N

Author : F.1dcn D . Nielson ,*i1:F.-B, 1967, No . 9

Biological Research Division

To : Dr . Murray Senkus October 3, 1967

Director of Research

Re : MONTHLY RESEARCH REPORT Period Covered :

Biological Research Division August 16 through
1967, No . 9 September 15, 1967

No . of Pages : .' 7

A . BIOLOGICAL ACTIVITY

I . Pharmacologic Screening

a ._ Acute Toxicity Studies_

Acute toxicity studies were run on 32 RJR compounds during this

period . Follow up studies will be undertaken on some compounds of
this series .
I
b ._ PharmacoloRic Screening_

Of 29 compounds which were screened for pharmacologic activity,

4 were tested on a secondary screen for .possible anti-reserpine activity .
Three of the four compounds antagonized the reserpine-induced,ptosis,
but increased sedation . Three compounds were tested for respiratory '
stimulating properties, one of which appears to have a fair degree of
activity . Two of the compounds depressed rather than stimulated
respiratory activity .

Picrotoxin iaas added to the list of standard analeptic compounds

for comparison with dihydromariolide and has, along with the other
compounds, compared unfavorably to dihydromariolide .

II . Fate of Smoke Constituents

The distribution of the C14 from palmitic acid-C14 and phenol-C14 was
determined in a number of rat organs following the administration of 15
microcuries of the compound either intraperitoneally, in a smoke aerosol,
or as a "vapor ." ' ,

The compound was administered intraperitoneally simply by injecting

15 uc of palmitic acid-C14 or phenol-C14 in an ethanol solution directly
into the peritoneal cavity of the rat . The rat was then placed in a
metabolism cage for four hours, sacrificed, and the individual organs
dissected and analyzed for C14 .

In the case of the smoke aerosol, the C14 compound was vaporized, mixed
with a smoke aerosol, and the C14-labeled smoke aerosol introduced directly
into the rat's lungs through a tracheal tube : Approximately f ifteen minutes
were required to administer the smoke aerosol, after which the animal was
.immediately sacrificed . -The-rat-hyperventilated-in-response to-the cigarette
smoke, forcing much of the smoke aerosol out of it's lungs .
 2

To administer the compounds as a "vapor," the compound was vaporized

and then forced into the rat's lungs in a stream helium through a tracheal
tube . The "vapor" was administered over fifteen minutes ; and in this case,
the rat did not hyperventilate .

The results of the distribution of C14 from palmitic acid-C14 are

shown in Table I and from phenol-C14 in Table II .
 3

TABLE I

DISTRIBUTION OF C14 IN RAT ORGANS FOLLOWING AD .IINISTRAZION

OF PALMITIC ACID-CN BY THREE DIFFERENT METHOD S

C14 activity expressed as dpm/g wet weigh t

Method o f
Administration Intraperitoneally* "Vapor Smoke Aerosol**

Brain 2,500 1,300 0

Kidney 24,000 12,000 56 0

Stomach 22,000 4,000 0

Skeletal Muscle 9,000 1,600 0

Heart 15,000 8,500 300

Lungs 21,000 15,000 850

Liver 87,000 7,100 400

*Sacrificed four hours after administration .

**Sacrificed ti15 minutes after administration . ~

TABLE I I

DISTRIBUTION OF C14 IN RAT ORGA .NS FOLLOIdING AD .`•iINISTRATION

OF PHENOL-C~+ BY THREE DIFFERENT METHOD S

C14activity expressed as dpm/g wet weight

'Method o f
-Administration Intraperitoneally* "Vapor"** Smoke Aerosol* *

Brain 0 2,430 0
Kidney 0 47,900 1,95 0

Stomach 3,180 - 32,600 0

Skeletal Muscle 920 9,600 0

Heart 2,980 14,500 0

Lungs 880 37,100 87 0
Liver 1,700 7,750 0

*Sacrificed four hours after administration .

**Sacrificed ti15 minutes after administration .
 4

These preliminary results show that the distribution of the C14 from
both phenol and palmitic acid is different, depending on whether the com-
pound is administered through the lungs or intraperitoneally . The high
level of C14 in the kidney following administration through the lungs is
particularly interesting .

The amount of C 14 in the tissues is much higher when the compound is

administered as a"vapor" as compared to in-the-smoke-aerosol . This is
believed to be due to the hyperventilation observed when the rat is exposed
to the smoke aerosol . -

The low uptake of C14 from the labeled aerosol creates a major
experimental problem since the levels obtained are not sufficiently high
to permit the design of experiments to determine the rate of removal of C14
constituents from the individual organs . A .larger animal, such as the dog,
would be more satisfactory since it's tidal volume to total lung capacity,
respiratory rate, etc ., are more like those of man ; and therefore, a higher
retention of the C14 could be expected .

III . Herbicides

a. Duckweed Tests -

Of-47 compounds tested, five killed duckweed at 50 ppm and will

be tested at lower concentration levels .-

b ._ Comoounds with Commercial Herbicide P~,ter~tial

Two compounds, SAA14G (1-decylpiperidine) and'SAA40A (1-decyl-4-

hydroxypiperidine), were reported by the USDA Station at Fort Lauderdale
to look extremely promising on three species of submersed weeds,
southern naiad, elodea, and coontail . The herbicidal action of SAA14G
is covered by German patent•1,032,023 assigned to Badische Aniline
und Soda-Fabrik on January 10, 1957 . The patent covers tertiary

amines of the type R1 N'R3 in which R1 is an alkyl group from

C8 to C10 and R2 and R3 are alkyl groups from C1 to C7 and also

nitrogen containing ring compounds, such as pyrolidines and piperidines .
It is not clear whether this patent would also cover SAA40A (1-decyl-
4-hydroxypiperidine) . It is recommended that a patent be applied for
compound SAA40A . Compound SAA40A is less active than SAA14G, but it
is still a fairly effective aquatic herbicide .

IV . Abscisin

a . Abscisin Derivatives

In tests against duckweed, the butoxyethyl ester of abscisin II

was as effective as abscisin as a growth inhibitor (94% inhibition
after seven days at 10 ppm) . This abscisin derivative will be further
evaluated by greenhouse tests .
 5

b ._ Effects of Abscisin Reported on Citrus Fruit Abscission

William C . Wilson of the University of Florida Citrus Er.periment

Station has reported that our preparation of abscisin showed good
activity in the "pan test" with "Valencia" orange explants . There
was 100% abscission in 72 hours at 50 ppm . However ; tests on
Calamondius (a small ornamental variety of orange) and on a small
"Valencia" orange tree produced no fruit loosening or dormancy .
Tests on these plants were made at rates up to 500 ppm . The citrus
tree has a thick cuticle, and it is not uncommon for cheinicals-to
get into some other plants but not into the citrus tree . Dr . Wilson
feels that abscisin should not be dropped from the study but feels '
that it will be necessary to find some way to get the compound into
the plant . Dr . William C . Cooper of the USDA Station at Orlando has
reported that he has had some success with citrus fruit abscission
at high .concentrations of abscisin . He is also concerned with the
problem of getting the compound into the tree .

It is apparent from the studies reported from workers outside

the Research Department, as well as from our own research results,
that one of the major problems in obtaining an effect of the abscisin
is to get the material into the plant to the point where it can
actively influence the physiology of the plant . It would be desirable
to synthesize radioactively labeled abscisin which then could be
utilized in studies to determine factors affecting its absorption into
the plant as well as to obtain basic information as to just how readily
it is moved once it gets into the plant . itself . Our results obtained
thus far seem to indicate that it does not-move rapidly through the
plant . Studies with C14-labeled abscisin might,&uggest a method of
getting this material to the point in the plant"wliere it can be most
effective .

- V . Insecticides

Of 56 RJR compounds screened- for insecticidal activity, two compounds,

SM61A and SM63A, showed significant activity . Both S:161A, bis-(diethyl,
N-[2-ethylamino]phenyl)-sulfuryl amide, and SM63A, 2,4,4,5,5,7-hexamethyl-
1-oxo-hexahydro-l-thia-2,7-diazepin, showed a 100% kill against the fruit
fly and the cigarette beetle at a concentration of .5% . These two compounds
will be submitted to the Entomology Research Division of the USDA in
Beltsville, Maryland, for further evaluation of their insecticidal properties .

B . STARCH BIOCHEMISTRY

I . Glucose Isomerase

Corn hull .hydrolysates and bagasse prepared by the Chemical Division

were evaluated as substrates for the production of glucose isomerase by
Culture 2453-2 . Yields up to about 65% of those obtained on Eastern Chemical
Company xylose were obtained . However, yields up to 88% of those obtained
on Eastern Chemical Company xylose were previously obtained on a hydrolysate
of_bagasse prepared by.Pfanstiehl : :.Company .by carbon :tireatment .•ion-exchange
and recrystallization . The last form of xylose may be adequate for
production purposes .
 6

Strain 2453 has been maintained in active culture only with considerable
difficulty . It does not yield readily to the classical methods of culture
storage . Therefore, reisolation and purification of this particular strain
must be done frequently . Reversion is relatively rapid . Work on the
isolation of other active glucose isomerase producers has been started in an
effort to find organisms producing high yields of glucose isomerase on xylose
and other substrates such as glycerol and ethanol .

The increase in the activity of glucose isomerase produced by treatment

with detergents has been studied further . Another anionic detergent, sodium
dodecyl sulfate, was found to have the same effect as sodium heptadecyl _
sulfate previously tested . However, non-ionic detergents such as Tween-60
(polyoxyethylene sorbitan monostearate) and Span-60 (sorbitan monostearate)
increased activity only slightly .

Customarily, the activity of glucose isomerase has been measured at

60°C . A twofold increase in the activity is experienced .if the assay is run
at about 75°C . The effect of the anionic detergents and the heat effect are
not additive, but they do compliment one another and point the way to
procedures to be used to increase the enzyme activity . When eight micrograms
of sodium heptadecyl sulfate per mg of cell weight are used to induce increased
isomerase activity at 75°C, the rate of activity is increased about six-fold
over the rate when incubated without detergent at 60°C .

II . Starch Liquefaction

Numerous syrups have been prepared from starch by the .starch-to-water

method for use by the Development Department in connection with their study
of the Japanese alkaline isomerization process . . The,~syxups ranged from
94 to 99 D .E .

A new process for the preparation of high D .E . syrup from starch by

means of a hydroheater and a single enzyme has been studied . High temperatures
(148 to 149°C) are used without amylase to liquefy-starch which is subsequently
saccharified in the usual manner'with amyloglucosidase . The liquefied starch
was changed to the extent that the iodine stain test was brown instead of blue,
indicating considerable depolymerization . At high pH values the starch
effluent gelled_quickly upon cooling, as expected . Therefore, efficient pH
adjustment and mixing of amyloglucosidase was difficult . At lower pH values
the liquefied starch remained fluid, although so viscous that mixing was not
readily possible .

III . Analytical Piethods for Carbohydrate Determination

The new method for the rapid enzymatic determination of isomerase was
reported previously to require a supply of xylitol dehydrogenase which is
not available commercially . It was known to be available from guinea pig
and rabbit livers . It has now been found to be readily available from frozen
beef liver . However, purification procedures are yet to be perfected .

During the past four weeks, 1,844 determinations of isomerase activity

-•were run--using-•the Technicon autoanalyzer .---•To- #acil .itate handling- .of . data
the teletyped sheets from the Mathatron 848 computer have been turned over
directly to the investigator for incorporation into theix records, thus,
reducing the manpower to make these determinations and to transcribe them .
_ -~
 7

C . MISCELLANEOUS

I . Sanitary Analysis of Food Products

Examinations of food•products for sanitary quality were continued .

This month 191 samples were examined for Production plants and the .Product
Development Division .

II . Beetle Control

In a series of experiments being conducted in our storage warehouses

in Kernersville comparing different insecticide treatments, the following
general conclusions previously reported still appear to be correct .

1 . Some insecticide is better than none at all .

2 . The DDVP aerosol is superior to other insecticides .

3 . The DDVP aerosol when used in sealed sections appears to be-

significantly more effective than when it is used in unsealed
sections .

4 . Effective control has been attained in an infested shed for one

year by sealing the shed, fumigating the infested tobacco, and
employing a DDVP aerosol system .

Distribution :

Dr . W . M . Bright Mr . Manford R . .Haxton

Dr . Murray Senkus Mr . Donald F . Mackintosh
Dr . Richard E . Farrar
Dr . Karol J . Mysels
Mr . E . H . Harwood
Dr . Eldon D . Nielson
,Library (2)
Dr . Charles W . Nystrom
Dr . William C . Squires

Submitted : October 3, 1967

Completed : October 3, 1967

From manusc ript :vlb

Approved :

0OT 5 1 967