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Author : Eldon D . Nielson . MRRB 1 966 No 7Biological Research Divisio n

To : D Murray Senkus-Director of Research

July 21, 196 6

Re : MONTHLYRESEARCHREPORTPeri od CoveredBological Rese arch Dvi si on June 27 through1966, No . 7J uy15 1966

No . of Pages : 4

A . BIOLOGICAL TESTIN G

1 . Smoke Mildness Studie s

A study is under way to determinewhether or not-protein analyses ofthe i mplanted Cambridge f ilter mght be a b et ter index of irritationthan

the presently used dry weight to wet weight ratio . Aprel imnary studyindicates this may be so .

In addition, studies are under way to determine more accurately theuptake of tissue exudate as a function of time after implantation of thefilters

II . Pharmacology of Mariolid e

IStudies are under way to compare the analeptic (respiration stimulating)activity of mariolide with commercially available analeptics . Preliminarydata indicate that doses of 2 mg ./kg . of mariolide and dihydromariolide,administered to rabbits, are sufficient to restore pentobarbital-inhibitedrespiration . Preliminary values for other analeptic agents are 2 mg ./kg .for Picrotoxin, 16 mg ./kg . Metrazole, and 15 mg ./kg . Benegride .

III . Acute Toxicity Studie s

Acute toxicity studies were made on 30 RJR compounds obtained fromthe Chemical Division . Some of these compounds demonstrate potentiallyinteresting pharmacologic properties, which will be reported in greaterdetail at a later date .

IV . Analysis for Tryptophan Metabolite s

a ._ o-Aminophenol s

A chromatographic column procedure has been developed whichpermits the determination of total o-aminophenols in a urine sample .This method is satisfactory since the o-aminophenolic functionimpartsthe carcinogenic activity to these,compounds .

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b . _ N - M e t h Y l n i c o t i n a m i d e

N-Methylnicotinamide can be determined with the newspectrophotofluorometer by measuring-a fluorescent-reaction productformed with methyl ethyl ketone . There are still minor difficultieswith the procedure, but it is reaching a stage where it can be

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employed for studies on tryptophan metabolites.

V. Effect of Dosing Rats with Tryptophan '

Rats were given 50 mg . of tryptophan, and 24-hour urine sampleswere collected and analyzed for o-aminophen ols and for kynurenine . Theyield of o-aminophenols in these preliminary experiments indicated that

rats excrete a smaller proportion of these tryptophan metabolites than

d o h u m a n s . This suggests that the metabolic pathway in rats for theformation of o-aminophenols is relatively inactive or that they are

f u r t h e r m e t a b o l i z e d o n c e f o r m e d . These preliminary results suggestt h a t t h e r a t m a y n o t b e a s a t i s f a c t o r y a n i m a l

. o n . w h i c h . . t o , p e r f o r m

experiments on the effect of smoking on tryptophan metabolis m.

VI . Plant Hormone Activit of Abscisin

a ._ Axial Bud DeveloQment

It was previously reported that axial bud development was

inhibited in plants immersed in nutrient containing abscisin.

This suggested that abscisin might serve as a sucker control agent.

T w o s e p a r a t e e x p e r i m e n t s i n d i c a t e t h a t - A t w o u l d n o t " b e a - s a t i s -

f a c t o r y s u c k e r c o n t r o l a g e n t .

b . _ E f f e c t o f A b s c i s i n o n L e a v e s - a n d F l o w e r s

An ornamental pepper plant, just beginning to flower, wassprayed with solution containing abscisin . After 48 hours,, theleaves started to yellow and began to fall off . After nine days,nearly all leaves and flowers had fallen from the plant . . Leaveswhich had not been sprayed were unaffected . The plant is nowshowing vigorous new leaf growth .

- - I n a s e c o n d e x p e r i m e n t , o n e o f t w o t e r m i n a l b r a n c h e s w a s

s p r a y e d w i t h a b s c i s i n . After two weeks, only three leaves hadf a l l e n f r o m t h e s p r a y e d b r a n c h ; b u t g r o w t h o f t h a t b r a n c h w a s

a l m o s t c o m p l e t e l y i n h i b i t e d . The_untreated branch grew rapidlyt o n e a r l y t w i c e i t s o r i g i n a l s i z e . It appears that the lowerlevel of abscisin used in this experiment was sufficient to retard

g r o w t h w i t h o u t c a u s i n g l e a v e s t o f a l l . This experiment demon-strated that abscisin is not translocated appreciably when

a p p l i e d t o a l e 1 f s u r f a c e.

These results suggest that it may be possible to dwarf

ornamental plants by applying a spray to leaf surface prior to

f l o w e r i n g . Additional tests are planned .

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B . BIOSYNTHESIS

I . Production of Amyloglucosidase

Yields of amyloglucosidase were as good (500 units per milliliter)

or better (up to 640 units per milliliter) when. 0 1 % f u m a r i c a c i d w a s

r e p l a c e d b y. 0 0 5 % s u c r o s e i n t h e f e r m e n t a t i o n m e d i u m . In addition,

t h i s p r o c e d u r e o f f e r s a c o s t a d v a n t a g e .

With present methods, amyloglucosidase can be produced at about$0 .05 per million units as compared to a cost of $0 .67 to $1 .09 permillion units for commercial amyloglucosidases . These are calculatedon the basis of assays of activity performed in our laboratories . Theestimate of our production costs in an unofficial "ball park" figure .

II . Evaluation of Enzymes

- W h e n 2 , 7 0 0 u n i t s o f a m y l o g l u c o s i d a s e _ w e r e a p p l i e d t o l i q u i f i e d

s t a r c h , h a v i n g a D E o f 1 4 , a 9 5 D E s y r u p w a s f o r m e d i n 2 4 h o u r s . Theestimated cost of producing this enzyme was six cents for the quantity

required to convert 100 pounds of dry starch . It is conceivable thatstill higher concentrations of amylogl ucosidase can be applied to

liquified starch to obtain proportionally higher rates of conversion

i n p r a c t i c a l a p p l i c a t i o n s .

I I I . Screening for Glucose Isomerase=producing Organisms

Several additional organisms capable of producing glucose isomerase

have . b e e n f o u n d . A screening program is being continued at an increasedr a t e .

Several organisms have been shown to produce glucose isomer ase on

substrates containing butyraldehyde, propionaldehy de, and starch . Afteran initial growth on xylose, a known isomerase producer has been found

to produce isomerase on substrates of ethylene glycol and propylene glyc ol

without,adaptat ion on xylose in the intermediate stages . Further exami-nation of these substrates and glycerol for use in an isomerase production

a r e c u r r e n t l y u n d e r s t u d y .

, Glucose isomerase, produced by cells of Culture 2453, was incubatedwith a 1 molar glucose solutio n at pH 7

. 6 5 u n d e r a u t o m a t i c p H c o n t r o l

at 60 C . for 24 hours . The product was centrifuged, filtered througha Seitz filter and clarified with carbon (Darco G-60), and concentratedto 83 .3% solids . The resulting syrup was light, straw colored and,according to gas chromatographic analysis, contained 48% fructose and54% glucose . The syrup had a "honey-l ike" taste .

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C . CARBOHYDRATE ANALYSIS

Because the screening program for organisms capable of producingglucose isomerase was limited by our ability to analyze cultural liquors,the colorimetric procedure for the determination of fructose was abandonedin favor of a similar -procedure using the Technicon Auto-analyzer . Thisincreased the capacity from about 50 samples per day to 50 samples perhour . With this change, the capacity for centrifugation becomes thelimiting factor in the number of analyses which can be performed-eachda y . This is a significant help in the research program .

Fructose and glucose have been satisfactoril y resolved on the gas

c h r o m a t o g r a p h a s T M S d e r i v a t i v e s . It is expected that the separationscan be improved and that this meth od can be used to verify fructose

a n a l y s e s m a d e b y t h e c o l or i m e t r i c p r o c e d u r e .

I n i t i a l t e s t s o n t h e s e p a r a t i o n o f d e r i v a t i z e d m a l t o d e x t r i n s

indicates that satisfactory separation of maltotriose and maltose was

o b t a i n e d . Some difficulty in the separation of G-4 and G-5 oligomersw a s e n c o u n t e r e d .

D . CIGARETTE BEETLE CONTROL

There has been some question about what time during the day thebeetle population leaves the hogsheads or other places and becomes apredominently flying population . . T h i s _ .information_is . _ . n e e d e d . , . b e c a u s einsecticide spraying is more effective when the population is flying .

Beetle population counts are usually made utilizi ng a suction

l i g h t t r a p . In this study, the light trap was connected to a fractioncollector so that beetle counts could be made on a time basis through-

o u t t h e d a y . Data collected over a 13-day period show that the beetlesa r e r e l a t i v e l y i n a c t i v e d u r i n g t h e d a y t i m e . They show the greatesta c t i v i t y b e t w e e n t h e h o u r s o f 6 p . m

. a n d 6 a .m . w i t h a p e a k a c t i v i t y

b e t w e e n 1 0 a n d 1 1 p.m .

Distribution :

E l d o n D . N i e l s o n

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W. M . Bright Dr . Fred T . WilliamsDr . M u r r a y S e n k u s .

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