Experimental Biology and Medicine

1. An egg-adapted strain of Theiler's mouse encephalomyelitis virus (TO) has been carried through 42 passages in vitro on embryonic chick tissue. Attempts to adapt the mouse passage virus directly to chick tissue culture were unsuccessful. Preliminary growth in embryonated eggs, followed by tests in mice of 20% embryo suspensions, facilitated the titration of tissue culture fluids. 2. One of 3 strains of approximately the same mouse-infective titer failed to adapt either to egg or tissue culture. 3. A representative strain of Theiler's mouse encephalomyelitis (TO) virus obtained from the American Type Culture Collection also adapted to egg and tissue cultivation.

1. Topical application of acetylcholine usually produced a local depression. The depression was not propagated, and was not due to changes of systemic blood pressure. 2. The local depression was often succeeded and sometimes masked by enhanced electrical reactivity. This action varied from a barely discernible increase to a sustained high voltage pattern lasting 30 minutes. The discharges were randomly distributed in time in some instances; in others, the activity was organized in distinct groups of paroxysmal spikes each group being accompanied by a distinct surface negative D. C. pulse.

Studies have been made to determine the effects of native heparin upon the lipemia of cream fed dogs. It was found that shock due to anaphylaxis or to the injection of certain histamine liberators sometimes causes heparin to appear in the blood of the animals. The presence of native heparin in the blood, however, does not necessarily coincide with clearing of a lipemia; fat clearing may occur during shock either in the presence or in the absence of detectable amounts of native heparin in the blood of dogs. In several experiments, the injection of dextran (M.W. 75.000) produced marked clearing of a heavy alimentary lipemia in the absence of heparin activity in the blood of dogs. Protamine sulfate caused the return of both visible turbidity of the plasma and of whole blood chylomicron counts to control levels after fat clearing due to shock. This occurred both in the presence and absence of significant blood heparin activity.

A bluish-violet color stable for 24 hours in a pH range of 5.5 to 8.0 results from addition of a solution of ferric ammonium oxalate to a solution of epinephrine or of suprarenal gland extracts. The transmittancy of the solution is determined at a wavelength of 555 mμ. The presence of ascorbic acid, uric acid or other naturally occurring substances found in adrenal glands does not interfere with the determination. This method is not applicable for the determination of epinephrine in blood or in drugs. The procedure is extremely simple and rapid and is capable of an accuracy of less than 1% to 5% as the greatest variation. The bluish-violet color that is obtained from minced autolyzing adrenal glands has been shown to be caused by a reaction between an epinephrine-like substance and iron products from the food chopper. A comparison of the specific extinction of various epinephrine standards and a note on the effects of dry storage is made (Fig. 1).

1. Monkeys and rabbits were injected intravenously with various amounts of polysaccharides (dissolved in 0.85% saline) prepared from the muscle stage of Trichinella spiralis and from adult Ascaris lumbricoides from pig. 2. The titer of the α₂ isoagglutinins detected at 37 °C in the serums of the animals was reduced to negative for a period of 24 hours, and remained lower than the titer before inoculation for a period of 96 hours. 3. The titer of the α₁ was also reduced, but not as much as the α₂ agglutinins. 4. The titer of the agglutinins acting at 6°C against A₁, A₂ human, sheep, and the animal's own erythrocytes was also reduced after inoculation of the polysaccharide. 5. The possibility of injecting these polysaccharides to inhibit autoagglutination of erythrocytes as seen in certain pathological conditions is mentioned.

Data have been presented suggesting that the enhancing effect of a heat labile component of canine serum on the agglutinative capacity of the canine isohemag-glutinin A is due to complement. Although guinea pig and human serum are ineffective, bovine serum, in most instances, will restore maximum activity to a previously inactivated system. Further data suggest that this enhancement, which appears to be unique so far as erythrocyte isoantibodies are concerned, is related to conglutination.

1. A microbiological method to assess the biological value of proteins is described. It implies the digestion of proteins with pancreatin and subsequent testing of the growth-promoting activity of the hydrolysate for S. faecalis, in a medium devoid of the 10 essential amino acids. The nutritive value of the protein digest is determined in a single assay by the amino acid liberated in a limiting concentration. 2. The results obtained with casein, egg albumin, gelatin, gluten, and zein are presented and their agreement with the biological values obtained by the rat growth method is suggested. 3. The growth promoting activity (biological valued) of casein and egg albumin is high and about equal; gelatin and gluten, in comparison, are much less active, 29 and 15%, respectively, and zein still less (2%). 4. At the limited growth level, casein is deficient in: lysine, arginine, isoleucine, and threonine. Egg albumin under comparable conditions is limited in lysine, arginine, histidine, threonine, isoleucine, leucine, and valine. Gelatin is low in tryptophane, isoleucine, leucine, methionine, and threonine. Gluten and zein are short in lysine. 5. The suitability of the method described for studying imbalances of amino acids is proposed.

High cortisone dosage in ad-renalectomized dogs induces marked polyuria and polydipsia. The 24-hour urine volume may exceed control values by 5-10 fold. The daily renal excretion of Na, Cl, and K is increased, but considering the quantity of water eliminated, the urine is very dilute. Pituitrin is effective in controlling the polyuria and polydipsia only when administered in large amounts. The normal water balance was not re-established until long after cortisone injections were discontinued.

1. The agar dilution and tube dilution methods of determining bacterial sensitivity are equally reliable. 2. Aureomycin-containing plates can be stored for 3 weeks without loss of potency; plates containing penicillin, bacitracin, streptomycin, chloramphenicol, and terramycin can be stored for 4 weeks without loss of potency. 3. The agar diffusion method of sensitivity determination is not fully reliable, as shown by poor correlation of the results obtained by that method and the tube dilution method of testing with some antibiotics.

Biliary and urinary excretion of radioiodine was studied following subcutaneous injection of tracer amounts of I¹³¹-labeled thyroxine in normal, hypo- and hyper-thyroid rats. Thyroidectomized and thiouracil-treated groups showed reduced biliary radioiodine elimination, as well as diminished bile volume during the 6-hour collection period. Hyperthyroid animals showed a marked increase, thiouracil-treated animals a decrease in urinary excretion of radioactive iodine over a 12-hour period. Radioiodine excretion in both bile and urine tended to be lower in the thiouracil-treated animals than in the thyroidectomized groups.

1. Cortisone is ineffective in preventing HN₂-induced leukopenia in rabbits. On the contrary, cortisone induces a greater leukopenia as a result of increased lymphopenia. 2. Administration of cortisone to rabbits receiving L-cysteine prior to HN₂ administration results in a more severe leukopenia and neutropenia than administration of L-cysteine and HN₂ alone. 3. The combined use of cortisone and HN₂ therapeutically may induce a dangerous depression of hematopoietic tissue.

1. L-lyxoflavin, D-galactoflavin and isoriboflavin markedly increase the growth response of Lactobacillus casei to suboptimal amounts of riboflavin, but do not promote growth in the absence of this vitamin. Appropriate differential assays indicated that lyxoflavin was not deposited in cells of L. casei under conditions where it stimulated growth, and actually decreased the concentration of riboflavin deposited in such cells. Thus it does not serve as a partial substitute for riboflavin in L. casei, but appears to enhance the efficiency with which limited supplies of riboflavin are utilized. When riboflavin is present in excess, lyxoflavin does not increase the cell yield or the growth-rate. At higher concentrations, lyxoflavin and galactoflavin inhibit growth by acting as competitive antagonists of riboflavin. Dichlororiboflavin and isoriboflavin are ineffective as inhibitors of this organism. 2. L-lyxoflavin promotes maximum growth of Lactobacillus lactis in the absence of riboflavin, and such growth continues indefinitely upon subculture. Differential assay with L. casei and L. lactis showed that cells of the latter organism grown with lyxoflavin contain no riboflavin, but do contain lyxoflavin; i.e., in this organism lyxoflavin can fill the essential metabolic roles normally played by riboflavin. Similar assays of tissues from rats grown on natural rations indicate that lyxoflavin does not occur naturally in significant quantities. 3. In chicks, lyxoflavin stimulated growth when fed at levels 2.5 to 10 times the amount of riboflavin in the diet. At ratios of lyxoflavin to riboflavin higher than 40 to 1. lyxoflavin inhibited growth. Amounts of lyxoflavin that inhibited growth on diets of low riboflavin content were non-toxic when the amount of riboflavin was increased. 4. Although the growth-responses observed in chicks when L-lyxoflavin is added to diets low in riboflavin may result in part from a sparing action similar to that found with L. casei, such a sparing action does not explain the growth effects of the analog observed in rations of high riboflavin content.

1. Sharply limited ablation of the arm or leg area of the precentral motor cortex in the macaque produces a complete or partial depression of the reflex myoclonic responses of the affected extremities as elicited after subconvulsant metrazol dosage. This occurs in chronic, as well as acute preparations. 2. When responses are present, there is a predominance of extensor muscle activity, with marked diminution of finger- or toe-flexor responses. A similar pattern is observed when generalized seizures are elicited by repetitive acoustic stimulation.

1. The preparation of a sensitive and specific Vi agglutination reagent by sensitization of human type O erythrocytes with Vi antigen isolated from E. colt has been described. 2. Vi antibody content of rabbit and human sera was determined by this method and the conventional bacterial agglutination test. The hemagglutination test provided a more sensitive measure of Vi antibody by a factor of 3 to 6 fold. Greater variability in Vi titers was encountered in testing the sera of typhoid carriers; however, both methods were in good agreement in the detection of Vi antibody in these sera. The advantages inherent in the Vi hemagglutination procedure are discussed.

1. Administration of PAS to rats over a 2-week period produced goiter associated with diminished I¹³¹ uptake by the thyroid gland. 2. No effect on the pituitary-adrenal axis was observed. 3. Elevation of liver glycogen concentration was noted in all PAS-treated animals.

A method is presented which permits the differentiation and enumeration of basophils from the counting chamber. Basophil counts were performed on 69 normal individuals and the range, mean, and standard deviation are reported by sex.

1. At plasma levels of 30 to 50 μg/cc, significant renal tubular secretion of pantothenic acid occurs. 2. It is probable that pantothenic acid is secreted by the same renal tubular transport mechanism involved in the transport of penicillin, phenolsulfonphthalein and PAH. 3. Probenecid can inhibit effectively the renal tubular secretion of pantothenic acid in man. 4. Probenecid enhances the plasma pantothenate levels obtained by the intravenous administration of sodium pantothenate in doses of 500 and 1000 mg.

During the course of studies involving adrenal slice incubations. it was noted that extracts prepared from the slices after incubation sometimes contained 3 compounds which migrated very slowly on paper chromatograms. At other times these would not be present, or if present, in amounts which could not be detected.

The possibility was considered that these might represent conversion products of one of the known steroids. Consequently, hydrocortisone was carried out with 2.2 gg of adrenal cortical tissue slices. Four and seven tenths mg of hydrocortisone were added to the incubating medium which was saline-phosphate (4:1), pH 7.3. extracts made from the medium and slices were purified by silica gel chromatography. The purified fractions were then chromatographed on filter paper using the technic of Bush 1 . In this particular in cubation, adrenal slice incubated without added hydrocortisone produced no detectable quantities of the polar compounds, whereas the extracts from the incubation with added hydrocortisone showed the 3 polar compounds on the paper chromatograms (Fig. 1). The hydrocortisone had previously been shown to be chromatographically homogeneous.

1. Amphetamine and its isomers are diuretic and natriuretic in starved, normal and salt loaded rats. 2. The glomerular nitration rate of starved rats is elevated by d-amphetamine. The elevation of nitration rate is sufficient to account for the diuresis and natriuresis observed without postulating a decrease in tubular absorption of water or sodium.

1. When Newcastle disease virus is mixed with penicillin, inactivation of the virus takes place. The extent of the inactivation depends both on the concentration of penicillin and the time of incubation of the mixture. Incubation of the virus in vitro for 3 hours with more than 5000 units per ml of crystalline penicillin G results in at least a 100-fold reduction of the infectivity of the virus and up to 2 million ELD₅₀ of the virus may be completely inactivated. 2. The virus inactivating property of penicillin may be destroyed by acid or alkali treatment but is not affected by heating. Penicillinase also inactivates the antiviral properties of fresh and heated penicillin. However, this effect is apparently not due to enzymic action. Virus previously inactivated by fresh or heated penicillin cannot be restored to its original potency when penicillin is neutralized by penicillinase. 3. Injection of 50000 units of penicillin mixed with the virus protects chick embryos and 10-day-old chicks against as many as 4000 ELD₅₀ of the virus. At this concentration, however, penicillin has no prophylactic or therapeutic activity.

1. Ascorbic acid has been found to have thiamine sparing action on the growth of L. jermenti 36. The addition of both ascorbic acid and thiamine gives a greater growth response than either one when added alone. 2. Addition of pyrithiamine inhibits the utilization of ascorbic acid for growth of L. jermenti 36 in a thiamine-free medium. This result shows that ascorbic acid may function in part by stimulating the synthesis of thiamine. 3. Addition of iodoacetate, 2,4-dinitrophenol or sodium fluoride to the thiamine assay medium containing either thiamine or ascorbic acid shows the difference in inhibition of growth of L. jermenti 36. These results indicate that ascorbic acid must also function in a different metabolic pathway as compared to thiamine in carbohydrate metabolism. 4. It was found that L. jermenti 36 was able to synthesize thiamine when ascorbic acid was present. With the presence of both ascorbic acid and thiamine in the assay medium, the cell was found to contain more thiamine than the cell grown in thiamine medium alone.

1. Enteramine (5 hydroxy-tryptamine) decreases the urine volume of hydrated dogs at doses which do not stimulate the smooth muscle and do not modify the systemic blood pressure. The lower doses (0, 1-0, 3 mg per kilo) subcutaneously or intravenously decrease the urine volume without affecting the GFR, while higher doses reduce also the filtration rate; both lower and higher doses increase the RPF. 2. The above described experiments suggest that the antidiuretic effect of enteramine in the dog derives principally from an increased reabsorption of water from the renal tubule.

Minced immature rhesus testicular tissue remained viable for a period of 8 weeks when refrigerated at 5°C in Hanks' basic salt solution containing 2.5% salt-poor human albumin. Some observations on the use of human albumin as a constituent of tissue culture media have been recorded.

1. The effects of a mild degree of induced-hyperthyroidism or vit. B₁₂ or both on growth rate were determined in 81 immature male mice fed a vit. B₁₂-deficient ration, in two experimental trials of 26 or 30 days duration. 2. Feeding .025% Protamone (iodinated casein) or 100 μg of vit. B₁₂/kilo of ration each increased the growth rate of the mice, and Protamone was more effective than vit. B₁₂ in this respect. Each substance increased food intake and enhanced the efficiency of converting food into body weight gains. When Protamone and vit. B₁₂ were fed together however, the favorable effects of either on growth were largely eliminated and efficiency of food utilization was reduced. No satisfactory explanation can be offered for this phenomenon at this time.

The effects of growth hormone, insulin, and cortisone acetate administration was determined on the change in body weight of rats during protein depletion and repletion. Neither the weight loss during depletion nor the gain in body weight during repletion was affected by the administration of these hormones. After pre-depletion weight was regained, however, the growth increment for animals administered growth hormone was larger than for the other groups.

Polyhydroxy compounds (D-ribose, pyridoxine hydrochloride, D-sorbitol hydrate); known to form complexes with boric acid, were tested for their effect on the teratogenic action of boric acid during development of the chicken embryo. It was found that complex formation reduces or abolishes the teratogenic qualities of boric acid. Triethanoalmine borate produced the same malformations as did boric acid. Injected sodium aluminate caused few developmental defects, but they seem to be related to those brought about by boron compounds. Our results support the working hypothesis that boric acid interferes with normal development by complex formation in ovo with potyhydroxy compounds, thereby producing symptoms resembling riboflavin deficiency. Additional evidence suggesting that in our material boric acid acted on coenzymes rather than on enzymes was presented earlier.

1. The average portal venous pressure of rats was 15 cm H₂O in the control period, 9 cm H₂O after bleeding to an arterial pressure of 45 mm Hg, and 14.5 cm H₂O immediatly after blood replacement. 2. Direct microscopy disclosed reduction in blood flow and narrowing of sinusoids, central venules and sublobular venules after bleeding from 0.7 to 1.0 cc/100 cm 2 of body surface. These changes became extreme after a hemorrhage of 2.1-2.4 cc/100 cm^-2. Replacement transfusion after 3 hours of shock restored normal vessel caliber and blood flow, whether recovery followed or not.

A delayed and retarded type of embryonic development occurred in 16.7% of the Beltsville Small White turkey eggs laid 54-224 days after the females were separated from males. This development in most instances could not be detected macroscopic-ally earlier than the 4th day of incubation. The embryonic membranes usually discontinued their growth after covering an area of the yolk equal to that occupied by a normal 2- to 3-day turkey embryo. In most instances no embryo could be detected macroscopically although serial cross sections showed a definite organization on the part of centrally located cells. Two living embryos were found in eggs laid 78 and 98 days respectively after the hens were separated from the males. These embryos, after 8 days of incubation had attained a size equivalent to a normal 4-day turkey embryo. The embryonic hearts were still beating and there was a well developed vascular system in both cases. A 7-day embryo, with a well-developed vascular system was also found in an egg which had been incubated 11 days. This egg was laid 195 days after the female had been separated from males.

1. A strain of Semliki Forest virus passaged intracerebrally in Swiss albino mice has been successfully transmitted intraperitoneally and intracerebrally to mongrel puppies. 2. Puppies exposed by intacerebral, intranasal, intradermal, intraperitoneal, and intracardial routes to Bwamba Fever virus and puppies exposed by intranasal, intradermal, and intracardial routes to Semliki Forest virus showed clinically no signs of nervous symptoms, and mice injected intracerebrally with brain suspensions from these puppies showed no evidence of Bwamba Fever or Semliki Forest disease.

1. Data are presented on 4 mice treated for 18 months with a diet to which propylthiouracil had been added. 2. Three of 4 mice had chromophobe adenomata of the anterior lobe of the pituitary gland. 3. All 4 mice had carcinomas of the thyroid gland.

1. A simple method has been developed for the recovery of FSH from residues resulting from the acid acetone extraction of swine pituitary glands. 2. The biological activity of the preparations obtained compare favorably with those of other investigators.

Total body water and blood volume were determined in mice with the hereditary obese hyperglycemic syndrome and in littermate controls. Only 12% of the excess weight of obese animals was found to be represented by water. Blood volume was not increased in obese animals. The total body water and percentage of weight represented by water in young obese animals was similar to those in non-obese animals of the same weight.

1. Glycogen contents of rat liver and leg muscle were unchanged after daily injection of acetoacetate and propionate in increasing amounts for 21 weeks. 2. Ability of very small amounts of insulin to promote glycogenesis in surviving hemidiaphragms was not inhibited by 50 mg % of acetoacetate. 3. A rapid hydrolysis of the glycogen of liver slices was unaffected by 125 mg % of acetoacetate in the buffer in the absence of glucose, but glycogenolysis was markedly decreased if both were present. 4. Acetoacetate in these concentrations does not appear to inactivate insulin, to inhibit glycogen storage in the muscle or liver tissue, or to affect appreciably the utilization of carbohydrate as measured by the glucose tolerance.

The responses to repeated identical doses of epinephrine in the same dog show marked variability. In general, cocaine does not sensitize the dog to the hypertensive effects of epinephrine injections. Neither do the doses of cocaine used, 2 and 5 mg7 kg, alter the level of the control blood pressure.

Suspensions of viable cells of Leptospira icterohetnorrhagiae (Wijnberg) were disrupted with an equal volume of a solution containing 0.1 M NaCl, 0.2 M sodium citrate, and 0.4% sodium desoxycholate. An immunologically- reactive, non-dialyzable substance (Fraction 1) was prepared by a procedure which avoided heating and use of alkalis and acids. The active principle was isolated in mixture with a non-dialyzable polynucleotide moiety. The organism of L. icterohemorrhagiae contained an appreciable amount of the polynucleotide substance and was composed of pentose—as well as desoxy-pentosenucleic acids. Crystalline ribo- and desoxyribonuclease altered the absorption spectrum of the nucleotide, but failed to inactivate the antigen. On the basis of the latter evidence it appeared likely that the polynucleotide was not the determinant of immunological reactivity. The active principle resisted the action of trypsin, hemi-cellulase and was not destroyed by heating for 1 hour at 100°C.

The adrenal cholesterol of pantothenate deficient rats was significantly depressed compared to that of control animals on a normal diet. After the stress of unilateral adrenalectomy, the cholesterol content of the remaining adrenal decreased immediately in both diet groups. By 24 hours after unilateral adrenalectomy, the adrenal cholesterol of the normal rats had returned to its initial value; in the deficient rats adrenal cholesterol remained depressed up to 7 days after the stress. The data are interpreted as evidence that the synthesis of adrenal cholesterol is decreased in pantothenate deficiency.

1. Feeding of 0.5 g of potassium penicillin per 100 lb of milk replacement to Holstein bull calves resulted in a decreased rate of gain in body weight and growth in height at the withers in this experiment. 2. Feeding of 0.1 g, 0.3 g, 0.9 g, and 2.7 g of procaine penicillin also resulted in decreased growth rates in terms of mean gains in body weight and mean increase of height at the withers in this trial.

1. By ultracentrifligation at 90,000 × g, a particulate fraction inhibitory to growth of transplantable lymphosarcoma, Ehrlich ascites tumor and Yoshida sarcoma has been obtained from small intestines of mice and rats. 2. Inhibitory properties of the fraction have been demonstrated by pre-inoculation exposure of tumor cell suspensions to the inhibitory material. 3. The intestinal fraction markedly suppresses oxygen consumption of tumor slices, slightly reduces the respiration of normal lymphoid tissues, and has no effect on respiration of other normal tissues tested; an initial respiratory stimulation was observed with ascites tumor, followed by marked depression. 4. The active component of the intestinal fraction appears to be an ethanol and petroleum ether-soluble lipid.

The well known diurnal fluctuation in the number of circulating eosinophils in mice was maintained in animals exposed to continuous cold. It is suggested that a diurnal cycle of activity is responsible for this variation and that the nocturnal activity, acting as a stress, elicits increased activity from the adrenal cortex which is already responding to the stress of cold.

The preparation of water of sufficient purity for critical biological work, and in sufficient quantity, is a perennial problem in biological research laboratories. It is generally agreed that the output of metallic stills such as those of block tin is likely to prove unsatisfactory. Quartz is expensive and the units ordinarily available have a low output and require constant attention. Pyrex glass has proved a satisfactory substitute for most work and there are many types of Pyrex stills available which give excellent results as to quality of product. Most of them, however, are far from automatic and are generally likewise of rather low output. The type of all-Pyrex still described here has been developed after considerable experience, and has been used successfully in preparing nutrients for tissue-culture studies in a number of laboratories. It is relatively inexpensive, and once set up requires a minimum of attention.

The outfit as we use it (Fig. 1) consists of two duplicate units, each made up of 4 items, 3 of which are standard. The units are 1) a standard stove-type heater, usually a 2-burner electric stove with 3-heat burners; 2) two standard flat-bottomed 2-liter distilling flasks (Fig. 2. 3) each with 34/45 ST female joint at the top and side-arm having a 24/40 ST male joint; 3) two standard 22 cm Fried-rich condensers, each with a 24/40 ST female connection at the top and standard drip point below, and 4) two constant-level inlet tubes. The inlet tubes∗ consist of 5 pieces (Fig. 1, 2, 3): first a) there is a feed unit consisting of a 34/45 ST male joint attached above to a 30 mm tubulation and below to one of 20 mm diameter.

1. Under the conditions of our experiments; the administration of cortisone to mice given external applications of methyl-cholanthrene in benzene or in carbowax 1500 was followed by some reduction of the early inflammatory response and by an increase in the incidence of epidermal tumor formation per number of animals, as compared with the results in the control experiments without cortisone injections. 2. Biopsy excisions performed in the methylcholanthrene-exposed skin areas appeared to increase still further the incidence of tumor formation in the mice treated with cortisone.

Rabbit liver mitochondria contain K which cannot be removed by repeated washings with NaCl solutions at 2°C. This K fraction exchanges with ambient K under appropriate metabolic conditions, indicating a dependence of K metabolism on aerobic oxidation. Orthophosphate depresses the mitochondrial K level. A newly observed poly-phasic action of DNP indicates a specific relationship between mitochondrial ester phosphate and K exchange.

1. Regarding the pregnant organism as a multi-compartment system the exchange rate of the water in amniotic fluid with the water of the maternal and fetal systems, was determined. By the application of trans-abdominal catheters, placed into the amniotic sac of pregnant women at term, amniotic fluid could be withdrawn at desired intervals without disturbing the continuity of the system. The mechanism of tracer (deuterium) exchange was demonstrated to be that demanded by theory, and the application of well known theoretic equations to the data so obtained permitted the calculation of absolute exchange rates. 2. In 2 experiments where the isotopic tracer was first placed in one compartment (mother) and then in the other (amniotic fluid) the exchange rates in both directions were found to be about 600 cc per hour. The observations of Flexner and his associates on the exchange rate of the water of the amniotic fluid are thus confirmed in principle.

The serum lipoprotein patterns of immature rabbits were determined by ultra-centrifugation at a density of 1.21. Two main peaks were resolved: one at -S 35 (20-40) and another at -S 7 (1-10). After castration, these animals were treated with diethylstilbestrol and with testosterone. Only testosterone altered significantly and regularly the lipoprotein pattern by the appearance of two abnormal components; one, -S > 70 and the second, -S (40-70); both disappeared on cessation of treatment.

Initial experiments indicate that when 9 parts of liquefied human semen are treated with 1 part of absolute glycerol and placed in an insulated box containing dry ice, it can be stored for at least 3 months and, after quick thawing in a 37°C water bath, it will show an average spermatozoal survival of 67%, a figure superior to those realized with faster freezing methods. Such revived spermatozoa show no detectable alterations in motility or morphology.

Repeated positive histoplasmin skin tests in normal medical students caused false positive reactions in the collodion agglutination test (57.1%) and in the complement-fixation test with histoplasma yeast phase antigen (78.6%) or with histoplasmin as antigen (85.7%). The response was relatively transient in the former 2 tests, and negative titers were usually restored by 21 weeks after the last skin test. However, positive results were noted as late as 21, 25 and 39 weeks in complement-fixation tests employing histoplasmin as antigen. A transient response was observed in complement-fixation studies with cross-reacting yeast phase biastomyces antigens (57.1%), but no sera reacted in the same test with coccidiodin antigen. A single positive skin test and repeated negative skin tests did not alter serologic results. Thus, repeated histoplasmin skin testing of skin-test positive persons may invalidate complement-fixation data for long periods with histoplasmin as antigen, and for much shorter periods with yeast phase histoplasma and blastomyces complement-fixation or histoplasmin collodion agglutination tests.

The effects of large doses of ACTH, cortisone acetate and desoxycorti-costerone acetate were investigated in the intact rhesus monkey. ACTH was without effect on hematocrit and the blood levels of glucose, non-protein nitrogen, amino acid nitrogen, inorganic phosphorous, sodium, potassium and chloride, although the adrenal glands were strongly stimulated as evidenced by adrenal gland weight and histological examination. Cortisone acetate and desoxy-corticosterone acetate effected a decrease in serum potassium and a lowering of the hematocrit without altering the levels of the other blood constituents. One of the 6 animals receiving cortisone suffered a significant weight loss, but hyperglycemia and elevated glucose tolerance curves were never observed. A transient polydipsia and edema were noted in the course of desoxycorticosterone administration.

The oxycellulose technic has been applied to the analysis of blood ACTH in children. ACTH was not present in detectable quantities in the blood of afebrile children without endocrine disease. ACTH was detected in the blood of untreated, but not of cortisone-treated, children with adrenogenital syndrome.

1. The effect of 2 different dosage schedules of cortisone acetate on guinea pigs infected with Rocky Mountain spotted fever of varying degrees of severity has been investigated. The overall mortality rate in 25 animals given daily intramuscular injections of 1 mg of the drug, starting on the day each received, by intraperitoneal injection, an inoculum of rickettsiae, did not differ significantly from the mortality rate in infected guinea pigs which received no treatment. The severity of the infection induced by different preparations of rickettsiae varied considerably; when this factor was taken into consideration, it appeared that cortisone acetate in doses comparable to those employed in human beings reduced the mortality rate in those animals with moderately severe infections. 2. In another group of 20 animals which received daily intramuscular injections of 25 mg of cortisone acetate for 5 days at the peak of the illness (fourth through the eighth day of infection) the overall mortality rate was identical with that in 20 infected animals not given the hormone. During the period of administration of this relatively huge dose of cortisone acetate, however, the fatality rate was significantly lower than that in the untreated animals.

Purified corticotropin prepared from hog anterior pituitary by the glacial acetic acid-oxycellulose method was found to be potent in adipokinin. Doses of 3 to 15 μg produced an increase of 40% in the hepatic lipid concentration of mice within three hours after injection. Pituitary adipokinin appears to be a principle distinct from the gonadotropins, thyrotropin, corticotropin, growth hormone, and intermedin.

1. Lethal total body x-irradiation (1000 r and 880 r) of rats produced the following changes in certain constituents of the liver or kidney. 2. As early as 1 day after irradiation, both inorganic and labile phosphate (ATP) in the liver were definitely subnormal. The liver inorganic phosphorus remained at this low level through the third day after irradiation while the labile phosphate continued to fall slowly. On the fourth day, both inorganic and labile phosphate in the liver rose; the inorganic phosphorus approached the control level where it remained until death. The labile phosphate, however, reached the control range on about the fifth day after irradiation and apparently continued to rise slowly until death. In the kidney, the changes which occurred in phosphate distribution were not pronounced. 3. The fasting of rats immediately following total body x-irradiation resulted in what appeared to be a retarded rate of glycogenosis. After 24 hours fasting, following radiation, liver glycogen values were 2% as compared with 0.1% in the fasted non-irradiated group. In adrenalectomized animals the liver glycogen content was also found to be higher after irradiation and fasting than the liver glycogen content found after fasting only. 4. A definite increase in the sulfhydryl content was found in the liver of fasted x-irradiated animals as compared with non-irradiated fasted animals. Removal of the adrenals produced a decrease in the sulfhydryl content of the liver of fasted animals. It was further reduced by irradiation. No significant changes were observed in the sulfhydryl content of the kidney of normal rats after irradiation. 5. There was no significant effect of x-irradiation upon the sodium, potassium or the water content of the liver and kidney up to 48 hours after irradiation with perhaps the exception of a slight increase in the sodium in the kidney.

The intravenous injection of colloidal carbon or saccharate of iron oxide produces, in the rabbit, an alteration in the reactivity to gram negative bacterial toxins which resembles the effect of cortisone, thorotrast and trypan blue. An intradermal injection of toxin in such animals causes a local reaction of hemorrhagic necrosis which is similar to the local Schwartzman reaction, and an intravenous injection causes the development of bilateral cortical necrosis of the kidneys resembling the generalized Shwartzman reaction. It is suggested that the colloidal materials may produce these effects by interfering with normal protective functions of the reticuloendothelial system.

(1) In dogs prepared under thiopental and then immobilized with C₁₀, the injection of d-amphetamine causes a progressive increase in the frequency of the EEG, with a corresponding decrease in amplitude. These effects are maximal within the first hour. (2) When unanesthetized dogs are gently roused from sleep and then fully alerted, there is a rise in the frequency of the EEG, accompanied by a decrease in amplitude. (3) When d-amphetamine is injected into unanesthetized dogs, there is an increase in motor activity, the peak effect being reached within the first hour. There is some evidence for an increase in the frequency of the EEG. (4) These findings suggest that the effects of d-amphetamine on the EEG of the dog are parallel to its effect on behavior.

The experiments presented here represent and attempt to obtain data on the comparative influences of short-term hormonal overdosage or endocrine insufficiencies upon electrolyte, carbohydrate, and lipid metabolism followed concurrently in the animal. The major purpose of these studies was to secure information on the metabolic patterns of the two latter foodstuffs which would enable an evaluation of the endocrine influences upon potassium of the endocrine influences upon potassium content and distribution in tissues. It is generally agreed that hormones modify the rates of metabolic reactions. It was our intent in Exp. I (Table I and Fig. 1) to obtain data concerning a number of constituents of liver and plasma in intact, adrenalectomized, and adrenalectomized-alloxanized which would allow a comparative evaluation of the metabolic status of the latter two types of animals with that of the intact rat. The two endocrine-deficient kinds of animals which were used in this comparative study were considered desirable for our purposes since it is well known that certain adrenocortical hormones and insulin have similar resultant influences upon certain phases of carbohydrate metabolism (glycogen formation) and are dissimilar in their resultant influences on other phases of carbohydrate metabolism (gulcose oxidation). There is also increasing evidence that adrenocortical hormones and insulin are antagonistic in their fatty acids to the liver and the mobilization of depot fat. It is apparent therefore that caution must be used in relating the level of tissue constituents to the effects of a specific hormone on intermediary metabolism or the absence of a gland in that the observed effects may be due to “unrestrained” influences of an antagonistic hormone(s). Examination of the data in Experiment I within this context suggests that in the adrenalectomized rat carbohydrate oxidation, and lipogenesis or neutral fat mobilization was greater than in the intact rat. As stated above, this might be interpreted as the resultant influence of lack of adrenocortical hormones or the unopposed activity of insulin. Parallel inspection of the data of the adrenalectomized-alloxanized group suggests that glucose oxidation in the adrenalectomized group was proceeding under the unopposed action of insulin since in the former group the evidence suggests an amelioration of this metabolic status during the condition of dual-glandular insufficiencies (adrenal and insulin). However, it is equally evident that the status of the metabolism of fat as evidenced by the levels of neutral fat and phospholipid in the liver was not altered in the absence of insulin and adrenal hormones from that found in the absence of adrenal gland secretions alone. Thus the changes in the levels of the various lipids in the two groups from those found in the controls of Experiment I mus be due mainly to absence of the hormonal influences of the adrenal gland. A more detailed interpretation of the liver lipid changes must necessarily be tentative in view of the uncertaintics regarding the metabolic interrelationships of the lipid fractions in the liver. It has been reported that in the depancreatized dog there is a marked increase in the turnover of liver phospholipids when exogenous insulin is withdrawn (22) . Since the liver phospholipids probably are not involved in fat transport (23) , the increased turnover suggested that some of the liver phospholipids participate in fat catabolism. In teh present experiments the decrease in liver phospholipids observed in the animals with glandular deficiencies suggests an elevated level of fat metabolism. The increase in neutral fat could be due either to mobilization of fat from depots or an increased rate of synthesis in response to the elevated level of fat metabolism.

Information concerning the effect of epinephrine on lipid metabolism is very meagre. Cori and Cori (24) deduced from their data that fat is the fuel for the increased metabolism due to epinephrine. An increase in liver phospholipids and fatty acids 2-3 hours after subcutaneous injection of epinephrine in rabbits was reported by Pollack (25) . There is increasing evidence however that insulin affects several phases of lipid metabolism. A marked reduction in the phospholipids of blood and liver in depancreatized dogs maintained with insulin was reported (26) (27) . A depression in lipogenesis in the alloxan-diabetic rat (28) and an increase inhepatic lipogenesis in the normal rabbit fed a high carbohydrate diet given insulin injection has been reported (29) . In general the evidence reported in the literature indicates that insulin's influence upon lipid metabolism is related through the effects of this hormone on glucose utilization. The data presented in Experiment II (Fig. 2) show that significant changes in the fractions of liver lipids followed the injection of insulin and epinephrine. However, the present data does not permit a definitive interpretation of a specific effect of either epinephrine or insulin on these lipid fractions because of the evidence recently reported that in the intact animal each of these hormones stimulated the secretion of the other hormone (30-32) ; the known effects which both of these hormones exert upon carbohydrate utilization and oxidation; and the increasing evidence of an influence of the latter processes upon lipid metabolism. A particularly puzzling aspects of the findings in this experiment is that in the shift in the liver lipid pattern from that in the intact untreated animal is essentially the same found in the adrenalectomized, the adrenalectomized-alloxanized rats, and the intact rat which received injections of epinephrine or insulin. It would appear that the same changes in fat metabolism in the liver occurred in all 4 types of experimental animals. Further investigation of the effects of insulin and epinephrine with the purpose of delimiting their specific actions on liver lipid metabolism is presented in the succeeding paper.

The data of Fig 3 show clearly that administration of intravenous glucose affected the level of the liver lipid fractions. Also the results show that the endocrine status of the animals exerted a definite influence on the effects of the glucose administration upon the liver fractions. In the adrenalectomized group the increase in total lipid content was accounted for by the increased neutral fat fraction alone. In the intact and the adrenalectomized-alloxanized groups the changes in the neutral fat and the phospholipid fractions were in opposite directions following the glucose infusions. It is noteworthy that the liver glycogen content of the intact and adrenalectomized rats was significantly increased after the glucose (Table III) and in both these groups the neutral fat content was increased. The evidence presented is suggestive that in the absence of insulin and adrenal gland secretions there was a decrease in the level of fat metabolism following the glucose infusion. Furthermore, the data presented in Table III is suggestive that carbohydrate utilization was apparently proceeding at an extremely slow rate in the adrenalectomized-alloxanized group.

There are several general aspects of the liver lipid data obtained in these experiments which deserve special comment. First, significant changes in the level of the lipid constituents can occur with much greater rapidity (30-60 minutes) than is generally recognized. Thus, it appears that fat metabolism is as responsive to the changing metabolic requirements of the organism as is carbohydrate metabolism. Secondly, the changes in the levels of phospholipid and neutral fat were always in opposite directions. When changes occurred in the cholesterol fractions, they parralleled those for the phospholipids. These interrelationships are probably a reflection of the functions of these constituents in fat metabolism. Neutral fat being the form in which fat is mobilized and transported, and the phospholipid and cholesterol esters constituents involved in some stages of fat catabolism in the liver. Thirdly, it is apparent from the present data that the level of total fat in the liver is not a dependable criterion in studies on the influence of hormone deficiency or excess on fat metabolism in the liver. Some of the shifts in lipid pattern reported here occurred without any change being evident in total lipid values. The total lipid values found in the various experimental groups reported here appeared to be the resultant of opposing changes in the different fractions, and whether the values were the same, greater, or less than the respective controls was due to the quantitative rather than the qualitative nature of the changes in the level of the lipid constituents. Finally, it should be emphasized that the level of the various lipids found in these experiments are all within the so-called normal range for the rat. Thus, the significant changes are not due to grossly abnormal conditions but rather represent “normal” shifts in the pathways of metabolism in response to changes in the hormonal pattern of the organism.

The effects of epinephrine and of insulin pretreatment on liver lipid partition, glycogen, water and electrolytes content, and certain plasma constituents were compared in adrenalectomized-alloxanized groups of rats given a glucose infusion. A similar pattern of changes in liver lipid partition was found in both pretreated groups compared to the non-pretreated controls. An increased liver potassium and lowered plasma potassium content was found in both pretreated groups. However, differences in liver glycogen content and glycemic level in the pretreated groups were sufficiently notable to suggest that common resultant effects on potassium mobilization probably were not produced by the same mechanism of action of both hormones.

1. Mammary tumors occurred in 12 or 20% of 60 agent-free strain C3H_f male mice implanted with pellets of cholesterol containing 10% of diethylstilbestrol. The average age at which the tumor arose was 15.1 months. Tests of cell-free filtrates of 5 of these tumors selected at random failed to give evidence of the presence of the mammary tumor agent. 2. Hepatomas occurred in 14 or 23% of the diethylstilbestrol-treated males at an average age of 22.6 months. This incidence is significantly less than that recorded for untreated strain C3H_f males of a comparable age, but greater than that recorded for untreated C3H_f females of comparable age.

An aldoheptose phosphate has been isolated from the polysaccharide of the somatic antigen of Sh. flexneri, type 3. The heptose moiety is identical with gala-mannoheptose and appears to be the enantiomorph of D-gala-L-mannoheptose.

1. In the rabbit, intradermally injected crystalline egg albumin and bovine gamma globulin are taken up by histiocytic cells both locally and in the peripheral and medullary sinuses of the draining lymph node. Considerable quantities remain extracellular and presumably diffuse away to be dealt with elsewhere in the body. Polymorphonuclear cells, though present in considerable numbers, are not found to contain intact antigen. 2. In the draining node many lymphoid cells containing antigen are seen in the peripheral portions of the lymphoid nodules. 3. Egg albumin disappears much more quickly than bovine gamma globulin from the injected skin site and the draining node. 4. Passive sensitization and to a greater extent sensitization with Freund's adjuvants result in a slowing of both antigens' disappearance from skin and node. Adjuvant sensitized animals also show speeding up and intensification of phagocytosis of antigen in both localities.

The relationship of bacterial growth to utilization of citrate and clotting of plasma has been determined. Clotting of citrated plasma may be used as a dependable and simple end point of citrate utilization by certain strains of bacteria. Fluoroacetate was found to prevent clotting by interfering with utilization of citrate by A. aerogenes and the enterococci. Cyanide had no effect on clotting by enterococci.

Under the experimental conditions employed in these studies, findings may be summarized as follows: a) The insulinhypersensitive hereditary hypopituitary dwarf mouse can tolerate only 3% of the dose of insulin that produces comparable symptoms in normal mice (2 units of insulin/kg of mouse). Higher dosages cause severe hypoglycemia followed by death. b) ACTH, adrenocortical extract (ACE), and cortisone act as anti-insulin agents as shown by their ability to increase blood-glucose levels and to reduce hypoglycemic convulsions. Desoxycorticosterone acetate and testosterone propionate exhibit no anti-insulin action. c) Factors that may be responsible for the hypersensitivity to insulin are discussed.

1. Subcutaneous administration of purified corticotrophin in gelatin; in phosphorylated hesperidin; and in gelatin plus phosphorylated hesperidin results in an enhanced effect of the hormone upon the adrenal cortex as measured by adrenal ascorbic acid. The combination of gelatin plus phosphorylated hesperidin has been shown to be most effective in extending the duration of effect of the corticotrophin. 2. The mechanism of action is attributed to the antihyaluronidase as well as the anti-proteolytic properties of the phosphorylated hesperidin.

1. A uniformly fatal encephalitis develops in 7-day-old rats following either intracerebral injection or intranasal instillation of epidemic keratoconjunctivitis virus. 2. Most of the 12-day-old rats tested were found to be susceptible to infection with epidemic keratoconjunctivitis virus but were not uniformly so since some animals in this age group survived. 3. Rats 3 to 4 weeks of age as well as rats 7 to 8 weeks of age apparently are refractory to epidemic keratoconjunctivitis virus since they did not develop a clinically apparent infection following intracerebral injection of the virus.

A soluble factor from group A streptococcal skin lesions prepared American Dutch rabbits for the generalized Shwartzman reaction. The reaction can be provoked in rabbits prepared with this material not only with toxins from Gram-negative organisms but also by reduced filtrates of streptococcal cultures with a high streptolysin O content. The tissue changes associated with the reaction brought about with these materials include myocardial necrosis and cortical necrosis of the kidney. Although these reactions simulate those observed in the classical generalized Shwartzman reaction, points at difference are discussed. It was possible to immunize rabbits against the preparative activity of the soluble factor by repeated injections of the streptococcal skin lesion extract.

Daily doses of 15-60 mg/kg of Viomycin administered for a period of 6 months did not appear to produce any evidence of renal toxicity as estimated by renal function studies (clearances). There were no serious alterations of the concentrations of serum sodium and potassium. These data suggest that the renal toxicity and altered electrolyte metabolism associated with Viomycin therapy may not be as serious as the earlier observations indicated. Therefore, we believe that further use of Viomycin with careful observations for signs of renal toxicity is indicated, particularly in patients unresponsive to the other more effective therapeutic approaches.

1. Lensectomy only and lensectomy combined with retina removal was performed in adult newts 4-8 days after the excision of the thyroids, the hypophysis or both. The results were compared with those in control experiments. Normally lens regenerates reach full shape and structure about the eighth week when cell nuclei of the primary fibers disappear. Rate of regeneration as well as growth of lens were distinctly diminished after retina removal. Early stages of retina regeneration proceeded for about 2 weeks before a lens vesicle was formed. Retina differentiation might be completed during the fifth week. 2. Measurements of lenses showed that regenerates developing in the presence of a regenerating retina were much behind those in eyes with the retina intact. Development rate in both kinds of eyes was very similar when eyes of normal and of gland-deprived animals were compared. In the latter, retina regeneration was not significantly retarded. The size of the lens regenerates at various dates after the eye operation was considerably less in gland-deprived animals. Gland removal seemed to increase the incidence of malformed regenerates and of cataracts. The incidence of cataract was also increased in normal controls after removal of the retina.

The virulence of 2 strains of Coccidioides immitis was evaluated by intracerebral inoculation in albino mice. The LD₅₀ for the 2 strains was found to be between 1 to 6 and 1 to 9 fungus particles.

1. Over 350 thioureas have been prepared in our laboratories and tested for antitubercular activity. 2. A number of disubstituted thioureas have demonstrated considerable protective and therapeutic activities in tuberculous mice and guinea pigs. 3. Little correlation existed between the in vitro and in vivo tuberculostatic action. 4. Many compounds possessed strong antifungal as well as antimycobacterial properties.

A method of measuring specific gravity, and thereby estimating total body water and fat content in intact animals is presented. The dilution volume of helium, in a closed chamber of known capacity containing the animal, is measured, and subtracted from the volume of the empty chamber. This gives the volume of the animal. Weight/volume gives specific gravity. No correction for residual air is necessary. The results on 10 cats by this method corresponded closely to results obtained by underwater weighing of each carcass.

A method is described whereby the ratio of FSH and LH present in human pituitary tissue can be ascertained in terms of well-known qualitative end points in the gonads of hypophysectomized rats of both sexes. Such end points, representing unit responses of FSH and LH, were chosen because they could be readily determined, and because they occurred at the same dosage of pituitary homogenate. It was thus determined that pooled homogenate of the pituitary glands of 3 adult men contained 100 units of FSH and 100 units of LH per pituitary, a ratio of 1:1. By comparison on an equal weight basis with pituitary tissue, the urine of adult men contained only minute amounts of gonadotropins, but proportionately, there was much more FSH relative to LH, in a ratio of much greater than 2:1.

1. Aureomycin-HCl in concentrations of 10-100 μg/ml inhibits the reduction to arylamine of the nitro groups of chloramphenicol and p-nitrobenzoic acid by cell-free extracts of E. coli (E-26). 2. Under similar conditions, terramycin has little effect on the reduction. 3. 2,4-dinitrophenol inhibits the reduction of the organic nitro compounds to the same extent as aureomycin. 4. Studies are in progress on the possible similarity in the underlying mechanism of inhibition by aureomycin and dinitrophenol.

1. The incorporation of car-boxyl C¹⁴ labeled glycine into the protein of human colon mucosa has been studied in vitro. The incorporation was inhibited by nitrogen and cyanide. There was no consistent effect from omitting calcium and/or magnesium from the buffer. Penicillin depressed the incorporation: aureomycin produced no consistent results. Homogenized tissue failed to incorporate even though viable bacteria were present. 2. A mucoprotein was isolated from colon mucosa and both carboxyl C¹⁴ glycine and inorganic S³⁵ sulfate were found to be incorporated into protein and mucoprotein. 3. Solution and reprecipitation of total crude protein removed the S³⁵ label, but not the C¹⁴ label.