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Fall I – Summary Project

TABLE OF CONTENTS:

BLOCK I: CORE CONCEPTS

CHAPTER 1: MOLECULAR BIOLOGY CHAPTER 2: CELL BIOLOGY CHAPTER 3: GENETICS CHAPTER 4: GENERAL HISTOLOGY CHAPTER 5: MUSCLE CHAPTER 6: NERVOUS SYSTEM CHAPTER 7: INTRO TO EMBRYOLOGY CHAPTER 8: ANATOMY & RADIOLOGY OF EXTREMITIES

BLOCK 2: CARDIAC, RESPIRATORY, RENAL

CHAPTER 9: ANATOMY, EMBRYOLOGY, & RADIOLOGY OF THORAX CHAPTER 10: CARDIAC CYCLE

CHAPTER 11: EKG

CHAPTER 12: VASCULAR MICROANATOMY AND BIOPHYSICS CHAPTER 13: BLOOD & HEMOSTASIS CHAPTER 14: RESPIRATORY HISTOLOGY CHAPTER 15: RESPIRATORY PHYSIOLOGY CHAPTER 16: RENAL EMBRYOLOGY & RADIOLOGY CHAPTER 17: RENAL HISTOLOGY CHAPTER 18: RENAL PHYSIOLOGY

BLOCK 3: GI, METABOLISM, NUTRITION, ENDOCRINE, REPRODUCTION

CHAPTER 19: ANATOMY & EMBRYOLOGY OF GI TRACT CHAPTER 20: GI HISTOLOGY CHAPTER 21: GI PHYSIOLOGY CHAPTER 22: METABOLISM CHAPTER 23: NUTRITION CHAPTER 24: ENDOCRINE HISTOLOGY CHAPTER 25: ANATOMY & EMBRYOLOGY OF REPRODUCTION CHAPTER 26: MALE REPRODUCTION CHAPTER 27: FEMALE REPRODUCTION CHAPTER 28: INTEGRATIVE SEMESTER TOPICS

SChapter One - Molecular Biology

BLOCK I – CORE CONCEPTS THREAD: BIOCHEMISTRY & GENETICS

CONTRIBUTORS: ALEX SWEENEY, ELIZABETH MCQUITTY & ZAINA AL-MOHTASEB

TABLE OF CONTENTS:

GENE I

RAM REDDY

GENE II

RAM REDDY

GENE III

RAM REDDY

GENE IV

RAM REDDY

GENE V

RAM REDDY

GENE VI

RAM REDDY

PROTEIN TAXONOMY PROTEIN II PROTEIN III PROTEIN IV PROTEIN V PROTEIN VI

MIKE SCHMID MIKE SCHMID MIKE SCHMID MIKE SCHMID MIKE SCHMID MIKE SCHMID

GENE I

- DNA synthesis (semi-conservative replication) RNA synthesis (transcription 5’-3’ with leading and lagging strand) protein synthesis (translation)

- C:G base pairs are harder to break because they are connected by 3 H-bonds. A:T pairs have 2 H-bonds…usually promoter regions/origins of replication are AT rich so that less energy is required to start replication.

- GC rich regions have a high melting temperature compared to AT rich regions

- DNA synthesis requires template, primer with 3-OH, building blocks (5’ dNTP’s), energy (p-p bonds), and DNA polymerase.

1. Mg and buffer also reqd. if done in a test tube.

2. 2 replication forks in prokaryotic replication (circular DNA).

3. DNA polymerase makes few mistakes due to 3’-5’ exonuclease activity

a) Other polymerases: taq polymerase (heat stable, non-proof

reading DNA dependent DNA polymerase), reverse transcriptase (RNA dependant DNA polymerase), telomerase (RNA dependant DNA polymerase)

i. Telomerase: prevents telomere shortening. Cancer cells become immortal due to telomerase activity. It uses its own RNA template to elongate DNA

4. Topoisomerase I relieves strain due to supercoiling (gyrase in bacteria

and Topo II in circular DNA)

- DNA is packaged into condensed chromatin (inactive genes) or extended chromatin (active genes)

- DNA is found in a:

1)

B form (Biological form – right handed, 10bp/turn)

2)

A form (right handed, 11bp/turn, ssDNA,

3)

DNA:RNA and RNA:RNA) Z form (left handed 12bp/turn, active chromatin)

-DNA repair can be achieved by endo and exo-nucleases - methylation is commonly used to distinguish the parent strands

- DNA can also be repaired by ligation of broken strands and using the second chromosome as a template.

- P53 prevents cell replication before DNA repair has occurred. Cells deficient in P53 can become cancerous.

GENE II

- siRNA’s are used as a defense mechanism in lower eukaryotes and plants

- -Dicer cleaves 21 NT long dsRNA from foreign RNA.

- -siRNA is amplified and spread

- -RISC pairs siRNA with viral mRNA for clevage

- -Host DNA is protected by methylation

- -For more info on siRNA, go to www.fuzzymittens.com/ms1 , click on the link for core concepts, and find the iRNA ppt. in the cell bio folder

- Plasmid cloning vectors

- -Plasmid contains origin of replication, antibiotic resistance gene and polylinker region (site where polylinker DNA insertion occurs).

- -New DNA, with sticky ends, is annealed to plasmid.

- -Amplification occurs with antibiotic (the plasmid should be resistant) as the selection marker.

- -You can prepare a genomic library in a plasmid vector.

- Lambda phage cloning vectors and yeast artificial chromosomes

- Both of these accommodate larger pieces of DNA.

- Hybridization or antibody usage helps you to identify the specific DNA insert.

- Genomic libraries vs. cDNA libraries vs. cDNA expression libraries:

- Genomic libraries have introns, promoters, non-transcribed spacer DNA, etc.

- cDNA contains only sequences complementary to post-modification mRNA (ready to be translated into protein)

- cDNA expression libraries are composed of cDNA’s with the requisite DNA sequences for expression in an artificial system

- DNA inserted into a cloning vector is isolated by using complimentary endonucleases

GENE III

2 ways to produce DNA probes:

1. Separate DNA strands and add radioactive dNTP’s

2. Use a polynucleotide kinase to transfer radioactive phosphate to the 5’ end of DNA.

DNA sequencing

1.

Chemical method (Maxam-Gilbert):

4 tubes used (A, C, G, T)

Use a chemical that will attach to the designated nucleotide and break the strand

Result: a bunch of different sized strands that are sequenced

DNA footprinting also uses chemicals to determine where specific proteins bind

2.

Enzymatic method (Sanger’s dideoxy)

Make 4 different vials (same as above) and include specific dideoxy, “chain terminating” nucleotides in each vial. Result: same as chemical.

Anti-viral drugs (AZT) can work by this same dideoxy method

3.

Automated sequencing

Uses fluorescent tags on dideoxy nucleotides.

The stability of a DNA hybrid is directly proportional to its base pair matching. More perfect hybrids are more stable, so you can separate less perfect hybrids with high temperature and NaCl concentration.

Blots (Southern uses DNA, Northern uses RNA, and Western uses proteins)

Electrophoresis to separate different size fragments

Heat applied to denature and separate all fragments (i.e. dsDNA ssDNA)

Radioactive probe (complimentary to target sequence) added to the filter and hybridization occurs.

The sample is exposed to film so that you can identify hybrids.

Restriction fragment length polymorphism is used to detect mutations in gene sequences. It involves cleavage of an isolated DNA fragment and identification of the digested pieces.

1. Southern blotting used

2. useful for prenatal screening, sickle cell detection and phenylketonuria

PCR – polymerase chain reaction is used to make copies of genetic info.

1. Heat to separate strands

2. Add primers to hybridize and add dNTP’s

3. Heat and use Taq polymerase to transcribe

4. Repeat

Variable numbers of tandem repeats (VNTR). Unique sequences outside of the coding regions of DNA are amplified for identification purposes

Transcription (5’-3’)

GENE IV

Synthesis of RNA from DNA templates.

Enzyme – RNA polymerase

Helicase and topo I are needed for accurate transcription

Promoters, enhancers, repressors and insulators are all used to regulate

Most regulation occurs at the level of initiation. Regulation can be negative or positive.

Prokaryotes have no nucleus so transcription and translation are coupled. 1 strand of Prokaryotic mRNA produces multiple proteins (polycistronic).

Eukaryotic transcription and translation occur in nucleus and cytosol, respectively. Eukaryotic mRNA is monocystronic.

All genes are present in every cell, but differential expression causes functional variation.

Methylation on promoters causes inactivation

Acetylation on histones causes activation

Both demethylation of promoters and acetylation of histones are necessary to activate gene transcription

Steroids hormones alter gene expression (look at Block 3: Endocrine)

Eukaryotic mRNA processing

Eukaryotic mRNA is capped (5’) and polyadenalted (3’). These modifications enable the mRNA to exit the nucleus.

Introns (70% of gene) are removed. Alternate and trans-splicing can give you many mRNA’s.

7 subunits of snRNA come together to make snRNP which identifies the splicing site.

GENE V

Overall: Translation and Translation Control

I. Translation = synthesis of proteins.

a.

mRNA to protein: need mRNA, ribosomes, tRNAs, amino acids, several protein factors, energy (ATP, GTP)

b.

tRNAs have 2 functions: 1. bind correct AA @ 3’ end; 2. bind triplet codon on mRNA using anti-codon segment.

c.

Wobble hypothesis: 3 rd position in anti-codon pairs loosely, so imperfect pairing occurs.

d.

Eukaryotic vs bacterial ribosomes: Es are bigger!

e.

Positioning ribosome correctly on the mRNA: in bacteria, 16S segment binds 3’ end of mRNA; in eukaryotes, ribosome starts at 5’cap and scans for start codon (AUG)

i.

Sometimes start at IRES = internal ribosome entry site.

f.

Initiation: initiation factors aid small ribosomal subunit, with initiator tRNA bound (always Met for AUG site), to find start codon; then large ribosome subunit binds, then next tRNA; now AA can be transferred and translation begins.

g.

Elongation: each new AA residue gets transferred to growing chain. (believed to be catalyzed by rRNA: a rare ribozyme action).

h.

Termination: Release factors are like tRNA but without an AA attached:

when RF binds to ribosome, protein is released.

i.

Polysomes – lots of ribosomes translating single mRNA at once; on RER surface (secretory proteins) or in cytosol (soluble proteins).

II. Post-translation: proteins are folded as they come out of ribosome (see cell biology lectures) and proteins undergo post-translational modifications (see biochem lectures).

III. mRNA stability and Translational Control

a. mutations:

i. silent – at protein level, no difference

ii. missense – protein still translated but with an AA substituted.

iii. nonsense – non-functional protein or truncated (mutation causes premature stop codon).

iv. Addition/deletion – add or delete single nucleotide

v. Frame-shift – generally results from addition or deletion.

b. Stability:

i. 5’cap: 5’-5’ linkage: protects mRNA

ii. UTRs – UnTranslated Regions at 5’ or 3’ end of mRNA confer stability and places for regulatory proteins to bind. Eg: transferrin (5’UTR) and its receptor (3’UTR): aconitase (itself inhibited by Fe molecules) binds both UTR regions, so in low Fe,

inhibits TR translation and stabilizes receptor mRNA to bring Fe into cell. Converse in Fe overload: TR translated and receptor mRNA degrades

Overall: Biotechnology

GENE VI

I. Recombinant proteins: (example: insulin)

a. Use plasmid to clone insulin gene into bacteria. Cell culture makes insulin; once purified is perfect replacement therapy.

b. Can get recombinant proteins in milk; expression of recombinant genes limited to mammary glands. Resolves some problems of making human genes ‘work’ in prokaryotes. These proteins get purified from the milk.

c. Recomb proteins in use today: TPA (tissue plasminogen activator), blood clotting factors, interleukins, enzymes for research, safer vaccines.

II. Use of transgenic mice in research

a. Humans and mice have similar genes and similar development.

b. Options: gene replacement, gene knock-out, gene addition.

c. Accomplish by growing mouse stem cells in culture, altering DNA, then introducing stem cells into early embryo; breed 2 nd generation to get pure-

breds.

III. Genetic therapy

a. ADA (adenosine deaminase) deficiency causes SCID (severe combined immune deficiency). Tx: take stem cells or Tcells from pt, add the ADA gene, then reintroduce patient’s cells into system.

b. Portions of HIV genome used as very effective vector. (used for ADA) Viruses successful at incorporating into genome – randomly or at LTRs (long tandem repeat segments).

c. Cloning sequence using viral vector: LTR – Enhancer – promoter – gene cDNA – poly(A) signal – LTR. (use promoter appropriate to target tissue).

IV. Tailor-made anti-cancer drugs:

a. Example is GLEEVAC: treats chronic myeloid leukemia. Disease caused by abnormal chromosome translocation resulting in a fusion kinase present only in cancer cells. GLEEVAC is tailor-made to inhibit this activity. Patient takes pill once a day until all blood cells should have turned over and all cells with mutation have died. Pt then cancer-free.

V. Oncogene discovery – from cDNA library. Can note cell populations with over-growth phenotype. Led to discovery of Ras oncogene.

VI. siRNAs

a. The low-down: siRNAs are ‘small interference RNAs’. In plants, double stranded RNA is chopped by an RNase (dicer) into fragments 23nts long. These mark other incoming RNA that is complementary so that dicer can cut them as well. RNA – dep RNA polymerase makes more of these short

fragments to function in future defense. Humans have dicer (but not the

RNA-dep RNA polymerase).

viral or cancer mRNAs in human disease.

Research is ongoing to use dicer to chop up

PROTEIN TAXONOMY

Overall: Protein Structure

I.

Amino Acid structure

a. All AAs are chiral with a side chain (giving specific AA), an amino group (NH2), a carboxy group (COO-) and an H. (exception is glcine with 2 Hs).

α carbon side chain R-CH-CO 2 - NH 3 + amino group
α carbon
side chain
R-CH-CO 2 -
NH 3 +
amino group

carboxyl group

II.

b. Learn your amino acids – they’re in your syllabus. (note that categories such as non-polar vs. polar, aliphatic, aromatic, uncharged polar and charged polar actually matter and are worth learning also).

Acid-Base behavior of AAs

a. The charged AAs are acids and bases; usually they are on the surface of a protein and form salt-bridges with each other.

b. Acid – proton donor; base – proton acceptor.

c. Henderson-Hasselbach: relates the pKa of a buffer and the ratio of acid and base present to the resulting pH of the solution.

pH = pK a + log ([A - ]/[HA])

d. Titration curves: pKa = mid-point along the flat segment of these curves: at this point, half of the molar amount of titrated group is hydrogenated, half is de-hydrogenated. Work any problems from there, depending on where you start (pH) and whether you’re adding acid or base.

III.

IV.

(pH) a nd whether you’re adding acid or base. III. IV. The peptide bond a. Peptide

The peptide bond

a. Peptide bond is planar; rotation only around alph-Carbon and side chain elements.

Primary structure

a. Each protein: unique number and sequence of amino acids coded from DNA to RNA to protein.

b. Sequence conservation: percent identity across species.

PROTEIN II

Overall: More Protein Structure.

I.

Key concepts:

a. Interactions between sidechains that are far apart in sequence cause protein to fold into compact shape.

b. Inferior of protein typically hydrophobic, compact with few holes.

c. Exterior typically hydrophilic and charged residues typ on surface.

d. Majority of N-H and C=O are involved in hydrogen bonds which conver secondary structure.

e. Specialized functional regions may form – catalytic sites.

II.

Secondary structure: - when sequence folds in regular, repeated fashion.

a.

Helix structures, sheets, turns, random structure.

III.

Super-secondary structure - Recognized sequences of known secondary

 

structures.

 

a.

helix-turn-helix, beta-meander, beta-alph-beta structure.

IV.

Tertiary structure

a.

3-D structure of single protein molecule; brings together different parts of primary and secondary structure.

V.

Quaternary structure - describes assembly of multiple enzyme subunits.

a. Hydrophobic interactions important; also, charge and H bond interactions.

b. Association of multiple subunits allows regulation of activity, info transmission (allostery and cooperativity), and creation of multiple assemblies using multiple subunit types.

VI.

Protein stability

a.

Proteins only marginally stable; this confers flexibility for enzyme activity.

VII.

Protein folding (key to Cystic Fibrosis and other diseases)

a. Mechanism directing protein folding not known.

b. Unfolded proteins will collapse then refold from compact structure.

c. Not all proteins refold spontaneously.

d. Molecular chaperones (bind unfolded proteins to prevent mis-folding)

 

and Foldases (in ER; forms disulfide bonds) aid process.

VIII.

Post-translational Modifications:

a. Disulfide bonds (2 Cys residues) – stabilize structure (eg: insulin has 3)

b. Phosphorylation (Ser, Thr, Tyr) – think metabolism control mechanisms

c. Hydroxylation

d. Glycosylation – alters stability, signal recognition

e. Carboxylation (like Acetyl CoA Carboxylase)

f. Fatty acylation/prenylation – increases hydrophobicity of protein

g. Proteolysis – all zymogens

Protonation/deprotonation – changes in pH cause this

PROTEIN III & IV

HEMOGLOBIN:

Has heme group bound by hydrophobic interactions, and iron as Fe 2+ , which binds oxygen.

• α 2 β 2 tetramer.

Positive cooperativity in binding causes greater “steepness” about the P 50 point; successive oxygens are easier to add. This increases the amount of oxygen bound in the lungs and the amount delivered in the tissues.

The Bohr effect: decrease pH, decrease Oxygen binding to hemoglobin; Hemoglobin gives up oxygen more readily at low pH; saturation curve shifts to the right because it is less oxygen bound/saturated even at the same PO2.

H+ takes the place of oxygen; hemoglobin is a proton carrier between the lungs and the tissues, important buffer.

CO2 also binds deoxyhemoglobin in tissues and is carried to lungs.

BPG binds deoxy Hb and inhibits Oxygen binding in presence of BPG. Fetal HB doesn’t bind BPG as well. Therefore, higher oxygen affinity than adult Hb.

BPG is an intermediate in glycolysis, which indicates working tissue in need of oxygen for oxidative phosphorylation after glycolysis.

pH 7.4 pH 7.0
pH 7.4
pH 7.0

MYOGLOBIN:

Lower pH releases oxygen Higher oxygen releases protons

Monomer, similar to one of Hb’s subunits

Shows saturation behavior at Oxygen binding site; increases linearly to p50, then begins to level off.

Releases oxygen at lower pO2 than Hb; takes oxygen to very starved tissues, ex. Red/type I muscle.

Enzyme kinetics:

Enzymes are catalysts to reach equilibrium faster.

Very specific-few substrates

Active site equals binding domain plus catalytic domain.

Enzymes are controlled by amount of enzyme, posttranslational modification, and quantity of substrates and products.

Isozymes are different proteins from different genes that catalyze same reactions, often in different tissues.

CPK found after heart attack (cardiac cells die or after muscle trauma); CPK-BB-brain and lungs. MB-heart MM-skeletal muscle (different isozymes can tell you which tissue has the problem).

At low substrate concentrations:

Velocity of enzyme reactivity is directly proportional to [S], then becomes limited by the concentration of the enzyme.

Km=[S] at ½ Vmax= Michaelis constant, perenzyme, sort of an indicator of enzyme’s affinity for its subsrate.

Michaelis-Menten:

V=Vmax [S]/Km+[S] (Usually expressed in umol/minute of substrate consumed)

V or Vmax/[E] = specific activity of enzyme.

V varies the most in the range where [S] is less than Km; therefore most enzymes in the body have Km close to physiological [S], so their activity can be finely regulated by changing [S].

Lineweaver-Burk double reciprocal plot:

Y intercept = 1/Vmax Slope = Km/Vmax

Inhibition by products or product analogs gives reversible inhibition. Bind to active site/occupy it.

reve rsible inhibition. Bind to active site/occupy it. Competitive inhibitor : effector binds E. Can be
reve rsible inhibition. Bind to active site/occupy it. Competitive inhibitor : effector binds E. Can be
reve rsible inhibition. Bind to active site/occupy it. Competitive inhibitor : effector binds E. Can be
reve rsible inhibition. Bind to active site/occupy it. Competitive inhibitor : effector binds E. Can be

Competitive inhibitor: effector binds E. Can be fought off by increasing [S] and thus Vmax does not change. Changes slope, Km/Vmax, by increasing [S] required to reach Km. (Either substrate or inhibitor binds)

Non-competitive inhibitor : effector binds E and ES; cant be fought off by increasing [S]
Non-competitive inhibitor : effector binds E and ES; cant be fought off by increasing [S]
Non-competitive inhibitor : effector binds E and ES; cant be fought off by increasing [S]
Non-competitive inhibitor : effector binds E and ES; cant be fought off by increasing [S]

Non-competitive inhibitor: effector binds E and ES; cant be fought off by increasing [S] because inhibitors and substrates bind to different sites. Changes slope and intercept.

Allostery: change enzyme function by changing enzyme shape

Cooperativity: allostery via substrate; + or – Produces sigmoidal kinetics, see hemoglobin.

+ or – Produces sigmoidal kinetics, see hemoglobin. Phosphofructokinase : an example of allostery. Term
+ or – Produces sigmoidal kinetics, see hemoglobin. Phosphofructokinase : an example of allostery. Term

Phosphofructokinase: an example of allostery.

Term

Expt'l hallmark

Structural cause

'zif

Cooperativity

"s-shaped curve" (n>1)

Allostery (using

[S]

"creates" new f'n

substrate)

enz.

Comp. Inh.

High [S] can overcome it (1/vmax same)

1.

S and I Bind at

[I]

"destroys" f'n

same site

enzyme

 

2.

allostery

Non-comp. Inh.

High [S] cannot overcome it

S and I bind at different sites

 

Allosteric act.

Enzyme "works better" esp at low [S]

Allostery (using

Activator "converts" enz to f'n form

activator)

Allosteric inh.

Enzyme "works worse" esp at low [S]

Allostery (as in 2.)

"Bohr effect" or BPG

PROTEIN V & VI

Catalysts by enzymes:

Specificity constant: efficiency of enzyme finding and converting substrate.

Specificity constant = Vmax/Km

Lock and key: specificity (the substrate fits specifically into the active site). Induced fit: binding substrate changes shape of enzyme and brings catalytic site into the right geometry for activity.

Enzymes lower activation energy required for transition state. (G act ) (don’t change overall equilibrium)

( ∆ G a c t ) (don’t change overall equilibrium) Catalysis mechanisms : 1. Organization
( ∆ G a c t ) (don’t change overall equilibrium) Catalysis mechanisms : 1. Organization
( ∆ G a c t ) (don’t change overall equilibrium) Catalysis mechanisms : 1. Organization

Catalysis mechanisms:

1.

Organization of substrates spatially:

mechanisms : 1. Organization of subs trates spatially: 2. 3. 4. 5. Solvation effects/microenvironment excluding
mechanisms : 1. Organization of subs trates spatially: 2. 3. 4. 5. Solvation effects/microenvironment excluding
mechanisms : 1. Organization of subs trates spatially: 2. 3. 4. 5. Solvation effects/microenvironment excluding

2.

3.

4.

5.

Solvation effects/microenvironment excluding water

Covalent catalysis/alternative mechanism

Coenzymes/vitamins:

Alternative (non-AA) side groups like aldehydes, ketones, metals, oxidizing and reducing agents.

Rational Drug Design:

The old way:

Accident

Screening

The new way:

Isolate target enzyme or receptor first

Screen compounds for binding inhibitors and denerage lead compound

Determine lead compound’s structure and a synthetic strategy for compound.

Optimize structure (ex combining bits for greater inhibitor efficiency to make a multisubstrate analog).

Penicillin:

to make a multisubstrate analog). • Penicillin: Old way: Inhibited cell wa ll crosslinking but was

Old way: Inhibited cell wall crosslinking but was susceptible to B lactatmase

New way: Cephalosporins are inhibited but not susceptible to B lactamase.

Substrate analogs: resemble substrates, 5 reactions. HIV blocked by dideoxy base which is incorporated into DNA and prevents further polymerization. Ex. AZT, DDI, DDC. These work because they don’t inhibit mammalian DNA polymerase, only RNA reverse transcriptase action.

DNA polymerase, only RNA reve rse transcriptase action. AZT (azidothymidine), DDI (dideoxyinosine) and DDC

AZT (azidothymidine), DDI (dideoxyinosine) and DDC (dideoxycytidine)

Multisubstrate analogs: More efficient. Cancer blocked by PALA, which inhibits pyramidine biosynthesis for DNA replication.

Transition state analogs: Very specific, very tight binding to enzymes. AIDS protease that activates viral preprotein “protease inhibitors.”

De novo design:

Get 3D structures (positive charge and hydrophobicity info) of target.

Use computer to screen known compounds, synthesize molecule.

Chapter Two – Cell Biology

BLOCK 1 – CORE CONCEPTS THREAD: CELL BIOLOGY/HISTOLOGY

CONTRIBUTORS: LAURA HANSON, MICHAEL STEWART, LAURA GARCIA, SAGARI PONNURU, ADVA BUZI & ROBERT DOMINGO

TABLE OF CONTENTS:

CELL MEMBRANES I CELL MEMBRANES II CELL MEMBRANES III

CYTOSKELETON JUNCTIONAL COMPLEXES

CELL ORGANELLES I CELL ORGANELLES II CELL ORGANELLES III CELL ORGANELLES IV CELL ORGANELLES V

CELL SIGNALING I CELL SIGNALING II CELL SIGNALING III CELL SIGNALING IV CELL MOTILITY

JEANETTE KUNZ JEANETTE KUNZ JEANETTE KUNZ

DAVID ROWLEY DAVID ROWLEY

RICHARD SIFERS RICHARD SIFERS RICHARD SIFERS RICHARD SIFERS RICHARD SIFERS

ERIC KLANN ERIC KLANN ERIC KLANN ERIC KLANN DAVID ROWLEY

CELL MEMBRANES I: STRUCTURE AND FUNCTION

PHOSPHOLIPIDS: BASIC STRUCTURE

I: S TRUCTURE AND F UNCTION PHOSPHOLIPIDS: BASIC STRUCTURE -most common kind is phosphoglyceride (aka

-most common kind is phosphoglyceride (aka glycerophospholipid) -is glycerol backbone + 2 fatty acids + [phosphate + head group]

WHY PHOSPHOLIPIDS ARE SUITABLE AS MAJOR COMPONENTS OF MEMB’S -amphipathic so in solution polar region is in contact with water, and non-polar region is away from water. -shape allows them to form monolayers, micelles, and bilayers -lateral fluidity HOW MEMBRANE FLUIDITY IS INFLUENCED

1. Longer acyl tail of fatty acids= increased Van der Waals interactions= higher Tm

2. More unsaturation= kinks that are harder to pack together= lower Tm

3. Cholesterol= bulky structure hard to pack and hard to move fast= wider range Tm

hard to pack and hard to move fast= wider range Tm DIFFERENT TYPES OF MEMBRANE PROTEINS

DIFFERENT TYPES OF MEMBRANE PROTEINS Peripheral: Soluble proteins that associate with head groups of membrane or other proteins.

Electrostatic interactions that are easily dissociated with high salt or change in pH

Integral: Insoluble proteins that penetrate or traverse the membrane.

Removed with detergents. May be single or multi pass (sequences of hydrophobic helices). Lipid-Anchored: Proteins with covalently attached lipid anchor in the bilayer

May be fatty acid or isoprenoid on the inner leaflet, or GPI on the outer FUNCTIONS OF MEMBRANE PROTEINS

1. Communication across membranes

2. Cell-cell and cell-matrix adhesion

3. Cell-cell recognition

4. Transport of compounds across membranes

FACTORS AFFECTING PASSIVE DIFFUSION

Membranes are differentially permeable and allow 1. Small nonpolar, 2. Hydrophobic, 3. Small polar uncharged molecules to pass through.

1. Flux is proportional to concentration difference across the membrane

2. Lipid solubility: partition coefficient= [concentration in bilayer]/[concentration in aq. soln.]

3. Membrane thickness and surface area (constant in animals)

4. Temp- higher temp= higher velocity of molecules

5. Size- bigger=slower

Rate of passive diffusion is described by Fick’s Law: J=-P(Co-Ci) (doesn’t apply to charged molecules) WATER FLUX BY OSMOSIS REGULATES TONICITY Ingested water will distribute between extracellular and intracellular compartments. Water will flow from lower solute concentration to higher until the osmotic pressures on both sides of the membrane are equal. Donnan Effect: If a cell contains concentrated amounts of large molecules, water will tend to flow into the cell and change the volume. Water can flow across membranes through the membranes or by

facilitated diffusion through water pores. The large molecules can’t pass, but Na+ and K+ ions can, so cells regulate their volume by pumping ions in the direction that will reduce osmotic pressure.

Na+ and K+ ions can, so cells regulate their volume by pumping ions in the direction
Na+ and K+ ions can, so cells regulate their volume by pumping ions in the direction

CELL MEMBRANES II: TRANSPORT

FACILITATED DIFFUSION VS. ACTIVE TRANSPORT Facilitated Diffusion (Passive transport)

Requires channels—simultaneously open to both sides; or carriers (permeases)—bind release

No energy utilized

Selective

Multi-pass with hydrophilic residues towards the channels

Saturable

Active Transport

Moves molecules UP conc. Grad.

Energy utilized

Saturable

Used to generate a membrane potential

Uniport or Co-transport (sym or anti)

Specific

PRIMARY ACTIVE TRANSPORT Uses the energy of ATP hydrolysis to make conformational change that transports the molecule through. #1 example: Na+/K+ ATPase

Pumps Na and K up their concentration gradients: 3 Na out for every 2 K in and every 1 ATP (antiport)

No ATP means Na and K gradients are lost (Ouabain, a steroid like drug, blocks the pump specifically and the gradients are lost)

Electrogenic (voltage gradient=stored energy)

Regulate osmotic balance, cell volume, and resting potential

• Electrogenic (voltage gradient=stored energy) • Regulate osmotic balance, cell volume, and resting potential
• Electrogenic (voltage gradient=stored energy) • Regulate osmotic balance, cell volume, and resting potential
• Electrogenic (voltage gradient=stored energy) • Regulate osmotic balance, cell volume, and resting potential

SECONDARY ACTIVE TRANSPORT Uses energy to establish a concentration gradient, then uses that gradient to transport other molecules up their concentration gradient. #1 Example: Na+/Glucose cotransporter

First step is Na/K pump

Na+ gradient from step one is driving force to transport glucose into the cell

ATP is indirectly used

Gut epithelial cells use this

Na+ goes down its conc grad into the cell, and glucose goes up

goes down its conc grad into the cell, and glucose goes up MANY DISEASES ARE CAUSED

MANY DISEASES ARE CAUSED BY TRANSPORT SYSTEM MALFUNCTION

1. Multi-drug resistance: ABC transporters that have highly conserved ATP binding domains, and are normally found in the liver, kidney, and intestine, and function to remove toxins. MDR gene is over expressed in certain cancers, and the ABC pumps pump out chemotherapeutic agents, causing drug resistance.

2. Cystic Fibrosis: ATP and cAMP sensitive Cl- channel becomes insensitive to cAMP. Cl- flux is disturbed in epithelia (eg lung).

3. Dropsy= Congestive Heart Failure: increasing intracellular Na+ will also increase Ca2+ levels which will increase force of contraction. Treatments will block or decrease the Na/K ATPase. Exs of drugs- Ouabain, Digitalis, Digoxin

CELL MEMBRANES III: ION CHANNELS

ION CHANNELS CAN SELECTIVELY ALTER MEMBRANE PERMEABILITY Non-gated channels are always open and are important in maintaining resting membrane potential. Gated channels are either open or closed in response to specific electrical, mechanical, or chemical signals. Ions flow rapidly when the channel is open (passive transport). Gated ion channels are categorized according to the kind of signal they respond to. The channels have specific amino acids that interact with the ions in

solution and determine which ions will pass (this is called the selectivity filter). Specificity isn’t explained by ion size or charge alone. BASIC TYPES OF ION CHANNELS AND HOW THEY ARE GATED

1. Ligand-gated: non-covalent, reversible binding of a specific ligand will directly or indirectly cause conf. Change in the channel. Ligands can be NTs that bind the extracellular face, or can be second messengers or enzymes that act on the cytoplasmic face of the channel (by binding or changing phosphorylation state). Ligand-gated channels allow rapid communication.

2. Voltage-gated channels: change in memb potential causes movement of charged regions of the channel and opens or closes the channel. These channels propogate electrical impulses in nerve and muscle.

3. Mechanosensitive channels: stretching or deformation of PM induces a change in the shape of the channel and closes or opens it.

IONIC BASIS OF MEMBRANE POTENTIAL AND ROLES OF ION CHANNELS Resting membrane potential = the difference in the ionic composition of the cytosol vs the surrounding fluid. (separation of charge=membrane potential).

Interior Na=15 K=150 Cl=10

Exterior Na=150 K=5

Ion channels allow the selective movement of the ions above (not the large anions) down their conc grad’s

creating a difference in electrical potential between the inside and outside of the cell. Extent of the electrochemical gradient determines the direction and extent of net charge movement.

large anions=65

Cl=110 large anions=.2

Resting potential depends mainly on K+ leak channels (passive movement) and the K+ gradient

MEMBRANE POTENTIAL VS. NERNST POTENTIAL VS. ACTION POTENTIAL Membrane potential= separation of charge/voltage difference b/w the inside and outside of the cell Nernst potential= equilibrium potential for any conc grad of a particular ion across a membrane, and is predicted by the Nernst Equation

across a membrane, and is predicted by the Nernst Equation Action potential= the membrane potential changes

Action potential= the membrane potential changes that occur during nerve impulse propogation

CYTOSKELETON

Cytoskeleton: Location Structure Function Diameter Microtubules Cytoplasm, 24nm (MT) cilia, flagella α & β
Cytoskeleton:
Location
Structure
Function
Diameter
Microtubules
Cytoplasm,
24nm
(MT)
cilia, flagella
α & β tubulin subunit
polymerizes to a helical MTs
(13 units/turn) “soda-straw
structure”. GTP cap at (+) end
grows faster than (-) end.
MTOC- microtubule
organizing center directs
polymerization. MAPs-
microtubule associated proteins
stabilize MTs by cross-linking
them into bundles
• Mitotic spindle: Roadway
for motors; + end attaches
Chromosomes; - at centriole.
Kinesin (- to +) Dynein (+ to -)
• Axon/Dendrites:
Roadway for motor proteins to
carry “cargo”
• Cilia/Flagella: 9 +2
arrangement of MTs, Dynein on
A tubule pushes B causing
bending
Microfilaments
Present in
Globular subunits (G- actin
monomer) organize into a
double stranded helix (F-actin
polymer). Actively
depolymerized and
polymerized in non- muscle
tissue. + end grows; - end
disassembles; termed
treadmilling. Capping actin w/
ATP stabilizes the polymer
• Muscle: forms
paracrystalline array with myosin
for contraction
5-7nm
(actin filaments)
every eukary-
otic cell
• Cell cortex beneath
membrane in most cells: aid
endocytosis, exocytosis, and cell
migration
• Cytoplasmic streaming-
associated with some organelle
movement
• Cytokinesis: associated
with myosin in mitotic cells
• Actin, like MTs, provide
force and motion via motor
proteins leading to contraction!
Intermediate Cell Type: Helical monomer wraps around another to form a coiled-coil dimer. Two coiled-coils
Intermediate
Cell Type:
Helical monomer wraps around another to form a coiled-coil dimer.
Two coiled-coils unite to form a staggered tetramer. Staggered
tetramers can unite. Then 8 staggered tetramers twist into an
intermediate filament.
10-12nm
Filament:
Keratin
Epithelium
Both Keratinized and Nonkeratinized
epithelium
Vimentin
Mesinchymal
cells
Fibroblasts, chondrocytes, macrophages,
endothelial cells, vascular smooth muscle
Desmin
Contractile
cells
Striated & smooth muscle (except vascular
smooth muscle)
Glial Fibrillary
Glial cells
Astrocytes
Acidic Proteins-
GFAP
Neurofilament
Neurons
Nerve cell body processes

Antimitotic Drugs Vinblastine: depolymerizes formed MTs, binds subunits of MTs and prevents polymerization by aggregating bound units into a paracrystalline array Taxol: accelerates formation of MTs and stabilizes them thus depleting the available tubulin for mitotic division Cochicine: colchicines-tubulin complex binds to growing (+) end of MT and stops MT growth.

JUNCTIONAL COMPLEXES

 

Structure:

Function:

Gap Junction

6 connexins create a connexon which is a pore connecting adjacent cells (2nm diameter)

Allow ions and molecules up to 1500 D flow down a gradient. Ca 2+ blocks gap junctions stopping flow

Tight junction (Zonula Occludens)

multi-protein complex btwn cells that binds directly to the integral occludin proteins and to the cytoplasmic actin cytoskeleton

Forms a seal around the cell which prevents flow of materials between epithelial cells (paracellular leakage)

Desmosome (Macula Adherens)

2

Disk shaped Cadherin

Provide firm adhesion between cells

transmembrane structures matched between cells. Attachment Plaque (12 proteins)

 

located on cytosolic side where intermediate filaments insert. Ca 2+ necessary.

 

Hemidesomosome

1

Disk shaped integrin

 

transmembrane structure that binds to the basal lamina (laminin and collagen type IV). Intermediate filaments attach to cytosolic side.

Bind the cell to the basal lamina

Adherent Junction

Junctional complex with Cadherin trans-membrane protein. Inserts into the cytoplasmic actin of the Terminal web

Encircles one cell and provides adhesion to neighbor.

Focal Contacts

Junctional complex between cell and basal lamina. Integrin is the transmembrane protein. Actin is the cytoplasmic anchor

Bind the cell to the basal lamina & ECM. Important for the motility of cells like fibroblasts. Not present in epithelial cells.

Facts to Know:

fibroblasts. Not present in epithelial cells. Facts to Know: • Desmosomes and hemidesmosomes attach to intermediate
fibroblasts. Not present in epithelial cells. Facts to Know: • Desmosomes and hemidesmosomes attach to intermediate
fibroblasts. Not present in epithelial cells. Facts to Know: • Desmosomes and hemidesmosomes attach to intermediate
fibroblasts. Not present in epithelial cells. Facts to Know: • Desmosomes and hemidesmosomes attach to intermediate
fibroblasts. Not present in epithelial cells. Facts to Know: • Desmosomes and hemidesmosomes attach to intermediate
fibroblasts. Not present in epithelial cells. Facts to Know: • Desmosomes and hemidesmosomes attach to intermediate

Desmosomes and hemidesmosomes attach to intermediate filaments like keratin, vimentin, or desmin; Tight junctions, adherent junctions, and focal contacts attach to actin.

Desmosomes and adherent junctions use cadherins; hemidesmosomes and focal contacts use integrins.

to actin . • Desmosomes and adherent junctions use cadherins ; hemidesmosomes and focal contacts use

CELL ORGANELLES I. THE NUCLEUS.

Heterochromatin-highly condensed / Euchromatin-less condensed (transcriptionally active)

Chromatin=complex of DNA, histones, and nonhistone proteins in eukaryotic cells. —the material of which chromosomes are made.

Histones = one of a group of small abundant proteins rich in positively-charges amino acids that form the nucleosome with the DNA of eukaryotes

Basic Unit of DNA packing is a Nucleosome:

o consists of the core histones (H2A, H2B, H3, and H4), the linker histone (H1), and DNA

Making ribosomes:

o

Requires nuclear transport in two directions: (1) to bring ribosomal proteins to nucleolus (2) to export newly assembled ribosomal subunits

o

A lot of cellular machinery is devoted to making ribosomes

The nuclear pore complex mediates membrane permeablility

NLS importin importin NLS
NLS importin
importin
NLS

Nuclear

Import

After import, importin recycles back to cytoplasm for another round of import.

In cytoplasm, importin binds import cargo, carries it through the nuclear pore complex, and dissociates (releasing cargo) in the nuclear interior.

o

All movement of macromolecules between the nucleus and cytoplasm occurs through these structures. Because of its large size, the NPC allows free diffusion of macromolecules up to (45-60 kDa)

o

Signals mediate transport <<Nuclear localization Sequence>> & <<Nuclear Export Sequence>>

o

These targeting signals on nuclear cargo are recognized

by soluble receptor/carriers called karyopherins (“nuclear carriers). <<Import

karyopherins = importins>> <<export karyopherins= exportins>>

o

Over 20 karyopherins exist in human genome. Each receptor recognizes a different type of targeting signal.

o

mRNA export: very tight relationship between the splicing and processing of a newly transcribed mRNA and its export

If one inhibits splicing, then export is inhibited also. In general mRNA export remains poorly understood because of complicated link between mRNA maturation and export

Relevance to disease: HIV Virus Life Cycle

o

Some HIV viral proteins contain NLSs. This allows viral nucleoprotein complexes to be actively imported into the nucleus of non-dividing cells.

o

Export of viral mRNA also uses host machinery.

o

Many viruses enter the nullius to gain access to the DNA replication machinery. The host machinery is used for the viral DNA to integrate into the genome.

CELL ORGANELLES II: THE ENDOPLASMIC RETICULUM

The rough endoplasmic reticulum: where proteins enter the secretory pathway

reticulum: where proteins enter the secretory pathway Three-dimensional reconstruction of a region of the smooth

Three-dimensional reconstruction of a region of the smooth and rough ER in a liver cell.

Alberts Fig. 12-38

All ribosomes are equal. The translated mRNA determines the free vs. membrane bound state. <<Proteins contain targeting sequences or “zip codes” that mediate

sorting to the correct internal organelle>>

o

Signal Sequence ER (Mediates translocation of nascent secretory and membrane proteins into the ER, the first compartment of the secretory pathway)

o

Signal sequences are stretches of 20 hydrophobic amino acids (# of AA required for a polypeptide to span a lipid bilayer)

Covalent modifications assist protein folding (conformational maturation) and stabilize native structure

o

Asparagine-linked glycosylation

o

Disulfide bonds (do not form in cytoplasm because of the reduing atmosphere (glutathione).

o

Some membrane proteins lose their transmembrane domain and gain a GPI anchor

Final modification-protein folds (aided by chaperones of the ER lumen) <<Correct folding, and release form chaperones, is necessary for exit from the ER…FIRST HALF OF THE CHECKPOINT>>

Fxn’s for the smooth ER

o

Abundant in liver cells, because it contains some of the membrane-bound enzymes used to degrade certain hormones and to neutralize noxious substances such as alcohol and barbiturates

o

When large amounts of certain compounds, such as the drug Phenobarbital enter the system, the smooth ER in the liver doubles in surface area in a couple of days. (This change reflects rapid synthesis of detoxification enzymes, and the need for more membrane in which to place them.)

o

Synthesis nearly all of the major classes of lipids, including phospholipids and cholesterol, required of r the production of new cell membranes.

o

Most cellular lipids are synthesized on smooth ER. They reach the other

membranes in the cell by one of three ways Vesicles bud off and move along cytoskeletal elements by motor proteins

to other membranes with which they fuse Diffuse to the rough ER which is continuous with the smooth Er

By transfer of proteins (to take lipids to organelles like mitochondria that don’t receive vesicular traffic from the ER

Smooth ER is common in cless engaged in steroid synthesis and lipid metalbolism because it contains some of the membrane bound enzymes required for these processes.

CELL ORGANELLES III: GOLGI & LYSOSOMES

Whether to release an ER-situated protein to the Golgi complex represents a crucial prost- translational checkpoint.

The efficiency of protein clearance, underlies many loss-of-fxn and gain-of-toxic-fxn disorders

o

Cystic Fibrosis: Most common fatal genetic disease in the US today (1/3300 live births)

o

Repeated chest infections result in progressive loss of lung fxn, the major cause of premature death.

o

The most common mutation leading to cystic fibrosis results from deletion of Phe 508 in CFTR.

o

The mutant protein slightly misfolds, is trapped in the ER, and then degraded.

o

The mutation does not appear to affect the ability of the CFTR protein to transport ions, indication that the mutant protein would probably fxn if delivered to the plasma membrane/

o

Alpha 1-antitrypsin deficiency: A genetic disease responsible for chronic obstructive lung emphysema

o

Also responsible for liver cirrhosis and is one of the most common genetic causes of childhood liver transplantation.

o

Both disorders are causes by mutations in the alpha 1-antitrypsin protein, Accumulation (in hepatocytes) of the undegraded mutant protein can lead to cirrhosis (gain-of-toxic-fxn disorder). Its hindered secretion is responsible for the lung disease (loss-of-fxn disorder).

Misfolded ER-situated proteins are dislocated into cytoplasm for degradation by proteosomes (prolonged duration of non-native structure = degradation).

Protein sorting signals specify: (1) ER retention (of native proteins) <<KDEL = retention signal>> (2) Golgi retention signal (different signals for cis, medial, & trans Golgi) (3) Signals for diverting secretory proteins to regulated secretory vesicles

Unless signals specify ER-retention, correctly folded proteins are delivered to the Golgi complex by default.

Golgi consists of flatted membrane-bound cisternae and resemble a stack of plates.

The tetanus toxin (protease) cleaves receptors used for the movement of synaptic vesicles in inhibitory neurons (blocks secretion of inhibitory NT = convulsive contractions of skeletal muscle lockjaw). Lysosomes.

Membrane bound organelle, acid pH (special coating on interior), degrades proteins, lipids, carbohydrates, DNA, RNA

The modifications of attached sugars divert most newly synthesized acid hydrolases out of the secretory pathway, and into lysosomes. (Mannose 6-phosphate (M6P) is a signal that targets newly synthesized proteins to lysosomes)

Lysosomal storage diseases: when a cell lacks one of the hydrolytic enzymes, lysosomes accumulate material that is normally destroyed

o

Tay-sachs disease is especially prevalent among Jews, particularly among those of Eastern European origin (1/30 reported chance)

o

Affected infants appear nml at birth (manifest relentless motor & mental deterioration & increasing dementia at 6 mths) death at 2-3 yrs

o

Results from an absence of hexoamionidase A (breaks down glycolipids)

o

Histological examination shows neurons ballooned with cytoplasmic vacuoles (distended lysosomes filled with glycolipid) >> progressive destruction of neurons

CELL ORGANELLES IV: MITOCHONDRIA AND PEROXISOMES

Mitochondria = generates cellular energy

Mitochondrial translocation machinery

“Translocator of Outer Membrane” “Translocator of Inner Membrane”
“Translocator of Outer Membrane”
“Translocator of Inner Membrane”

OXA complex mediates insertion into

innermembrane.

Signal sequence for mitochondrial import (cytochrome oxidase)

Chaperones are required both in the cytosol and inside the mitochondria to achieve protein import

# and placement of sorting signals dictates site of protein residence

When mitochondria fail, less energy (ATP) is generated

with the cell and cell death follows.

Damage is most notable in the following organs/tissues brain, heart, liver, skeletal muscle, kidney and endocrine system

Peroxisomes fxns = oxidation of fatty acids and other lipids, oxidation of purines, amino acids, hydroxyl acids, and other metabolites, biosynthesis of cholesterol, bile, acids, ether based lipids, contain large amounts of catalase which can convert excess hydrogen peroxide into water

Peroxisomal protein import (soluble matrix proteins only): 2 import signals (PTS1 and PTS2) [no detected targeting signal on some peroxisomal proteins = unknown mechansism]

Peroxisomal proteins are detected in vesicles that bud from the ER, and are claimed to represent immature peroxisomes

Multiple human diseases have been linked to peroxisomal disorders

o Zellweger’s syndrome (inherited genetic disease): general defect in importing proteins into peroxisomes…exhibits “empty” peroxisomes…individuals die son after birth

Peroxisomal pathway is not completely understood

o No chaperones exist in the peroxisomal matrix

CELL ORGANELLES V: ENDOCYTOSIS

o

Protein trafficking from the plasma membrane

 

o

Phagocytosis “cell eating” (large particles) i.e antibody activated phagocytosis

o

Endocytosis (a) pinocytosis “cell drinking (b) Receptor mediated (active uptake of external & plasma membrane proteins)

o

Transcytosis (one side of cell to other)

o

Endocytosis: at the cell surface clathrin-coated pits and vesicles cycle between soluble (disassembled) and membrane-associated states. (adaptin proteins bind both clathrin, and the cytoplasmic tails of certain receptors clustering those receptors in coated pits.

 

o

Caveolae-another type of vesicle that buds from the plasma membrane that clusters GPI-linked membrane proteins

o

Transcytosis: In polarized cells, “tight junction” complexes are responsible for preventing the diffusion of membrane proteins between apical and basolateral surfaces limiting transcytosis to select substances.

 

o

Transcytosis is utilized for the uptake of maternal antibodies by nursing infants

o

Autophagy: protein trafficking distinct from plasma membrane that leads to lysosomal degradation.

o

General strategy used by cells to destroy their own organelles

o

Active in hepatocytes, but especially during period of amino acid starvation

o

The engulfing membrane may originate from ER or Golgi

o

Very little is known about the mechanics

o

also used in the destruction of proteins accumulating in the cytosol (aggressomes) that failed to be degraded by proteasomes (**potential modifier of the several gain-of-toxic-fxn diseases)

CELL SIGNALING I

Three basic

categories of signaling molecules

 

Category

Endocrine

Paracrine

Chemical neurotransmitters

Produced by

islets or glands

many types of cells

neurons

Released

into blood

not in the blood

After action potential

Action

diffusely

locally at nearby target cells

Locally at nearby neurons, gland, or muscle cells

throughout body

Examples

Hormones

Growth factors, prostaglandins, FA derivatives

 

Time

Slow acting

Fast acting

Fast acting

Endocrine

Three different categories:

1. steroid (i.e. androgens, estrogens, glucocorticoids, mineralcorticoids, vit D) -small derivatives of cholesterol -hydrophobic so when in blood must be carried by carrier proteins -lipid soluble/ not water soluble -receptors are intracellular / bind the steroid and DNA

2. proteinaceous (proteins and polypeptides) -hydrophillic and water soluble -receptors on cell surface

3. amino acid related (i.e. epinephrine) -receptors on cell surface and have

extracellular domain that bind hormone Neurosecretory cells in the hypothalamus couple the control of the nervous system to the endocrine system

1. Stimulating parvicellular neurons results in release

of releasing and inhibiting factors from the hypothalamus that affects the secretions of the pituitary.

2. Magnocellular neurons extend axons to the

posterior pituitary and release oxytocin and vasopression directly into circulation.

Paracrine

Growth factors:

-small polypeptides with receptors on cell surface of target cell Functions:

1. Mitogenic: stimulate cell proliferation

2. trophic: stimulate cell survival and growth

3. chemoattractant: gradient of the factors will illicit cells to follow

4. pleiotropic: multiple effects

Nerve growth factor:

If there are too many neurons for the amount of target cells around, NGF will not be released by those cells and certain neurons will die resulting in a match in the number of neurons and cells.

Histamine:

-Example of a paracrine signaling molecule that is not a polypeptide. -IgE leads to histamine release (degranulation of mast cells); histamine is a vasodilator Eicosanoids -oxygenated bioactive derivatives of 20-carbon polyunsaturated fatty acids -diffuse across cell membrane and bind to surface receptors on nearby cells -an example is prostaglandins which when released from a damaged cell bind to nociceptors (sensory nerve endings which relay pain to the CNS), they do this by reducing depolarization necessary for an action potential Nitric Oxide (NO) -gas which can be extracellular messenged (diffuses across membrane) -vasodilator by relaxing smooth muscle of blood vessel wall (binds to guanylyl cyclase which converts GTP to cyclic GMP) Nitroglycerine is converted to NO in the body

Autocrine

: cell responds to its own signaling molecules

Example: Activation of T-helper cells

1. Antigen bound to surface of Antigen Presenting cell binds to receptor on helper T cell

2. T Helper cell stimulates APC cell to secrete IL 1

3. IL 1 binds to receptor on T-helper cell which activate synthesis and secretion of IL 2 and expression of IL 2 receptors on its membrane

CELL SIGNALING II

Types of synaptic transmission:

1. Electrical: ions travel through gap junctions (i.e. cardiac muscle and smooth muscle)

2. Chemical: transmission between neurons (within the synapse)

Connections between neurons:

neurons (within the synapse) Connections between neurons: (a) Axodendritic (b) Axosomatic (c)

(a)

Axodendritic

(b)

Axosomatic

(c)

Axoaxonic

Not pictured: axosecretory (onto capillary) and neuromuscular (onto muscle)

Criteria for true neurotransmitter:

1. found in presynaptic neuron

2. enzymes required for its synthesis are found in the neuron

3. stimulation of presynaptic neuron leads to NT in the cleft

4. applying NT to postsynaptic neuron gives same result as stim of presynaptic neuron

5. way to terminate the neuron must exist

6. drugs that block synthesis of NT or its reaction should block the effects of stimulation

7. drugs that block the degradation or reuptake of the NT must prolong the action of the NT

StepsSteps InvolvedInvolved inin SynapticSynaptic TransTransmmiissionssion 1. action potential drives down the axon 2.
StepsSteps InvolvedInvolved inin SynapticSynaptic TransTransmmiissionssion
1. action potential
drives down the axon
2. Ca 2+ channels open
and Ca 2+ enters the
presynaptic terminal
3. vessels fuse with
presynaptic
membrane
4. NT is released and
will have its effect on
postsynaptic neuron

Postsynaptic potentials:

(EPSP = excitatory postsynaptic potential / IPSP =

excitatory postsynaptic potential)

1. influx of Na + - EPSP

2. influx of Cl - - IPSP

3. efflux of K + - IPSP

4. influx of Ca 2+ - EPSP

Amplitude of these is relatively small and degrade across distance

Spatial summation: summation from several sources Temporal summation: summation of potentials that follow each other in time

Ionotropic receptor – binding of the NT will allow passage of cations or anions Na + channel: inflow of Na + ions will cause postsynaptic cell to become positive or “excited” Cl - channel: inflow of Cl - ions will cause postsynaptic cell to become negative or “inhibited”

Metabotropic receptor– do not contain the ion

channel Take longer to affect ion channels -G protein will be result in modulation of an ion channel that is close or far -Na + and Cl - channels are the same as above; K + channels will cause an outflow of K + and the cell will become negative or “inhibited”

Removal of NT: either degraded in the cleft or reuptaken into the presynaptic neuron via pumps

Other NT info:

1. NT and receptor must match for result

2. NT is synthesized from precursor molecules

3. NT is packaged into vesicles

4. Leaks of NT from vesicle is degraded

Other receptors:

Autoreceptors: receptors on neurons are activated by the NT that are released from themselves (usually feedback inhibition) Presynaptic: receptors located on axon terminals and activated by NT from another neuron (usually decrease release or synthesis of NT)

CELL SIGNALING III

Types of Receptors:

1) Steroid hormone recep tor, composed of:

1. COOH-terminus domain – contains hormone binding site

2. NH2-terminus domain – involved in activation of transcrip tion

3. middle-domain – contains DNA binding site

nac t ive form, Hsp90 is bound to DNA binding site

*in i

and the complex can bind to specific DNA sequences

… when steroid hormone binds, Hsp90 dissociates

2 ) Adhesion molecules

- activate cells through ce ll-cell or cell-matrix interactions; involves signal transduction cascades

3 ) Ion channel-linked receptors (Ex. Nicotinic Ach receptor, GABA, AMPA, and NMDA)

- binding of NT causes an allosteric change in the receptor (channel) channel opens to all ow ion flow

Nicotinic Ach: in neuromuscular jxn.; if Ach is bound for too long, channel will close (desensitization)

AMPA: permeabl

Glutamate binds to the AMPA receptor (ligand gated) Na comes in depolarizatio n NMDA

(ligand and voltage-gated) channel opens

e to Na and K; NMDA: permeable to Na, K, and Calcium also

Oth

GABA: GABA is the major inhibitory NT in CNS; receptor type A= ionotropic, type B= metabotrophic

GABA: GABA is the major inhibitory NT in CNS; receptor type A= ionotropic, type B= metabotrophic

er substances tha

t affect GABA receptor (only when GABA is bound also!):

1. Barbiturates increase duration of channel opening

2. Benzodiazepines increase frequency of channel open ing

 

3. Ethanol

4) G

-protein-cou

pled receptors (7 transmembrane α-helices with G-protein α, β and gamma subunits)

**slow response but good for signal amplification (1 NT can affect many channels) α subunit is most important binds GTP to carry out its function, hydrolyses GTP to G DP to turn off signal

5 ) Catalytic receptors (Ex. Receptor Tyrosine Kinase) Ligand binds conformational change in receptor au tophosphorylation now tyrosine kinase can phosphorylate something else

P rotein Kinase (catalyze transfer of phosphate from ATP to substrate protein)

Pro

have regulatory and catalytic regions

catalytic region has binding site for AT P; substrate has consensus sequence (signals kinase to phosphorylate)

regulatory doma

in often contains pseudosubstrate sequence (binds and inhibits catalytic domain)

te

in

Phosphatase (dephosphorylate proteins)

not as specific as kinases; do not have consensu s sequence; can be inactivated by corresponding kinase to amplify phosphorylation

In tracellular signaling Proteins:

1. adaptor molecules bring 2 signaling molecules next to each other to facilitate the reaction

2. anchoring proteins help accomplish specificity

Concept o

f Divergence vs. Convergence

one NT could have multi ple effects (divergence) OR several NTs could have the same end effect (convergence)

this means that there is a very co

mplex circuitry of signaling

drugs can have wanted effects on one cell, but unwanted effects on another cell

CELL SIGNALING IV

Calcium as a 2 nd messenger:

- increases in [Ca] can cause dramatic changes within a cell

- normally, [Ca] is low inside the cell (due to calcium pumps, which require ATP)

- [Ca] can increase via: 1. activation of NMDA receptor 2. release from intracellular stores via activation of G-protein signaling cascade (involves Phospholipase C) Calmodulin (the principal Ca binding protein)

- binding of Ca results in conformational change activates CamKII (calmodulin dependent protein kinase II) Diacylglycerol (DAG)

- activates Protein Kinase C

Overall Pathway:

signal molecule G-protein receptor activates PLC PIP2 hydrolyzed to produce IP3 (which opens Ca channels on ER to increase [Ca]i) and DAG (which activates Protein Kinase C)

Role of Calcium in NT release:

Action potential arrives at pre-synaptic terminal voltage-gated Ca channels open Ca enters cell Ca mobilizes vesicles for docking and release

a) Synapsin binds vesicles to the cytoskeleton CamKII phosphorylates synapsin to release the vesicle from the cytoskeleton

b) Synaptotagmin docks vesicles at the active zone Calcium has a direct effect on synaptotagmin, resulting in vesicle fusion & NT release

**Ca channels are located very close to where synaptic vesicles need to be released, so NT release happens very quickly (200µs after Ca channel activation) ** [Ca]i can be altered by pre-synaptic inhibition (cell becomes more neg; inhibit Ca influx) or by pre-

synaptic facilitation (depolarization increases Ca influx).

Ca means signal transmission.

Calcium in Memory Formation (in Hippocampus)

Long Term Potentiation (LTP) = cellular event that is thought to contribute to memory formation

depends on activation of NMDA receptor (NMDA channel opens in response to glutamate binding AND strong depolarization of the post-synaptic cell)

NMDA channel opens Calcium influx activation of numerous kinases (including PKC, CamKII, tyrosine kinase, etc)

Early phase LTP

Can result from:

1. more AMPA receptors on post-synaptic membrane

2. phosphorylation of receptors so that channel stays open for longer

3. more NT release

4. structural changes

Late phase LTP

* includes changes in gene expression Ca/calmodulin adenylyl cyclase cAMP cAMP kinase CREB-1 affects gene expression synthesis of effectors/ regulators that are responsible for long-term changes (i.e. creation of new synapses!)

CELL MOTILITY

requires signaling, reorganizing of cell adhesion systems, and alterations in cytoskeleton

Signaling: chemotactic gradient

Reorganization: cell polarity changes, lamellipodium – extension of migrating cell’scytoplasm, breaks in attachments to ECM

Neutrophil Cell Motility

1. Chemotactic gradient signal neutrophil to reorganize

2. Neutrophil reorganization: triggered by surface receptors

- polarization of cell organelles

- Lamellipodia – actin filaments polymerized at cell edge to “reach”; serves as an extension

3. new membrane mass inserted at leading edge of lamellipodia

4. chemotactic receptors steer cell ex. F-MLP and f-met cause neutrophil to move towards these signals

Diapedesis: passage of cell between endothelial cells into underlying connective tissue Margination: ex. Neutrophil attachment to inner vessel wall by binding selectin

binding increases increases calcium cascades inside endothelium

Neutrophil’s integrins bind ICAMS to increase adhesion so can slow down to enter space between endothelial cells

Movement across cell wall:

Focal adhesion – sites where integrins of neutrophil bind to vessel wall

Thrombin and histamine—released to increase permeability of endothelium

Act through CD31 that alters the cytoskeleton of the endothelial cells

Regulatory Mechanisms:

inside-out signaling – change of conformation of integrins by phosphorylation that decreases binding

Receptor alterations:change in number of receptors, change receptor conformation

Leukocyte Deficiency Disorder:

- rare autosomal disorder

- neutrophils not have adhesion molecules to follow chemotactic gradient to infection

Type 1 LAD: cells not bind ICAM 1 on endothelial cells ecause cytoskeletal proteins not active —diapedesis not occur Type II LAD: neutrophils do not express ligands for E or P selectins on endothelial cells defect in cell rolling along vessel walls

Chapter Three – Genetics

BLOCK 1 – CORE CONCEPTS THREAD: BIOCHEMISTRY & GENETICS

CONTRIBUTORS: ANN MARSHBURN, MEGHA PATEL, THERESA WILLIS & ANNIE WEYAND

TABLE OF CONTENTS:

MITOSIS

CARLOS BACINO

MEIOSIS

CARLOS BACINO

INTRO TO MEDICAL GENETICS MEDICAL GENETICS II MEDICAL GENETICS III MEDICAL GENETICS IV

LORRAINE POTOCKI LORRAINE POTOCKI LORRAINE POTOCKI LORRAINE POTOCKI

CYTOGENETICS I CYTOGENETICS II

CARLOS BACINO CARLOS BACINO

MITOSIS

Interphase G1 – machinery of cell prepares for division S – synthesis stage; DNA replication G2 – pre-mitosis; centrioles replicate

Mitosis Prophase

Microtubule spindle forms

Chromatin condenses

Nuclear envelop disappears Prometaphase

Chromosomes begin to migrate

MTs begin to contact kinetochores Metaphase

Sister chromatids align at metaphasic plate

CDK1 kinase

Protein that

signals mitosis

to begin

3 Classes of Microtubules

1. astral: form an aster around centrosomes

2. kinetochore: attaches to and directs chromosome

3. spindle: overlap at metaphasic plate to form a skeleton

Force on kinetochores from each pole is equal and opposite Anaphase

Sister chromatids separate

Chromosomes migrate to opposite poles Telophase

Nuclear envelop reassembles

Cytoplasmic MTs reassemble

Cytokinesis

Actin & myosin filaments make a contractile ring around cell

Division occurs equidistant from MT asters

Contractile ring disappears after mitosis

cut2 and PDS1 Proteins that, when degraded, signal anaphase to begin

Anaphase Lag

An abnormal separation of sister chromatids resulting in abnormal # of chromosomes in daughters

MEIOSIS

MEIOSIS: process of cell division in the maturation of sex cells

Summary: replication of DNA (diploid with 2 chromatids per chromosome, or 2n and 4c), recombination (or crossing over) between homologues, followed by 2 divisions Result: generation of haploid gametes genetically distinct from each other and from the original parent cell

Meiosis I: reduction division Result: The number of chromosomes (and DNA content) are reduced to 1n, 2c

Sources of genetic diversity

1. genetic recombination, or crossing over, occurs during prophase I period and results in the actual physical exchange of portions of

chromosomes between maternally and paternally derived chromosomes of a homologous pair

2. independent assortment: resulting gamete has varying ratio of maternal and paternally- derived chromosomes; 2 n different types of gametes

could be formed (where n is the haploid number of chromosomes)

Stages of Prophase in Meiosis I:

Leptotene: Chromosomes become visible Zygotene: chromosome pairs with homolog into a synaptonemal complex Pachytene: crossing over occurs Diplotene: The homologues repel; joined only at the chiasmata. Diakinesis: last stage of prophase I; crossing over has completed, nuclear envelope has completely disintegrated by this stage, centromeres attach to spindle fibers, chromosomes condensed Dictyotene: only in female meiosis; oocytes are frozen until puberty. Meiosis will be completed in the female only after fertilization with sperm.

Meiosis II Result: No DNA replication and DNA is reduced to 1n, 1c

OOGENESIS: process of egg formation

Result: mature ovum + 3 polar bodies Steps:

1. Primary oocyte is frozen in Meiosis I until stimulated for ovulation in puberty

2. Completion of Meiosis I forms secondary oocyte + 1 st polar body

3. Secondary oocyte immediately begins Meiosis II and freezes at metaphase II by cytostatic factor

4. Meiosis II completes upon fertilization to form mature ovum + polar body

Purpose of the asymmetric divisions: preserve the nutrient-rich cytoplasm necessary to sustain the egg until implantation into the uterus

SPERMATOGENESIS: process of spermatozoa formation

Result: 4 functional haploid spermatids which later (inside the seminiferous tubules) differentiate to produce highly motile sperm. Differences from oogenesis: even divisions, meiosis begins at onset of puberty, process is continuous and never freezes *greater number of cell divisions of the male germline before the gamete formation leads to higher mutation rate in males than in females.

FERTILIZATION

1. Capacitation: process that makes sperm capable of fusing to an egg; occurs as sperm migrate through the female reproductive tract

2. Binding of the sperm to the zona pellucida: induces the sperm to release digestive enzymes that enable the sperm to bore its way through; the

plasma membranes of the sperm and egg then fuse, and the sperm nucleus enters the egg cytoplasm

3. Digestive enzymes: modify the glycoprotein network of the zona pellucida so additional sperm are no longer able to bind to the egg

NONDISJUNCTION: Failure of a pair of homologues fail to separate (disjoin)

-In meiosis I, one daughter cell will have two of the chromosomes and the other will have none (2:0 segregation) -In meiosis II, at fertilization (if the other gamete is normal), the conceptus ends up trisomic or monosomic. -Trisomy 21, 18, 13 most common -Frequency of trisomies increases with advancing maternal age

INTRODUCTION TO MEDICAL GENETICS

Selected Single Gene Disorders:

1. Achondroplasia (ACH) - most common type of dwarfism.

Autosomal Dominant, 1:26,000-40,000 births

Traits include average-sized trunk, short arms and legs, lumbar lordosis, normal life span and intelligence.

Problems include compression of spinal cord and airway obstruction, apnea, and hydrocephalus.

Gene: 2 mutations on FGFR3 in >99% of ACH patients

2. Cystic Fibrosis (CF) - most common Autosomal Recessive disease in Caucasians.

(1:25 are carriers of 1 copy of the gene)

Autosomal Recessive, 1:2,500 births

Most common fatal genetic disease in US today.

Problems include thick, sticky mucus that clogs lungs, infections, obstruction of pancreas.

Gene: CFTR (Na, Cl transporter), hundreds of mutations in this gene that lead to the disease

3. Sickle Cell Disease (SC) - most common inherited blood disorder in the US

Autosomal Recessive, 1:500 African Americans have the disease, 1:12 are carriers

Disorder of hemoglobin resulting in anemia, sickling crisis, risk of infection and organ damage.

Gene-Beta Hemoglobin, mutation causes structurally abnormal Hb, called HbS. (Substitution of valine for glutamic acid)

4. Breast Cancer - 2 nd major cause of cancer death in American women.

Genes - BRCA1 on chromosome 17 and BRCA2 on Chromosome 13

Mutations on these 2 genes leads to increased risk of developing breast and/or ovarian cancer.

These genes are thought to participate in repair of radiation-induced breaks in DNA strands and the mutations are thought to disable the mechanism.

Treatment includes careful monitoring and possibly drug or surgical therapies.

GENETICS II, III & IV

 

Features of Transmission

 

Representative Syndromes

 

Single allele is sufficient

Achondroplasia

Vertical transmission patterns, with male-to-male transmission possible

o

Characterized by short stature, average- sized trunk, short arms and legs, and a slightly enlarged head with a prominent forehead.

Number of affected males and females in population will be roughly equal

Affected children born to non-affected parents may be explained by

o

100% penetrance at birth

o

80-90% are new mutations

o

differences in penetrance

o

Caused by mutation in the FGFR3 gene.

o

variability of expression

Marfan Syndrome

Autosomal Dominant

o

new mutation in either the germ line or in somatic cells of the embryo

o

Characterized by tall, thin body habitus, long fingers, extensible joints, aortic root dilation, floppy heart valves, and dislocated ocular lenses

o

gonadal mosaicism.

 

o

50% are new mutations

o

Caused by mutation in the fibrillin gene.

Huntington Disease

o

Characterized by abnormal body movement (chorea), loss of cognitive skills, and psychiatric disturbances.

o

Disease is progressive and fatal.

 

o

Example of age-dependant penetrance. Affected individuals are normal through the first several decades of life, but disease shows 100% penetrance by age 65.

Neurofibromatosis Type 1

o

Characterized by tumor growth along peripheral nerves, patches of brown pigment on the skin, bone deformities, and learning disabilities

o

50% caused by new mutations

o

Wide variability of expression

 

Two mutant alleles required, so both parents of affected child are considered obligate carriers

Horizontal pattern of affected individuals, often with unaffected parents

Cystic Fibrosis

o

Characterized by chronic infections, progressive lung damage and loss of functional lung tissue, failure to produce adequate pancreatic digestive enzymes, and progressive loss of pancreatic function

Autosomal Recessive

New mutations are very rare

Number of affected males roughly equals number of affected females

o

Most common AR condition in Caucasian population

o

Caused by mutation in CFTR gene.

Consanguinity increases risk

Tay-Sachs disease

Majority of inborn errors of metabolism are AR conditions

Gaucher disease

Hereditary Hemochromatosis

   

B-thalassemia

Phenylketonuria

Sickle Cell

 

No male-to-male transmission

Incontinentia Pigmenti

X-Linked Dominant

Generally more severe in hemizygous males than in heterozygous females

X-Linked Hypophospatemic Rickets

One X-chromosome in each somatic cell in a female undergoes random inactivation, causing females to be functional mosaics

In some disorders, inheritance of the mutant gene causes prenatal lethality in hemizygous males

 

A new mutation can give rise to observable phenotype in both males and females

 

No male-to-male transmission

Duchenne and Becker Muscular Dystrophies

X-Linked Recessive

Almost all affected individuals are male

o

Caused by mutations in the dystrophin gene

o

Duchenne is considerably more severe

Females are obligate carriers if they bear more than one affected offspring, bear one affected offspring and have a male relatives with the same condition, or are the offspring of an affected male

New mutations only give rise to observable phenotypes in the male

o

Characterized by ongoing muscle cell degeneration, which leads to elevated levels of CPK

Fragile X Syndrome

o

Most common inherited cause of mental retardation

o

In families with fragile X syndrome, males in later generations are more likely to be affected -- anticipation

   

Hemophilia A

Mitochondrial

Very wide variety in expression

LHON

Exclusively maternal inheritance

MELAS

MERRF

NARP

Keams-Sayre Syndrome

 

Refers to genetic traits or disorders determined by combinations of multiple genes and their interactions with the environment and other factors such as DNA methylation

Neural tube defects

Multifactorial

Isolated cleft lip or cleft palate

Pyloric stenosis

Congenital heart defects

Coronary heart disease

Although these disorders are obviously familial, there is no distinct pattern of inheritance within a single family

Insulin dependent diabetes

Schizophrenia

 

Autism

CYTOGENETICS I

Overview:

Chromosomal abnormalities are more common than generally thought. They are present in over 50% of 1 st trimester abortions and 7-10% of a