PURIFICATION OF DIPHTHERIA TOXOID BY CONTINUOUS ELECTROPHORESIS ON FILTER PAPER

A separation of purified diphtheria toxoid in a continuous electrophoresis apparatus has been achieved and resulting components have been.collected in quantities sufficient for testing and elaboration. The simple and inexpensive apparatus constructed of materials readily available is described and some factors concerned with the separation of the components are discussed. The results of the biological tests carried out on the components are outlined.

THE ANTIBACTERIAL SPECTRUM OF USTILAGIC ACID

Ustilagic acid is shown to be relatively inactive against common Gram-positive and Gram-negative pathogenic bacteria and against Mycobacterium tuberculosis. Serum and urine levels in rabbits following oral administration are much lower than the concentration required for in vitro inhibition of most bacteria tested. Human serum depresses the antibacterial effect of ustilagic acid in vitro. The drug had no effect on the course of experimental infection in mice.

NUCLEASES IN THE CEREBROSPINAL FLUID I. RIBONUCLEASE IN NORMAL, NEUROLOGICALLY NORMAL, AND IN PATHOLOGICAL CSF'S

Ribonuclease (RNA-ase) was investigated in 800 individual CSF samples. The uranylacetate precipitation technique of MacFadyen was used, followed by total acid soluble phosphorus determinations (King) before and after incubation. Normal and neurologically normal CSF's showed low RNA-ase activity. The values ranged under 20% substrate depolymerized in 24 hr. A few exceptions showing high values were encountered: one specimen each of acute nasopharyngitis, 25%; infectious mononucleosis, 28%; and myositis, 30%. The meningitis CSF's showed a wide range of RNA-ase activity, 0-50%. Poliomyelitis specimens exhibited uniformly the highest values, 15-52%. This high RNA-ase activity paralleled chronologically the chromatolysis of the motoneurons and is of special significance. In the syphilis group only the neurosyphilis samples presented moderately high activity, but rarely attained the levels observed in meningitis and in poliomyelitis. In miscellaneous neurological conditions the highest activity was found in a specimen from a case of genuine epilepsy. The findings are discussed in detail.

FURTHER STUDIES ON LIPOTROPISM IN THE DOMESTIC DUCK (White Pekin)

On choline-free diets containing 10, 18, and 28% casein, the total liver lipids of ducklings were found to be 34.8, 25.0, and 13.8% respectively. Supplementation of the 10% casein diet with 0.5% methionine increased the liver lipids. The same amino acid has no effect at the intermediate level of casein while it is lipotropic at the 28% casein level. In similar experiments with a purified soybean protein (low in methionine) fed at levels of 12, 21.7, and 27.9%, the liver lipids were 31.8, 26.6, and 27.6% respectively. These values do not differ significantly from each other. Methionine was found to be lipotropic at any of these three levels of soybean protein, and more so as the level of the soybean protein increased in the diet. From these results, it is concluded that the purified soybean protein used is a nonlipotropic protein, while casein is definitely lipotropic in ducklings. The lipotropic activity of methionine is closely related to the quality and quantity of the protein in the diet. On high fat diets containing 22, 32, and 42% fat, choline had no lipotropic effect. The average liver lipids for all groups ranged from 11 to 15%. High fat diets are less inducive of fatty livers than high carbohydrate diets. Ducklings fed a fat-free, choline-free basal diet presented livers with an average total lipids of 18%. Supplementing the basal diet with 1% cholesterol increased the liver lipids to 22%. Inositol is lipotropic either in the presence or absence of cholesterol, but choline was much more effective under similar conditions. The two lipotropic factors have a synergistic action and the reduction of the liver lipids is primarily on the glyceride fraction. Cholesteryl ester was at its lowest value in the group fed the basal diet supplemented with inositol only.

ON THE NUTRITIONAL REQUIREMENTS OF YOUNG CHILDREN WITH PARTICULAR REFERENCE TO CALCIFICATION

Two nutritional surveys have been carried out on 158 children of one to six years in Halifax, including dietary records, anthropometric measurements, roentgenograms of the bones of the hand, and laboratory tests. Observations were repeated after six months. Deficiencies in calories, calcium, and vitamin D occurred most frequently on the basis of the Canadian dietary standard. Seventy-eight per cent of the children received less than 1 gm. of calcium per day. The ratio Ca : P averaged 0.77. The consumption of vitamin D varied widely and was less than 400 I.U. in 47% in the first survey and 84% in the second. The average intake was less than 200 I.U. per day. From data of repetitions it was apparent that half of the children showed adequate growth and calcification on substandard diets while 30% did not. It was evident that normal maturation of bone could take place on intakes of vitamin D of 25 to 150 I.U. and of calcium of less than 1 gm. Calcium was adequate at 0.2 to 0.7 gm. per day in some cases but not in others.

THE ISOLATION OF BACTERIOPHAGES FOR MYCOBACTERIA WITH REFERENCE TO PHAGE TYPING OF THE GENUS

Two hundred and eight acid-fast microorganisms were used in this investigation. ' Methods of isolating bacteriophages from soil, positive sputa, stool specimens, and from lysogenic strains are described. Eleven phages isolated from soil, their adaptation to other strains, and their characteristics are presented. Classification of these phages by plaque type and size, range of activity, cross-resistance tests, serological grouping, and morphology (some) are dealt with in detail. The phage preparations showed activity for 22 (44%) of 50 saprophytic strains tested. None were active against 120 human, 14 bovine, and eight avian tubercle bacilli. Though phage adsorption occurred onto some pathogenic strains it was not followed by phage multiplication and liberation. A tentative phage typing scheme is presented and discussed.

INDUCED VARIATION AND STRAIN SELECTION OF PENICILLIUM CHRYSOGENUM IN RELATION TO TITER OF NATURAL PENICILLINS

Penicillium chrysogenum strain Wisconsin Q176 was used as the parent for a sequence of six experiments in combined irradiation and selection of strains yielding high titers of penicillin with N-(2-hydroxyethyl)-phenylacetamide as the precursor for benzylpenicillin. Altogether, 124 colonies were tested. The six strains chosen as best in their respective experiments were compared. Visible changes were found in color and amount of sporulation and in loss of the deep yellow pigment characteristic of fermentation with Q176. Under standard conditions of test, the peak titer of total penicillin increased in a stepwise fashion from 1285 units/ml. to 2575 units/ml. With appropriate medium, the best strain produced nearly 3000 units/ml., of which more than 95% was benzylpenicillin. In the absence of added precursor, the percentage of p-hydroxybenzylpenicillin produced by new isolates increased from 13% of 600 units/ml. to more than 30% of 1000 units/ml. The amount of penicillin F was quite constant, while that of other types was not changed with any apparent regularity. The change in reaction during rapid production of penicillin differed in the degree of acidity noted. When other common precursors for benzylpenicillin were used, about 50% more total penicillin was found than in the control without precursor. The total titer was never as high as with suitable amounts of the particular precursor which had been used in isolating the strains. With each irradiation experiment, the strain deemed best showed increased ability to convert the chosen precursor to benzylpenicillin.

THE LIPID COMPOSITION AND WATER CONTENT OF BRAIN, HEART, LUNG, LIVER, GUT, AND SKIN IN THE HOST COMPONENT OF THE ALBINO RAT–WALKER CARCINOMA 256 DUAL ORGANISM

The carcass of albino rats bearing Walker carcinoma 256 contains, at or near death of the dual organism, concentrations of lipids and water altered in the direction of the concentrations of these constituents in the tumor component. The objective of the investigation herein reported was to find the extent to which brain, heart, lung, liver, gut, and skin participated in this shift of lipid and water levels of the host carcass. Total lipid, neutral fat, total fatty acids, total cholesterol, ester cholesterol, free cholesterol, and phospholipid of these tissues were measured by oxidative micromethods in 23 albino rats bearing Walker carcinoma 256 at or near death of the dual organism. The results were expressed as gm. per 100 gm. dry weight of tissue and compared with corresponding values in 23 littermate albino rats without tumors, and differences with a P value of 0.01 or less were noted. Brain could be proved to take no part in the lipid and water shift in the carcass. Heart, lung, liver, and gut participated in the increased water shift. Liver participated in the increased free cholesterol shift. Skin and gut participated markedly in the decreased total lipid, neutral fat, and total fatty acids shift. Gut participated in the increased phospholipid shift. There were no other significant changes in lipid or water levels. These data are further evidence of the metabolism-directing influence of Walker carcinoma 256 on the host.

THE COMBINATION OF HEPARIN AND BOVINE SERUM ALBUMIN

The complexing of heparin and bovine serum albumin in acetate buffer at pH 5.0 was studied by electrophoretic techniques. A stable complex with a heparin : albumin mole ratio of 1 : 2 was found, though there was evidence for the existence of unstable complexes with a higher heparin : albumin ratio.

THE PREVENTION OF LIVER NECROSIS BY ALPHA-TOCOPHEROL, METHIONINE, AND CYSTEINE BY ORAL AND PARENTERAL ROUTES

Groups of 10 male weanling rats of the Sprague-Dawley strain were fed a basal necrogenic diet supplemented with alpha-tocopherol, DL-methionine, and L-cysteine. The amount of supplement contained in the daily ration was administered parenterally to similar groups of rats. All three antinecrogenic agents employed exerted some action when given either in the diet or by injection. Alpha-tocopherol completely prevented the development of liver necrosis by both routes. On an equisulphur basis, methionine was obviously more effective than cysteine by either method of administration. The experiment threw no light on the question of the site of action of these compounds. The low antinecrogenic potency of cysteine compared to methionine or to cystine has posed a problem yet to be explained.