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Author: Eldon D. Nielson MRR-B, 1966, No. 8

Biological Research Division

To: Dr. Murray Senkus August 25, 1966

Director of Research

Re: MONTHLY RESEARCH REPORT Period Covered:

Biological Research Division July 18 through1966, No. 8 August 19, 1966

No. of Pages: 5

A. BIOLOGICAL TESTING

1. Smoke Mildness Studies

Further experiments have been run to determine whether adrenal

hormones play an important role in the irritation response of the filter

implantation test. The following observations were made: (1) hydro-

cortisone admnistered to intact rats on the test had a negligible

effect on the test results; (2) removal of the adrenals of the test

animals greatly increased the irritancy response; (3) replacement

therapy in adrenalectomized rats did not improve the increased

irritancy effect; (4) animals subjected to the same surgery but

without removing the adrenals (sham-adrenalectomy) responded the

same as did adrenalectomized animals.

These results showthat the irritancy tests as conducted in our

Laboratories are not affected by the anti-inflammatory adrenal hormones.

Since this test was adapted froman anti-inflammatory test, these results

clearly demonstrate a difference between the terms inflammation, ascommonly employed, and irritation, as employed in these studies. It

is believed that this lends increased validity to the concept that

this test actually measures irritancy as distinguished froman

inflammatory response.

Additional evaluations were made on G-7 cigarettes containing

magnesium malate or magnesium citrate. Results continued to be in

partial agreement with those obtained on a smoking test panel. Malate

and citrate salts appear to be most effective in reducing the irritancy

at higher solids levels.

In experiments employing filters containing menthol alone, there

appears to be no anti-irritancy effect of the menthol. However, menthol

does appear to decrease the irritancy of cigarette smoke solids.

II. Pharmacology of Mariolide and Dihydromariolide

The respiration stimulating activities (analeptic activity) of

mariolide and dihydromariolide are being compared to those of commer-

cially available analeptics. In studies conducted on rabbits, mariolide

and dihydromariolide are superior to bemegride, metrazole, picrotoxin,

and a new Robin's analeptic drug, doxapram. At the present time,

dihydromariolide in rabbits appears to be the best analeptic agent

of all; however, the evaluations are continuing.

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III. Acute Toxicity Studies

LD50's were determined in mice on an additional ten RJR compounds.

One compound previously demonstrating interesting responses on the acute

toxicity test has been studied further. This compound, SB16A, demonstrated

apparent central nervous systemstimulation on the acute toxicity test.

This compound has a mild analeptic effect but a strong influence on theelectrocardiogram and causes a marked drop in blood pressure in experi-

mental animals. Further tests wll be conducted on this interesting

compound.

IV. Herbicidal Activity

Of 28 new RJR compounds tested in the duckweed test, only one

compound demonstrated significant activity. Compound SAA40A showed

activity at 5 ppm comparable to that of previous active compounds.

A total of 30 compounds were tested for herbicidal activity against

eight different plant species in the greenhouse. Although some physio-

logical activity was demonstrated, none of these compounds showed enough

activity to warrant further investigation.

V. Plant Hormone Activity of Abscisin

a._ Effect on Ornamental Pepper Plants

It was previously reported that leaves of an ornamental pepper

plant, when sprayed with abscisin, turned yellow and dropped from the

plant. New leaves have now developed on the branches from which the

yellowed leaves dropped; and the plant has flowered and developed

fruit, indicating a complete recovery.

b._ Effect on RiQeninRof Tobacco

The plant hormonal effects of abscisin suggested it might speed

the ripening of tobacco. A number of experimental approaches were

carried out in order to determne if this were so. In no case was

there any evidence that abscisin affects the ripening of tobacco

leaves.

B. STARCH BIOCHEMISTRY

I. Screening for Glucose Isomerase-producing Organisms

The regular screening method employing a xylose substrate for the

selection of organisms capable of producing glucose isomerase was applied

to 106 new isolates. The highest conversion rate obtained was about 10%.

Evidence of the production of glucose isomerase on substrates other

than xylose was found in several cultures. For example, culture No. 37

was grown on several substrates as indicated in the following table:

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FOR*tATION OF GLUCOSE ISOMERASE

BY CULTURE NO. 37 ON VARIOU SUBSTRATES

Carrying Medium ist Liquid Culture2nd Liquid Culture

% Conversion

of Glucose

to Fructose

TGE* Agar Slant 2% Xylose 2% Glycerol 3.0

" " 2% Starch 1.0it it

2% Xylose 2.4

"o f

2% Dextrose 1.0

2% Glycerol 2% Glycerol 2.3it

" 10

*Tryptone, glucose, extract agar

It is apparent that the amount of glucose isomerase produced after

adaptation on xylose was relatively loweven when the final culture

medium was xylose, dextrose, starch, or glycerol. However, when the

first and second stages of liquid culture were on glycerol, the yield

of glucose isomerase was three to fivefold previous production. Simlar,

but less dramatic, results were obtained from 12 other organisms grown

on substrates other than xylose; namely, propionaldehyde, butyraldehyde,

and propylene glycol. The cultures are being re-examined currently.

The production of glucose isomerase by culture 37 and other bacteria

was of particular importance. Al pertinent publications and patents

available to us specify the use of xylose as a substrate for the pro-

duction of an enzyme, properly designated as xylose isomerase, which

wll also convert glucose to fructose. The above stated evidence,

when properly confirmed, appears to form the basis for a broad-range

patent on the production of a substance which could be properly

designated glucose isomerase.

Current studies are aimed at confirming the above results and the

development of methods for more efficient production of glucose isomerase

by these organisms. Progress in these studies and the screening program

have been severely curtailed by the limted analytical facilities available

at the present.

II. Production of Glucose Isomerase

Xylose-grown, lyophilized cells of culture 2453 were applied toVeltose at 60° C. and pH7.65 with a small amount of MgCl2 for 24 hours.

The analyses indicated that nearly 50% of the glucose was isomerized and

that some maltotriose was degraded. The reaction was run with automatic

addition of alkali to maintain the pH When these cells were applied to

98.6 DE corn syrup, they converted 56.6% of glucose present to fructose

in 24 hours.

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Work is being continued to establish the optimal conditions for the

production of isomerase by ten other organisms which produce isomerase

on a xylose medium

III. a-Amylase Production

Preliminary studies on production of a-amylase have so far revealedno culture capable of producing higher yields of a-amylase than B. subtilis

obtained fromNRRL. This culture is being studied for the purpose of

establishing a method of assay for a screening program to be startedsoon.

IV. Starch Cooker-enzyme Converter Process

The continuous starch cooker has been assembled and has been used

successfully to produce liquid starch preparations having 24-28% dry

substance. By incorporating a-amylase into the system, it was found

that it will withstand momentary exposure to steam and prolonged

exposure (up to three hours) to temperatures of 85-87° without

destruction of its hydrolytic properties. Subsequent application of

amyloglucosidase, of our own manufacture, yielded syrups approaching

100 DE after 24 hours of incubation.

V. Carbohydrate Analyses

An attempt is being made to set up an analysis for amyloglucosidase

activity using the Technicon Auto-Analyzer. The method could permit

analyses of 60 samples per hour once set up. It may also be possible

to analyze samples without prior centrifugation by use of an automatic

filter built into the Technicon Auto-Analyzer System The Auto-Analyzer

is being used to considerable advantage to determine glucose isomerase

activity. This month, 1,730 samples were analyzed.

C. CIGARETTE BEETLE CONTROL

1. Time of Flight of Cigarette Beetle

It was previously reported that the flying beetle population outside

the hogsheads in the warehouses was greatest between the hours of 6 p.m

and 6 a.m wth peak activity from10 to 11 p.m This investigation was

continued for an additional 16-day period and shows the same general trend

as reported previously. The peak activity was recorded to occur one hour

earlier, perhaps a reflection of the change in day length. During this

period, the flying beetle population has increased markedly with trapcounts increasing from 1,500 on June 22 to 14,300 on July 28. This

increase is typical of the population buildup during the summer season.

II. Effectiveness of DDVP Aerosol Dispensing System

An automatic DDVP aerosol dispensing system was recently installed

in a sealed storage shed in Kernersville. The effectiveness of this

dispensing system was tested by placing cages of beetles in exposed

areas and between stacks of hogsheads throughout the shed. This

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systemwas found most effective in killing beetles in that all beetles

were killed when exposed for a two-hour period, even 24 hours after the

DDVP was dispensed.

III. Microwave Power Required for Killing Cigarette Beetles in

Hogsheads

Six small cloth bags containing all the life stages of the cigarette

beetle were placed in appropriate positions within each of 12 hogsheads

containing 900 pounds of burley stems. The hogsheads were then shipped

to Raytheon where they were exposed to different levels of microwave

power. It was hoped that it would be possible to determine the minimum

power necessary to kill beetles. The results were not conclusive.

It was apparent fromthese studies that little is known about the

factors causing the death of the beetles during the microwave treatment.

For example, in areas where high temperatures were obtained within the

hogsheads, beetles survived, whereas in other areas, where lower tempera-

tures were obtained, a complete kill of beetles occurred. In order to

obtain more basic information about the effect of microwaves on cigarettebeetles, a supply of cigarette beetles is being raised so that a cooperative

study with a group at Raytheon can be undertaken. Such factors as the

electrical properties of the beetles in a field of mcrowave energy

and the optimum microwave frequency for killing the beetles will. be

determined.

e ^ ( 4 ' ^ ' ) . k z ^ - "Eldon D. Nielson

Distribution:

Dr. W M Bright

Dr. Murray Senkus

Dr. Richard E. Farrar

Mr. E. H. Harwood

Dr. Eldon D. Nielson

Library (2)

Dr. Charles W . Nystrom

Dr. William C. Squires

Mr. Donald F. Mackintosh

Mr. Manford R. Haxton

Submitted: August 25, 1966

Completed: August 25, 1966

From manuscript:pws

Approved:

V,. S.

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