Experimental Biology and Medicine

Generally, it has been found that cis and trans isomers of 1,2-tertiary aminocyclanols and their esters, studied largely in the cyclohexane series, are capable of exhibiting convulsant activity in the mouse and the cat. From graded activities in the mouse it is observed that with few exceptions each cis isomer of a given cis-trans pair is more potent than its corresponding trans isomer. Within the tertiary amino compounds as a class, it appears that convulsant activity in the mouse, which is roughly paralleled in the cat, runs in the increasing sequence alcohols < phenylacetate esters < di-phenylacetate esters ≤ benzilate esters. The cis quaternary alcohols 3 and 7 are active in cats and mice, whereas the trans isomers 4 and 8 are strikingly inactive in cats. Further, the probability that active and inactive members of a quaternary cis-trans pair (e.g. 7 and 8, and 4 and 3) reach the same receptor centers in the cat is indicated by the observations that 8 protects against the action of 7, and 4 against 3. Finally, the quaternary cis and trans benzilates 18 and 19 appear to show strong species specificity, with high activity in mice (cis better than trans) and low activity in cats.

Injection of homologous spinal cord in Freund's adjuvant into 7-day-old cockerels retards body growth with disproportionate retardation of comb and testicular growth. Preliminary microscopic examination of combs revealed decreased mucoid matrix of the intermediate layer of the dermis in experimental animals, suggesting a hormonal mechanism (1). Freund and co-workers (2) reported production of aspermatogenesis in guinea pigs by injection of homologous testicular tissue and adjuvant. In studying the effects of other tissues, these investigators found mild testicular damage and aspermatogenesis after injecting homologous CNS tissue and adjuvant into guinea pigs. The effects of these injections on secondary sex characteristics were not studied. They believed that, although CNS and testes shared a common antigen, the testicular injury was secondary to physio-pathologic changes associated with meningo-encephalitis (loss of body weight and other symptoms). In our chickens the incidence of encephalomyelitis was very low. Although body weights were retarded in these animals and in animals receiving liver-in-adjuvant, ratio of comb weight to body weight was lower only in the experimental group, indicating a disproportionate retardation of comb growth. It appears that some factor more specific than decreased body weight and changes associated with encephalomyelitis is operative. Whether it is a common antigen shared by CNS tissue and testes or whether it is inhibition of hypothalamic-hypophyseal function is purely speculative.

In the force-fed intact rat, treatment with small daily doses of cortisone or hydrocortisone or their acetates and Δ¹ analogues had slight, if any effects on serum Na and K but augmented urinary excretion of water (urine volume), Na and K, suppressed body weight gain, and caused reduction in adrenal weight. Hydrocortisone acetate and prednisolone were most effective of 10 steroids tested in causing adrenal atrophy. Treatment with 9α-fluorohydrocortisone resulted in marked increase in serum Na, severe decrease in serum potassium, marked loss in body weight, no change in urinary sodium, and mild increases in urinary volume and K. Corticosterone, 11-dehydrocorticosterone, or Δ^1,4-pregnadiene-17, 20, 21-triol-3, 11-dione (Δ¹ U), in the doses employed, failed to affect water and electrolyte balance. These 3 compounds tended to suppress adrenal weight and, excepting Δ¹U, caused slight reduction in body weight gain. In general, renal responses were similar to those found previously in adrenalectomized rats.

Tetrahydro S was excreted in amounts ranging from 7 to 44 mg daily in 5 patients with adrenocortical carcinoma exhibiting excessive production of Porter-Silber chromogens. Dihydro S was presumptively identified in the urine of one of these patients. The amount of tetrahydro S in 4 patients with adrenocortical carcinoma with normal levels of Porter-Silber chromogens was likewise normal.

1) Acquired tolerance to isolo-gous male skin may be produced in female mice of the A and C₅₇ Bl (subline I) strains by injection at birth of living spleen cells taken from isologous adult male donors. The results show that by this procedure sex-determined incompatibility observed between male and female mice of these 2 strains may be overcome. 2) These observations are interpreted as providing evidence for the immunological basis of male skin graft rejection by isologous females.

The S E polyoma virus derived originally from strain AKR mice can be passed repeatedly from tissue culture to tissue culture. It is relatively stable when stored at −70°C, −20°C or 4°C for 404 to 169 days and it resists the action of ether, glycerol or trypsin. Heating to 80°C destroys or inhibits its tumor inducing property and this property is impaired by heating at 60°C for 60 minutes or 70°C for 30 minutes. It withstands lyo-philization as indicated by induction of multiple tumors in hamsters 1 to 3 days of age with reconstituted lyophilized virus or with the fluids of cultures infected with reconstituted dried virus.

Incubation of rat thoracic duct lymph at 37°C did not affect lymph cholesterol partition. Lymph also failed to cause esterification of added cholesterol when included alone, or with oleic acid and sodium taurocholate. Likewise, there was a lack of hydrolysis of added cholesterol oleate, with or without sodium taurocholate inclusion. Adjustment of the pH of incubation digests containing cholesterol or cholesterol oleate did not result in changes in the ratio of free and esterified cholesterol. From the present data it is apparent that rat thoracic duct lymph does not possess synthetic or hydrolytic cholesterol esterase activity.

Syrosingopine is a less potent stimulant of gastric secretion than is reserpine.

The effect of cortisone administration upon the hexosamine-collagen ratio (H/C) of several tissues was investigated. The H/C of skin, sternum, trachea, lung, aorta and vena cava was significantly reduced when a 20% loss in weight was induced by cortisone. When injections were discontinued and the original weight was regained, H/C of sternum and vena cava returned to starting level. This was nearly true of lung and aorta. Skin lagged behind considerably in this respect. Multiple biopsies were taken from skin following cortisone administration and withdrawal. When depression of H/C was greatest, recovery lagged the most. The findings are pertinent to the question of the general nature of cortisone effect upon connective tissue and the reversibility of this effect.

Additional evidence is presented for the existence of a new factor essential for utilization of mevalonic acid. This factor is present in autoclaved extracts of fresh liver from several species but absent from auto-lyzed liver. It is heat stable but is lost on dialysis or exposure to pH 0.5 and to a lesser extent by exposure to pH 11.0. A variety of biological compounds has been examined for activity. No compound thus far studied, including coenzyme A, will replace natural material.

The trichloracetic acid filtrate of rat pancreatic juice contains an inhibitor of trypsin which does not inhibit chymotrypsin. The inhibitor is inactivated by trypsin and chymotrypsin.

1) By use of peritoneal lavage, removal of Sr⁸⁵ and Ca⁴⁵ from bone has been studied relative to time of injection of radioactivity. Earlier studies had shown that parathyroidectomy or addition of citrate to lavage rinse had no effect on removal of radiocalcium injected 24 to 48 hours prior to start of lavage. In these experiments, similar results with radiostrontium substantiated earlier studies. In addition, experiments in which radioactivity, both calcium and strontium, remained in the animal 2 to 3 weeks before lavage procedures were done. In these experiments, parathyroidectomy reduced amount of radioactivity removed, while addition of citrate to the rinse increased it. Under these situations, therefore, changes in rate of removal of radioactivity paralleled changes in removal of total calcium. 2) These experiments are additional evidence that calcium is removed from bone by 2 processes, one possibly an equilibration phenomenon between fluid media of body and those portions of bone with which it is in closest contact. The second is removal of calcium from deeper areas of bone, under the influence of hormone of the parathyroids.

1) Living HeLa cells were incubated in various concentrations of ribonu-clease and deoxyribonuclease and their ultraviolet absorption characteristics were followed for 6 hours by means of time lapse motion pictures obtained with the ultra-violet flying spot television microscope. 2) No change in ultra-violet absorption images of cells was detectable as long as 2 hours after the time that replicate cultures demonstrated a loss of affinity of cytoplasm for basic dyes and the nuclei were negative with Fuelgen stain. 3) Living HeLa cells react to incubation in ribonuclease by first shrinking slightly and then expanding their cytoplasm markedly. 4) Prolonged incubation of HeLa cells in either enzyme eventually produces collapse and pyknosis of all of the cells in the culture.

The antibiotics kanamycin, paromomycin and neomycin were shown to have essentially the same activity in vitro against strains of Staphylococcus aureus and of various Enterobacteriaceae. Bacteria made resistant to any one of these 3 antibiotics by subcultures on that antibiotic also exhibit virtually complete cross-resistance to the other two. Organisms isolated from infected sources do not show significant cross-resistance between streptomycin and any of these 3 antibiotics. However, strains made resistant in vitro to either kanamycin, paromomycin or neomycin show moderate increases in resistance to streptomycin, and strains made resistant to the latter exhibit only minor increases in resistance to the other 3 antibiotics. With all 4 antibiotics, corresponding parent and resistant variants of the staphylococci were of the same phage type, and resistant variants of K. pneumoniae and E. coli retained their serological specificity. Fecal organisms isolated from patients during oral treatment with paromomycin or kanamycin were resistant to the antibiotic administered and showed moderate to marked resistance also to the other one and to neomycin.

It is hoped that a means has now been provided, through use of hamsters, to study more intensively the incitant of this disease and to determine its possible relationship to the toxic principle reported by Stein-haus(4) in D. andersoni eggs. Ecologic and/ or genetic factors may be influential, but it is pertinent that reared progeny of paralysis-producing ticks have now induced experimental disease. In spite of successful transmission by partially fed ticks from paralyzed animals in many of the above experiments, individual female ticks that produced paralysis in 3 child patients of Drs. Barmeyer and Adlerson of Missoula completed engorgement and mated on 2 dogs and 1 hamster, respectively, in 2 to 4 days without reproducing paralytic signs in these hosts. These observations indicate that hamsters are the animals of choice for the study of tick paralysis.

Chronic treatment of rats with levorphanol produced profound polydipsia and polyuria. These observations are of interest since levorphanol and its close relative, morphine, are known to produce antidiuretic responses on acute administration.

Methionine (presumably as a methyl donor) accelerates floral abscission in tobacco. IAA retards abscission. When the two factors are added to developing ovaries in combination, in general the effect is one of diminishing the effect of either when added alone. However, high concentrations of IAA completely overcome the accelerating effect of methionine.

The technic of stop-flow analysis has been used to localize the site of renal sodium reabsorption which is affected by adrenalectomy and aldosterone. Following adrenalectomy, the distal tubule was not able to reduce sodium concentration to the low value achieved during stop-flow in normal dogs. Following aldosterone administration this distal reabsorptive capacity was restored. No conclusion could as yet be made regarding the effects of aldosterone on the proximal tubule.

1) Poliomyelitis virus was fed to 4 groups of 5 monkeys each which were subsequently caged with an equal number of normal animals. 2) All of those fed virus became infected as evidenced by either the development of paralysis or virus excretion accompanied by an antibody rise. 3) Two of the contact monkeys became paralyzed and although one of these and 10 others excreted virus briefly, antibodies developed and were demonstrated only in the 2 paralyzed animals suggesting that sub-clinical infection did not occur.

1. Maleuric acid produced mitotic inhibition in onion root-tip cells and an increase in proportion of mitotic cells in meta-phase, but little or no cytoplasmic or nuclear abnormality was observed. 2. A single intraperitoneal injection of maleuric acid into mice bearing the Ehrlich ascites tumor resulted in marked cytoplasmic blebbing of the tumor cells. Mitotic abnormalities produced by the treatment persisted for prolonged periods. 3. The affected tumor cells showed essentially normal patterns of free amino acids.

1. A massive hemorrhagic reaction was observed at site of subcutaneous injection of large amounts of egg albumin in guinea pigs sensitized with egg albumin incorporated in water-in-oil emulsions containing killed M. tuberculosis or killed M. butyricum. No hemorrhages were seen in guinea pigs sensitized with paraffin oil adjuvants without mycobacteria, although these animals had circulating antibodies in amounts comparable to those in guinea pigs sensitized using mycobacteria. 2. Guinea pigs sensitized by intracutaneous injection of egg albumin in paraffin oil adjuvants with or without mycobacteria underwent a protracted but usually non-lethal anaphylactic shock after a subcutaneous eliciting injection of egg albumin. On the contrary, in guinea pigs sensitized by intracutaneous injection of egg albumin in saline alone, death occurred in 39 to 66 minutes and at necropsy there were manifestations of a combination of symptoms of acute (pulmonary emphysema) and protracted shock. In accordance with previous work, prolongation of the anaphylactic syndrome was associated with the presence of antibody in the circulation. 3. Protracted anaphylaxis did not cause grossly apparent hemorrhages at sites of tuberculin reactions to PPD. Intravenous injection of S. typhosa or E. coli endotoxin 24 or 48 hours after the intracutaneous injection of PPD caused hemorrhages at the sites of tuberculin reactions.

1) FHA (9α-fluoro-11β-hydroxy-Δ⁴-androstene-3,17 -dione) was an effective sodium retaining substance but was inactive in thymolytic and anti-inflammatory tests. This is the first instance of a highly active sodium retaining substance in the C₁₉ series. 2) Introduction of fluorine at the 9α-position in 11β-hydroxy-Δ⁴-androstene-3,17-dione resulted in a compound essentially inactive as an androgen while the uterotrophic activity was intensified as compared to the non-fluorinated steroid.

Incorporation of S³⁵O₄ ² into the mucopolysaccharide of rachitic cartilage, and its subsequent release, were enhanced by administration of parathyroid extract when given before, with, or after the labelled sulfate. The mechanism of operation of parathyroid hormone in this regard is unclear, but these observations reinforce the possibility that the action of the hormone on bone is mediated by its influence, direct or indirect, on the metabolism of bone mucopolysaccharide.

Methods of differentiating rachitic tibial sections that do not contain nuclei of crystallization in the preosseous cartilage from those that have been exposed to the calcifying solution but are below the threshold of visibility of the silver stain test are presented. These exposed sections are regarded as “nucleated” because they exhibit properties similar to those where calcification is visible with the silver stain test. “Nucleated” sections calcified in vitro while the non-nucleated sections showed complete inhibition when subjected to the following: (a) incubation in a calcifying medium (Ca × P = 50) containing a mixture of iodoacetate and magnesium ions; (b) shaking in distilled water at room temperature for half an hour, followed by in vitro calcification at a Ca × P product of 50; and (c) heating in distilled water at 76–77°C for 10 minutes, followed by in vitro calcification at a Ca × P product of 50. Calcification in vitro of “nucleated”sections took place at a lower Ca × P product than that needed for in vitro calcification of fresh, nuclei-free sections. It is suggested that the absence of nuclei should be established in studies of the nuclei-forming mechanism.

1) Antigenicity of a polymer of glutamyl peptide has been tested in 24 human volunteers. The polymer was of molecular weight and dimensions suitable for use as a plasma volume expander in humans. Skin tests, precipitin tests and complement fixation tests were performed on all subjects. 2) The results show 18 subjects with completely negative reactions by all 3 tests. There were 4 skin test reactions of dubious significance and one of real significance. There was one significantly positive serological reaction in a person showing a minimal skin reaction.

Rabbits given injections of res-erpine and fed a high cholesterol diet showed significantly lower mean serum total cholesterol levels and less lipid deposition in aorta, liver, spleen and adrenals than non-treated animals on the same diet. No variation was observed in rabbits treated with reserpine on a stock diet. Some rabbits treated with reserpine showed a kidney lesion similar to that described in rats treated with serotonin.

A mitosis stimulating factor in serum obtained from unilaterally nephrectomized rat on the 2nd postoperative day was investigated. This mitosis stimulating factor was heat stable at 56°C, non-dialyzable, organ specific, but not species specific. The probable nature of this factor is discussed.

TPN⁺ content of erythrocytes of primaquine-sensitive males is greater than that of erythrocytes of normal males. The increase in TPN⁺ may be explained by two known factors: 1) decrease in glucoses-phosphate dehydrogenase activity, and 2) increased glutathione reductase activity in primaquine-sensitive erythrocytes. The mean values of DPN⁺ content of erythrocytes in the two populations studied were different, but the values showed considerable overlap. Our current studies concern the effect of drug administration on both oxidized and reduced coenzymes in normal and primaquine-sensitive erythrocytes from both males and females.

1. Cholic acid prevents mobilization of elevated liver cholesterol. 2. β-sitosterol did not increase rate of mobilization of accumulated mouse liver cholesterol. 3. β-sitosterol reduces in large measure the effect of cholic acid on liver cholesterol mobilization.

Multiple tests on solutions to which have been added known numbers of various bacterial species, suggest that 1000 or more organisms/ml must be present before the U.S.P. rabbit pyrogen test will yield a positive result. The potentiality is inferred of a simple culture procedure as a screen test for pyrogenicity of solutions to be employed for parenterl infusion.

Acute pharmacologic comparisons have been made of N, N-dimethyl, 2- chloro-2-phenylethylamine (DMEA) and N, N-dimethyl, 2-chloro-2-phenyl-1-methylethyl-amine (M-DMEA). The 2 differed in immonium ring stability in vitro, speed of onset and degree of adrenergic blocking activity, and production of atropine-resistant salivation. There was no relationship between duration of effects and in vitro life of intermediate ethyl-eneimmonium forms.

1) A trace dose of cholesterol-4-C¹⁴ incorporated into a well-balanced test meal has been fed to rats. The labeled cholesterol is rapidly absorbed i.e. 50 to 70%. The newly absorbed cholesterol is then held up by the cells of the small intestinal mucosa, the halflife of the newly absorbed cholesterol in these cells being approximately 12 hours. From the intestinal wall the cholesterol enters the chyle, the peak specific activity of which is reached about 9 hours after the feeding. 2) The newly absorbed cholesterol of the thoracic duct lymph in the intact animal is selectively taken up by the liver and is only subsequently mixed in a metabolic sense with the cholesterol of blood and other tissues. 3) As a consequence of the delay of the absorbed cholesterol in the intestinal wall and the liver, blood cholesterol activity time curve reaches its maximum about 16 hours after the feeding. This delay in blood activity peak is prolonged to the second day if amount of cholesterol fed is increased to 10 mg.

Intravenous injections of the surface active agent Triton WR1339 into rabbits caused a marked increase in serum neutral fat as well as in cholesterol and phospholipids. A large percentage of this increase was found in the lipid fraction which rose to the surface when serum was centrifuged at 20,000 g for 2 hours or more. Administration of Triton to rabbits rendered hypercholesterole-mic by cholesterol feeding decreased the percentage and absolute value of total cholesterol remaining in the subnatant fraction after highspeed centrifugation. After several injections, concentration of cholesterol in the subnatant dropped well below the pre-injection value in spite of increase in total serum cholesterol concentration. It is suggested that an increase in percentage of the cholesterol associated with chylomicrons together with a probable decrease in the fraction associated with lipomicrons might be factors in Triton-induced inhibition of development of atherosclerosis in cholesterol-fed rabbits.

Asian or “A” viruses isolated at the State University of Iowa in the recent epidemic were tested against selected paired human sera. All sera which had a 4-fold rise in titer by the complement fixation test also had a significant rise in titer by the hemagglutination inhibition test when several “A” viruses were used as antigens. Neither viruses nor sera were chemically treated. There was a distinct variation in antibody response to “A” viruses when tested by hemagglutination inhibition. Possible causes of this variation have been discussed.

Epinephrine and norepinephrine stimulate production of nonesterified fatty acids from rat adipose tissue when incubated in vitro in rat plasma, presumably by stimulating the hydrolysis of neutral fats within the tissue. Incubation under nitrogen markedly inhibits this effect of epinephrine and appears to increase production of fatty acids from control tissues. The d-isomers of epinephrine and norepinephrine are much less active in this system than the physiological 1-isomers. indicating a specificity in tissue response. Some implications of this lipolytic activity are discussed.

The lack of antigenicity of purified pneumococcal polysaccharide and dextran in guinea pigs has been demonstrated. Both the methods used for immunization and detection of antibody have been shown to be quite good for the other systems.

A macroglobulin was purified from sera of individuals with rheumatoid arthritis which agglutinated sensitized sheep erythrocytes and fixed gamma globulin-latex particle suspensions. Tentative evidence for the homogeneity of a preparation was furnished. The isolated macroglobulin is a component of the rheumatoid factor. The latter is a mixture of closely related macroglobulins.

1) Granulomas were induced subcutaneously by 5 mg pellets of cotton wet with turpentine in 90 male albino mice. Thirty-five of these mice were segregated, 55 were in groups of 5 each. Granulomas of grouped mice weighed 19% less than those of segregated mice. These results were interpreted to mean that crowding increased adrenocortical activity sufficiently to have a suppressive effect on inflammation. 2) Granulomas were induced in rats with cotton and cotton plus turpentine. Cotton plus turpentine approximately tripled the weights of the induced granulomas over those resulting from cotton alone.

A comparative susceptibility study of kidney cultures derived from primates and non-primates, indicates that ECHO-10 virus has distinctive biological characteristics which differentiate it from the other human enteroviruses.

HeLa cells and chick embryo fibroblasts were grown on glass and harvested under various conditions. Trypsinization and scraping were each compared with alcoholether denaturation. Trypsinization was found to be less efficient as a means of obtaining material for chemical analysis although less damaging to cell viability than scraping. Alcohol-ether denaturation in situ was the preferred means of harvesting cells for chemical analysis.

1) Further evidence for species specificity and for lack of organ or cell type specificity of an antiserum to chick embryo heart cells has been obtained from tissue culture studies. 2) Passive sensitization of cells in tissue culture could not be adequately demonstrated and attempts to test the reactivity of endothelium or smooth muscle were unsuccessful.

Benzimidazole and 2-ethyl-5-methyl benzimidazole inhibited the growth of 2 B₁₂-requiring algae, Euglena gracilis, strain Z, and Ochromonas malhamensis, and of 4 B₁₂-synthesizing bacteria, Rhizobium meliloti, R. trifolii, R. leguminosarum, and R. japonicum. Benzimidazole inhibition was complete at 5 × 10^-3M concentration. The 2-ethyl-5-methyl compound inhibited the algae at 5 × 10^-3M concentration; rhizobia were more sensitive. Although benzimidazole inhibition of E. gracilis was not competitive, there was, at a 10⁸:1 ratio of inhibitor to vitamin, a relation to amount of growth which could be expressed graphically as a straight line.

1. Passive cutaneous anaphylaxis of the guinea pig has been successfully used to study thyroid autoantibodies of rabbit, guinea pig, and man. 2. The thyroid autoantibody proved to be specific for the homologous antigen (thyroid extract and purified derivative) and did not give cross reactions with thyroid extracts of other species. 3. Five human sera with high titer of thyroid autoantibody were studied. The antibody was completely neutralized by human thyroid extract or human thyroglobulin, but was not significantly affected by any of a variety of other organ extracts or of iodinated amino acids, again indicating the specificity of this type of antibody.

1) Supernatant fluid from 105,-000 g centrifugation of Ak leukemic tissues caused a multiplicity of tumors and a variety of non-neoplastic lesions in 87.5% of AkR mice injected shortly after birth. 2) Evidence reported here, and obtained by others (notably Gross, Stewart et al.) suggests that lesions described here, are caused by a pluripotent agent (to be called P virus) which is distinct from that which causes leukemia (to be called L virus). 3) The relationship between P and L viruses is conjectural. On the basis of evidence presented, we suggest as a working hypothesis that the P virus is of the parasitic type, is very small, and spreads by natural means. It is usually masked, and may be inhibited in tissue extracts by some macromolecular particles. The L virus is larger, has a more limited host range, and may be related to the genome of the leukemic cell.

Recent quantitative studies of immunological tolerance have focused attention on the need for special technics of bleeding and injection of embryonic and neonatal animals. Billingham and Brent(1) found that a higher proportion of tolerant mice was produced when homologous cells were injected by the intravenous route into the newborn than by other routes of administration. Their method of intravenous injection requires considerable skill and is not suitable for bleeding neonatal mice. Terasaki and Cannon(2) have devised an ingenious technic for cross-transfusion of blood in embryonic chicks. An intravascular method of injection for rat embryos of 13 to 17 days was described by Grazer and Clemente(3) but it has a relatively high mortality rate. The intracardiac technic described here for bleeding and injection of newborn rats and mice was accompanied by mortality of less than 5% in over 600 newborn rats, and 40 newborn mice. This technic has been advantageously employed in studies of transplantation immunity (4), and heterologous tumor cell tolerance.

Methods and materials. The animal is immobilized on an injecting board ventral side up (Fig. 1) with masking tape. It is important to stretch the subject as it is masked down, which helps to bring the heart closer to skin surface. The injecting equipment consists of a mounted Luer Lock Tuberculin syringe connected to a 15-inch length of P 50 polyethylene tubing by means of a plastic tubing adapter (Clay Adams size A). The 15-inch length can be conveniently calibrated for volumes up to 0.1 ml. A small length (1/2inch) of P 10 tubing is slipped inside the distal end of the P 50 tubing to serve as an adapter for the 30 gauge needle stock which is obtained by cutting off the hub of a 30 gauge needle.

Vaccinia virus particles have been counted accurately and reproducibly in dilutions of 10% extracts of infected egg chorioallantoic membrane and rabbit skin by the agar sedimentation method. Standard deviation of these counts ranged from 10 to 14%. Comparison with concentrated, purified vaccinia virus, of counting by sedimentation and spray droplet methods gave results which are in excellent agreement. Various factors important in the accurate counting of vaccinia virus in crude tissue extracts are discussed.

A method is described for collecting intermittent samples of adrenal venous blood. This method is applicable to rats, dogs, cats and other laboratory animals. It has the advantage of involving no manipulation, tension or interference with adrenal hemodynamics during periods of collection or when the adrenal effluent is being returned to the animal through a cannula placed in an ipsilateral abdominal vein.

Conversion of glucose to lactate by ascites tumor cells derived from the Ehrlich carcinoma, Crocker sarcoma 180, and Freund sarcoma is stimulated by 4,6-dinitro-o-cresol under both aerobic and anaerobic conditions. Degree of stimulation increases as temperature is lowered from 37° to 15°, although the absolute rate of lactate production diminishes. Aerobic lactate production is stimulated to a greater degree than anaerobic and the former exceeds the latter under certain conditions. The implications and possible mechanisms of these phenomena are discussed.

Polyvinyl sponges inserted under the dorsal skin of guinea pigs fill with cellular connective tissue. Dry weight of the granulation tissue, concentration of protein, desoxyribosenucleic acid and acid mucopolysaccharide at intervals between 1 and 12 weeks are reported. Both glucosamine and galactosamine were present in the mucopolysaccharide fraction. Evidence for the presence of 3 types of mucopolysaccharide components was obtained by chromatography and carbazole/orcinol ratios. A decrease in carbazole/orcinol ratios suggested an increased proportion of chondroitin sulfate B in the older granulomas.

A new sulfonamide, Madribon. exerted high protective activity in 12 experimental bacterial infections produced by grampositive and gram-negative organisms. The frequency of drug administration could be reduced to a single dose in all 7 infections in which this technic was tried.

A study with growing chicks and adult roosters indicated that dietary saponin will depress blood plasma cholesterol previously elevated by feeding low protein levels in presence and absence of dietary cholesterol. It is suggested that complexing of saponin with cholesterol secreted in the intestinal lumen makes less cholesterol available for reabsorption from the intestinal tract.

1) Previous observations on the toxicity of plasma from animals in irreversible hemorrhagic shock were confirmed and extended by the isolation of a toxic polysaccharide fraction from this plasma which cannot be isolated, under identical conditions, from normal plasma. The ability of this polysaccharide fraction to elicit responses characteristic of bacterial endotoxins, and to convert the reversible to the irreversible state of hemorrhagic shock has been demonstrated. The ability of bacterial endotoxins, in turn, to elicit the lesions characteristic of shock, was reviewed. 2) Although the entire quantity of toxic polysaccharide circulating in the irreversibly shocked animal is innocuous to a normal animal, only a fraction of the total is sufficient to inflict lethal injury to an animal with its defenses already depleted by reversible shock. The mounting evidence for the presence of endotoxemia in shock provides a logical basis for incriminating the loss of functional integrity of the R.E. system in the development of irreversibility to transfusion in hemorrhagic shock, and for an understanding of the phenomenon of variation in resistance to shock under different conditions. The development of irreversibility to transfusion in hemorrhagic shock in the germ-free animal is discussed in the light of these considerations.

Chromatography on silicic acid is a powerful tool for separation of blood lipids. In one experiment, about 20 different lipids may be detected; some of these remain unidentified.

1) Plasma was obtained from hemorrhagic anemic rats and from rats injected with cobalt for 26 days for determination of erythropoietic activity. Cobalt concentration of plasma from cobalt treated rats was lowered to normal levels by dialysis against isotonic phosphate-cyanide solution of pH 7.5. The level of cobalt in plasma was determined by modification of the nitroso-R-salt method. 2) Plasma to be injected was concentrated 6.6 times in vacuo. The erythropoietic activity was tested, using reticulocyte changes as index of activity, in hypophysectomized rats injected subcutaneously with 1 cc of concentrated plasma for 3 consecutive days. Reticulocyte counts were made 24 and 48 hours after end of injections. Plasma from anemic animals caused increase of reticulocytes to 61.5 and 151% (24 and 48 hours respectively) above values obtained from injections of plasma from normal rats. Injections of “cobalt treated” plasma increased the level of circulating reticulocytes to 36.4 and 54.5% above values obtained from injecting plasma from normal rats for similar time intervals, and demonstrates that erythropoietic activity of plasma is increased in rats receiving prolonged cobalt therapy, and that the activity is not due to increased levels of cobalt.

1. Cardiac hypertrophy was produced by exercise in albino rats. 2. There was a significant increase in concentration of glycogen in rat hearts with cardiac hypertrophy.

1) It was shown that 3 intravenous injections of thorotrast every 48 hours could induce an effective reticulo endothelial blockade in mice over 1 week. 2) S³⁵ labelled mouse plasma proteins were transferred intravenously to 2 groups of 5 normal mice and 5 mice which then received such blockade treatment. 3) The average half life of labelled plasma proteins was 37 hours in the normal and 30 hours in the blocked mice. 4) The significance of these findings, as related to a possible function of the reticulo endothelial system in catabolism of plasma proteins, is discussed.

Rats fed a magnesium deficient diet develop a leucocytosis during the hyperemic phase of the syndrome. Mononuclear leucocytes and neutrophiles contribute to the leucocytosis but eosinophiles are elevated relatively more, being increased over 1,000%. Possible relationships to the development of the leucocytosis are discussed.

An assay for the ulcerogenic property of steroids is described. The test compound is administered subcutaneously once a day for 4 days during which the animals are fasted in individual cages. At autopsy, the incidence of animals showing ulcers is noted, severity is estimated, and number of ulcers per rat is counted. These 3 values are combined and expressed in terms of ulcer index ranging from 0 to 20. It was shown that response is directly related to dose and that Δ¹-cortisol is 2 to 3 times as ulcerogenic as cortisol. Hypotheses to explain this effect of steroids are mentioned, namely, increased secretion of gastric juice, antiphlogistic property of steroids favoring necrosis of the mucosa, and decrease of mucus formation in steroid-treated animals.

Insulin was injected in different doses and at different rates into either the femoral or the portal vein of anesthetized dogs. No qualitative differences were noted between the 2 routes of administration: in all cases insulin caused a decrease in concentration of blood glucose and potassium and an increase in concentration of pyruvate and lactate. Intravenous carbutamide and tolbutamide caused a decrease not only in blood glucose, and potassium, but also pyruvate and lactate. It is suggested that the drugs may stimulate release of insulin from the pancreas, and that when a minimum (permissive?) amount of insulin is available and glycogenolysis is not accelerated, as in severe diabetes, they may also inhibit glucose production by the liver or its utilization by the muscle. If this hypothesis is correct, prolonged therapy with sulfonylurea-like drugs should be attempted with caution, for continued stimulation of the B cells with sulfonylurea may lead to their exhaustion (1,30) and suppression of hepatic glucose production may be a sign of tissue damage.

The steroid-17-spirolactones (I and II) have been shown to have progestational activity in the estrogen-primed immature rabbit.

1) Improved methods for preparation of formalinized red blood cells and a simple method using formalin to bind proteins to these cells yielding a stable, highly sensitive antigen have been described. 2) Cells prepared by these methods give agglutination patterns almost as distinct as those obtained using fresh cells, they are not agglutinated by traces of formaldehyde and are stable on storage at 3–5° C. 3) When cells were coupled with sulfanil-azo-groups a convenient and sensitive antigen for the estimation of antibody against this hapten was obtained.

Sex differences in control blood values of box turtles are presented. Mild to toxic doses of cobalt, including mineral supplement and injections of crude liver extract failed to stimulate erythropoiesis. Marked reticulocytosis was induced by repeated bleeding. High reticulocyte counts persisted for 3 weeks following the last bleeding and 4 months were required for restoration of normal erythrocyte values. Blood formation was altered by injections of folic acid antagonist, aminopterin. Arrested maturation of blood cells was detected in form of enlarged marrow blast cells as early as 5 weeks and anemia and leucopenia followed after 6 weeks treatment.

1) Spermine inhibited growth of several species of yeasts, Saccharomyces and Debaryomyces species being the most sensitive. The antimycotic effect of spermine was more pronounced at an alkaline pH and was antagonized by inorganic salts. The adsorption of spermine by yeast cells was significantly smaller than by Staph. aureus. 2) Spermine retarded the death rate of washed cells of S. cerevisiae suspended in tris buffer. A pronounced killing effect of spermine was observed when glucose was added to the buffer.

Incorporation of acetate-1-C¹⁴ and P³² labeled phosphate into the cholesterol and phospholipids of plasma, liver, and aorta of chicks treated with Δ-4-cholestenone was measured. The response of the chick differed from that of the rat in that there was an enhancement of the inclusion of the radioactive labels into their respective products in plasma and liver. A significant decrease in aortic cholesterol content and specific activity was noted.

The foregoing data show that pure cultures of micro-colonies can be isolated readily from smooth strains of Br. abortus. These G cultures can be maintained for several generations if subcultures are incubated aerobically. When fresh transplants are incubated in an atmosphere of 10% CO₂ reversion to the large colonial form occurs. Mice can also be easily infected with G colonies and the small forms can be recovered from the tissues, though the infection is of low pathogenicity and of relatively brief duration. The practical significance of these observations in animal and in human brucellosis under natural conditions cannot be ascertained at this time. Huddleson and Baltzer (2) did report the presence of G colonies of Br. abortus in a culture of stomach contents of an aborted bovine fetus, and Hall and Spink(1) isolated Br. abortus G colonies from a human patient. On several occasions we observed the presence of small colonies in cultures of Br. melitensis isolated from tissues of mice. It is suggested that a more intensive study should be made of fluids and tissues of animals and man for presence of small colonial forms of brucellae.

An acute infection with strain 19, Br. abortus was established in mice. Simultaneous treatment with cortisone enhanced the infection, but did not interfere with the formation of brucella agglutinins. However, cortisone did not cause the severe necrosis of the hepatic parenchyma and proliferation of brucellae that had been previously observed in studies with a more virulent species of Brucella.

Improved objective technics for assay of lactogenic and mammogenic hormones have been developed. The amount of lactogen and mammogen has been estimated in several different pituitary preparations by these assay methods. Wide differences in lactogen/mammogen ratios between preparations were demonstrated. These observations are taken to indicate that the pituitary hormone stimulating mammary gland growth (mammogen) is different from the hormone stimulating milk secretion (lactogen).

1. It has been demonstrated that retrobulbar injection of type A botulinus toxin in rabbits followed in 15 minutes by systemic administration of type A botulinus antitoxin produces a dilated fixed pupil in the eye on the injected side. 2. The dilated fixed pupil results from a block of the parasympathetic efferent nerves synapsing in the ciliary ganglion. This block is produced by the botulinus toxin. 3. Such a block persists in the intact animal at least 6 months without interference to nerve transmission of sympathetic or somatic pathways elsewhere in the animal.

Heretofore, complementary pairs of growth-factor dependent mutants of Streptomyces coelicolor have interacted to yield colonies which grow readily in the absence of added supplements. Recently however certain combinations have been found which did not interact successfully. This failure of nutritionally dependent mutants to form growth-factor independent growth was not the result of allelism, but of a compatibility system controlling heterokaryon formation. The compatibility system was apparently determined by two factors, one of which probably arose by mutation.

1) The anti-progestational activity of 4 estrogens—estrone, estradiol, estriol, and stilbestrol—administered subcutaneously along with progesterone into Clauberg rabbits has been demonstrated by estimation of endometrial carbonic anhydrase content and simultaneous measurement of uterine G/M ratio. 2) All these estrogens inhibit the effect of progesterone on carbonic anhydrase content and G/M ratio of the endometrium. Intensity of inhibition depends upon dosage. 3) The anti-progestational activities of these estrogens are approximately the same. 4) When administered alone, these estrogens produce no significant change in either carbonic anhydrase titers or G/M ratios of the endometrium. 5) The linear relationship with negative slope which is obtained between the logarithmic dose of the estrogens and the endometrial carbonic anhydrase content of the progesterone-treated Clauberg rabbit suggests the usefulness of the carbonic anhydrase method as an assay procedure for anti-progestational activity. 6) Comparison of the log-dose-response curves of progesterone with and without estradiol indicates that estrogen may depress reactivity of the endometrium to progesterone rather than neutralize or inactivate progesterone in the body.

Purified erythropoietin has been subjected to mild acid hydrolysis. This mucoprotein thus has been further characterized and shown to lose erythropoietic activity upon hydrolytic removal of neuraminic acid.

Atheromatous changes developed on the intimal surface of nylon and orlon thoracic-aortic vascular replacements in 2 of 6 dogs with induced chronic hypercholesterolemia. In both animals high serum cholesterol concentrations were maintained for over one year before the changes became evident. This is the first report of induced atheromatous change in a synthetic vascular prosthesis and may shed some light on the mechanism by which such changes occur.

L- and D-phenyllactic acids were shown to be interchangeable in replacing L-phenylalanine for L. casei 280–16A, but not interchangeable in functioning as a D-α-hydroxy acid source for this organism. It was concluded that L. casei 280–16A cannot racemize phenyllactic acid, and it was suggested that utilization of L- and D-phenyllactic acids in place of L-phenylalanine for this organism is mediated by separate (L- and D-) phenyllactic dehydrogenases.

Evidence has been obtained by in vitro studies that human post-heparin and endogenous plasma lipolytic activity (lipoprotein lipase) are inactivated during the process of triglyceride lipolysis. Addition of heparin to supernatant plasma after removal of lipoprotein lipase activity by Ca₃(PO₄)₂ adsorption occasionally restores its lipemia clearing activity. This indicates that heparin is a component of the enzyme, and that its dissociation from the apoenzyme readily occurs.

Daily oral administration of 150 mg of cobaltous chloride to 6 normal human subjects caused development of increased erythropoiesis characterized by erythrocyte increases ranging from 0.5 to 1.19 M (average 0.96 million) cells/mm³ of blood within 7 to 22 days. The high red blood cell counts returned to normal within 9 to 15 days after cessation of cobalt administration.

Twenty-seven white male Wistar rats weighing 450 g at beginning of the experiment were given a diet that induced a state of hyperlipaemia and produced vascular injury. This diet, arrived at as a result of several pilot experiments, proved to be very effective, and yielded the following incidence of coronary lesions: Lipomatous coronary changes 88%. Atheromatous coronary changes 81%. Atherosclerotic coronary changes 63%. Coronary occlusion 33%. Cardiac infarcts 14%.

Progestins found capable of maintaining pregnancy in rats castrated on the 8th day after insemination included progesterone, 17α-hydroxyprogesterone acetate, 17α-ethyl-19-nortestosterone, desoxycorticosterone acetate, 9α-bromo-11-ketoprogesterone and 6α-methyl-17α-hydroxyprogesterone acetate. The most potent compound in this respect was 6α-methyl-17α-hydroxyprogesterone acetate. Compounds which failed to maintain pregnancy include 17α-ethinyltestosterone, 17α-ethinyl-19-nortestosterone, 17α-ethinyl-17-hydroxy-5(10)-estren-3-one, and 17α-methyltestosterone. With the exception of 17α-hydroxyprogesterone acetate, all compounds which maintained pregnancy when administered concomitantly with estrone also maintained pregnancy when administered alone. Higher doses were required, however, for comparable results. Fair correlation between reported potency in the McPhail or Clauberg rabbit endometrium assays and pregnancy maintenance potency existed only for 6α-methyl-17α-hydroxyprogesterone acetate, 9α-bromo-11-ketoprogesterone and desoxycorticosterone acetate.

A study of properties of extracts from cultures of 160 non-pathogenic fungi has shown that 4 cultures produce non protein materials able to lyse plasma clots and fibrin, both human and bovine source; at least one shows some specificity against human plasma clots.

Two modifications of the “George Wright” canine tracheal divider have been made. They are: 1) substitution of polyethylene for brass tubing, and 2) addition of prongs to the divider head. Inadvertent pulmonary artery obstruction is prevented, and the divider does not slip past the carina.

Rats fed a diet with a lactose content similar to human milk (43% of the calories) grow at a reduced rate. Normal growth was attained with an equivalent mixture of glucose and galactose, thus implicating the intact disaccharide and suggesting that the adverse effect is of intestinal bacterial origin. Chlortetracycline, and penicillin to a lesser degree, increased growth rate. Carbomycin, bacitracin, polymixin, sulfathalidine and a dihydrostreptomycin-polymixin-pectin-adsorptive clay mixture were ineffectual despite their relatively low solubility. The characteristic low-body fat of the lactose-fed rat was not affected by chlortetracycline administration.

1. Lymph and lymphocyte outputs from thoracic and right lymphatic ducts of male rats have been measured. 2. Rate of flow and lymphocyte output of the thoracic duct was 1.8 ml/hr/kg and 42.9 million lymphocytes/hr/kg; right lymphatic duct values were 0.3 ml/hr/kg and 5.7 million lymphocytes/hr/kg. 3. Utilizing these data a Daily Replacement Factor for blood lymphocytes of 4.6 was obtained for the anesthetized rat. 4. These results have been compared with similar findings reported for other species.

1) Vit B₁₂ binding capacity of normal human saliva has been estimated using Co⁶⁰vit B₁₂ and exhaustive dialysis. It varies from 13 to 80 mμg/ml, with a mean of 31 ± 10.2 mμg/ml. 2) These values appear constant for the individual over a period of hours to days. 3) No correlation was found between vit B₁₂ binding capacity and salivary specific gravity, refractive index, optical density at 280 mμ, leukocyte count, ABO blood groups or secretion of ABO substances in saliva.

In supine, anesthetized dogs breathing 100% O₂ the alveolar-arterial O₂ difference is unaltered when ambient pressure is increased from 1 to 2.5 atm. The results can be explained in terms of constant admixture of systemic venous blood with fully oxygenated pulmonary capillary blood. It is likely, but not proven, that previous estimates of arterial O₂ pressure at high barometric pressures are too low.

1) Thyroid hormone secretion rate (TSR) was determined in lactating and non-lactating rats by administration of graded levels of L-thyroxine and measuring by radioiodine technics, subsequent suppression of thyroidal I¹³¹ output. 2) The average TSR of 2.2 μg/100 g L-thyroxine for lactating rats during the period of intense lactation (8–14 days postpartum) was significantly greater than the average of 1.3 μg/100 g for non-lactating rats of the same strain, age and weight range. These data are in accord with the concept that a hyperthyroid state is generally desirable for optimal milk secretion.

In 5 patients with Cushing's syndrome due to adrenal cortical tumors, urinary 17-ketosteroids were fractionated by column chromatography and identified by infrared spectroscopy. The findings were: 1. One patient with benign adrenal cortical adenoma had a low value of total urinary 17-ketosteroids. The only steroids identified were 11-oxygenated 17-ketosteroids. 2. Four patients with adrenal cortical carcinoma showed elevated urine 17-ketosteroid levels. All 4 had absolute increases in the quantity of 11-oxy-17-ketosteroids. All showed increases in ratio of etiocholanolone to androsterone. Only one showed a marked increase in amount of dehydroisoandrosterone. 3. Certain similarities to and differences from the 17-ketosteroid pattern of patients with Cushing's syndrome due to bilateral adrenal hyperplasia are pointed out. It is suggested that the urinary 17-ketosteroid excretion pattern in patients with Cushing's syndrome due to adrenal carcinoma may not always be sufficiently characteristic to point to a diagnosis of tumor as opposed to hyperplasia of the adrenal cortex.

Absorption of 5-HT, including that present in banana pulp, is quite rapid and fairly complete. Conversion to 5-HIAA and renal excretion of this metabolite are accomplished without delay. In evaluating abnormally high levels of 5-HIAA in human urine the dietary history is of some significance. Urines obtained more than 8 hours after ingestion of bananas should show little influence of exogenous 5-HT administration.

Sympathomimetic agents produce a severe lactate acidemia in the alligator. Although the lactate concentration often exceeded the initial alkali reserve, over 90% survived by withdrawing sodium from sources outside the extracellular fluid. The extracellular osmotic pressure rose sharply during the acute acidotic phase and fell when the lactate was metabolized. There was no evidence that K contributed to the defense against this acidemia.

1) A. niger growth was prevented when DNB at a final concentration of 2.5 × 10^-5 M was added to the culture medium at any time up to 30 hours' incubation. When DNB was added at any time later, some additional growth occurred. The DNB effect appeared to be limited to molds and was static rather than cidal. 2) Growth inhibition by DNB was overcome by synthetic mixtures of amino acids, but not by ammonium nitrogen, vitamins or minerals. 3) Nitroaryl compounds can be reduced to relatively nontoxic arylamines by a sulfhydryl-dependent, pyridine nucleotide-linked enzyme from A. niger. 4) The possible significance of these findings is discussed.

Fungal phenolsulfatase of Mylase P was purified by fractionations with ethanol, zone electrophoresis on cellulose, and treatment with aluminum hydroxide gel. Related to the activity of the original Mylase P the enzyme was purified approximately 1900 times. On the basis of purification of the protein of Mylase P approximately 230-fold purifications were achieved.

A study of biosynthesis of cholesterol in vitro from radioactive acetate in animal blood was undertaken. Degree of incorporation of acetate into cholesterol was higher in calf blood than in pig blood samples. Cholesterol synthesized in rat, rabbit, cow, and piglet blood samples had comparatively a very low radioactivity.

Commercial neomycin and its fractions A, B and C possess a curarimimetic action as found when employing sciatic-gastrocnemius preparation of frogs. This action is more pronounced with commercial neomycin and fraction B than with fractions A (Neamine) and C. The blockade induced by neomycin, fraction B and C, but not by fraction A. was effectively antagonized with neostigmine or edrophonium in proper doses. Ether anesthesia did not potentiate this action in the frog when neomycin or its fractions B and C were used.

Intraperitoneal administration of radioactive phosphorus to Rana esculenta resulted in an appreciable incorporation of the isotope in the phosphoprotein fraction of liver and very much less in the corresponding fraction of ovary. The presence, in liver, of a metabolically active phosphopeptide, was demonstrated. These findings were discussed in relation to the site of synthesis of phosphoproteins in the amphibian.

Rate of disappearance of injected I¹³¹-labeled TMV was more rapid from spleen, an important site of antibody production, than from liver, which produces little or no antibody. It was concluded, therefore, that persistence of the virus is not in direct relation to the tissue's antibody-producing capacity. The need for studies at cellular level was indicated. In regenerating liver, total radioactivity was not significantly influenced by increased mitotic rate. This was interpreted as indicating that virus localized in liver may be retained during cell division.

Glucose - cyclo - acetoacetate (G.C.A.), a precursor of ascorbic acid in germinating legumes and which on hydrolysis gives rise to a reducing dienol glucose, is formed during germination from glucose and acetoacetate. Thiamine and pantothenic acid accelerated biosynthesis of GCA and their antivitamins depressed such synthesis. GCA has thus been shown to be an intermediary product towards biosynthesis of ascorbic acid during germination of legumes.