B, 1967, No. 1 Author: Eldon D. Nielson Biological Research Division To: Dr. Murray Senkus February 6, 1967 Director of Research Re: MONTHLY RESEARCH REPORT Period Covered: Biological Research Division January 2 through 1967, No. 1 January 20, 1967 No. of Pages: 3 _BIOLOGICAL TESTING L Smoke mildness studies have continued on competitive brand cigarettes. In order to improve the statistical reliability of the test, it was decided to use a greater number of animals per test. This necessitated the prepa- ration of two Canbridge filters for implantation under the skin of the experinental animals. When this was done, it was observed that there was a greater difference between the two separate WINSTON Cambridge filters than was consistent with previous experience. Since the ciga~ rettes employed for the preparation of the filters were randomly selected, it was decided that the experizents should be repeated employing only cigarettes selected on a weight and draft basis. It appears that selecting the cigarettes on this basis has reduced the variability which had given such problens previously. All future brand comparison studies will be made on the basis of wéight and draft selected cigarettes. 11. Pharmacology of Mariolide-type Compounds, Analeptic activity has previously been reported for mariolide, dihydromarfolide and other compounds. The studies previously reported were performed in rabbits. It will be recalled that dihydromariolide performed better than cormercially available compounds in the rabbit in the previously reported work. Studies have now been undertaken to determine the analeptic activity of these compounds in the mouse. In these studies, a lethal dose (actually an LDgg) of phenoberbital uas injected into a series of animals. Five minutes later, these animals are injected with relatively large doses of The nunber of animals surviving is a reflection of the analeptic agent. In these studies as well the protection afforded by the analeptic agent. as those in the rabbitc, dihydronariolide has proved to be better than mariolide and cownercially available analeptic agents. These studies are continuing. 0828 gzog5STARCH BIOCHEMISTRY I. Glucose Isomerase Three authentic strains of Arthrobacter aurcescens, flavescens and vreafaciens were compared to our Culture 2453 for taxonomic purposes. Although the cultures were similar, Culture No. 2453 differed signifi- cantly, suggesting that it has not been previously characterized. These three species and Culture No. 262, the strain of Arthrobacter oxydans used in the hydroxylation of nicotine, were examined for ability to produce isomerase. All but Arthrobacter ureafaciens produced significant anounts of glucose isonerase when grown on a xylose medium. This brings to six the total nunber of cultures capable of producing glucose isomerase which are not known to be covered by patents or other publications. Some difficulty is still being encountered in the maintenance of the Asonerase producing ability of Culture 2453-2, In spite of this, a study of the conditions required for maximum enzyme production in small agitated fermentors is being continued. Several attempts have been made to disrupt cells of Culture 2453 by means of lysozyme and sonic treatment. Evaluation of these methods awaits further studies. HL._Carbohydrate Analyses During the last month, about 680 samples were analyzed for fructose on-the Technicon AutoAnalyzer after 24-hours incubation of enzyme and glucose. The time lag of two or three days in reporting results has placed a severe Limitation in our enzyme study program. A concerted effort is being made to devise a short-term assay for glucose isomerase activity. With the acquisition of new equipment, it is anticipated that results may be returned within six to eight hours of the time the samples are submitted. This would greatly facilitate the development of pro- duction nethods. Current efforts are aimed at establishing a measure of enzyme activity on the bases of a unit per volume of culture medium, unit of dry-cell weight and unit of wet-cell weight. Progress has been good, and it is expected that this standard will be established, within the next few weeks. A standard unit of isomerase activity has been established as that quantity of enzyme which will produce 1 gram of fructose from glucose per minute. The standard conditions employed for this assay are: one-hour incubation of 1-4 mg. wet weight of cells per ml. of 1.0M glucose solution in 0,1M phosphate buffer containing 0.01 MgCl, at pH 7.0 and at 60° C. L828 gzocs-_ MISCELLANEOUS: ‘Sat ary Analysis of Food Products 4 total of 303 tests on a wide variety of food products vas examined during the month. No Salmonella contaminations were found. — bbb abhn “Eldon D. Nielson Distribution: Dr. W. M, Bright Mr. Manford R. Haxton Dr, Murray Senkus Mr. Donald F. Mackintosh Dr. Richard E. Farrar Dr. Kerol J. Mysels Mr. E,W. Harwood Dr. Eldon D. Nielson Library (2) Dr. Charles W. Nystrom Dr. Willian C. Squires Submitted: February 6, 1967 Completed: February 6, 1967 . From manuscript :pws Approved: M.S. FEB YY 1967 7078 60S