflux.
o Loose: quantity over quality (needs lots of transporters, not too picky
about equal amounts).
• Know that four important substances - water, O2, CO2, and urea are never
pumped, but always move passively down their concentration gradients:
o You can open and close aquaporin channels to allow the water to come
in or go out, but it always flows along its gradient.
• Understand the relative roles of the G.I. tract (minimal) and kidney
(extensive) in excreting non-volatile metabolic wastes and regulating ECF
composition:
o GI tract excretes (in feces) largely things you couldn't absorb in the
first place. GI tract absorbs pretty much everything it can that comes
in to it indiscriminately (thus its small role in excreting waste).
• However, GI tract does excrete dead red blood cells (excreted
into the GI tract from the liver), which is significant.
o Kidney concentrates waste very well in the urine- gets rid of
approximately 0.5 moles of non-volatile (nongaseous) metabolic waste
a day, mainly end-products of nitrogen metabolism (urea) and
protons.
• Given two of the following, be able to calculate the third: apical membrane
potential, basolateral membrane potential, trans-epithelial potential:
o Trans-epithelial membrane potential = basolateral membrane potential
- apical membrane potential.
 • Notice trans-epithelial is abbreviated PD.
o One thing to remember here is that the membrane potentials of the
apical and the basolateral membrane will be different (different ion
permeabilities, etc).
o The other thing to remember is that membrane potentials are always
written in the form of describing the inside of the membrane with
respect to the outside-- ie, in a membrane potential is -10 mV, the
inside of the cell is 10 mV more negative than the outside.
• Know the basic process by which some epithelial cells secrete (rather than
absorb) fluid.
o There's a chloride-selective channel in the apical membrane in some
epithelial cells, cAMP-gated on the inside of the cell to open and allow
the efflux of Cl- back out of the apical membrane (which takes Na+
and water with it).
o This process is driven by receptors on the basolateral side of the
epithelial membrane that are triggered by acetylcholine to open those
gates.
o The reason the Cl- channels excrete Cl- instead of intaking it is that
there's an non-electrogenic pump on the basolateral side that uses
Na+ leakage to import Cl- into the cell.
o Effectively you're putting watery/serous substance out into the
epithelial secretions (mucus, etc).
• This is the basis for cystic fibrosis: the chloride channel isn't
properly implanted in the cell membrane, and thus no dilution
of mucus secretions are possible.
• This is also the basis for cholera: the cholera toxin gets into the
cell and opens the Cl- channel without regulation- which leads
to a massive efflux of water out the apical membrane into the
epithelial system (diarrhea, etc).
• Understand the main routes of excretion of metabolic wastes - CO2 and urea,
in particular.
o CO2, unsurprisingly, is excreted in the alveoli of the lungs.
o Urea is excreted in the urine after being picked up out of the blood in
the kidneys.

Action Potential I
Friday, November 16, 2007
9:02 AM

Action Potential I, 11/16/07:

• Understand how the passive electrical properties of axons render them poor
conductors of electrical signals over distances greater than a few millimeters:
o The electrical resistance in cytoplasm is high- also the electrical
insulation provided by the cell membrane is poor. Unassisted passive
electrical conductance would allow the signal to completely decay
before it reached its target.
• Describe the analogy between an electrical 'booster station' and an action
potential:
 o Essentially, at various "stations" along the axon, a power source (the
gradient of Na+, that is, potential chemical energy) 'boosts' the
electrical signal back its original strength, based on the 'readings' of
the signal strength by voltage-sensing receptors.
• In axons this 'reading' is done by voltage-gated sodium
channels.
• Know how changes in membrane resistance, membrane capacitance, and
internal (axial) resistance affect the passive spread of voltage along an axon:
o Okay. The cell membrane acts as a capacitor between the ICF and the
ECF. Notice that as soon as current reaches the ECF it is effectively
lost-- it's no good for signal transmission.
o The ion channels in the cell act as resistors that slow the flow of
current from the ICF to the ECF.
o The resistance of the cytoplasm in the axon itself acts as a resistor
that slow the flow of current along the ICF (in the direction you want
the impulse to go, namely down the axon).
o What this means if you want an axon to propagate quickly:
• (a) You want the capacitance to be low (high capacitance
delays the flow of current down the axon, since it's the first
place your current goes).
• (b) You want there to be lots of resistance to current leaving
the cell (thus closing the ion channels).
• (c) You want as little resistance as possible to current traveling
down the axon.
• The way the cell does this is (a) wrap the axons in myelin,
which effectively increases the thickness of the membrane and
thus drops the capacitance, speeding up the electrical
propagation; (b) close ion channels to pump up resistance to
current leaving the cell; and (c) increase the diameter of the
axon, which increases current flow and effectively lowers
internal (along-the-axon) resistance.
 If this doesn't make much sense to you, you're in good
company, or at least in mine. Just remember: thick
membranes = low membrane capacitance = faster AP
transmission. Fewer on channels = high membrane
resistance = faster AP transmission. Larger-diameter
axon = lower internal resistance = faster AP
transmission.

Action Potential II, III

Monday, November 19, 2007
9:00 AM

Action Potential II + III, 11/19/07:

• [Features of APs:]
o (1) Propagated
o (2) Vm reverses
o (3) Brief (<1 ms)
o (4) Threshold
 o (5) Refractory
o (6) Accommodation
• Describe the positions of the activation and inactivation gates in sodium
channels during an action potential:
o Sodium activation gates (m gates) are 'normally' closed at rest; they
open (thus making the membrane more permeable to sodium) in
response to a small depolarization (ie inside of the cell gets more
positive).
• Notice that the m gates are on the outside of the cell.
o Sodium inactivation gates (h gates) are 'normally' open at rest; they
close (thus making the membrane less permeable to sodium) in
response to the large depolarization that occurs after the m gates are
opened. Notice there's a lag time between the point at which the m
gates open and the point at which the h gates close (which allows the
flood of Na+ to fully depolarize the cell to generate the AP).
• Notice that the h gates are on the inside of the cell.
o Notice that there are also voltage-gated K+ channels that allow the cell
to repolarize much faster after firing an AP (allow K+ out of cell to
restore Vm after influx of Na+). These open around the time the AP
signal is at the peak of its intensity (ie. when the cell is approaching
maximum depolarization).
• Understand that intracellular concentrations of sodium and potassium do not
change much after a single action potential:
o The flow in of Na+, and the flow out of K+, don't put much of a dent in
the overall stores of both of those ions in the cell. Therefore an axon
can fire multiple times before it needs to equilibrate its Na/K levels
with the Na-K pump.
• [Length constant λ = length along axon it takes for amplitude of electrical
signal to decay to 37% of its original strength. In cellular axons this is about
1 mm.]
• [Time constant = time it takes for an axon's signal to reach its maximum
strength.]
• Understand the role of the sodium/potassium pump during the action
potential:
o During the AP, the Na-K pump does pretty much nothing important.
It's needed down the road to make sure that the Na-K concentrations
are back to their proper levels (see accommodation below), but in the
short term it's more or less irrelevant.
• Describe the mechanisms underlying the refractory period of the action
potential:
o Recall that there are two refractory phases: the absolute, during which
the cell will not fire an AP no matter what the stimulus, and the
relative, during which the cell needs a much larger stimulus than
normal to fire another AP.
o This is due to the fact that the h gates (Na inactivation gates) require
some time before they can reopen and allow Na into the cell (thus
even if m gates open, the Na+ can't get into the cell).
• During the absolute refractory phase, the h gates are more or
less all shut.
• During the relative refractory phase, the h gates are not all
shut, but enough of them are that it will require more
 depolarizing stimulus to trigger the Na+ positive feedback
cycle.
o Notice that the K+ channels also contribute to the refractory period: it
takes them a little while to close after repolarizing the cell, which help
make the cell briefly hyperpolarized during the refractory period and
makes it harder for the cell to become depolarized enough to trigger
another AP.
o Notice that the refractory period is the basis for the unidirectionality of
an AP: as the impulse travels down an axon, it can't travel backwards
because the portion of the axon just behind it (the one that just fired
the AP) is in its refractory period.
• (note: here I use "unidirectionality" to refer to the fact that an
AP can't go backwards. Remember, thought, that if you start an
AP in the middle of an axon, it goes in both directions down the
axons. One way of thinking about this is that the direction of
conduction down an axon isn't unidirectional, but the direction a
given AP travels is.)
• Describe the mechanisms underlying accommodation of the action potential:
o Accommodation: If a progressively larger current is applied to the
cell slowly, often an AP won't fire-- if the depolarization is slow, the
inactivation (h) gates have enough time to close before the AP has a
chance to get going.
• This is the basis for the problem with hyperkalemia: you wind
up with a slow depolarization of the membrane that prevents
APs from firing by slowly activating the h gates and preventing
sudden depolarization by positive feedback.
o [Not needed but interesting: As cells fire APs, they become
progressively less able to generate more APs. This has something to
do with the concentrations of intra- and extracellular Na+ and K+,
which are becoming more and more imbalanced with every AP (every
time an AP fires, a lot of Na+ comes into the cell, and a lot of K+ goes
out).
o This means that the more APs a cell fires, the more steadily
depolarized it becomes. This, in turn, means that after a certain point
of depolarization, the h gates (inactivation) are shut, and stay shut,
meaning that the cell can't fire more APs until the Na-K pump restores
its normal concentrations.
o When this happens depends on the axon's diameter. Basically, the
diameter of the axon determines its surface-area-to-volume ratio: the
larger the axon, the smaller this ratio, which means that its Na-K
balance is disturbed relatively less per AP than a smaller-diameter
axon.
• Thus: large-diameter axons accommodate more slowly; small-
diameter axons accommodate more slowly.]
• Define threshold for an action potential:
o AP threshold is the point at which the incoming current of Na+ equals
the outgoing current of K+ (as Na+ comes in, K+ wants to come out
more strongly). If the incoming current (Na+) gets just a little higher,
the cell will enter the positive-feedback loop of depolarization.
o A more intuitive way of thinking about it is that the threshold is the
point at which just a hair more depolarization will probably make the
 cell fire an AP and just a hair less depolarization will probably make
the cell return to normal polarization levels.
• Understand the explosive, positive-feedback nature of the rising phase of the
action potential:
o If a cell is depolarized past its threshold point, the depolarization of
the cell causes Na+ channels to open (m gates), which causes the cell
to depolarize even faster, which causes more m gates to open, etc,
etc.
• Describe how action potential propagation relies on voltage-gated sodium
channels acting like molecular 'booster stations'
o See "Action Potential I." Effectively the passive propagation of the AP
is sufficient to depolarize the next segment of axon enough to cause
that second segment's m gates to open, causing another AP-- and so
on down the axon.
• [Basis for anesthestic: block m gates so that depolarization can't happen.]
• [Notice: Difference between small and large nerve fibers:]
o Small (ie pain and temperature fibers):
• Slower AP conduction.
• Have a high threshold to extracellular stimulation (harder to
trigger AP).
• Lower safety factor: easier to block m gates with anesthetic.
o Large (ie touch receptor and positional fibers):
• Faster AP conduction.
• Have a lower threshold to extracellular stimulation (easier to
trigger AP).
• Higher safety factor: harder to block m gates with anesthetic.
• Know why action potential propagation is much slower than the velocity of
light:
o Aside from the fact that it relies on the movement of ions (not
massless particles), the fact that it's moving through a fluid with
resistance as opposed to through a vacuum or through low-resistance
copper wire, and the fact that it relies on a slow threshold-gated signal
for propagation? No idea.
• Know what myelination does to axonal membrane resistance and membrane
capacitance, and why these changes increase conduction velocity
o Myelination effectively increases the capacitance of the axon (making a
signal go faster, no waiting for the capacitance to build) and increases
the membrane resistance (making a signal go farther, no leaking of
ions out of the membrane).
• Describe refractoriness, and explain how it prevents an action potential from
reversing its direction of propagation:
o See above, under "refractory period."
• Understand why demyelination can slow or block action potential conduction:
o Well, if myelination allows the AP to go faster through increased
capacitance, demyelination can make an AP go slower. Likewise, if
myelination allows an AP to go farther through increased membrane
resistance, demyelinated can make it go a smaller effective distance,
which may not be far enough to reach the next node of Ranvier and
set off the next AP.
• Describe the effect of extracellular calcium ions on action potential threshold:
o Ca2+ in the ECF normally remains bound to negative charges on the
outer surface of the cell.
 o However, in hypocalcemia, [Ca2+] in the ECF goes down, which allows
some of those negative charges to go unbound. This makes the
outside of the membrane, at particular places where the Ca2+ is thin,
selectively more negative.
o As the outside of the membrane gets more negative in places, the
potential difference across the membrane at those places goes lessens
(ie, it depolarizes), thus triggering the m gates to create an AP without
normal signaling.
o Note the difference between hyperkalemia and hypocalcemia:
• Hyperkalemia: excess of [K+] in ECF causes gradual
depolarization of the entire membrane, which allows h gates to
shut and stay shut, preventing APs from being generated.
• Hypocalcemia: insufficiency of [Ca2+] in ECF caused localized
depolarization of the outside of the membrane, which causes m
gates (which are on the outside) to open and cause APs to be
generated erratically.
 [First sign of this is often "Trousseau's sign": contraction
of the flexors in the elbows and wrists.]
• Know the effect of axon diameter on conduction velocity, threshold to
extracellular stimulation, safety factor for conduction, and likelihood of being
myelinated
o Axons with a larger diameter conduct impulses more quickly.
• Interesting aside: notice that, given unlimited space, we'd want
all of our axons to be huge-diameter to get really quick
information to our brains. But given limited space, there's a
tradeoff between size of axon (faster signal) and number of
axons (more information in a slower signal). So some of our
axons
are really big (the get-out-of-the-way-of-the-bus axons) and some are
smaller but more numerous (the ponder-the-meaning-of-life axons).
o Safety factor: the fact that we have a lot more Na+ m gates
than we really need to get to threshold and generate an AP of
sufficient size in a given axon.
• Means faster conduction is possible (depends on # m
gates).
• Also means can send APs down multiple branches of an
axon.
o Threshold to extracellular stimulation: As mentioned, large-
diameter axons have a lower threshold to stimulation, while small-
diameter axons have a higher threshold.
o Smallest axons are unmyelinated: actually it winds up being
more efficient this way.
• Know the mechanisms by which calcium, glucose + insulin, Na/K ion
exchange resins, and renal dialysis counteract the signs of hyperkalemia:
o See "Membrane Potential III."

Unit III
Monday, November 26, 2007
6:39 AM
Secretory Pathways I + II
Monday, November 26, 2007
6:39 AM

Secretory Pathways I + II, 11/26/07:

• Describe the 3 mechanisms of protein transport

• Gated transport: requires a specific receptor protein to carry a folded
protein from the cytosol into the nucleus through the pore complex.
• Transmembrane transport: requires a translocator protein or protein
complex (ie translocon) to move proteins across membrane.
• Vesicular transport: membrane-bound transport; requires adaptor and
coat proteins.
• List the major functions of the ER:
ο Synthesis of lipids
ο Control of cholesterol homeostasis
ο Synthesis of proteins on membrane bound ribosomes
ο Co-translational folding of proteins and early post-translational
modifications
ο "Quality control": protein degradation and turnover.
 Describe co-translational translocation:
ο Translocon: Protein-conducting aqueous channel that spans the
rough ER membrane.
ο Protein sorting signals: most proteins are "tagged" with a region
(primary sequence or tertiary structure) that forms a "recognition
patch": this tells the cell how to package the protein and where to
send it.
• Notice that there's a specific 5' sequence that signals that the
transcript is going to be pulled into the ER; if it's absent, the
protein remains soluble in the cytosol.
• The 5' sequence that signals ER translocations causes the
ribosome translating that protein to attach to the membrane of
the ER (thus causing rough ER vs smooth ER).
ο Co-translational translocation: as mRNA is translated, it's moved
through the translocon into the ER lumen to be cleaved by signal
peptidases and folded.
ο The 5' signal sequence binds to the SRP (signal receptor particle) in
the ER membrane; this opens the translocon and the nascent
polypeptide is threaded through the translocon into the lumen as it's
translated (the 5' signal sequence is cleaved off almost immediately so
that it doesn't make up a part of the final protein).
• SRPs: have a multi-methionine "pocket" that binds to a wide
variety of 5' signal sequences.
• 5' signal sequences: variable; often mainly nonpolar.
ο Notice that the translocon is regulated on the luminal surface as well
by binding protein BiP: it can expel proteins as well as admit them.
ο If the nascent protein is meant to be a membrane protein, the protein
contains another region (aside from the 5' signal sequence) that
interacts with the translocon: a "stop-transfer sequence" which stops
the transfer of the protein into the lumen-- thus the protein remains
"stuck" with a transmembrane domain in the ER membrane, one end
 of the protein in the lumen, and the other end of the protein in the
cytosol. The "stuck" protein then translocates out of the translocon for
further packaging.
• Quick note about which end ends up where: the positively
charged end of the transmembrane domain (whichever that is)
winds up in the cytosol (outside the ER) and the negatively
charged end of the domain winds up in the ER lumen.
ο Notice that these transmembrane proteins can span (traverse) the
membrane many times, depending on the number and location of
start- and stop-transfer domains.
ο Asparagine-linked (N-linked) glycosylation: as a polypeptide is
threaded through the translocon, an enzyme (oligosaccharyl
transferase) catalyzes the glycosylation of select asparagine residues
in the protein ("core glycosylation") with the motif N-X-S/T.
• Part of N-linked glycosylation involves trimming the attached
glycosyl groups to properly package the protein for transport to
the Golgi apparatus.
• The trimming process is what signals the ER that a protein is
ready to be transported elsewhere.
 List the major functions of the Golgi:
ο Synthesis of complex sphingolipids from the ceramide backbone
ο Additional post-translational modifications of proteins and lipids
ο Proteolytic processing (protein cleavage)
ο Sorting of proteins and lipids for Golgi compartments
ο Morphology: notice that the Golgi complex can be bigger or smaller
(more or less cisternae) depending on how much packaging and
modification needs to happen in that cell.
ο How the Golgi apparatus transports proteins/lipids through itself:
• Depends on the type of things transported. Sometimes it's by
moving them from cisterna to cisterna with small vesicles;
sometimes it's by actually moving or modifying the entire
cisterna that contains the material ("cisternal progression").
• Which one used depends on the nature of the material-- if it
won't fit inside a vesicle, tend to use cisternal progression.
ο Notice that N-linked glycosylation is finished in the Golgi: this, again,
is a signal to move the protein to a new location
ο "Proproteins": proteins that undergo proteolytic cleavage late in
processing (Golgi).
ο One package sorting mechanism: by thickness of the package's
membrane (thicker -> plasma membrane; thinner stays in the ER
membrane, that sort of thing).
ο Other sorting mechanisms:
• There are some. They're complex. See the handout if you're
curious.
ο Apical membrane: tends to be thicker than basolateral membrane.
 Describe vesicular transport:
ο Vesicular "coats" are assembled at the site of vesicle formation: these
"sort" the proteins or lipids to be moved (different coat molecules
"select" various types of cargo using adaptor proteins), and aid in
pushing the vesicle out the side of the membrane.
 οNotice that there are adaptor binding motifs in cargo: thus it's
probably more accurate to say that the cargo "selects" the coating and
adaptive molecules.
• Process: Cargo attracts a certain type of adaptor protein; the
adaptor proteins then attract a certain type of coat.
ο Notice that you need SNARE proteins: essentially, the vesicle has to be
able to recognize and merge with the target cell membrane. Recall
that VAMP SNAREs are found on vesicles and SNAP and syntaxin
SNAREs are found on target membranes.
• Notice that GTP-binding proteins (Rab proteins) determine
specificity of docking location with the SNAREs on the outside of
the vesicle.
• These GTP-BPs can also regulate coat assembly (all kinds).
ο Once a vesicle's budded off from the ER, it's moved to the Golgi via
the cytoskeletal network. The motor proteins that move them are
dynein and kinesin.
• There's a "vesicular-tubular cluster" that pre-packages the
proteins before they get to the Golgi.
 Name 3 well-studied vesicle coats:
• Clathrin coats: highly structured and symmetrical, transport from
outside of the cell to inside (endocytosis) or lysosomal transport
to/from Golgi.
• COPI coats: traffic from Golgi to ER.
• COPII coats: traffic from ER to Golgi.

• [Important notes not in the LO's:]

o lumen of organelles = interior of organelles; exocytosis from the
lumen leads into the cytoplasm.
o Exocytic pathway: getting proteins to the organelles they're going to.
o Endocytic pathway: getting proteins, within their target organelles, to
their sites of action.
o Notice that the interiors of lymphocytes are almost entirely ER, like
most cells that produce massive amount of proteins (in this case
antibodies).
o ER cisternae: grooves or spaces between folds of the ER.
o GPI-linked proteins: proteins that will eventually end up on the outside
of the cell's plasma membrane. Glycosyl-phosphatidyl-inositol link
(GPI) links the protein to the cell membrane (the primary sequence of
the protein itself doesn't bind to the membrane).
o KDEL protein: protein for retrieving improperly transported packages.
o Thing to keep in mind: a protein sequence is essentially differentiated
information. As such, it contains not only information about its primary
function but only information about where it should go and how it
should be packaged to get there.

Cytoskeleton
Monday, November 26, 2007
6:40 AM

Cytoskeleton, 11/26/07:
[This one has lots of stuff in it and I doubt I really understand it all-- so take with
caution.]

• Discuss the concept of a cytoskeleton.

o Set of intracellular structures that orders the inside of a cell as well as
organizing it in the extracellular environment.
o Specifically: provides cell shape, mechanical strength, locomotive
structures, plasma membrane support, spatial organization of
organelles, and intracellular transport "roads".
• Describe two types of cytoskeletal elements, their properties, their functional
roles, and their protein composition.
o All cytoskeletal elements: polyprotein structures.
o Three types: microfilaments (listed on slides as "actin filaments"),
microtubules, and intermediate filaments. The latter two are discussed
here.
o Microtubules: hollow tubular structure, very flexible, don't stretch.
Primarily provide the scaffold for spatial organization and movement of
organelles, and also the movements of cilia and flagella. Outer
diameter 25 nm. Often clustered near the nucleus (used during
mitosis).
o Intermediate filaments: ropelike structure (more details below),
very strong, primarily provide mechanical strength to the cell.
Distributed primarily throughout the plasma membrane.
o Microfilaments will be discussed later.
o Microtubules:
• Can't keep tensile forces (no stretching).
• Made up of a protein called tubulin- specifically alpha and beta
tubulin.
• Tubulin has GTP binding regions, both between alpha and beta
subunits and on the outside of the beta subunit.
 Notice that the GTP between the alpha and beta
subunits never gets hydrolyzed, just the one of the
outside of the beta.
• In microtubules, tubulin assumes a linelike, polymerized alpha-
beta dimer configuration. Provides a polarity to the microtubule
structure (plus end is the end at which polymerization takes
place [the end with the free GTP, or the end with the exposed
beta subunit], minus end is the other one [the end with the
exposed alpha subunit]).
• This line of tubulin subunits wraps itself into a tube or solenoid
to form the microtubule.
• As the tubulin ages, the GTP in individual dimers at the minus
end is hydrolyzed to GDP, and these dimers fall off the end of
the tubule, acquire a new PO4 in the cytosol, and are
incorporated into the tubule again at the plus end.
• This means the plus end is more stable (has GTP, dimers can
add); the minus end is less stable (has GDP, dimers can fall
off). Note that certain cellular processes can destabilize the plus
end.
• Notice that the minus end tends to be around the nucleus; the
plus ends grow out from there into the cytosol.
 • Microtubules "hold" the Golgi apparatus in a particular location
relative to the nucleus. This stabilizing mechanism also doubles
as a transport pathway. (see below, "motor proteins".)
• Discuss cytoskeletal dynamics and the role of certain proteins and drugs in
actin and tubulin polymerization/depolymerization.
o Certain drugs interact with microtubule structure and, due to
microtubules' role in mitosis, block cell division (thus are compounds
of interest in cancer treatment).
• Colchicine and vinblastine (toxic plant extracts) interfere with
tubulin polymerization at the plus end.
• Paclitaxel ("Taxol") binds to and stabilizes the microtubule. The
net effect of this is to cause aggregations of microtubules,
which shuts down effective function during mitosis.
• Explain the concept of mechanoenzymes. Explain the mechanisms of tubulin-
based movement and intracellular transport.
o "Motor proteins": dyneins and kinesins. Associated with microtubules;
use them as tracks to drag cargo to target organelles.
o These proteins convert ATP into mechanical energy by conformational
changes.
• Kinesins: 2 head domains and a tail. The heads contain ATPase:
effectively the enzyme "walks" its heads down the microtubule
and pulls the tail (and cargo) behind it. By hydrolyzing ATP, the
enzyme shifts conformation to swing the trailing head group
around to attach in front of the leading head group.
 Notice that the kinesins only ever travel from minus to
plus ends of the microtubules.
 Notice also that the dyneins effectively travel the same
way but only ever go from plus to minus ends of the
microtubules.
• Tail domains bind cargo.
• Discuss the role of microtubules in mitosis.
o Three types of microtubules in the mitotic spindle: astral microtubules
radiating from centrosomes (place centrosomes in center of daughter
cell, help pull two centrosome halves apart), kinetochore microtubules
connecting chromosomes (plus ends bind to specific site of centromere
in chromosomes), and overlap microtubules (plus ends bind to each
other on opposites ends from each side of the dividing centrosome).
• Kinetochore action is driven by "minus-directing motors": the
chromosomes are pulled in the minus direction (away from the
middle), and the plus ends depolymerize between them as the
centromere separates (thus no remnants left over).
• Bare-bones: Astral and overlap microtubules pull centrosomes
apart (plus-directed motors, kinesins); kinetochores separate
chromosomes during this process (minus-directed motors,
dyneins).
• Explain the motion of cilia and flagella.
o Cilia primarily move in a switchlike fashion back and forth; flagella
usually move by progressive wavelike contractions.
o Microtubule-mediated: cilia and flagella contain a microtubule core
called an "axoneme": two microtubules in center, surrounded by a ring
of microtubules connected by mechanoenzymes (dyneins). The
 dynein-mediated movement is what bends or moves the cilia or
flagella.
• Most cells have a cytoskeletal microtubule network called a monocilium or
primary cilium: the "non-motile" portion of this network seems to be involved
in sensory perception.
• Describe how intermediate filaments are anchored in the plasma membrane.
o Effectively you have dimers of filaments; tetramers are formed by
antiparallel dimers.
o This means intermediate filaments have no polarity (there's two plus
and two minus ends at each side of the tetramer.
o Tetramers become polymerized into twisted bands of eight tetramers:
ropelike, very strong (32 filaments per intermediate fiber "rope").
o These anchor to the plasma membrane predominantly by attaching to
desmosomes or hemidesmosomes. Both of these are types of
junctions, and serve as the basis for cellular mobility/stability. See
"Epithelia" notes for more details.
• Discuss the cytoskeleton in the context of disease processes and as a drug
target.
o Epidermolysis bullosa simplex: Keratin mutation that affects
intermediate filament stability. IFs can't provide mechanical strength:
thus a very slight impact on the skin produces skin lesions and dermal
bleeding. Effectively the epidermis is ripped away from the underlying
subcutaneous tissue with extreme ease due to abnormal intermediate
filament connectivity.
o Charcot-Marie-Tooth syndrome: Neurofilament mutation causes
peripheral neuropathy. These mutations are also associated with Lou
Gehrig's disease.
o Kartagener syndrome: wide variety of symptoms (respiratory disease,
male infertility, left/right symmetry mismatch, etc)
• Turns out to be a monocilium (cilia/flagella) defect- sperm can't
swim, lungs can't clear mucus, cilial direction that establishes
left/right symmetry in early gastrulation is disturbed, etc.
Problems in outer dyneins of monocilium.
o Dyneins (minus-end directed motors, can transport into centrosome)
often shuttle viruses (which can attach to them) into the nucleus or
along peripheral nerves into dorsal root ganglia.

Clinical vignettes
Monday, November 26, 2007
6:41 AM

Clinical vignettes:

Lung Cancer, 11/26/07:

• The students will learn that lung cancer is a major global and national health
problem. The epidemiology of lung cancer will be discussed. You should be
able to give an estimate of the incidence of lung cancer and the prognosis.
 o More than 200,000 new cases in 2007. More than 160,000 deaths the
same year. Five-year survival rate is about 16%.
• What are the symptoms from lung cancer? You should be able to tell the main
symptoms.
o …Anyone have any ideas here besides the obvious "hey, you've got
this cough and this mass on your X-ray"?…
• What are the main subtypes of lung cancer? At least 4 main subtypes should
be known.
o Notice four stages of lung cancer: I through IV (5-year survival gets a
lot better the smaller the stage at diagnosis). Ranked by progression
of metastasis.
• I: local cancer
• II and III: regional cancer
• IV: distant cancer
o Non-small-cell lung cancer: 80-85% of total
• Adenocarcinomas on the rise (60-70%, peripherally located
tumors)
 Arise from glandular structures
• Squamous-cell carcinomas (centrally located in bronchi)
• Large-cell carcinomas (undifferentiated, rare)
o Small-cell lung cancer: 15-20% of the total: strongly related to
smoking.
• General principles for diagnosis:
o Not a lot of good diagnostic or treatment options for lung cancer.
o Local cancer only (in lung): Stage I
o Nearby lymph node invasion: Stage II
o Multiple cancer sites in lung: Stage III
o Distant cancer sites (liver, bone marrow, adrenal glands, etc): Stage
IV
o Tumor size correlates with poor rates of survival, as does number of
invaded lymph nodes and how centrally they are located.
• What are the general principles in staging and treatment of lung cancer and
what is the prognosis related to stages?. You should know the main
differences in treatment depending on stage and know some estimated
figures for the different stages as there are significant differences in
prognosis dependent on stage.
o Treatment depends on the stage of cancer:
• I and II: Surgery (60-70% or 40-55% cure rates, respectively).
• IIIA: either chemotherapy or surgery (10-25% or 35% cure
rates, respectively).
• IIIB and IV: Chemotherapy and supportive care (very low cure
rates).
o Adjuvant chemotherapy (given after surgery) seems to lower death
rates from lung cancer significantly, by about 10%.
• Adenocarcinoma of the lung is rising in incidence, especially among women
and never-smokers. This subtype have specific characteristics both related to
demographics, location, and sensitivity to certain agents. You should be able
to know some specifics about this subtype.
o Smokers: lung cancer seems to occur mainly through Ras signaling
pathway.
 o Non-smokers: lung cancer seems to occur mainly through EGFR
(epidermal growth factor receptor) signaling pathway.
• Newer treatment strategies for cancer is moving away from ”every treatment
fits everyone” to” individualized” therapy. What does that mean?
o Problem is: some patients have no positive response to chemo and
have to deal with all the side effects anyway. Molecular profiling should
allow targeting of therapy that actually works for individual patients.
• ie: if there's a mutation in EGFR, treat with EGFR therapy (see
below).
• What is molecular targeted therapy? Give some examples.
o Agents that work against particular biochemical pathways (see
Herceptin, probably Gleevac from last unit).
• What is the role of Epidermal Growth Factor Receptor (EGFR) signaling in lung
cancer?
o EGFR pathway: seems to be significant for carcinogenesis, activated a
number of ways; affects angiogenesis (to supply vasculature to
tumor), proliferation, anti-apoptosis.
• Know that inhibition of the EGFR signaling pathway has been successful in the
treatment of many cancers, and the students will learn about EGFR inhibition
in lung cancer, especially adenocarcinomas.
o Antibodies can be used to attack the EGF receptor protein, specifically
at the receptor binding site and the tyrosine kinase domain.
o Also can use EGFR tyrosine kinase inhibitors: non-injected; pill form,
no bone marrow toxicity.
• Note that EGFR proteins are members of the HER family (HER1
= EGFR protein).
o Mutation in EGFR's tyrosine kinase domain commonly results in
adenocarcinoma.
o Can use immunohistochemistry or FISH (copy number) to detect
abnormalities with EGFR (thus higher, 'personalized,' rate of response
to EGFR therapy).

LO's as stated in class: Magnitude of lung cancer as health problem,

types/staging/treatment/prognosis, future cancer therapy: EGFR pathway inhibition
(molecular targeted therapy)

Cholera, 11/28/07:
• Understand the key clinical features and treatment of cholera infection.
o Features:
• Sever, watery diarrhea
• Low-grade fevers and drowsiness
• Fatigue
• Decreased urination and dehydration
• Skin tenting without lesions
• Tachycardia
• Muscle cramps
• Nausea, vomiting
• Hypovolemic shock
o Treatment:
• Oral rehydration therapy. Notice can also use IV rehydration.
  Rehydrate, maintain hydration, feed early to make sure
nutrition doesn't get too poor.
• Mainly you're just replacing fluids and electrolytes until the
body can kick the microorganism out. Notice can use antibiotics
(azithromycin) to quicken this process.
• Just an odd side note: you use azithromycin to treat both
cholera (overactivation of CFTR) and cystic fibrosis (inactivation
of CFTR). Weird.
• Understand the actions of the cholera toxin's A and B subunits.
o Recall: the bacteria isn't the problem, it's the toxin that the bacteria
secrete.
o A subunit: active site; B subunit: transport/bindng molecule
o B subunit binds to the GM1 ganglioside receptor on enterocyte surface
o A subunit cleaved off and endocytosed:
• Binds to Gs proteins, inactivating their GTPase activity (thus
always turned on) -- stimulates production of cAMP.
• cAMP activates CFTR (cystic fibrosis transport regulator)
chloride channel in apical membrane.
 Notice that the Na-K pump on the basolateral side may
also be shut down.
• Perpetual CFTR activation leads to massive chloride efflux.
• The efflux of chloride draws water with it, causing diarrhea.
• Since the tight junctions in apical enteric cells are relatively
loose, you can lose a whole lot of water and sodium very
quickly paracellularly as they follow the chloride.
• Understand the physiology behind oral rehydration solutions (ORS).
o Effectively ORS is just water, sugar, and salt (plus potassium and
citrate in WHO formula). Homemade = 8 teaspoons sugar, 1 teaspoon
salt in one liter of water.
o Small amounts can be absorbed quickly; small sips can rehydrate
patients even when emesis follows.
o Glucose helps Na+ uptake (and therefore water retention) in enteric
system, also provides nutrition.
• Notice can replace glucose with rice powder, which may reduce
severity of diarrhea by adding amino acids to improve sodium
uptake in enterocytes (counteract Cl- and water efflux).
• Understand why someone with cystic fibrosis may be relatively protected from
cholera symptoms.
o Cystic fibrosis shuts down the CFTR receptors; heterozygotes for cystic
fibrosis seem to be partially protected from the effects of cholera
toxin.
• Another odd side note: the notes suggest cystic fibrosis genes arose as a
genetic partial defense against cholera-- but CF genes are by far most
common in European and Ashkenazi Jewish populations, whereas the cholera
bacteria is thought to have originated in India. Something don't sound right
there.

Cystic Fibrosis, 11/30/07:

• Note that a major diagnostic test for CF is the chloride level in sweat ('sweat
type'). Large levels of Cl- indicate homozygosity for CF (CF is autosomal
recessive).
• Review the genetics and underlying protein defect in Cystic Fibrosis (CF).
o Most common lethal genetic disease in Caucasians- median predicted
survival is 35 years.
o CF gene codes for CFTR protein: acts as an ion channel in apical
epithelia controlling movement of water and ions in and out.
o CF is a large gene in chromosome 7. Lots of different known
pathological mutations in the gene.
o Mutations in CF tend to cause either:
• (1) no protein synthesis
• (2) misfolding of protein leading to degradation before reaching
apical cell surface
• (3) altered conductance such that the protein can't be conveyed
to the apical surface, or
• (4) partial loss-of-function such that the CFTR proteins don't
work as well as normal.
• Review the multiorgan involvement in persons with cystic fibrosis.
o (Tends to show up in tubular epithelial systems:)
o Sinuses (chronic sinusitis)
o Lungs (main problem: chronic bronchitis and airway
infection/inflammation.)
• Chronic lung problems lead to loss of lung function
o Pancreas
• Most CF: pancreatic insufficiency; need to supplement with oral
enzymes.
• ~25% develop cystic fibrosis-related diabetes (requires insulin
injections)
o Intestine
• Most CF: intestinal closure (can't eat or excrete feces)-
"meconium ileus".
 -tends to be diagnostic for CF
o Liver
• Minority of CF patients, but second leading CF cause of death
(after lungs).
o Ductus deferens
• Male infertility (sperm are viable but can't get out).
• Notice that male heterozygotes can also have problems with
this.
o Sweat glands
• Problem with salt-losing dehydration (see below; effectively
can't reuptake Cl- through sweat glands).
• Review current understanding of pathophysiology underlying CF lung disease.
o Lung disease:
• Pseudomonas and Staphylococcus aureus are the most
common pathogens found in CF lungs (since the mucus is an
ideal breeding ground for bacteria).
• In lung: CFTR normally excretes Cl- to move water into the
mucus layer overlaying the lung epithelium (like in the
intestine). With defective CFTR, this water secretion doesn't
happen.
 In addition to this, normal CFTR also downregulates
epithelial sodium channels. Without CFTR, have elevated
 levels of import of Na+ into epithelial cell, which further
removes water from the lumen (and the mucus) into the
epithelial cells, exacerbating the thickness of the mucus.
o (Notice that in sweat glands, the CFTR defect causes a problem in
getting chloride into the epithelial cells, as opposed to in the lung and
intestine, where the defect causes a problem in getting them out.)
o Treat (lungs) with airway clearance therapy, antibiotics to keep lungs
clear, inhaled mucolytics (break up the mucus), brochodilators,
enzyme replacement, vitamin A/D/E/K replacement, azithromycin used
chronically.
o Therapeutic approaches: eventually need lung transplant. Gene
therapy not working yet, but "protein rescue" seems promising: in
principle, could correct misfolding or get nonconducted CFTR proteins
to the cell membrane to work properly.

Muscular Dystrophy, 12/6/07:

[Thanks to Andrew Brookens for supplying lecture notes for this one. --jcr]
• Describe the genetic basis for, and clinical features of, Duchenne's Muscular
Dystrophy:
• X-linked recessive inheritance
• Frequency: 1/3500 males
• “Climb up themselves” (“Gower” sign, showing early, ~1 year)
• Wheelchair bound by 12; die in 20s due to cardiorespiratory failure
• 1/3 from spontaneous (non-inherited) mutation
• 10 year old falls 30-40x/day
• Genetic defect in DMD: dystrophin protein (forms the “steelbelt” supporting
the muscle cell membrane) is broken down due to a premature stop codon in
its gene.
• Dystrophin is the largest gene known: on Xp21, 79 exons long,
2.4Mbases.
• Dystrophin links t-acting component on one side, then to
transmembrane complex on plasma membrane that enables force to
be conveyed to outside matrix
• DMD therapy:
• Surgical tendon releases/scoliosis surgery
• Corticosteroids prolong independent ambulation (8-24mos), but with
side effects
• Supportive care
• Future treatments:
• Clinical trials for exon skipping in Netherlands: the oral agent PTC124
reads through the premature stop codon in dystrophin.
• Stem cell transplant: replace dystrophin function.
• Myostatin antibody (Johns Hopkins)
• AT1 antagonist (Losartan) in mice
• Method of diagnosis: serum creatine kinase recognizes DMD early, without
muscle biopsy.

Nuclear Transport
Tuesday, November 27, 2007
8:03 AM

Nuclear Transport, 11/27/07:

• Describe the 3 mechanisms of protein transport.

o Gated transport: requires a specific receptor protein to carry a protein
from the cytosol into the nucleus through the pore complex.
o Transmembrane transport: requires a translocator protein or protein
complex (ie translocon) to move proteins across membrane.
o Vesicular transport: membrane-bound transport; requires adaptor and
coat proteins.
• List the major functions of the nucleus.
o Sequester cellular DNA
o RNA transcription and processing
o Contains nucleolus: site of ribosomal RNA synthesis, processing,
subunit assembly
• (how big nucleolus is depends on the number of ribosomes
needed)
• On a micrograph, the big dark thing in the nucleus is the
nucleolus.
• Describe the nuclear pore complex.
o Pore complex: network of large nuclear membrane-spanning
structures that control entry to and exit from the nucleus.
• The nuclear membrane is double-layered; the pore complex
penetrates both.
 Inner layer: binding sites for chromatin and the nuclear
lamina (structural supports consisting of intermediate
filaments).
 Outer layer: continuous with the ER membrane.
• Because this is the case, proteins that are
translated into the ER membrane (through the
translocon) are only one membrane layer (the
inner nuclear membrane layer) away from the
inside of the nucleus.
 Note that the inner and outer nuclear membrane layers
are continuous at the pores of the pore complex.
• 4 structural building blocks of a pore:
 Column subunits (pore wall)
 Annular subunits ("spokes" extending in toward the
pore's center)
 Lumenal subunits (transmembrane, hold membrane
open and anchor pore to membrane)
 Ring subunits (cytosolic and nuclear faces of the
complex; on the outer and inner surfaces of the nuclear
membrane)
• Very large structures: ~125 million daltons (30X larger than
ribosome).
• Roughly 3000-4000 pores per mammalian cell nucleus
• 9 nm diameter channel, but can expand to accommodate larger
transports.
• Describe nuclear import/export.
 o Molecules less than 5000 daltons can freely diffuse through the pore
complex; everything else has to be gated in by a specific receptor
protein.
o Nucleoporin proteins (lots of hydrophobic repeats) line the central pore
transporter channel to interact with import/export receptors.
o Common imports: histones, DNA/RNA polymerases, gene regulatory
proteins, and RNA-processing proteins
o Common exports: tRNAs and processed mRNAs
• Export of mRNA: part of mRNA processing is to attach a whole
bunch of proteins to it (among others, mRNA exporter
proteins). Without all these proteins, it won't exit the nucleus.
Note that a lot of the nuclear-exit-signal proteins fall off to be
recycled as it leaves the nucleus.
o Ribosomal proteins: imported into nucleus to be assembled with
rRNAs, then exported.
o Nuclear Localization Signals (NLS): sequences in proteins (primarily
positively-charged lysines and arginines) that allow those proteins to
be picked up by a nuclear import/export receptor. Notice the sequence
doesn't have to be contiguous.
o Nuclear import/export receptors:
• Soluble, cytosolic proteins; bind to both NLS on transported
protein and to nucleoporins in the pore channel.
• Exportins export; importins import. Two things: one, they need
to be on the right side of the membrane to work, and two, this
does take energy (GTP); selective placement of GTPases and
GDP phosphorylases on different sides of the nuclear
membrane allows a concentration gradient to occur, which
helps the proteins to get back to the side that they work on.

Protein Degradation
Tuesday, November 27, 2007
9:00 AM

Protein Degradation, 11/27/07:

• Recall that one of the functions of the ER is "Quality Control"-- that is, protein
folding and degradation.
o If a protein isn't folding correctly, chaperone proteins can help refold
it; however, if the protein still can't get it right (due to mutations or
what have you), it needs to be chopped into little bits and reassembled
(don't try this at home).
• Basically, if you have a bunch of misfolded proteins, they can
aggregate and cause real problems (ie apoptosis) for the cell.
• About 30% of all new proteins need help from a chaperone to
fold right; about 30% of those can't be folded correctly and get
sent to the cellular trash can.
• This trash can is called the proteasome: it chops the proteins
up into amino acids so it can be resynthesized.
o ER quality control:
 • Provides optimal (oxidizing) environment for folding and
oligomeric assembly; also has a lot of chaperone proteins,
folding enzymes, and folding sensors (proteins that detect
misfolding).
• Note that the proteasomes are primarily in the cytoplasm and
not in the ER themselves; therefore proteins marked for
degradation need to get out into the cytoplasm again.
 BiP (binding proteins) can export proteins out of the ER
(back through the translocon) to be taken to the
proteasome.
• List 3 mechanisms for endocytosis.
o Endocytosis (uptake of materials into the cell): major route for cell-
specific drug delivery and gene replacement therapy.
• Clathrin-coated vesicles: use SNARE proteins on vesicles to
merge lipid surface of vesicles with plasma membrane.
 There's effectively a string of lowered-pH steps in the
cell to take endocytosed stuff to the lysosome to be
degraded into its component parts. Drugs can either be
active once degraded by the lysosome or be picked up
by their targets somewhere in this process.
• Caveolae: cholesterol-rich vesicles budding from membrane-
evidently have signaling functions. Not a lot of elaboration here.
• Fluid-phase: stuff can move in and out of the membrane freely.
Not a lot of specifics here, either.
• Describe two types of molecular chaperones.
o Chaperone protein example: heat shock proteins (Hsp's).
• Hsp70 family: these can bind to exposed hydrophobic domains
to reform and prevent aggregation.
• Hsp 60 family: there form large barrels which envelope
misfolded proteins to refold them without aggregation.
• Describe the proteasome and protein degradation.
o The proteasome subunits are popular proteins: make up about 1% of
cellular protein complement. Dispersed throughout the cytosol and the
nucleoplasm.
o Structure: has a central cylinder containing the protease active sites
and a cap on either side to detect ubiquitinylated proteins (recall that
ubiquitin is the molecular protein tag with which a cell marks proteins
for degradation).
o General idea: proteins are tagged with ubiquitin (four ubiquitin protein
attachments are required to degrade a protein) and transported to the
proteasome, where it's shoved into the central protease cylinder and
degraded.
o So the entire process looks like: misfolded proteins are detected in the
ER, BiP-shoved out the translocon into the cytosol, tagged with
ubiquitin, and degraded in the proteasome.
o Non-proteasomal methods of protein degradation:
• Lysosome: degrades all cellular components transported to it.
 The lysosome can degrade pretty much every damn
thing in the cell; it's basically a big acid pit.
• Autophagy: digests entire organelles for energy
 Cell does autophagy primarily under conditions of
extreme starvation or illness, but it also can be used to
 degrade very long-lived proteins or senescent
organelles. Can also be used to remodel cell during
differentiation in development.

Cell Signaling Overview I

Tuesday, November 27, 2007
9:56 AM

Cell Signaling Overview I, 11/27/07:

• Understand the basic principles of cell-cell signaling. Know all the forms of
signaling and their uses in organisms (when organisms uses contact-
dependent versus endocrine signaling, etc).
o Ligand, or signal molecule, gets to target cell, where it binds with a
receptor. If no receptors are around, no signal is transmitted.
o Notice that a single ligand can bind to multiple receptors for different
effects, and multiple ligands can bind to a single receptor. Allows for
greater variety of responses to stimuli.
o Forms of signaling:
• Contact-dependent signaling: signaling molecule never leaves
its cell of origin; therefore the signaling cell and the receptor
cell need to come into physical proximity to work. An example
would be immune response.
• Contact-independent signaling: signaling molecule leaves its
cell of origin.
 Autocrine signaling: one cell type is signaling itself
(release ligand, bind the same type of cell)-- example is
growth factor.
 Paracrine signaling: the target cell is very nearby.
Ligand relies on passive diffusion to get to receptor cell.
 Endocrine signaling: target cell is distant. Ligands are
called hormones; travel through bloodstream until it
finds receptor cell. More on this under "Epithelia."
 Neuronal signaling: long-range, rapid signaling; can't
use endocrines (too slow)-- send APs down axons to
synapses.
• Know all the types of extracellular signaling molecules and how they relate to
the forms of signaling.
o Water-soluble ligands:
• Usually peptides.
• Can't cross plasma membrane without transport
• This means it needs a plasma membrane transporter both from
its cell of origin and at its cell of reception; usually bind, at their
site of action, to plasma-membrane-bound receptors (see
below).
• Generally faster action.
• Example: neurotransmitters (neuronal signaling).
 o Water-insoluble (lipid soluble) ligands:
• Can cross membrane easily, but usually needs to bind to a
carrier protein of some kind to be taken through cytosol.
• Usually bind to intracellular receptors (also called nuclear
receptors) at their site of action (see below).
• Generally slower action.
• Example: steroid hormones (endocrine signaling).
o Gaseous ligands:
• Can cross water or lipid boundaries; easily diffuses, no need for
transporters.
• Very short-range (paracrine signaling).
• Example: nitrous oxide (regulates blood pressure, etc)
• Understand the different types of receptor molecules and know the types of
cellular processes that they regulate. Understand the concept and functions of
intracellular signaling molecules.
o (1) Plasma-membrane receptors: respond to water-soluble signaling.
Generally work by activating signal transduction inside the cell. 3 main
types:
• Ion channel receptors:
 Gated channels-- usually closed until ligand binds, at
which point gates open and particular ions travel
through.
 Ion channel receptors largely regulate channels for
Ca2+.
• Notice that one main type of Ca2+ channel takes
up calcium into the endoplasmic reticulum (see
"Signaling: Calcium" for details).
• G-protein coupled receptors:
 Cytosolic side binds to ligand; intracellular side binds to
a G protein complex (heterotrimeric G proteins, which
bind GTP; more on this under "Signaling: Receptors").
 This protein complex is normally bound to GDP at its
alpha subunit; when the ligand binds, the alpha complex
binds GTP instead of GDP and dissociates from beta and
gamma subunits.
• Both the alpha and beta-gamma subunits can
serve as signaling agents inside the cell.
 Notice this allows a variety of responses (different
trimeric proteins) to one ligand in a variety of tissues.
• Receptor kinases:
 Often growth factor and insulin receptors.
 Cytosolic side binds to ligand; intracellular side has a
kinase domain on it (tyrosine kinase)-- it will PO4 other
proteins with particular tyrosine residues.
• Most receptor kinases act as dimers: once they're
both bound to ligand, they will phosphorylate
each other, turning on their signaling: once that
PO4 is added, it creates a binding site for a set of
adaptor proteins, which bind to the receptor
kinase dimers and set off a signaling cascade.
• Lots of variety in receptor action here as well:
can PO4 other proteins, can have a lot of
 different potential adaptor proteins to bind to the
binding site, etc.
o (2) Intracellular/nuclear receptors: respond to lipid-soluble signaling.
• Generally work by activating transcription of specific genes. It
does this largely by activating proteins which activate
promoters upstream of transcription start sites.
• These proteins are also referred to as transcription factors.
• Steroids: bind to receptor proteins, inducing a conformational
change which allows the receptor protein to release its inhibitor
protein and bind its activator protein (entire complex: receptor-
ligand-activating protein); this complex can interact with
promoter regions to activate transcription.
• Understand the concept of signal integration (one signal-many functions and
many signals-one function).
o Example: calcium ligand can bind to a variety of kinase receptors
depending on the type of cell.
o Just remember that no signaling agent or pathway is ever, ever simple
- a healthy signal paranoia - and you'll be fine.

Cell Signaling Overview II

Tuesday, November 27, 2007
9:56 AM

Cell Signaling Overview II, 11/27/07:

• Describe principle types of detectors of extracellular signaling molecules.

o Ligand or voltage-gated ion channels (water-soluble ligands)
o G-coupled protein receptors (water-soluble ligands)
o Enzyme-linked receptors (ie receptor kinases) (water-soluble ligands)
o Nuclear receptors (lipid-soluble ligands)
• List other "tools" of signaling pathways, including at least three 2nd
messengers.
o Second messengers: small molecules released within the cell in
response to the binding of the first ligand; can bind to other
intracellular signaling molecules.
• Calcium: first ligand opens ion channel to admit Ca2+; calcium
then binds a variety of other things inside the cell.
• cAMP (cyclic adenosine monophosphate): generated by
adenylate cyclase
• IP3 (inositol triphosphate)
• DAG (diacylglycerol): generated by phospholipase C (PLC)
• NO (nitric oxide): generated by nitric oxide synthase (NOS)
• (notice that, for example, calcium can trigger the production of
NOS, which triggers NO, which triggers.. etc, etc. Thus can
have second messengers activating third messengers activating
fourth messengers, and so on, though the third and fourth
messengers can also act as second messengers. The cell has no
idea that it's screwing up all the cool categories we invent for it.
Again: develop a healthy distrust of simple signaling pathways.)
o Other signaling steps:
 • Protein modification (PO4ation, ubiquitinylation, etc)
• Protein-protein binding/targeting
• GTP/GDP exchange (G-proteins coupled to receptors, small
GTPases like ras)
 Note that GTP does not seem to act as a second
messenger: I think it's the fact that GTP changes the
configuration of proteins which then act on other
proteins that makes the difference. That is, GTP itself is
acting as more of a helper than an agent in the
signaling.
 G-proteins: see "Signaling: Receptors."
 Ras proteins: see "Receptor Tyrosine Kinases."
• Describe at least three mechanisms for signal termination (including
phosphodiesterases).
o Uptake, breakdown, or diffusion of original signaling molecules
o Desensitization of receptor
o Termination initiated by another signal (phosphorylation,
dephosphorylation, etc)
o Termination-dedicated enzymes: ie phosphodiesterases (PDEs) which
break down the second messengers cAMP/cGMP.
o Some kind of feedback inhibition: ie, the bound ligand or receptor acts
in such a way as to undermine its ability to stay bound or stay active
(G-proteins gradually lose their GTP-bound active state).
• Evaluate a "pathway" for amplification and termination.
o Amplification depends on signaling cascades: one signaling molecule
can create a large number of signals, which in turn can create an ever
larger number of second signals, etc.
o Positive feedback mechanism (ie. calcium-induced calcium release):
very quick but very dangerous signaling pathway (can get out of
control rapidly). Usually coupled with a negative feedback mechanism
past a certain threshold to keep it under control.
• Identify "nodes" (such as calcium) and "modules" in a signaling pathway, and
evaluate the potential for crosstalk in signal transduction.
o Nodes: points in a network that receive multiple inputs and/or multiple
outputs. Calcium is evidently the most extensive node in signaling.
o Modules: groups of components that function together, often
physically assembled to form complexes. Ie: G-protein heterotrimeric
complex would presumably form a signaling module, as would
negative feedback mechanisms.

Mitochondria and Peroxisomes

Wednesday, November 28, 2007
8:00 AM

Mitochondria and Peroxisomes, 11/28/07:

• Know the origin and basic structure of mitochondria.

o The current theory is that they showed up by way of endocytosis of
oxidative-phosphorylating bacteria by our distant ancestor(s).
o Structure: has a double membrane (only other organelle with a 2x
membrane is the nucleus)-
• Outer membrane: semi-permeable, regular ol' membrane.
 These have TOMs (Translocases of Outer Membrane:
large, non-gated channels allowing protons, etc, to
equilibrate with the cytosol).
• Inner membrane: less permeable, forms the folds or cristae
inside the mitochondrion, contains the cellular machinery
responsible for ox-phos.
 These have TIMs (Translocases of Inner Membrane:
specific, receptor-based protein channels allowing
various required proteins selectively in).
 Four protein complexes in the cristae: their purpose is to
pass electrons from NADPH to various electron acceptors
(ending in oxygen to form H2O), generating energy with
each transfer.
• As I understand it, the reason for all these
transfers is to minimize the amount of energy
wasted through each step- if you just took
electrons/protons off NADPH and stuck them on
oxygen, there would be a large energy release,
most of which would be lost to heat. By breaking
it down into a bunch of smaller steps, it's able to
keep more of the total energy as ATP. Not
necessary, and possibly not even correct, but
interesting.
 Space inside the inner membrane = matrix. Contains the
mitochondrion's own DNA (recall that the vast majority
of proteins that actually function in the mitochondrion
are derived from the nucleus, though).
 As these electrons are transferred, the protons (from
NADPH -> NADP+) are pumped out of the matrix, at
which point they can leak out of the outer membrane.
• This creates a chemical gradient on H+ with less
concentration inside the matrix than outside it.
• Notice this also creates an electrical gradient with
more negative charge inside the matrix than
outside it.
• Essentially this is a killer drug target: if you
disrupt one of these gradients, you can kill cells
quickly. The reason for this:
• The H+ gradient, chemical and electrical,
is used by channel proteins (F0) to allow
protons to rush through the inner
membrane into the matrix and be used by
another protein (F1 or ATP synthase) to
generate ATP.
• There's an ATP-ADP exchanger in the
inner membrane, run by concentration
gradients of both those compounds, that
keeps the concentration of ADP in the
matrix high.
 • Notice that an alternative method uses creatine
kinases. Effectively you keep a phosphate group
on creatine (usually in muscles); when required,
you can quickly transfer off the PO4 onto ADP to
make more ATP in conditions of high energy
consumption.
o Notice that mitochondria also function in targeted apoptosis (as wh.en
differentiating your fingers from each other or destroying DNA-
mutated cells). When "death receptors" are triggered on the cell's
surface (or irreparable DNA damage is detected), the mitochondria
release cytochrome C, which releases caspases (real nasty proteases
to destroy cell's protein complement).
• Understand the basic principles of electron transport and ATP production in
mitochondria.
o See above.
• Know the basic functions of peroxisomes.
o Small, membrane-bound organelles.
o Specialized form of lysosome: involved in degradation of fatty acids
and amino acids.
o Peroxisomes generate, and thus also have to deactivate, a lot of H2O2
(hydrogen peroxide). They deactivate it with an enzyme called
catalase.

Signaling: Receptors
Wednesday, November 28, 2007
8:47 AM

Signaling: Receptors, 11/28/07:

• Draw the membrane topology of a G protein-coupled receptor and identify the

basic structural characteristics that mediate ligand binding and coupling to G
proteins.
o Well, I'm not going to draw the topology here, but basically you've got
a barrel-shaped receptor protein with a ligand binding domain on the
outside of the membrane, then a heterotrimeric G protein complex
binding domain on the inside of the membrane, bound to the
heterotrimer plus GDP. The G protein complex is made up of three
subunits, alpha, beta, and gamma. The beta-gamma subunits are
pretty much always stuck together, and in their GDP-bound state
they're also stuck to the alpha subunit.
• [Basic note: "Agonist": ligand that activates receptor. "Antagonist": ligand
that inactivates receptor.]
• Explain how G protein-coupled receptors activate hetero-trimeric G proteins
and diagram the GTP-hydrolysis cycle of G protein signaling.
o When the ligand binds, a conformational change drives the GDP on the
alpha-beta-gamma complex to be exchanged for GTP, and the
complex becomes "activated".
o When activated, the alpha subunit (bound to the GTP) and beta-
gamma complex break apart, separate from the membrane receptor,
 and go their separate ways within the cell to serve as signaling
molecules ("transducers") to other targets ("effectors").
• Notice that you have a lot of different possible responses to
this- this is just a basic signaling mechanism and can be used
for just about any conceivable purpose.
o The system is "reset" to get back to GDP-bound trimers by the
hydrolysis of GTP on the alpha subunit to GDP; when the GTP becomes
GDP, the alpha subunit returns back to the membrane receptor and
binds up the beta-gamma complex to reform the inactive state.
Obviously this can be helped by particular enzymes that trim
phosphates off the GTP on the alpha complex (ie GTPases), but it will
also happen spontaneously, providing a built-in time limit for G-protein
signaling.
o Some uses of G protein-coupled receptors:
• Adrenergic receptors: bind norepinephrine. Muscarinic
cholinergic receptors: bind acetylcholine.
• Notice that what kind of ligand a receptor binds to has
comparatively little impact on what its eventual effect pathway
in the cell is. However, what the receptor protein is bound to on
the inside of the membrane does have a lot of impact. The
reason for this is that the receptor's G-protein binding domain
on the inside of the membrane can interact with different G-
protein complexes depending on what tissues the receptors are
in.
 That's not to say that the ligand and the eventual effect
are unrelated. Generally norepinephrine receptors are
involved in sympathetic reactions and muscarinic
receptors are involved in parasympathetic reactions. But
notice that sympathetic and parasympathetic reactions
can have very different effects in different tissues (see
below).
• G proteins: Gs stimulates adenylyl cyclase to form cAMP; Gi
inhibits adenylyl cyclase to stop the formation of cAMP; Gq is
involved in other methods of calcium transmission.
• For example, beta-adrenergic receptors in the heart (triggered
by norepinephrine) are bound to Gs proteins, which stimulates
the formation of cAMP, which eventually leads to calcium influx,
which increases heart rate contraction.
 Beta-blocker drugs: block this effect; decrease heart
rate.
• However, alpha-adrenergic receptors in the periphery (still
triggered by norepinephrine) are bound to Gq proteins, which
binds phospholipase C to activate a kinase pathway that ends in
calcium influx in smooth muscle, causing vasoconstriction,
increasing blood pressure and shunting blood away from the
periphery towards the body core.
• Muscarinic cholinergic receptors in the heart (triggered by
acetylcholine) are bound to Gi proteins, which inhibit cAMP
formation, causing open Ca2+ channels in heart muscle to close
(stopping influx) and slowing the heart rate.
 • Other muscarinic cholinergic receptors in the heart are also
bound to Gi proteins, but these open potassium channels to
regulate the membrane's potential (and thus excitability).
• As far as Gi is concerned: the reason the level of cAMP goes
down when its synthesizing enzyme is turned off is that cAMP is
continually degraded to AMP by phosphodiesterases (PDEs).
 Notice that certain drugs inhibit PDEs (like caffeine)--
this blocks the degradation of cAMP, which keeps cAMP
activity (stimulation) high.
 Viagra and Cialis target PDEs which degrade cGMP, for
similar effects in smooth muscle.
• Notice that beta-adrenergic receptors in the lung (not heart)
also act through Gs pathways, but the increased cAMP causes
the smooth muscle in the lung to relax (bronchodilation) rather
than contract.
• Notice that muscarinic receptors in the lung act through Gq
pathways, causing influx of Ca2+ and causing
bronchoconstriction.
• Describe the function of second messengers in receptor signaling and give
two examples for how they are generated by activated G proteins.
o See above, Gs and Gi. cAMP is a second messenger.
• Explain how receptor activation leads to signal termination through receptor
desensitization.
o Desensitization: response to overexposure of receptor to ligand.
o How this works: in the active state, the beta-gamma complex recruits
kinases to phosphorylate the receptor protein such that an "arrestin"
protein binds to the receptor, not allowing the receptor to bind more
inactive G proteins to activate them.
o In addition, once arrestin binds to the receptor, the receptor is
endocytosed, inactivating it until either the phosphorylation is removed
to restore the receptor (resensitization) or the receptor is degraded by
lysosomes. With long-term exposure, the receptors are more often
degraded than resensitized.
o This means that after an activation, the receptor effectively is taken
out of the loop and can't activate any more G-protein signals; with a
lot of activation over time, the number of receptors in the membrane
goes down as they are degraded.
o Opiates, for example, over time will reduce the number of receptors
available to produce their analgesic effect.
• Give two examples of drugs that act through modulating different steps in a
receptor-G protein-second messenger signaling cascade.
o See above re caffeine and Viagra, also below re cholera and whooping
cough.
• [Other notes: Pertussis toxin (whooping cough) modifies the Gi protein to
prevent the inactive G protein to be able to be activated-- thus inhibits Gi
proteins.
o Whooping cough: no activation of Gi proteins.
• By contrast, cholera toxin modifies the Gs protein to prevent the active G
protein from being able to be deactivated-- thus perpetually keeps Gs
proteins activated.
o Cholera: no inactivation of Gs proteins.
Signaling: Calcium
Wednesday, November 28, 2007
9:48 AM

Signaling: Calcium, 11/28/07:

• Understand the functions of cyoplasmic Ca2+ ion buffers and how these
buffers affect cytoplasmic Ca2+ signals:
o These bind free Ca2+, convert to inactive form. They usually involve
free carboxyl groups on negatively charged amino acids.
o They protect against enormous influxes of calcium and also make sure
local influxes of calcium are restricted from spreading around. Their
other function is to make sure calcium doesn't remain active in the cell
for long (inactivated and extruded again)-- limits Ca2+ both spatially
and temporally.
o Buffers are also present in endo/sarcoplasmic reticular lumen: allows
greater storage of (temporarily inactivated) Ca2+ in those organelles.
• Understand the routes by which extracellular Ca2+ enters the cytoplasm.
Understand the routes by which Ca2+ moves out of the ER/SR into the
cytoplasm. Understand the routes by which Ca2+ is extruded from the
cytoplasm (a) into the extracellular space and (b) into the lumen of the
ER/SR:
o Note that calcium doesn't need to be synthesized-- it's already present
at high concentrations extracellularly. This means the signaling
pathways for calcium are extremely rapid (just need to let it in).
o Other sources/sinks of calcium (sources: from whence calcium comes.
Sinks: to where it shall return.): nuclear envelope, mitochondria,
endo/sarcoplasmic reticulum.
o Most cells: a voltage differential of about -60 mV across the cell
membrane. This serves as another driving force to get Ca2+ into the
cell.
o Routes to enter cell cytoplasm:
• Passive via ion channels:
 Voltage and ligand-gated Ca2+ channels in the plasma
membrane to uptake Ca2+ into cell. Also "store-
operated" channels (dependent on existing store of
Ca2+).
 Endo/sarcoplasmic reticulum and nuclear envelope: IP3
receptors and ryonodine receptors moves calcium from
E/SR/nuclear membrane to cytoplasm.
 Mitochondrial pores ('uniporters')- dependent on Ca2+
concentration.
o Routes to get out of cell cytoplasm:
• Active transporters against gradients (slower movement of
Ca2+):
 Ca2+ pumps use ATP to move calcium into extracellular
space or into lumen of E/SR.
 Can also use energy from sodium leak into cell to pump
calcium out of the cell.
 • Learn what EF hands and C2 domains are. Learn the identity of the
archetypical protein that contains EF hands. Learn the identity of the
archetypical protein that contains a C2 domain. Learn whether these domains
are present in other proteins.
o See below for greater detail. C2 domains, when bound to calcium, bind
to cell membranes; they're contained in protein kinase C and
synaptotagmin proteins. EF hand domains are found in calmodulin
proteins and cause them to effect calcium-mediated modulation of
other proteins. These are, in fact, present in other proteins
(parvalbumin and troponin C for EF domains, for example).
• [Some functions (effectors) of Ca2+ signaling:]
o Ion movement: changes voltage across membrane (depolarization of
neurons and cardiac muscles)
o Protein Kinase C (PKC) activation:
• Has a C2 domain: when bound to calcium, this domain tends to
fuse with membrane and have activate phosphorylation effects.
(not much detail here)
o Synaptotagmin activation:
• Also has a C2 domain, which causes vesicles within cell to fuse
with membrane and release cargo (like neurotransmitters).
o Calmodulin activation:
• Strongly conserved genes in plants, animals.
• Contain four EF-hand domains that bind calcium.
• When activated by calcium, sticks to and modulates ion
channels, protein kinases, phosphatases, and
phosphodiesterases.
• [Notice that maintained Ca2+ influxes in the cell can activate negative
feedback activation that stops the Ca2+ influx: RyR protein channel in ER to
allow Ca2+ efflux into the cytoplasm]

Stem cells and cellular diversity

Thursday, November 29, 2007
8:01 AM

Stem cells and cellular diversity, 11/29/07:

[general notes; learning objectives are kind of FUBARed.]

• Differentiation is normally irreversible but exceptions exist, primarily

fibroblasts in connective tissue.
• Differentiation is driven by different combinations of molecular cues.
o Notice that the same signal combination will have different outcomes
in different cell type lineages.
o Lineage: pedigree of a differentiated cell. Describes how related cells
are, predicts similarities at level of gene expression, and predicts how
easily external cues or mutations may turn one cell type into another.
 • Hematopoeitic lineage (blood cell lineage): a bunch of different
blood- and lymph-related cells, all derived from originally
quiescent stem cells.
o Potency/developmental potential:
• Potency (the number of different fates a stem cell can have) is
restricted by commitments during differentiation- every time
one branch of the 'family tree' is chosen, other branches close
off as options.
 Totipotent: have unlimited potential, can give rise to any
cell type. Zygote is the only human example of this.
 Pluripotent: can give rise to many, but not all, cell
types. Embryonic stem cells are an example: can
become just about anything aside from trophoblasts,
which aren't found in the adult human body anyway.
 Multipotent: can give rise to a small number of cell types
(hematopoietic stem cells, etc). Most adult stem cells
are examples.
o Commitment:
• An irreversible lineage choice has been made and other fates
are no longer permitted.
• Notice that commitment precedes overt differentiation; that is,
a stem cell is committed to become a certain cell before it
actually becomes that cell.
• This happens largely because the genes that trigger
development along one path inhibit the genes that trigger
development along other paths.
• Underlying mechanisms of stem cell development:
o (1) Lineage-specific transcription factors (reversible, but not often)
• "Master regulators"; activate genes necessary for differentiated
phenotype, repress genes necessary for alternate lineages, and
maintain their own transcription.
o (2) Epigenetic chromatin modifications (stably transmitted to daughter
cells, very difficult to reverse)
• DNA methylation: CpG dinucleotides near a promoter are
methylated to mark that gene for expression or silencing
• Histone tail modifications: methylation, acetylation,
phosphorylation; create docking sites for chromatin
modification protein complexes. This does two things:
 Changes packing of chromatin (opens or closes
chromatin loop) to make transcription either more or
less accessible.
 "Template-dependent histone modifications": recognize
histone modifications, duplicate them on all new
histones. Maintains fidelity of histone modification on all
histones in cell.
o (3) Regulatory microRNAs:
• Recently discovered; not too much known, seems to be
(untestably) important

• Tissue renewal and stem cells:

 o Adult stem cells are in action all the time: renewing tissues in the
intestines, creating sweat gland/hair follicles/skin/sebaceous glands in
dermis, etc.
o Properties of adult stem cells:
• Can repopulate tissue and self-renew.
• Generally rare in the body: not a whole helluva lot of them.
• Give rise to diverse progeny (multipotent), but not very widely
diverse (not pluripotent).
• Self-renewal: generally, during mitosis, one daughter cell will
differentiate and one will continue on as a stem cell. However,
it can also make two new stem cells or two differentiated cells.
 Notice that long-term depletion of stem cells contributes
to aging.
• Tend to reside in specialized niches in the body, in contact with
regulatory cells which keep them quiescent, self-renewing, and
undifferentiated. Without the contact with regulatory cells, they
will differentiate and proliferate.

• Stem cells and disease:

o Some ideas that leukemia is a stem cell disease that results from the
displacement of functional stem cells by abnormal, leukemic stem
cells.
o Aging: aging stem cells are more easily mobilized from their regulatory
cells (thus no longer as self-renewing).
o Difference between adult and embryonic stem cells:
• Embryonic stem cells are pluripotent: can become any variety
of tissue type in the body, unlike adult stem cells. They are also
rapid proliferators.

Serine and threonine kinases

Thursday, November 29, 2007
9:03 AM

Serine and Threonine Kinases, 11/29/07:

• [Once again: kinases are proteins that phosphorylate other proteins. Notice
that this doesn't say anything one way or the other about what effect this will
have on the activity of the PO4'd protein.]
• Describe a phosphorylation reaction (including which amino acids can be
phosphorylated) and explain how it can affect a phosphorylated protein.
o Possible phosphorylation targets: serine, threonine, tyrosine.
• Notice that Ser and Thr have very similar structures to each
other, but different from Tyr, which is why there's generally two
types of kinases (Ser-Thr vs Tyr).
o This is because they have hydroxyl residues that can be replaced with
a phosphate group (OH makes nucleophilic attack on gamma
phosphate of ATP)
 o The PO4 is generally donated from ATP; the donation is catalyzed by
the kinase.
o PO4'd proteins can be thus activated, inactivated, made to sing the
national anthem, whatever.
• List at least two other types of secondary protein modification.
o If I understand the point correctly, this seems to just be asking (once
again) to run through the various types of protein modifications that
exist: ubiquitinylation, acetylation, glycosylation, etc.
• Explain the structure of an ATP molecule.
o Adenine (double loop), ribose, triphosphate. At the risk of stealing
Jeff's thunder:

• Explain how protein kinases can be classified and describe examples.

o Based on which residue they phosphorylate (Ser-Thr-Asp)
o Based on their substrate protein
o Based on their activating stimulus
o Based on their phylogenic relationship (certain families of related
kinases)
• Describe the structure/function of a protein kinase and principles of their
regulation (including: alternation between "open" and "close" conformation in
all kinases, and requirement for activation loop phosphorylation in some
kinases).
o Protein kinases have two lobes: one small, one large.
o ATP binds in cleft between lobes. Substrate mainly binds to large lobe.
o "Activation loop": region of many kinases that needs to be
phosphorylated before the kinase itself is activated. Notice that some
kinases can phosphorylate themselves.
o "Glycine-rich loop": region that presses down on ATP to correctly
position the gamma phosphate to bind to substrate. Has two
conformational states:
• "Closed" state = phosphate transfer state. Transfer happens
very quickly.
• "Open" state = ADP-for-ATP exchange state. Happens more
slowly.
o Active conformations of most kinases are very similar (nonspecific)-
problem for specific drug interactions. However, the inactive
conformations are less similar- better specificity as drug targets.
• Generally in the inactive state (not the same as the "closed"
state of the glycine-rich loop), the activation loop or the ATP
binding cleft is characteristically distorted- these can be
targeted.
• Notice also that the substrate binding domain can also be used
to bind antagonists to inactive kinase proteins.
• Some examples:
 o CAMK II and calcineurin: promote long-term potentiation or
depression, respectively, of synaptic strength (memory/learning).
o Notice that rapamycin blocks mTOR (ser/thr kinase) to block T cell
proliferation (thus is an immunosuppressant).
o Cyclosporin (first choice of immunosuppressant in transplants) blocks
calcineurin, thus blocking the expression of IL-2 (inflammation
cytokine).

Extracellular Matrix and Cell Adhesion

Thursday, November 29, 2007
9:42 AM

Extracellular Matrix and Cell Adhesion, 11/29/07:

• Summary of the general idea here: You've got proteoglycans. They attract
water and form a extracellular gel in which fibrous proteins and multidomain
proteins are embedded. The fibrous proteins provide a scaffold for cells as
well as imparting fibrous/elastic properties to tissue. The multidomain
proteins attach to both fibrous proteins and to CAMs (cell adhesion
molecules) on the surfaces of cells. The CAMs are transmembrane proteins
which attach extracellularly to other CAMs or multidomain proteins and
intracellularly to signaling molecules inside the cell (information can flow both
ways).
o Types of fibrous proteins: collagen, elastin
o Types of multidomain proteins: fibronectin (found all over), laminin
(found in basal lamina)
o Types of CAMs: cadherin (attaches to other cadherins, form calcium-
activated 'zippers,' homodimeric), immunoglobulin (attaches to other
Igs, form non-calcium-activated 'zippers', monomeric), integrin
(attaches to ECM proteins, has intracellular signaling end).

• Discuss the contributions of the ECM to cell and tissue function:

o Essentially, need an substance to which cells can attach, both for
motile purposes and also because non-carcinogenic cells can't grow
without something to attach to.
• Carcinogenic cells can grow without an attaching surface (ie in
agar) due to mutations in adhesion signaling pathways.
o Dr. Pfenninger seems to really be harping on this idea that adhesion is
not a simple, glue-like process: cells 'stick' to very specific types of
materials, and different cells stick to different things. He describes it
as a combination of mechanical adhesion and cellular communication--
that is, the cell 'knows' what it's attaching to and can respond
appropriately.
o Also use the ECM to form functionally distinct compartments in the cell
and to allow cells to act in concert with each other.
• Define the four major classes of ECM components and their properties:
o (1) Glycosaminoglycans (GAGs), often linked to proteins to form
proteoglycans:
• GAGs: large, unbranched disaccharide polymers. Typically
contain an amino sugar and a sugar that's substituted with a
 carboxy group (acidic sugar), sometimes with a sulfhydryl
group. What this means: GAGs are negatively charged.
• Proteoglycans are just GAGs linked to a "core protein".
• Note that these proteoglycans or GAGs can aggregate together:
due to their negative charge and solubility, they form a kind of
gel as space-filling molecules in the cell.
o (2) Fibrous proteins (collagen/elastin):
• Collagen is the most abundant (25% of total protein weight in
H. sapiens); tough polymers that provide tensile strength in
connective tissues.
• Elastin, as the name implies, is elastic protein found in a
variety of tissues.
 Recall that alpha-1-antitrypsin deficiency involves an
inability to control the breakdown of this protein by
elastases.
o (3) Multidomain adaptor proteins (fibronectin and laminin):
• Fibronectin: dimeric glycoprotein (proteins with saccharides
attached to their backbones) linked by disulfide bonds. Can
bind a variety of proteins (collagen, heparin, each other, etc).
 More importantly, can also bind integrins (principal
adhesion molecule on cell surfaces).
 Found all over the body.
• Laminin: found predominantly in basal lamina (ECM that
provides the "ground" for epithelial cells). Heterotrimeric form
in the shape of a cross (proof of intelligent design!). Cross-links
with itself pretty good.
o (4) Water and solutes:
• Predominantly found in the "gel" between cells: allows easy
diffusion of nutrients and growth factors between cells.
o Notice that the ECM, like anything else, needs to be broken down and
remade frequently. This job falls to extracellular proteases:
• (1) matrix metalloproteases (MMPs)
• (2) serine proteases
• Notice these can be used in a targeted manner to allow
development of tissues or cell migration or invasion of nearby
regions.
• Define two types of fibrillar proteins and at least two types of multidomain
adapter proteins of the ECM:
o See above (collagen and elastin, fibronectin and laminin).
• Discuss the role of adhesion in cell function and survival in metazoans
o As said: need to adhere to each other and not all fall down in a heap;
need something to adhere to in order to survive; need to form distinct
compartments in cell.
• Define and describe at least three different types of cell adhesion molecules
(CAMs) and their ligands:
o Cadherins:
• Single-pass transmembrane glycoproteins.
• Found as homodimers.
• Activated by calcium: when activated, cadherins on one surface
"zip up" with cadherins on the other surface (homophilic
binding reaction)
o Ig (immunoglobulins):
 • Single-pass transmembrane glycoproteins as well.
• Unlike cadherins, operate as monomers, and binding doesn't
require Ca2+.
• However, the Ig domains on these proteins do bind to Ig
domains of Ig proteins on other surfaces.
o Integrins:
• Heterodimers (alpha and beta subunits): lots of different types
of integrin, both alpha and beta, so can have lots of different
combinations for binding to different things.
• Has a ECM-binding domain at its end: can bind to laminin,
fibronectin, collagen, etc.
• Notice that the intracellular "tails" of integrins attach to various
things inside the cell that can respond to the attachment:
 Can be attached to catenins (tumor suppressor),
cytoskeleton, etc.
• Discuss the role of CAMs in signaling:
o CAM extracellular portions can interact with signal proteins to tell the
cell what it's attached to. (outside-in signaling)
o This can go the other way: the cell can interact with the CAM to
change or get rid of its extracellular adhesion. (inside-out signaling)
• Describe proteins associated intracellularly with CAMs:
o Cytoskeletal proteins to anchor the CAMs in the cell membrane
(mechanical linkage): often actin-binding proteins.
o Signaling proteins: often protein kinases or GTPases.
• mentioned in lecture: (1) tyrosine kinases, (2) src, (3) protein
kinase C, (4) Rho GTPases.
• Discuss the ECM and cell adhesion in the context of disease processes
o Cell adhesion molecules, since they often control apoptosis and
proliferation, are frequent targets for cancers and viral oncogenes (ie
v-src).
o Notice that cell adhesion is also intimately involved with immune
response (mainly integrins): thus problems with CAMs can lead to
compromised immune function.
• "selectin": CAMs that "catch" leukocytes coming out of the
bloodstream. Mutations in this protein can cause obvious
problems.

Receptor tyrosine kinases

Thursday, November 29, 2007
10:59 AM

Receptor tyrosine kinases, 11/29/07:

• [Functions: cell growth, motility, metabolism, cell survival, differentiation]

• Describe the mechanism of receptor tyrosine kinase (RTK) activation.
o As mentioned by Dr. Prekeris, RTKs are activated by dimerization;
when two nearby RTKs in the membrane are bound by their ligands,
they undergo a conformational change, bind to each other, and co-
activate by phosphorylating each other.
 • Ie: for these RTKs, their substrates (PO4 targets) are other
RTKs.
• Once activated, they can also act as catalysts for other
reactions.
o Notice that this doesn't have to be homodimerization: two different
kinds of RTKs can dimerize to generate a different signal than a dimer
formed of two of either kind.
o RTKs generally trigger complex kinase cascades that result in changes
in gene expression.
• Explain the molecular mechanism of stimulation of ras GTPase by RTKs.
o All RTKs activate ras signaling.
o Ras proteins: membrane-bound switches regulated by GAPs and GEFs
(see below). Involved with cell proliferation.
• In the "off" state, it's bound to GDP; in the active state, it's
bound to GTP.
• In GTP-bound (active) form, Ras proteins sets off proliferative
cascade.
• Generally, Ras proteins can switch themselves off (intrinsic
GTP->GDP activity)
• GAP: GTP activating protein (increases its ability to switch itself
off). Inactivating mutations in GAPs can cause cancer since ras
proliferation is unchecked.
 GAPs: turn Ras proteins off (tumor suppressor genes).
• GEF: Guanine nucleotide exchange factors: helps exchange
"spent" GDP for fresh GTP (thus activating Ras proteins).
Increase-of-function mutations in GEFs can lead to cancer since
Ras protein is constantly being activated again after it
inactivates itself.
 GEFs: turn Ras proteins on (oncogenes).
o Mechanism:
• Dimerized RTKs bind to and activate Grb2, an adaptor protein;
this has a SH3 binding domain which binds to Sos, which is a
GEF (Ras activator).
• Sos being bound, it's brought into proximity with Ras proteins,
which are located in the cell membrane like the RTKs.
• Essentially RTKs activate Ras proteins by changing the location
of GEFs, not by catalytic activity. (Movement of proteins into
proximity causes proliferation).
• [Epidermal growth factor receptor (EGFR): overexpressed in a variety of
tumors; good drug target for cancer treatment.]
• Describe mechanism of action of two main classes of RTK-targeted anti-
cancer agents (antibodies and TKI's).
o Antibodies: Recognize extracellular domain of RTKs.
• Effectively act as competitive antagonists: prevent ligand from
being able to bind to the extracellular receptor domain.
• Nicely specific to particular RTKs.
o TKIs (tyrosine kinase inhibitors): inhibit tyrosine kinase activity of
RTKs.
• This works by competitively binding to the ATP binding pockets
of specific RTKs. No ATP, no phosphorylation, no activation of
RTK dimers.
 • Recall that the "closed" or un-ATP-bound TK has a specific
conformation that allows more specific drug targeting--
however, there's still enough similarity that TKIs will inevitably
have multiple TK targets.
• List tumor cell characteristics that predict clinical response to EGFR-targeted
therapeutics.
o Patients with non-small-cell lung cancer show only a 20% response to
TKIs.
• The 20%: usually non-smokers, women, Asian descent.
• When you've had a mutation in EGFR to make the activation
domain much more active, the response to TKIs tends to get
better.
 Why this is: it's thought that with an improved or more
efficient EGFR proliferation pathway, the cell may be
"addicted" to that pathway-- which would make the cell
much more vulnerable to TKIs, which target that
pathway.
• Notice this also applies to patients with abnormally high levels
of EGFR in their tumor cells: again, may be "addicted" to this
pathway.
• Describe mechanism of resistance to TKI's caused by EGFR somatic
mutations.
o There are secondary (after first treatment) mutations in EGFR that
cause decreased response after an initial improvement when treated
with the TKI.
o Essentially, seem to be selecting for EGFR proteins that have
mutations that block TKI binding.
o Note that there are drugs that can inhibit these mutant EGFR
proteins-- since we know what the mutations are, we can use the
sequence to target inhibitory drugs.

• Important note: Dr. Thorburn kindly filled out his own LO's in his handouts;
might check them out for a slimmer version of all this verbiage.

Epithelia I, II, + III

Friday, November 30, 2007
7:55 AM

Epithelia I, II, +III, 11/30/07:

• State the various structural arrangements, classifications, and functions of

epithelial tissues, and state their general structural relationships (orientation)
to connective tissue, blood vessels, muscle, and neurons (peripheral nervous
tissue).
o Make up "business ends" of many organs.
o Most cancers derived from epithelia.
o Epithelia: tissues that line all surfaces of body, internal (gut, glands,
tubes, ducts, etc) and external (skin). Note this includes a variety of
internal organ linings.
 o Functions: (1) barrier to microorganisms and toxins; (2) selective
transport into and out of body; (3) biochemical modification of
molecules and metabolites (detoxification in liver); (4) specialized
reception of stimuli (ie taste receptors); (5) self-renewal.
o Properties:
• (1) epithelial cells are highly adherent to each other and form
'sheets,' often wrapped to form tubes;
• (2) most epithelial cells have polarity: the surface that faces
the lumen is called the apical surface, and the surface that
faces opposite the lumen (towards the connective tissue) is
called the basal surface.
 Note that polarity allows unidirectional transport (in
either direction) through epithelial cells for specific
substances.
• (3) Structure found in all epithelia, underlying the basal
surface: the basal lamina layer.
• (4) All epithelia are attached on the basal side to connective
tissue beneath the basal lamina.
 Inside the connective tissue is vasculature. This is
important because epithelial cells have no inbuilt
vascular supply. Blood needs to diffuse through the
connective tissue to reach the epithelia. This will be
important when we get to epithelial self-renewal.
• Notice that the cells lining the vascular system
are not called epithelia; they're called endothelia.
Just a side note.
 Also within the connective tissue: muscles and nerves.
o During development, primitive epithelial cells become mesenchymal
cells and migrate through the body to form new regions of epithelia
(epithelial-to-mesenchymal transition).
• The reason this is significant is that this is also what some
tumors do-- reactivate the mesenchymal transition, migrate
through the body, and metastasize their little evil hearts out.
o Types of epithelial cells:
• Single sheet of cells (simple) vs. multiple stacked sheets of
cells (stratified).
 Notice an exception: certain columnar stacks are called
"pseudostratified" (cell nuclei are in all kinds of places,
not in rows)
 Also a "transitional" stack type, which seems to be more
or less like regular ol' stratified. Evidently when you
stretch it, it gets wonky.
• Also classified based on cell shape: cuboidal (like cubes),
columnar (like columns), squamous (just kind of flat and
messed-up looking).
 Notice that stratified cell shapes are classified based on
the outermost layer of epithelia.
• Describe the cellular basis for apical-basal polarity of epithelial cells and
describe the functions of epithelial polarity.
o Molecular/protein composition on apical side is different from that on
the basal side.
 • On the face of it this seems weird-- if membrane proteins can
run around throughout the lipid membrane, why don't they
equal out on both sides? Answer is that there are tight
junctions between epithelial cells that prevent apical membrane
components to getting to the basal side, and vice versa.
o Interestingly, the cytoskeletons of epithelial cells are also polarized,
which make the organelles inside epithelia polarized as well (have
directionality).
• State the different cell junctions that connect epithelial cells to one another
and to the basal lamina, and describe their key components and functions.
o Tight junctions: hold adjacent epithelial cells together. Made of
transmembrane proteins; most common such proteins are called
"occludens".
o Tight junctions wrap all around the cell; prevent flow of molecules
from the apical to the basal side.
o Basically the tight junctions are the basis of impermeability of
epithelia: it forces substances in the lumen (water, ions, etc) to go
through the cells as opposed to between them to get into the basal
surfaces.
• As noted last unit, there are epithelia that are 'looser' than
others; ie, their tight junctions are a little less tight (need to hit
the gym).
• Looser epithelia are found primarily in the intestines (as noted
by Betz): allows for quick, massive transport of water and ions
between cells (paracellular transport).
 Important note: tight junctions can be loosened or
tightened depending on what substances are being
transported (the intestinal epithelia gets a lot looser in
the presence of glucose, for example).
 Notice also that when tight junctions are loosened,
substances on the basal side of epithelia can leak out
into the lumen as well (simple concentration-driven).
o Notice that there are punctiliar, specific regions between cells that are
also strongly adherent. These are called either desmosomes or
adherence junctions and are caused by a variety of specialized
proteins, including cadherins.
• Cadherins, recall ("Extracellular Matrix" lecture), bind with each
other in the presence of calcium.
• These provide a way to regulate the tightness of specific cell
junctions.
• Notice that desmosomes have no barrier function: they're just
there to bind the adjacent cells together.
o Third type of cell junctions: gap junctions. Essentially a small "tunnel"
or channel between intracellular regions of two adjacent cells. Function
is to selectively allow the flow of small molecules (like signaling
molecules) between cells (speed up broad response to stimuli).
• State the types and functions of the different cell surface modifications on
epithelial cells.
o Microvillae: "pouches" on the apical side of epithelial cells: increase
surface area of apical surface. Filled with actin, generally.
o Cilia: "hairlike" structures, move substances by rhythmic motion. Made
up of microtubules powered by dynein motors.
 o Stereocilia: found in cochlea of ear; seem to be important in stimulus
reception.
• Describe basal laminae by stating their basic components, the basis of their
diversity, their functions, and their structural relationship to epithelia and
other tissues.
o Structure: a thin sheet made up of interlocking proteins. Some
proteins are common to most basal laminae (ie type IV collagen).
Some proteins are unique to particular laminae in particular tissues.
o Functions of the basal lamina:
• (1) promote attachment of the epithelia to the underlying
connective tissue.
 Two kinds of attachment:
• Hemidesmosomes (no relation to desmosomes,
irritatingly), which contain integrins that provide
membrane attachments to the epithelial cells and
also other protein complexes that link the
connective tissue to the lamina. Effectively
hemidesmosomes are a kind of protein 'hitch'
that hooks the lamina to both the epithelium and
also the connective tissue.
• Notice that in some tissue, you have specialized
hemidesmosomes called focal adhesions which
connect the basal lamina to intracellular
components inside the epithelial cells. This allows
signaling communication between the lamina and
the epithelium, which allows the lamina to
influence the development or regulation of the
epithelium.
• (2) Regulate epithelial cell biology (through focal adhesion
signaling).
• (3) Barrier function: barrier to movement from epithelial layer
to connective tissue. Generally not very good at this.
• (4) Sort of a subset of (3)- specialized types of basal lamina
can act to filter specific molecules, particularly in the
glomerulus of the kidney.
• Compare and contrast exocrine and endocrine glands in terms of their
development, general structure, and functions. For both types of glands, trace
the path that a secreted molecule must take from its synthesis to its
destination, and describe all the barriers/structures the molecule must cross
en route.
o Exocrine glands secrete agents molecules onto epithelial surfaces
(inside or outside the body).
• Developed as simple invaginations of the epithelium (still in
contact with epithelial surface).
• Synthesize a variety of substances.
• Notice that exocrine secretions have to cross the apical
membrane to get to the 'outside' of the epithelial cell.
• "Secretory units" = invaginated pouches that function as
exocrine glands. These make the secretions and transport them
across the apical membrane, from which they flow down the
narrow part of the invagination ("channels") to reach the
outside of the epithelium.
 • Notice that sometimes you don't get all this invagination junk
and you just have single cells within the epithelial layer that
produce secretions and transport them out the apical
membrane. The mucus-producing cells in the intestine are a
well-populated example of this.
• Types of exocrine secretions:
 (1) mucus secretions: mucusy. You know.
 (2) serous secretions: watery. Sweat, saliva, etc.
o Endocrine glands always secrete agents into blood.
• Developed as invaginations of the epithelium that are pinched
off from the epithelial surface; surrounded by blood vessels in
the connective tissue, the secretions from endocrine glands go
out into the vascular system.
• Synthesize hormones.
• Notice that endocrine secretions (generally) have to cross the
basal membrane, as well as getting through connective tissues
and blood vessel surfaces to make it into the bloodstream.
 Exception: thyroid endocrine hormones secrete through
the apical membrane, mature in the lumen of the gland,
and then are transported across both membranes out
again.
• Describe the epithelial to mesenchymal transition during development.
o See above (epithelial cells transition to mesenchyme, travel in the
body, and transition back to epithelium to set up epithelial regions in
other locations).
• Describe how epithelial tissues are maintained and regulated, and describe
the properties, functions, regulation and development of epithelial stem cells.
o Most epithelia have self-renewing potential via specialized stem cells.
o These are stored in "crypts" or pouches at the bottom of epithelial
sheets.
o Stem cells are more or less as described by Dr. Hooper on Thursday.
o Tightly regulated: few of them, spread out, slowly dividing.
o Notice that there is a polarity of cell proliferation (in skin, towards the
apical "outside")-- new cells push up old cells and old cells migrate
towards the apical direction. Cells become further differentiated as
they migrate.
o Regulatory proteins of epithelial stem cells: Wnt proteins.
• Wnt proteins in colon inhibit differentiation by binding at the
cell surface and setting off a signaling cascade. Notice that they
also promote cell division (which goes along with a general
stem cell principle: more differentiation, less division).
• Normally, APC protein inhibits the Wnt signaling pathway in the
colon.
• If APC is knocked out, differentiation stops and cell proliferation
is activated.
 APC mutations -> colon cancers
• Similar proteins in lung, but opposite pathway: in lung, Wnt
promotes differentiation and inhibits division.
• State the general terms for epithelial-derived cancer, and describe how
defects in epithelial cell regulation can contribute to cancer.
o Carcinoma (general term for all cancer of epithelial origin)
o Adenocarcinoma (term for cancers derived from glandular epithelia)
 o Interestingly, high levels of cadherin activity correlate with higher
survival rates in cancer, and low levels of cadherin with lower survival
rates-- this could be from (a) tighter connections between epithelial
cells lead to less metastasis of epithelial cancer cells, or maybe (b)
cadherins can act as signaling molecules to sense cancer and respond
appropriately.
• Describe how tissue sections are made and visualized for histological
(microscope) examination, both for general staining and for staining with
antibodies (immunohistology). Distinguish what general stains visualize from
what immunohistological staining techniques visualize. (NOTE: info on this
objective is found in the Intro/Epithilia lab PDF file, and will be discussed in
class.)
o Tissue sample is cut out, put in saline buffer, and "fixed" (solutions
that preserve large cell structure). Notice that carbohydrates and lipids
tend to leak out in this process.
o The sample is sliced very thinly and, usually, stained to create a two-
dimensional image.
• Stains can be very general (bind to lots of different
macromolecules), or very specific. Notice that anything that
doesn't bind to the stain will not show up dark on the stained
image.
o Evans' tip: Find nuclei. These tend to be big dark blotches. They will
tell you where individual cells are.
o Immunohistochemistry/immunofluorescence: methods of detecting
presence of particular proteins in pathology slices- information about
quantity and location of protein.
• Use antibodies: antibodies have high affinity (bind tightly) and
specificity (just bind target) for certain types of proteins, or
antigens.
• Method: after fixing and staining, apply "primary antibody"
(antibody that sticks to the protein you want to look at). Wash
off the excess, then add "secondary antibody" (antibody that
sticks to the primary antibody, which is also linked to either a
fluorescent tag or an enzyme that produces a colored product).
Then you wash off the unbound secondary enzyme, and look at
either the fluorescence or the color against the background of
the cells.
 Secondary antibodies that fluoresce:
immunofluorescence. Secondary antibodies that use
enzymes to produce color: immunohistochemistry.
•

Connective Tissues I + II
Monday, December 03, 2007
8:01 AM

Connective Tissues I + II, 12/3/07:

• State the types, origins, and functions of the different cell types found within
connective tissues.
 o Immune system function: connective tissue provides a leukocyte-filled
barrier to pathogens coming through the epithelium.
o Cells that make ECM:
• fibroblasts, chondrocytes, osteoprogenitors (all derived from
mesenchymal or connective stem cells).
• Fibroblasts differentiate into adipocytes or smooth muscles, or
can shift into osteoprogenitors or chondrocytes.
• Chondrocytes make cartilage, osteoprogenitors make
osteoblasts (which eventually turn into osteocytes).
• Notice fibroblasts are capable of division and can interconvert
into other kinds of cells as needed.
• Obviously, fibroblasts need to be highly regulated due to their
potential for uncontrolled division.
o Cells not made in connective tissues but present in ECM:
• lymphocytes (make antibodies when activated; activated
lymphocytes are called 'plasma cells'.)
• neutrophils/eosinophils (undetailed immune cells)
• mast cells (start out as 'basophils' in blood, involved in
inflammatory response)
• macrophages (start out as 'monocytes' in blood, phagocytic
'scavenger' cells)
 These can break down and restructure tissues by
engulfing other cells.
 Notice also that they can act as signaling centers:
synthesize and secrete signaling molecules that direct
restructuring of tissue.
 Can trigger blood vessel formation (angiogenesis).
• This is why tumor growth is aided by
macrophages- promote new blood supply to
tumors.
• Describe the components of the extracellular matrix, their functions, and how
they are organized in different connective tissues. Describe the structural
relationship between connective tissue and epithelia, blood vessels, muscles
and nerves (this will be best understood after you have studied all of these
tissues).
o General structure: Extracellular fibers embedded in gel-like mix called
the "ground substance."
• Fibers: collagens and elastins.
 Collagens:
• Fibrillar collagens: make long, thick composite
fibers, or fibrils (primarily Type I collagen). Lots
of tensile strength.
• Fibril-associated collagens: make thin fibers
connecting basal lamina to fibrillar collagens and
fibrillar collagens to each other.
• Networking collagens: form thin sheets that
provide the scaffold for the basal lamina (Type
4).
• General unit of collagen is the triple helix; these
helices pack together (for both width and length)
to form fibrils.
 • The assembly into triple helices takes
place outside the cell; proteases cleave
both N- and C-ends of the collagen
molecules, which enable them to form
helices.
• The N-cleaved ends can be detected in
blood and urine-- an indicator of metabolic
activity in bone and other connective
tissues.
• N-cleaved ends are called "N-telo
peptides."
• Recall that non-hydroxylation of proline residues
of collagens (caused by deficiency of vitamin C)
leads to scurvy.
 Elastins:
• As you'd expect, these are elastic fibers, cross-
linked to form "rubber bands."
• These fibers are supported by proteins called
fibrillins; it's mutations in fibrillins that cause
Marfan's Syndrome.
• Marfan's susceptibility to cardiac problems
has to do with the reduced elasticity of
their major vessels.
• Ground substance:
 Composed largely of proteoglycans (proteins +
glycosaminoglycans, or GAGs-- see "Extracellular Matrix
and Cell Adhesion"):
• Essentially a protein core and massive
carbohydrate side chains.
• The carbohydrate chains are negatively charged,
attracting water and hydrating the ground
substance.
• Because ground substance is hydrated, solutes
can move through it.
• Notice that GAGs are also involved in storing and
regulating polypeptides, like growth factors, that
diffuse near them.
 Also composed of GAGs unlinked to proteins (most
common kind is hyaluronic acid) and a lot of proteins
(signaling proteins, proteases, etc).
• For the proteins that form extracellular fibers, describe their types, their
properties, and how they are made and assembled in the extracellular matrix.
o See just discussed-- collagen, elastin, fibrillin. Note that the N-telo
ends are cleaved outside the cell (thus detected in blood and urine).
• Describe the basis and functional consequences of connective tissue diversity.
o Basis: stem cells that can differentiate into many kinds of basic cells,
difference in the function and ECM secretions of those differentiated
cells.
o Functional consequences: more sketchy. "We have working connective
tissue," I guess. Could also mean that from one type of cell
(mesenchymal stem cells) we get a massive diversity of connective
 tissue, dependent on differentiating signaling and signals that tell
those post-differentiated cells to produce different proteins.
• Describe how connective tissues are regulated upon tissue growth, use or
injury. Describe the events that occur following wounding or during edema.
o Tissue growth, or use: not sure what he means here. They can be
remodeled at just about any point by proteases and stem cells. Injury
is covered under the next point.
o Wound response: generally signaled by platelets from torn blood
vessels.
• Inflammation phase: mast cells activated to secrete various
molecules, among them histamines and chemoattractants
(polypeptides that attract migratory cells).
 These go into the blood vessels and cause an influx of
normally vascularly-located immune cells (leukocytes
and macrophages) into the ECM at the site of the
wound.
 Macrophages, in addition to 'eating' damaged tissue and
bacteria, trigger angiogenesis in the wound (though this
often doesn't transpire until the tissue actually begins to
heal in the next phase); also triggered are cytokines,
which attract the cells involved with the next phase.
• Proliferation/development phase: Fibroblasts are attracted by
cytokines and other signals; these divide and increase their rate
of ECM secretion.
 Fibroblasts in area begin to repopulate damaged
epithelium and connective tissue (if basal lamina is
damaged extensively, end up with scar tissue).
 Fibroblasts also differentiate into myofibroblasts: exert
pressure on area to restrict blood loss and close the
wound. These generally die off after the wound has
healed.
• Maturation/remodeling phase: Effectively a continuation of the
development phase, in which the fibroblasts replace temporary
collagen laid down to close the wound with more permanent
collagen oriented for maximal tensile strength.
• [Bone functions: structural support, calcium/PO4 homeostasis, houses
hematopoeitic system]
• For cartilage, describe its cellular and extracellular composition, its structural
properties, and how it is organized. State the functions of cartilage tissue.
o Cartilage: serve as template for bone or as resilient/pliant support
system.
o Mesenchymal stem cells form chondrocytes, which secrete
cartilaginous ECM.
o These chondrocytes become cocooned in "lacunae" of their own
secretions. Notice that they can still divide in lacunae (matrix is
flexible) and grow out the cartilage (interstitial growth, see below).
o The chondrocytic matrix is covered with a hard outer layer called the
"perichondrium", which contains the mesenchymal stem cells that
gave rise to the chondrocytes in the interior.
• This means that cartilage can grow in two different ways:
appositional growth, in which the perichondrium lays down new
layers of cartilage on the surface, and interstitial growth, in
 which the cocooned chondrocytes in lacunae continue to divide
and secrete (growing cartilage from within the existing
cartilage).
• This is significant largely by contrast: bone matrix is much less
flexible and cannot grow interstitially (bone lacunae can't shift
apart to accommodate internal growth like cartilaginous
lacunae).
o Note that cartilage is avascular-- it has to derive nutrients and
oxygen from peripheral vessels.
• Describe how cartilage grows during fetal and child development.
o All I can figure from the notes (I don't think he mentioned this in
class) is that chondrocytes arise from mesenchymal stem cells during
fetal development and do what they always do (interstitial and
appositional growth, with chondrocytes secreting cartilaginous ECM
and becoming encased in lacunae) from that point on.
• State the characteristics that distinguish the three basic types of cartilage.
o Hyaline cartilage: full of hyaluronic acid (recall, that's the GAG type
that isn't bound to proteins). Fairly sparse and irregular collagen fiber
matrix.
• Hyaline is the type of collagen that will ossify in long bones (see
next section).
o In particular locations, hyaline also has a lot of elastic tissue (earlobes,
etc). This is called elastic cartilage.
o Fibrocartilage: often found where tendons attach to bone or in joints.
Much more densely packed with fibrous collagen; tougher.
• State the different cell types found in bone. For each cell type, describe their
functions, their origins, and describe how they are organized in bone tissue.
o Bone cells: also formed (except for osteoclasts, see below) from
mesenchymal stem cells, which form osteoprogenitor stem cells which
can then self-renew and also produce osteoblast secretory cells.
o Osteoblasts assemble on the surface of forming bone; they secrete a
watery, loose ECM characteristic of bone called osteoid (contains
collagen, but not dense).
o Like chondrocytes, osteoblasts become cocooned within their own
secretions; when they're fully enclosed, osteoblasts transform into
osteocytes, which have vastly reduced secretory activity but which
send out processes that contact their osteocyte and osteoblast
neighbors to form gap junctions (signaling function).
• These long processes form canaliculi (sing. canaliculus) through
bone matrix.
• This seems to be involved in sensing the current status of the
bone matrix and regulating osteoblast activity depending on
that status.
o Notice that osteoblasts also trigger the mineralization of the loose
original bone ECM: they secrete Ca2+- and PO4-filled matrix vesicles,
which break open in the ECM as their contents form crystallized
calcium phosphate ("hydroxyapatite"). The released hydroxyapatite
gives the bone ECM its extreme hardness; it also serves as the main
storage site of calcium in the human body.
o As mentioned, due to this hardness, there is no interstitial growth in
bone (matrix is too inflexible to allow it)-- bone only grows by
 appositional growth at the surface due to division and secretion of
osteoblasts and osteoprogenitors.
o Completely different lineage of osteo- cells: osteoclasts, derived from
monocytes in blood vessels (as are macrophages).
• Osteoclasts are effectively bone-matrix-specific macrophages:
when directed, they 'chew up' and degrade bone matrix and
release liberated calcium into newly loosened ECM, where it can
get back into the bloodstream.
• Functions of osteoclasts:
 Degrade cartilage (for ossification of 'template' cartilage)
as well as bone (for remodeling and calcium
mobilization)
 Stimulate angiogenesis (like macrophages).
• As these tunnel through bone, they bring blood
vessels with them (note that bone is
vascularized).
 Innervation: Nerves follow osteoclast-formed channels
in bone matrix.
• Describe the composition of bone extracellular matrix. What are the functions
of the different components (only those discussed in class and in the lecture
notes)? Describe where these extracellular matrix components are made, and
how they are deposited to form bone matrix.
o See above. Loose collagenous bone ECM is secreted from osteoblasts,
which also secrete the matrix vesicles that contain the hydroxyapatite
that will mineralize the original, soft ECM. Osteoclasts resorb the
mineral matrix and excrete the inorganic ions inside into the
reloosened ECM, from where they can get back to the bloodstream.
Note that bone ECM is vascularized and innervated due to osteoclast
action.
o Not real specific, but about as specific as the lecture and notes.

Connective Tissue III

Tuesday, December 04, 2007
9:04 AM

Connective Tissue III, 12/4/07:

• Describe the two different processes that lead to bone formation. Describe
how long bones grow in length and in width.
o Intramembranous ossification:
• During development: mesenchymal stem cells condense (come
together and proliferate) and form layers with surrounding
connective tissue. Some of the stem cells differentiate into
osteoprogenitors and then to osteoblasts, which secrete bone
matrix that's vascularized/innervated/remodeled by osteoclasts
and mineralized by osteoblasts.
• Effectively this is the simple route of bone formation: direct
formation of bone from mesenchymal stem cells without a
cartilaginous intermediate.
• Most of the flat bones form this way.
o Endochondral ossification:
• Initial, 'template' cartilaginous 'bones' are ossified and replaced
with actual bone.
• Recall that the perichondrium of cartilage contains
mesenchymal stem cells which continue to create new
chondrocytes.
• At some point, a signal is received at around the middle of the
diaphysis (center of long bone) which tells the mesenchymal
cells to begin producing osteoprogenitors instead of
chondrocytes. This begins a transitional wave that transforms
the perichondrium to a periosteum.
• Formation of periosteum induces two events: calcification of
cartilage, and the destruction of chondrocytes embedded in the
cartilaginous matrix.
 Chondrocytes tend to get very large before they're killed
off (apoptosed)-- this is called hypertrophy, which is an
indicator that chondrocytes are becoming apoptotic prior
to ossification.
 Osteoprogenitors/osteoblasts send signals to bring
osteoclasts to the cartilage-- the osteoclasts show up,
see the calcified cartilage, and begin to degrade it and
engulf the apoptosed chondrocytes.
• Note that osteoclasts bring blood vessels and
nerves with them while this is going on.
 After the osteoclasts have done their thing, the
osteoblasts set in to lay down bone matrix and
calcification around the vessels that have just invaded
the nascent bone.
 Notice that the cartilage is still growing (appositional and
interstitially) wherever it can while this is going on.
 The secondary ossification centers, one at each end
(epiphysis) of the bone, form while the primary
ossification center is still being vascularized and
mineralized. These progress in the same manner as the
primary.
 Note that there's a layer of cartilage left behind (the
epiphyseal or growth plate) between the diaphysis and
epiphysis.
• This cartilage continues to grow interstitially in
the direction of the epiphysis (towards the ends
of the bone); the primary ossification center
'chases' this cartilaginous growth, ossifying the
new growth as it occurs.
• If you're looking at a histology slide of
ossifying cartilage, can always tell which
layer is the cartilage by looking at 'lines'
of proliferating chondrocytes pushing out
in the direction of the end of the bone.
• Interstitial growth of cartilage is what drives the
length of long bones; width of bone is driven by
appositional growth of the periosteum (which
 completely replaces the perichondrium along the
edge of the bone).
• Most of the skeleton (including all the long bones) are formed
this way.
• Describe the sequence of events that occur in bone remodeling.
o Periosteum forms on the surface of cartilage.
o Osteoblasts calcify the cartilage; chondrocytes undergo hypertrophy
and apoptose.
o Osteoclasts degrade the cartilage, engulf chondrocytes, lay down new
blood vessel and nerve pathways.
o Osteoblasts make bone matrix and mineralize it around vessels and
nerves.
• Describe how bone formation and remodeling is regulated.
o [Endosteum: inside surface of bone (inner surfaces of osteoclast-
derived tunnels).]
o Bone remodeling (osteoclast activity and subsequent osteoblast
secretions) occurs immediately upon formation of new bone, and also
recurs almost continuously throughout life.
• Notice that the activation of osteoclasts is tightly linked to the
activation (ie production and secretions) of osteoblasts.
o In early bone, the coordination of vessels/nerves with matrix is
disorganized and random; over time, the constant remodeling leads to
a particular division between compact and spongy bone.
• Compact bone: On the edge of the bone; dense, multi-layered,
no trabeculae.
• Spongy (or 'cancellous' or 'trabecular') bone: Deeper inside the
bone, form honeycombed networks of endosteal surface, filled
with bone marrow.
• Notice that both flat and long bones show this pattern (spongy
vs compact).
o Mechanisms of bone-remodeling-signaling:
• Short-range: Bone morphogenetic proteins (BMPs) released at
bone site.
 Don't travel through bloodstream.
 Effectively instruct other cells to lay down and remodel
bone.
 Significant pathophysiologically due to FOP disease
(Fibrodysplasia ossificans) that causes ossification to
occur in loose connective tissue. Caused by the BMP4
gene being fused to an abnormal promoter; this causes
it to be expressed in lymphocytes (from where it acts on
fibroblasts/mesenchymal stem cells to form
osteoprogenitors and bone).
 Can also use Wnt and Notch proteins to do short-range
signaling (no details needed).
• Long-range: Endocrine hormones, particularly calcitonin (lay
down more calcified matrix: Ca2+ out of bloodstream, build up
bone) and parathyroid hormone (reabsorb more calcified
matrix: Ca2+ into bloodstream, break down bone), as well as
steroid hormones (no details).
 • Mechanical stress: as per anatomy, putting stress on bones
leads to different cell activity in bone remodeling-- thickens
bone (greater appositional growth) in particular directions.
• Neuronal stimulation: CNS regulates bone remodeling. A "who
knows" category.
• Describe how defects in bone remodeling leads to disease.
o See FOP disease above.
• Describe how calcium is deposited and resorbed from bone matrix, and how
regulation of bone cells controls the levels of blood calcium.
o See calcitonin and parathyroid hormone, above.

Cytoskeleton II
Tuesday, December 04, 2007
5:48 PM

Cytoskeleton II, 12/5/07:

[Notice that I wasn't present for some of this lecture and have tried to fill in those
LO's from the notes. Dr. Pfenninger's notes being what they are, the following may
not be particularly complete or comprehensible.]

• Describe (reviewing material from Cytoskeleton I) the three types of

cytoskeletal elements, their properties, their functional roles, and their
protein composition.
o (1) Microtubules: long, flexible, polarized polydimers (alpha and beta
tubulin subunits). Involved in mitosis as well as in ciliar and flagellar
movement and transport inside the cell. Added to at plus end (end
with exposed beta subunit); partially GTP-regulated. Little tensile
strength. Express mechanical activity through motor proteins (dyneins
and kinesins) that can shuttle cargo down them or use them as levers
(as in mitosis). Note that microtubule polymerization requires GTP.
o (2) Intermediate filaments: long, thick fibers, assembled in nonpolar
bundles of 32 filaments per bundle. Assist in mechanical support and
strength. Found both inside the nucleus (nuclear lamins) and outside
(eg. keratin). Lots of different possible protein sources.
o (3) Microfilaments: mainly, polarized polymers of actin. Polymerized G
(globular) actin strands dimerize to form F (filament) actin. These
have lots of actin-binding proteins associated with them to assist in
polymerization (both of G-actin with other G-actins and of F-actin with
other F-actins), which usually takes place at the plasma membrane
and at the anchored minus end (plus end pushes out with
polymerization). Note that microfilament polymerization ("nucleation")
requires ATP.
• Discuss cytoskeletal dynamics and the role of certain proteins in actin
polymerization/depolymerization.
o As mentioned, F-actin polymers dimerize to form the basic unit of
microfilaments. Notice that certain drugs stabilize F-actin, causing
abnormal aggregation.
 o ARP proteins: catalyze actin polymerization at the minus-end
(elongating the plus-end). Effectively these act as "growth bases"--
plug into nuclear membrane and elongate actin into the cytosol.
o Cofilins: catalyze actin depolymerization, reversing ARP protein action.
o Rho family of proteins: signals having to do with particular
assemblages of actin.
• Rho: involved with stress fiber formation (contractile bundles).
• Rac: involved with "veil" formation at the border of the cell
(important in cell locomotion, see below).
• Cdc42: involved with forming filopodia (fingerlike extensions of
the cell).
o Basically, the cool thing about actin is that it's a scaffold that can be
assembled and taken down again very quickly, all over the cell. Want a
protrusion of the cell towards a food source? Use actin. Figured out a
food source isn't friendly and need to retract the protrusion? Actin all
the way. Feel like rolling your way down the lamina to the nearest
single-celled pub? Actin's your man.
• Explain the concept of mechanoenzymes. Explain the mechanism of actin-
based movement and contrast it to tubulin-based movement.
o Mechanoenzymes: enzymes that convert chemical stored energy into
motion.
o Actin-based movement: actin-myosin contraction, described here as
"sliding-filament" motion; it involves "winding a spring" as the target
of ATP use (more on this under "Muscle I, II, + III").
o Tubulin-based movement: two-headed dyneins and kinesins "walk"
down the tubulin chain; ATP is used to swing the lagging head around
in front of the leading head.
• Discuss the actin cytoskeleton in the context of disease processes.
o Actin makes up the core of microvilli (thus problems result when no
actin).
o Essential to amoeboid movement (see below): needed for axonal
development, leukocyte migration; required for cancer metastasis.
• Describe two types of locomotion of mammalian cells.
o Swimming motion propelled by cilia or flagella (ie sperm).
o Crawling movement over a surface (amoeboid motion, much more
common).
• Discuss the concept and the key steps of amoeboid locomotion.
o Essentially: the leading edge of the cell "pouches" forward, driven by
polymerization of actin filaments branching off from other actin
filaments (ARP proteins bind to filaments instead of nuclear
membranes and drive elongation of new microtubules from them), and
adheres the pouch to a new section of surface. Then it pulls the bulk of
its cell body towards the pouch (possibly with actin-myosin) and un-
adheres and retracts its lagging end (by depolymerizing the branched
actin filaments there).
o Steps called:
• Protrusion (pouching forward)
• Attachment (adhering to new surface)
• Traction (pulling forward)
• Detachment (retracting of lagging surface)
• Discuss the relationship between cell adhesion and the cytoskeleton in
amoeboid locomotion.
 o Well, if the thing can't adhere, it can't pull itself forward, can it?
• Describe some of the molecules critical for amoeboid locomotion.
o Actin, obviously. Then there's ARPs to polymerize actin and cofilins to
depolymerize it again at the lagging edge. Adhesion proteins
(cadherins, Igs, integrins) also critical. Notice the Rho family of
proteins for signaling particular actin formations (like the filopodia or
outpouching) and the WASp proteins (among others) involved in
pushing those filopodia forward in protrusion.
• Discuss cell motility in the context of developmental and disease processes.
o 2 diseases for this:
• Wiskott-Aldrich Syndrome: X-linked non-clotting disease--
caused by problems with protrusion of the leading edge of
amoeboid cells by WAS protein (WASp). (Platelets are formed
from castoffs of these protrusions.)
• Lissencephaly: neuronal disease-- non-migration of neuronal
axons leads to a smooth cortical surface and mental
retardation.

Muscle I, II, + III

Wednesday, December 05, 2007
3:39 PM

Muscle I, II, + III:

[I missed all four lectures on this day (family). So, again, I'm going mainly by notes
here. Though I have to say that I'll go by Caldwell/Tseng notes before Pfenninger
notes any day of the week and twice on Sundays.]

[Thanks to Andrew Brookens for supplying lecture notes on this one too. --jcr]

• Be able to explain the structural basis of skeletal muscle contraction by

constructing a sarcomere:
o Ok. Draw two lines. These are Z lines. The actin (thin) fibers poke out
from them. Draw a line in the middle. This is the M line. The myosin
(thick) fibers poke out from it between the thin fibers (which don't
extend all the way to the M line, but do extend enough to have some
cross-over with the myosin). Ta-da, a sarcomere. Line up a whole
bunch of them in a row. Now you have a myofibril. Repeat that a lot in
an orderly fashion. Now you have a striated muscle.
• -What is the molecular structure of the sarcomere and the arrangement of
contractile and linker proteins? How does this structural organization relate to
contraction?
o Basics: Thin filaments are made up of actin chains with tropomyosin
stuck to them and troponin stuck to the end of tropomyosin. Thick
filaments are made up of myosin chains that have big globular loops
('heads') on them that interact with actin.
• -What is a myofilament? Define a myofibril. What is the relationship between
myofibrils and the sarcoplasmic reticulum?
o A myofilament is a polymerized strand made up of either myosin or
actin/tropomyosin/troponin.
 o A myofibril is a bunch of sarcomeres placed end to end. Each myofibril
is covered with its own sarcoplasmic reticulum (see below).
• -How are connections of muscle contractile proteins made to surrounding
connective tissues, and how do they contribute to contractile
force/movement?
o Dystrophin connects actin to the surface membrane. Titin links myosin
to the Z line (centering the thick filaments). Nebulin does something
similar for the thin filaments. Alpha-actinin crosslinks actin fibers.
o The idea is that there's "passive" tension in a muscle fiber, presumably
generated by attaching everything in its original configuration, that
gives the cell something to return to after contraction. These proteins
help maintain that configuration.
• Be able to discuss the physiological and biochemical basis of skeletal muscle
contraction:
o (1) Influx of calcium binds to troponin.
o (2) Bound troponin changes the configuration of tropomyosin.
o (3) The shifted tropomyosin exposes binding sites for myosin on the
actin filament.
o (4) The actin filament binds to the myosin and the "spring" tension
pre-loaded into the myosin releases, shortening the sarcomere by
about 10 nm.
o (5) ATP binds to myosin, allowing actin to be released.
o (6) ATP is hydrolyzed to ADP + Pi, pre-loading the "spring" tension
into the myosin fiber by shifting its configuration a little.
o (7) Repeat steps 4-7 as long as there's Ca2+ and ATP around.
o (8) Once the calcium supply runs out (Ca2+ is being pumped out while
this is going on), troponin goes back to its original configuration, as
does tropomyosin, causing actin to no longer be able to bind to
myosin.
• - How do contractile proteins work and how are they regulated in skeletal
muscle?
o More or less discussed this already. The myosin heads binds to and
ratchets down actin with ATP activity.
• -Describe the regulatory proteins (where are they located and how do they
respond to changes in calcium concentration).
o Regulatory proteins: means troponin and tropomyosin.
o As mentioned, troponin sits at the end of tropomyosin and binds
calcium.
o Tropomyosin sits on actin and covers the myosin binding sites until
troponin is triggered by Ca2+.
• - What is the length of a sarcomere in resting muscle, contractile muscle, and
muscle that is stretched almost to the point of injury (tearing)? How does this
clarify the ambiguity of the question – “what is the length of a sarcomere?”
o Length of a sarcomere in resting muscle: around 2.4 µm.
o Length of a sarcomere in stretched muscle: around 3.6 µm.
o Length of a sarcomere in contracting muscle: unspecified. Less than
2.4 µm.
o Sarcomere is constantly stretching and contracting as it's used, thus
length shifts.
• What is the molecular basis of skeletal muscle diversity (fast and slow fibers)
and what is the value of having this additional complexity?
 o Molecular basis: the different skeletal muscle types have different
mixes of oxidative and glycolytic energy-producing frameworks.
• Fast-twitch muscles tend to be centered around glycolytic
energy production (quick bursts of energy, less sustainable).
Used for things you need quick bursts of energy for (ie.
catching the last beer can after you knock it off the table).
• Slow-twitch muscles tends to be red in color due to their high
myoglobin content (high oxygen load); they have all that
oxygen because they primarily use oxidative phosphorylation to
generate ATP for contraction. Tend to be used for sustained or
postural movements (ie. assuming a relaxed and attractive
mien with that beer can).
o Usefulness: Well, obviously, if you can catch the beer can but not relax
with it, you won't attract a mate on account of no one wants to date a
spaz. If you're too slow to catch the beer can in the first place, you
won't attract a mate because catching beer cans is just one of those
intrinsic abilities that's very attractive. Only with the right mix do your
slightly intoxicated genes get a chance to propagate.
• Describe the key structural and physiological features of cardiac muscle and
how they are similar to and different from skeletal muscle (sarcomere,
regulatory proteins, events involved in a single contraction and relaxation,
response to injury).
o Intercalated disks: only found in cardiac muscle. Two functions: hold
adjacent muscle cells together and allow gap junctions (see next
point).
o Mononucleated cells vs multinucleated in skeletal muscle.
o Actin-myosin contraction and relaxation is the same as skeletal muscle
(though note (a) that smooth muscle contraction is quite different and
(b) that cardiac AP excitation pathways are subtly different from their
skeletal counterparts, see below).
o Cardiac cells cannot regenerate damaged tissue like skeletal muscle
does (no satellite cells). So be careful with that double cheeseburger,
bucko.
• What is the role of gap junctions in each of the three muscle types (skeletal,
cardiac and smooth)?
o Gap junctions only occur in cardiac muscle fibers and some types of
smooth muscle. Their role in cardiac muscle is to ensure that cardiac
contractions occur rhythmically throughout the heart muscle by
allowing selective passive of signaling molecules; they may also form
the basis for synchronous contractions of gastroenteric smooth muscle
(ie. peristalsis). Also link adjacent cells together, though this is usually
associated less with gap junctions and more in the intercalated discs
that they're found in.
• What is the molecular basis of familial hypertrophic cardiac myopathy? What
proteins are mutated? Where are the mutations most likely to occur in the
proteins?
o Familial hypertrophic cardiomyopathy (FHC) occurs as a result of
mutations in cardiac muscle tissue. Usually the mutations are located
in the cardiac myosin heavy chain, where it interacts with actin and
ATP; occasionally a mutation will occur in troponin instead.
• Why does smooth muscle appear smooth? What are the key structural and
physiological features of smooth muscle and how do they compare to skeletal
 and cardiac muscle (contractile proteins, regulation of contraction, cell
organization)?
o Smooth: because there's no striations (ie. the sarcomeres aren't nicely
lined up like cardiac and skeletal muscle).
o Structural: smooth muscle cells are extremely thin-diameter, spindle-
shaped cells. Classically these are the "involuntary" muscles of the
body (ie. digestive muscles).
o Contractions: a little different from the other two:
• Instead of having calcium bind troponin as the catalytic step for
actin to bind myosin, in smooth muscle calcium binds
calmodulin (remember this one? With the EF hand domains?),
which binds calmodulin kinase (CAMK), which phosphorylates
the myosin chains, causing actin and myosin to bind and the
ratchet action to begin.
• This is a lot slower than skeletal/cardiac contractions.
o Notice that smooth muscle cells are mononucleated, like cardiac cells
and unlike skeletal cells.
• How does skeletal muscle develop (single cells going to multinucleate cells)?
o You start out with individual myoblast cells (muscle precursors). These
merge together into long chains during development. Predictably, the
reason you get multinucleate cells from single cells is that the single
cells decide to cohabitate.
• Explain the reason for the transverse-tubule system (t-system) in skeletal
and cardiac muscle.
o Essentially you need a way to get the membrane surface signal (the
AP) to get to the sarcoplasmic reticulum so it can trigger the release of
calcium and cause contractions. Having the AP be transmitted
throughout the SR would be too electrically laborious, so those wacky
cells decided t-tubules were the way to do it.
• How is excitation-contraction coupling accomplished in skeletal muscle?
o Ok. The AP's coming down the membrane, continuing down the t-
tubule. When it gets down to the end of t-tubule (which sits down on
the edge of the sarcoplasmic reticulum of the muscle cell), it runs into
a particular kind of receptor system, consisting of two parts:
• DHP receptor: voltage-gated receptor
• RyR (ryanodine receptor): Calcium channel in the
sarcoplasmic reticulum.
o Current theory: voltage hits DHP receptor, causes it to shift in such a
way as to cause the RyR channels to open and allow calcium to flow
out into the muscle cell, causing contraction.
• How is excitation-contraction coupling accomplished for cardiac and smooth
muscle? Why is the t-system not required in smooth muscle?
o Cardiac:
• Similar to skeletal, but here the difference is that the voltage
seems to release calcium before the RyR-equivalent channels
will open-- the cardiac calcium release channels (to trigger
contraction) open in response to a calcium signal. I think
they're not entirely sure how this works.
o Smooth:
• Don't need t-tubules at all: calcium released at the cell surface
can just go ahead and diffuse through the whole cell very
quickly. The reason this works for smooth muscle (but not for
 the others) is that smooth muscle cells are so small and thin. If
you tried this with skeletal muscle, you'd get weird
asymmetrical contractions as the parts of the muscle that got
calcium first would contract first and the other only later, as the
calcium diffused. Obviously you don't want that, and you really
don't want it in cardiac muscle-- thus t-tubules.
• What is the molecular basis of malignant hyperthermia? - What protein
contains the mutation? - Why does the temperature rise? - Why is the disease
not noticed until surgery? - What type of compound would you use to prevent
the temperature rise? Why wouldn’t induced muscle paralysis solve the
problem?
o Wow, that was a mouthful. Break it down:
• Basis is a mutation in the calcium release channel proteins in
the sarcoplasmic reticulum.
• This mutation causes inhaled anesthetic to trigger the release
of calcium from the SR; this causes ATPases to pump calcium
back out, and a vicious cycle is initiated in which the heat
generated by the pump just keeps rising, causing
hyperthermia.
• Patient's muscles also become rigid during this time (muscles
firing at speed).
• It's not noticed until surgery because most of us don't jaunt
around inhaling halothane all day.
• Dantrolene blocks calcium release from the SR; thus,
administered as a prophylactic, it prevents malignant
hyperthermia.
• Muscular paralysis (by which is meant blocking APs) would have
nothing to do with this because this isn't AP-generated Ca2+
release-- it's triggered by inhaled anesthetic.
• -Starting with an action potential in a motor neuron, explain the processes
required to have a skeletal muscle undergo a single contraction and relaxation
(a twitch).
o (1) AP in motor neuron travels down to the synaptic area.
o (2) AP causes a release of acetylcholine (neurotransmitter) at the
synapse.
o (3) ACh binds to ACh receptors in the muscle fiber, opening ion
channels in the muscle fiber and causing depolarization (muscular AP).
o (4) Muscle AP propagates down the fiber.
o (5) AP goes down into t-tubules on its way.
o (6) DHP receptors at ends of t-tubules sense voltage change and open
the RyR channels in the SR, releasing calcium into the cytosol of the
muscle.
o (7) Calcium binds to troponin, causing tropomyosin to shift and actin
to bind to myosin and contract.
o (8) Calcium and ATP drive contraction as long as signal persists and
calcium/ATP are present.
o (9) Calcium pumps (using ATP) move calcium back into the
sarcoplasmic reticulum; troponin, released from calcium, relapses and
causes tropomyosin to cover up the myosin binding sites on actin. The
muscle relaxes.
• -Where do motor nerve terminals associate with skeletal muscle fibers and
what is the distribution of cells innervated by one motor neuron in a muscle?
 o Motor nerve terminals (synapses) are located around the center of
skeletal muscle fibers; the AP propagates in both directions from
there. (note that the contraction of the muscle is not the same as the
AP to the muscle-- the former depends on t-tubule-triggering of
calcium release and occurs more or less at the same time throughout
the muscle, as you'd like for maximum contraction.)
o The distribution of innervated cells varies greatly depending on the
particular neuron and the particular muscle, but is generally called the
motor unit whatever its size.
• Fine motor actions tend to have small-size motor units (you're
triggering less muscle contractions with one AP); coarse motor
actions tend to have larger motor units.
• Notice that a single "muscle" (ie thigh muscle, triceps, etc) may
have lots of different motor neurons innervating it, all attached
to differently-sized motor units.
• What is a muscle motor unit and what does the average size of a motor unit
in a muscle tell you about the function of that muscle?
o Pretty much covered this already. If the average size of a motor unit is
small, that muscle is probably used largely for fine movement; if larger
motor units, probably mainly gross movements.
• - If you add the motor unit sizes of all the motor neurons innervating a single
skeletal muscle, is this sum greater than, less than, or equal to the number of
muscle fibers in the muscle?
o Should be equal-- normally, no muscle fiber is innervated by more
than one neuron (though see next point) and no muscle fiber isn't
innervated by any neuron at all. (yes, it's a double negative, all you
English majors out there. All two of you.)
• - Following nerve injury and muscle reinnervation or in someone in the early
stages of ALS (amytrophic lateral sclerosis: motor neurons are dying), the
sum of motor unit sizes can be greater than the number of muscle fibers –
how would you explain this?
o Probably has something to do with muscle fibers being innervated by
more than one neuron during the regenerative process.
• How is tension graded in cardiac and smooth muscle (two mechanisms)? How
is this different from the gradation of tension for skeletal muscle?
o Cardiac and smooth muscle: predominantly graded by cell length and
neurotransmitter/hormone receptor activity.
o Skeletal muscle: Doesn't change length (anchored at both ends)-- thus
its tension is graded differently (see below).
• What is the role of “satellite cells” in skeletal muscle development and repair?
What are the physiological and structural responses to exercise (or lack of
exercise) on skeletal muscle (number of cells and size of cells)?
o Satellite cells: specialized stem cells that produce new skeletal muscle
cells. These new cells, instead of replacing the muscle cell that's there,
will fuse with it to form a larger muscle fiber. This is useful in both
development of muscle during exercise and repair of damaged cells.
o Exercise:
• Exercise does not add more muscle fibers. What it does is
increase the size of the muscle fibers you have.
• Atrophy, likewise, reduces the size of the fibers, not their
numbers.
 • - How is skeletal muscle tension graded and regulated (the two major
mechanisms), and what is the basis of muscle fatigue?
o Skeletal muscle is tension-graded by the frequency of action potentials
fired and the number of motor units recruited for the contraction.
• Note 'tetanic' tension at which the muscle can't contract any
farther.
o Muscle fatigue: involves four different parts of the contractile reaction:
• (1) K+ builds up and Na+ is reduced in the t-tubular network;
this reduces its ability to propagate APs and trigger calcium
release from the SR. This problem is corrected quickly (K-Na
balance restores in seconds).
• (2) - (4) Involves buildup of Pi (from ATP hydrolysis) and a
drop in pH (6.5 from 7- pH drops due to lactate production
after glycolysis under conditions of insufficient oxygenation).
Notice that we don't really know why these happen, just how.
 (2) Pi and H+ buildup inhibits calcium release from the
sarcoplasmic reticulum.
 (3) Likewise, Pi and H+ inhibit the binding of calcium to
troponin.
 (4) They also reduce the contractile force exerted by the
myosin-actin binding.

Vasculature
Friday, December 07, 2007
7:59 AM

Vasculature, 12/7/07:

• Describe the structure, organization, and function of the basic layers of blood
vessel walls:
o Three layers surrounding lumen:
• Tunica intima: endothelial layer closest to the lumen of the
vessel.
• Tunica media: middle layer, composed of elastic tissue,
smooth muscle, or collagen.
• Tunica adventitia: outer layer, composed of
collagen/collagenous tissue.
 In large arteries, these often contain smaller vessels
running through them; these are called vasa vasorum
(vessels of vessels).
• Discuss the morphological characteristics that distinguish the different types
of blood vessels:
• [In largest arterial vessels, like the aorta ("elastic arteries"):]
o Tunica intima:
• endothelial cells (at the interface with the lumen)
• Stuff just outside the endothelial layer but still in the intima:
 fibroblasts
 connective tissue
 myointimal cells (responsible for laying down fibrous
plaque in atherosclerosis)
 o Tunica media:
• Lots and lots of smooth muscle cells and, particularly, elastic
fibers
o Tunica adventitia:
• Vasa vasorum (supplies blood and nutrients to outer parts of
larger vessels)
• Loose collagenous connective tissue, elastin
 This is loose so that leukocytes (white blood cells) can
exit the blood vessels and get out into the connective
tissue.
• [In more distal but still large arteries ("muscular arteries"):]
o Less tunica intima volume
o Shows characteristic "inner" and "outer" elastic laminae that form
the boundary of the tunica media, containing smooth muscle cells
(note less elasticity as you get farther from the aorta on account of
there's less arterial pressure farther on).
o Adventitia doesn't show vasa vasorum once you get to a sufficiently
small size-- the blood from the lumen can diffuse well enough on its
own.
• [In smaller muscular arteries:]
o Small (3-4 layers) tunica intima
o In the tunica media, the outer elastic lamina disappears, but the inner
remains.
• Notice the smooth muscle remains thick-- dilation and
constriction extremely important by this point.
o Adventitia: quite thin, can blend into surrounding tissue.
• [In smallest arteries immediately adjacent to capillaries ("arterioles"):]
o Tiny tunica intima, a few layers of smooth muscle, and a little
collagenous tissue on the outide rim.
o "Gatekeepers" for the capillaries (can constrict and shut off blood flow
to them).
• [In veins:]
o Still have a tunica intima, media and adventitia. Media is much, much
smaller than those of similarly sized arteries.
o Note that you can find valves or "flaps" in both veins and lymphatic
vessels.
o Occasionally a few smooth muscle cells in the media, but not many.
o Notice that the shape of the lumen often looks "collapsed" (acircular).
• [In lymphatics:]
o Also have irregular lumen (acircular), but have extremely thin walls
(just a thin endothelial layer, no media or adventitia) compared to
veins.
• [Important note: the endothelial cells of the tunica intima are pretty much
universally stratified squamous in shape (need to have things diffuse through
them quickly, need to be thin). If it's cuboidal or columnar, odds are good it's
not a vessel endothelium.]
• [Another note: size of red blood cells is always about 7-8 microns.]
• Explain the structure and function of the different types of capillaries:
o All capillaries have relatively wide lumens and, thus, slower blood flow.
 • This means that regulation of diameter is particularly
important, since it's easier to close off or open up capillaries
completely than larger vessels.
o Capillary structure:
• Small endothelial layer
• Specialized layer wrapped around endothelium: pericytes.
 If tissues are damaged, pericytes can generate smooth
muscle cells; mechanism not clearly understood.
o Three different types of endothelial layers:
• Continuous (standard) endothelium: continuous wall of
lumen; can have pinocytosis (vesicle-bound transport) across
it, but no free flow.
• Fenestrated endothelium: small windows in lumen, allows
plasma to freely diffuse through endothelium.
• In spleen and to some extent in the liver, can see
discontinuous endothelium: large holes in lumen or between
adjacent endothelial cells, allows entire red blood cells to
diffuse out of endothelium.
• Outline the unique functions of post-capillary venules
o Post-capillary venules: Where leukocytes interact with the endothelial
walls (actually, they break them selectively down, a process called
diapodesis) and leave the blood; also the action of histamines
regulating permeabilities of blood vessels occurs at the post-capillary
venules. Slow blood flow.
• Describe how blood flow is regulated in capillary beds
o As mentioned: smooth muscle "gateways" in arterioles.
• Discuss the general structure and functional significance of arterio-venous
shunts, portal systems, pampiniform plexus, anastomoses, and end arteries:
o Arterio-venous shunts: connecting vascular passages between
arterioles and post-capillary veins (controlled by smooth muscle
sphincters).
o [Metarteriole: System of vessels leading from arteriole through
capillary beds to the post-capillary veins. Also controlled by smooth
muscle sphincters.]
o Portal systems: From a capillary bed to a capillary bed, ie. hepatic
portal system. If that system is any indication, these probably aren't
primarily used for oxygenation of the second capillary system in the
chain.
o Pampiniform plexus: Countercurrent heat exchange between arteries
and veins. Say you're a scrotum. (did you say it?) You're hanging
around, so to speak, outside the body and you're a little cold for a
tissue system. What you don't want is for all that good warm blood
coming out to you to get cold and carry that cold back into the body,
cooling down the core. So you run your arteries right next to your
veins on the following philosophy: if the veins coming in are cold, and
the arteries going out are warm, then the incoming blood in the veins
will be warmed up (thus preventing core cooling) and the outgoing
blood will be cooled down (thus minimizing its heat loss). That
arrangement is a pampiniform plexus.
o End arteries: arteries that supply blood to a region that isn't supplied
by any other artery-- eg. kidney/lung arteries.
 o Anastomoses: come on, seriously, you better know what anastomoses
are. How did you pass anatomy?

Vocabulary
Artery, elastic; Artery, muscular; Arteriole; Aorta; Capillary; Capillary, continuous;
Capillary, fenestrated; Capillary, discontinuous; Tunica intima; Tunica media; Tunica
Adventitia; Post-capillary venule; Diapedesis; Muscular vein; Venous valves