Experimental Biology and Medicine

Amphotericin B was shown to have a high order of in vitroactivity against T. congolense, whereas only limited activity was observed in vivo. The limited activity was attributed to the rapid phagocytosis of amphotericin B by cells of the reticuloendothelial system (RES) with resulting lowered concentration free in the blood stream. Marked potentiation of activity was observed when intravenous treatment with amphotericin B was preceded by the blockade of the cells of the reticuloendothelial system (RES) with suitable agents such as thorium dioxide or triamcinolone acetonide.

The erythrophleum alkaloids, cassaine and coumingine, affect active transport of potassium by human erythrocytes as do the cardiac glycosides. At low concentrations both groups of compounds slightly increase K transfer from plasma to cells: higher concentrations inhibit the process. These effects probably reflect activation and inhibition of a membrane ATPase which others have implicated in the active movements of cations in erythrocytes. Erythrophleum alkaloids and cardiac glycosides also share similar effects on mammalian hearts. In view of the structural differences between the 2 classes of compounds, and their biological similarities in 2 kinds of tissue, it is suggested that previous views on minimal structural requirements for digitalis-like activity in hearts and erythrocytes be revised.

Cassaine sulfate was supplied by Dr. Robert Dowben from stock originally obtained from Prof. Ruzicka. Coumingine was donated by Dr. A. C. Keyl. We would like to thank both for their generosity. It is a pleasure to thank Frl. Rosemary Rottenberg for expert and willing technical assistance.

In dogs, susceptibility of the intestine to pooling of blood and tissue fluid during the portal hypertensive phase of endotoxin shock is diminished in younger animals. Pressure changes in large and small vessels of the mesentery and changes in intestinal weight per increment in portal pressure of young dogs given endotoxin were similar to the changes previously observed during mechanical elevation of the portal pressure suggesting analogous mechanisms are involved. Thus, during the early period, lesser effects of endotoxin (1 mg/kg) upon the amount of pooling in intestines of immature dogs may be due to an increase in arteriolar resistance, in contrast to the circumstances in older animals in which a decline in arteriolar resistance appears to occur after endotoxin administration.

The goitrogenic effect of raw soybeans was seen in significant increases of both fresh and dry weights of thyroid glands when a diet containing 60% of beans was fed to rats, and a significant increase in the dry weight only of the gland when a diet containing 35% of raw soybeans was fed. The goitrogenic effect of 35% or 60% of raw soybeans in rat diets was obliterated by adding casein to the diets. This result was not attributable to iodine in the casein.

The metabolic fate of DL-Tryptophan-7a-C¹⁴ in a human with multiple myeloma was studied. Only 5% of the administered C¹⁴ was excreted as respired C¹⁴O₂ and 14% in the urine in the first 24 hours. O-aminophenol-2-C¹⁴ was isolated from the urine.

Alpen and Cranmore (1) and Erslev (2) have concluded that erythropoietin increases red cell output by accelerating the rate of stem cell differentiation into erythroid precursors. Observations on erythropoiesis in polycythemic mice by Gurney, et al.(3) have supported this conclusion. However, observations by several investigators (4-7) seem to indicate that the action of the erythropoietic humoral factor (s) is not limited to regulation of stem cell differentiation. Experiments reported here tested the effect of erythropoietin on later phases of erythroid formation and the results suggest an action here as well as in the early stem cell phase.

Methods. Female Sprague-Dawley rats, weighing 180 to 200 g, were made polycythemic by one or several transfusions of packed homologous red blood cells. Each transfusion equaled 50% of the animal's original red cell mass. At various intervals following the initial transfusion the polycythemic rats were given a single injection of human urine erythropoietin (8) or purified sheep erythropoietin (9).‡ Subsequent changes in erythropoiesis were assessed by reticulocyte counts (10) and measurement of the 18-hour erythrocyte radio-iron incorporation(8). Erythroid elements per 1000 nucleated cells were enumerated in smears of marrow from control polycythemic animals.

In 3 experiments, polycythemic rats were given single intraperitoneal injections of 2 ml of urine concentrate on either the 5th, 15th or 31st day after their first transfusion. Control polycythemic rats were injected with saline. At the end of each successive 24-hour period following erythropoietin or saline injection, representative animals from each group were killed and the reticulocyte counts, hematocrits and erythrocyte radio-iron incorporation were determined. The radio-iron was injected 18 hours before the rats were killed. All groups given urine concentrate included at least 5 rats.

Attempts have been made to confirm the findings of Metcalf with respect to the lymphocytosis stimulating factor. Extracts of a lymphoblast grown in vitro behaved in a similar manner to thymic extracts in the tests used. The test method is subject to fluctuations which yield conflicting data.

In slide chambers the number of macrophages formed closely paralleled the number of monocytes present in the initial suspension. When most monocytes were removed by glass beads very few macrophages formed. When lymphocytes were removed by irradiation the number of macrophages formed corresponded to the number of monocytes at the start. Intermediate stages between lymphocytes and macrophages were never observed, whereas cells intermediate between monocyte and macrophage were regularly seen.

Measurements of relative hemolytic activity of varying mixtures of complement, red cells, and hemolysin with added heterologous protein reveal inhibition or apparent partial complement fixation in the absence of known previous immunization. The added protein in these studies is either hemoglobin or human gamma globulin. This inhibition is absent when the hemoglobin and complement are derived from the same specimen of blood. These data reveal an important possible source of error when quantitative estimates of antibody formation are made using complement fixation tests.

I would like to acknowledge gratitude to Drs. A. E. Lewis and G. R. Biskind for invaluable suggestions during the development of this project and for extensive aid in preparation of this manuscript.

The effect of mammalian ACTH on in vitro secretion of skate (Raja erinacea) interrenals was investigated directly. Inter-renal weights also were compared between males and nonovulating and ovulating females. ACTH increased interrenal output of BT and UV measurable substances in both sexes. Preincubation output rates in the absence of ACTH were substantially greater than those during subsequent incubation. A strong but not perfect correlation was observed between body and interrenal weights in both sexes. F-test analysis for statistical significance of differences between means of covariance-corrected interrenal weights showed that those of males were significantly less than those of either combined or ovulating females but not different from those of nonovulating females. Ovulating females had significantly heavier interrenals than nonovulating females.

Four experimental procedures have been tested for ability to increase ESF concentrations (as measured by Fe⁵⁹ uptake) and increase tumor growth. It was concluded that ESF extracts prepared by a modified method of Borsook or the method of White and procedures which increase endogenous ESF production (partial hepatectomy and cobalt chloride administration) are capable of stimulating Novikoff hepatoma growth in rats.

It was possible to demonstrate the existence of a potent reduced diphosphopyridine nucleotide oxidase in all Neisseria. This enzyme and alcohol dehydrogenase were increased in relative penicillin-resistant isolates of N. gonorrhoeae. Another potent enzyme found was diphosphopyridine nucleotide-linked lactic dehydrogenase. Some organisms contained much less LDH and DPNH oxidase than N. gonorrhoeae isolates. Reduced triphosphopyridine nucleotide oxidase was present only in small quantities in all organisms investigated. An acetate oxidizing enzyme system was present in large quantities in E. coli and S. aureus, in small quantities in N. sicca and TV. catarrhalis, and in traces in a few isolates of N. gonorrhoeae. The system was absent from N. meningitidis. For the above enzymes, the Thunberg technic with tetrazolium salt as the electron acceptor was employed. All Neisseria contained a cyanide sensitive, aerobic cysteine oxidase and a cysteine desulfhydrase. It is hoped that the present investigation will aid in identification and classification of Neisseria.

These results were obtained with old stock cultures of N. gonorrhoeae. When a large number of fresh isolates was assayed only partial correlation between penicillin resistance and enzyme activity was observed.

Inhibition of hemagglutinin formation in mice was demonstrated with single doses of 2.5, 5, 10, 15 and 20 mg/kg of TG. If the drug was administered 18 or more hours after primary immunization, maximal block of antibody synthesis occurred. The minimal effective dose was 2.5 mg/kg injected 18 hours after antigenic stimulation. The same amount of drug given either before or after was ineffective. The timing was so specific that it permitted the design of a model system in which simultaneous inhibition of anti-human hemagglutinins was obtained without significant interference with the formation of anti-sheep hemagglutinins. The significance of the data are discussed.

The technical assistance of Mr. John Niedra is gratefully acknowledged.

Immunologic unresponsiveness directed against delayed but not immediate hypersensitivity to protein antigen can be elicited in adult mice by exposing them to a buffer solution of antigen within a certain short, critical time after they have been vaccinated with the same antigen in water-in-oil emulsion. Further study of this phenomenon may improve understanding of and control over the mechanisms of induction of delayed and immediate hypersensitivities.

The concentrations of total zinc and copper in sera from 100 non-pregnant hospital patients were examined particularly in relation to the CRP reaction. Although the concentration of serum zinc tends to be slightly lower in the presence of CRP, the decrease does not correlate with the intensity of the CRP reaction and/or with the degree of hypercupremia. The data demonstrate that serum zinc and copper concentrations are independent variables.

My thanks are due to Mrs. Betty S. Grogan for technical assistance.

Ten-day-old chick embryos are extremely susceptible to infection with pathogenic coagulase positive staphylococci and the LD₅₀ of the strains studied was less than 10 bacteria. More than 10⁴ coagulase negative staphylococci were necessary to produce a 50% fatality rate in 10-day-old chick embryos.

The author gratefully acknowledges the advice of Drs. George Gee Jackson and Mark H. Lepper and the technical assistance of La Verne Anderson.

An anaerobic, gram-positive, spore-forming bacillus has been isolated from the cecal contents of conventional mice. This organism causes reduction in size of the enlarged cecum usually associated with axenic mice. It has been identified as Clostridium difficile. A combination of 2 strains of Bacteroides isolated from cecal contents of conventional mice was also effective in this respect.

By repeated continuous flow curtain electrophoresis of human serum, 2 distinct phenyl acetate hydrolyzing esterases were purified 120 and 850 times. One esterase was heat-labile and inactivated by EDTA, while the other was heat-stable and required EDTA for activity. EDTA-inactivation could be prevented by manganese. The methods for purification and assay are described and the results discussed.

Filtrates of normal human plasma that had been boiled for 30 minutes were administered by gastric tube to groups of normal rats. Ten daily doses each equivalent to 2 ml of the original plasma per 100 g body weight induced a 30% increase in the recipients'thrombocyte counts. Smaller doses, i.e., 0.5 or 1 ml per 100 g body weight, did not promote thrombocytosis. All other hematologic measurements in these animals remained stable. These findings provide additional support for the hypothesis that thrombopoiesis is subject to humoral regulatory control.

1) Normal rabbits injected intra-articularly (i.a.) with 0.5 ml Duck Anti-Rabbit Kidney Serum (DARKS) twice weekly for 6 weeks developed a progressive monoarthritis following a latent period averaging 10 weeks. Control rabbits treated similarly with Normal Duck Serum (NDS) failed to develop arthritis. 2) Rabbits with a remote history of immunization against duck serum developed an accelerated arthritis within one week following i.a. injections of either DARKS or NDS. 3) Nonimmunized animals preconditioned with isotonic NaCl or KC1 given instead of drinking water, developed accelerated arthritis within 2 to 4 weeks following i.a. injections not only of DARKS but of normal duck, calf or horse serum as well. 4) Circulating anti-duck serum precipitins always preceded development of arthritis induced by i.a. injections of duck serum. 5) The arthritic changes consisted of villous hypertrophy and infiltration of the synovium with lymphocytes and plasma cells. This was followed by fibrinoid necrosis of the synovium and articular cartilage and by formation in the joint cavity of an exudate of proteinaceous material, round cells and polymorphonuclear leukocytes.

1. Simultaneous addition of 1-phenylephrine (PE) and 1-isoproterenol (ISU) to the rabbit aortic strip significantly augmented contractions above the mere additive responses produced by each drug alone. 2. Addition of atropine to the bath fluid permitted an increase of ISU dose range which, together with PE, produced such augmented contractions. 3. The findings are correlated with ISU vasomotor reversal in the whole animal.

I am indebted to Dr. Theodore Koppanyi for his interest and encouragement.

Horses immunized by injection of live Edmonston measles virus in Freund's Arlacelmineral oil adjuvant produced measles antisera of high neutralizing antibody content and in large amount. The response to aqueous measles vaccine was very poor. The antiserum was devoid of bothersome nonspecific substances and was not toxic for human cells in tissue culture. The economic and scientific utility of horses for preparing anti-sera against viruses was discussed.

1. Percentages of total amino acids in viable tissues of the Walker carcinosarcoma 256 (rat), Sarcoma 180 (mouse), and Taylor tumor (mouse) have been determined by both column and paper chromatography. 2. Values obtained by column chromatography agreed satisfactorily with those determined in the same laboratory by microbiological assay while the majority of values determined by paper chromatography were in poor agreement with those of the other two methods. 3. There was no significant difference in the total amino acid content of tumors from rats or mice.

Hemagglutination of monkey red cells was demonstrated with a concentrated measles tissue culture antigen. The antigen was destroyed by trypsin and its activity was significantly reduced by ether. Heat inactivation at 60°C for 10 minutes destroyed the infectivity, while hemagglutinating activity was significantly reduced only after one hour. A small non-infectious hemagglutinating unit was also demonstrated. Both hemagglutinating particles are antigenic and their activity was inhibited by specific antiserum. The small particles appeared during viral multiplication in the cell. Receptors on the red cells were of a different nature than those of myxoviruses.

Rabbit antibodies against adult rat kidney were found to be fixed in the kidneys of new-born rats. Since new-born rats do not develop clinical disease when injected with anti-rat kidney antibodies, this suggests either that the antigens giving rise to cytotoxic antibodies are not present at this early date or the new-born rats are deficient in complement. Six weeks after new-born rats are so injected they still have the rabbit globulin (antibody) in the glomeruli. However, rabbit globulin was demonstrated in the glomeruli on the medullary side of the cortex and not in those of the periphery indicating that new glomeruli in the peripheral region become functional as the animal matures. The increase in functional glomeruli may be the reason for the observation that new-born rats injected with anti-rat kidney serum do not develop clinical disease.

Effect of oxytocin, lactogen, and hydrocortisone acetate upon mammary gland involution was studied in rat. LGH retarded involution to a significant degree, by maintaining DNA 48% above control group. HCA at levels of 500 and 1000 μg retarded involution significantly when DNA was related to final BW. OXT by either subcutaneous or intraperitoneal administration was not effective in retarding involution. These hormones, however, appear to maintain some secretory activity of involuting mammary glands. Data also suggest OXT has little influence upon release of lactogen from anterior pituitary.

This report is an account of the adaptation of EAV to L-M 929 cells with identification by complement-fixation methods. Infected cultures show cytopathic effects which may be measured by both TCID₅₀ or plaque-counting methods. The failure of the virus to propagate in L cells (original) in early experiments is unexplained. During this period the cells were grown in horse and human sera which may have been inhibitory. The possibility that the L-M 929 strains could be directly infected by hamster-propagated EAV has not been excluded. The possibility of contamination of the L-cell strain used in this study with HeLa cells has been considered. The special chromosomal marker of the L-M 929 strain permits precise identification and has not changed, according to Hsu and Merchant(6). Furthermore, YELP has proven very toxic to the HeLa cell line, and cell growth is highly unlikely in this medium.

Intravenous guanethidine enhances the pressor effect of norepinephrine. These experiments show that the increased pressor response in dogs resulted from augmentation of the vasoconstrictor action of norepinephrine rather than from higher blood flow. In humans the increased pressor response was the result of both increased vascular tone and higher flow.

Treatment of female mice with S,2-aminoethylisothiourea-DiHBr (AET) before 50 r whole-body X irradiation gave some protection to their reproductive capacity when compared to irradiated controls, increasing the length of the reproductive period, the number of litters per female and the number of offspring per female.

Crayfish (Orconectcs virilus and Procambarus clarkii) were scalded at 76°C for one to 3 minutes. A toxin was found in the blood of scalded crayfish, which, upon injection in normal crayfish, killed 91.1% of the recipients. The toxin taken from a scalded crayfish is a heat stable, non-dialyzable substance.

Four rheumatoid factors obtained from the sera of 3 patients with rheumatoid arthritis, a sample of 19.5 S gamma-globulin and a sample of 7 S gammaglobulin from healthy donors were analyzed for their qualitative and quantitative amino acid content.

Westphal's lipid A assayed in mice and hamsters enhanced non-specific host resistance against the activities of viruses such as was demonstrated earlier for bacterial endotoxins and for other materials of microbial origin. Lipid A, given intracerebrally into mice, suppressed neurotoxicity of concentrated influenza A virus and reduced the activities of neurotropic influenza (NWS) and herpes simplex viruses as measured by survival and by survival time. Given subcutaneously, it was active against influenza A virus and, following intraperitoneal injection, was active against encephalomyocarditis virus but showed slight if any activity against measles in hamsters or neurotropic (NWS) influenza virus in mice. It was inactive against Lansing type II poliovirus and Coxsackie B3 virus when given intracerebrally and intraperitoneally, respectively. Greatest activity was in the suppression of PVM virus, even when administered up to 72 hours following viral infection. The lipid A did not appear to suppress proliferation of neurotropic NWS influenza virus even though it did reduce the clinical consequence of such infection at low virus dose. Lipid A is discussed against the background of other host resistance factors.

1. Ultrathin sections of bone marrow, spleen, lymph nodes, and mediastinal tumors from 11 rats with leukemia induced by mouse leukemia virus (Gross), were examined in the electron microscope. 2. In organs of all 11 leukemic rats, 2 types of particles could be observed. One type, described as virus particles (990 A average diameter), with an electrondense center (630 A average diameter), surrounded by a less dense zone, and limited by another membrane, not always clearly defined. 3. The other type, described as vesicular or doughnut-type particles, with an electronlucent center, surrounded by an inner membrane or ring staining strongly with uranyl acetate (490 A average diameter) and an outer ring or membrane (840 A) reacting less with the uranyl acetate stain. Frequently, but not always, another membrane (580 A diameter) could be seen between the inner and outer membranes. 4. Both types of particles were found in megakaryocytes of bone marrow and spleen. 5. Virus particles were also observed in intercellular spaces of lymph nodes and mediastinal tumors, and occasionally within cytoplasmic inclusions of cells present in these tissues. 6. Characteristic cylindrical structures were observed in megakaryocytes of bone marrow and spleen of leukemic rats. 7. Neither virus nor doughnut-type particles could be found in either bone marrow, spleen, thymus or lymph nodes of 12 normal rats.

1. The per cent tumor inhibition, change in carcass weight, and concentration of nonprotein methionine in the liver, resulting from administration of ethionine to normal, castrated, and testosterone-treated castrated male rats, has been determined. 2. Tumor inhibition by ethionine, and the toxi-city of this substance, was greater in castrated than in normal or testosterone-treated castrated male rats and closely resembled that observed in female rats. 3. The concentration of free nonprotein methionine in the liver of castrated male rats was similar to that found in female rats and differed from that observed in normal male or testosterone-treated castrated male rats. The presence of a methionine conjugate was indicated in the liver of castrated male and normal female rats following ethionine administration while such a conjugate was lacking in the liver of normal male or testosterone-treated castrated male rats. 4. It was concluded that the sex difference observed in tumor inhibition, toxicity, and concentration of nonprotein methionine in the liver of ethionine-treated rats was mediated by the androgens.

Effects of adding a mixture of methionine and phenylalanine or a mixture of indispensable amino acids lacking histidine to diets containing 6% of fibrin on rate of food intake and on the blood amino acid pattern of rats have been determined in short term experiments. Amino acid imbalances induced by both of these procedures caused a reduction in rate of food consumption within a few hours after the feeding period. Feeding diets containing additional methionine and phenylalanine to protein-depleted rats caused a fall in concentration of the group of amino acids (leucine, isoleucine, valine and histidine) limiting growth. Similarly, a rapid fall in concentration of the most limiting amino acid, histidine, occurred in the blood of rats trained to accept single daily feeding and then fed diets containing an amino acid mixture lacking histidine.

Succinic dehydrogenase has been demonstrated histochemically in T. cruzi and L. leproide - leptomonad form. The protozoa were stained for the enzyme with nitro blue tetrazolium in presence of brilliant cresyl blue. Optimal requirements for the reaction have been described. Enzyme activity is indicated by blue deposits of diformazan in granular form in the cytoplasm. The reaction is weak in absence of calcium and bicarbonate and is inhibited by malonate. Spirotrypan had some inhibitory effect on the tetrazolium reaction in T. cruzi both in vitro and in vivo.

Extracts of rabbit and rat testes readily formed glucose-6-P (measured by NADP⁺ reduction in presence of excess glu-cose-6-P dehydrogenase) from ATP and glucose in presence of 0.06 M arsenate, which inhibits phosphoglucomutase approximately 90%. These findings are considered to indicate that contrary to the reports of Akaeda, glucose-6-P is the primary phosphorylation product in this tissue.

Complement fixation tests with liver and spleen antigens derived from various animal species showed no increase of circulating tissue antibody levels in rats suffering from runt disease. Complement levels, on the other hand, were markedly decreased and degree of reduction appeared to be related to severity of the disorder.

The turnover of Sr⁸⁵ was determined for four patients by wholebody counting for a period of 130 days following administration of the isotope. The data obtained were expressed as power functions. Two patients then received Sr lactate, and the other two Ca gluconate as dietary supplements for a period of 110 days. Sr⁸⁵ turnover was determined for this period and again expressed as a power function for each patient. While the data suggested that the addition of Ca gluconate to the diet somewhat decreased Sr⁸⁵ excretion and the addition of Sr lactate slightly enhanced excretion, no statistically significant changes in the long-term turnover rate of Sr⁸⁵ were observed.

Adrenalectomy stimulated incorporation of radioactivity from adenine-8-C¹⁴ into total nucleic acid and the RNA of isolated rat diaphragm. Moreover, adrenalectomy increased the actual amount of nucleic acid and of RNA that could be extracted from rat diaphragm. Cortisone administration (2 mg/day) was without effect on incorporation of C¹⁴ from adenine into nucleic acid or RNA, nor did it influence their amount.

A rapid and simple method is described for preparing specimens of an intra-erythrocytic parasite for morphological examination in the electron microscope. The technic includes dehemoglobinization of the erythrocyte by dialysis against a hypotonic solution of a volatile salt. The hemolyzed stromata are transferred to specimen grids by gravity sedimentation. Three principal artifacts associated with the method of preparation are noted. The complex structure of A. marginale as reported by previous workers is confirmed and extended by these results. A possible relationship between certain observed forms and the parasite is suggested.

While the National Animal Disease Laboratory was located at Beltsville, Md., blood samples for this work were supplied by Drs. D. W. Gates and T. O. Roby and Mr. James Caudill, of the Beltsville Parasitological Lab., Animal Disease and Parasite Research Division, Agri. Research Service, U. S. Dept. of Agriculture. The author gratefully acknowledges their assistance.

Young adult male rats exposed to a single dose of 450 r or 900 r total body x-irradiation exhibited a highly significant reduction 1, 3 and 6 days post x-irradiation in the serotonin concentration of the blood, spleen, brain and small intestines. No significant reduction in tissue serotonin levels occurred in rats exposed to 900 r total body x-irradiation and administered 2-mercapto-ethylguanidine intraperitoneally at a level of 200 mg per kg of body weight 20 minutes prior to x-irradiation exposure, paired the binding of serotonin not only to platelets but to various tissues as well has not, however, been excluded.

1. Proteins of bone and cartilage from skull, femur, rib and vertebrae of female rats, Wistar strain, age 5 weeks and 2 1/2 years, respectively, were fractionated into TCA supernatant of base soluble protein, nonscleroprotein, soluble collagen, insoluble collagen, glycoprotein and elastin. 2. With an increase in age, there was a decrease of protein and hexosamine in TCA supernatant of base soluble protein, nonscleroprotein and soluble collagen of both bone and cartilage. 3. The amount of insoluble collagen in skull, femur and rib did not change with age; in vertebrae it increased and in cartilage, it decreased. 4. Certain glycoproteins, not present in the connective tissues studied, other than bone and cartilage, decreased in bone and increased in costal cartilage wtih increasing age.

Naturally occurring Eaton PPLO was shown to propagate on an agaryeast extract-horse serum medium. The agent was recovered directly on agar from 12 of 13 serologically positive patients with pneumonia. Eaton PPLO was not recovered from 14 serologically negative pneumonia patients. All Eaton PPLO isolates produced small homogenously granular colonies which could be easily differentiated from the usual “fried egg” or “nippled” colonies produced by non-Eaton oral PPLO. The distinctive colonial morphology of Eaton PPLO may ultimately prove useful for its presumptive identification.

Streptococcal hyaluronic acid was biosynthetically labeled with C¹⁴ from acetate-1-C¹⁴. Approximately 75% of the label was in the acetyl of N-acetyl glucosamine. After intravenous injection, C¹⁴-labeled streptococcal hyaluronic acid was rapidly cleared from the plasma. Electrophoresis disclosed some binding of hyaluronic acid to plasma proteins. The maximal rate of respiratory C¹⁴O₂ loss and urinary excretion of hyaluronic acid did not occur until the 2nd and 3rd hour following intravenous injection. Only traces of intravenously injected hyaluronic acid appeared in the synovial space, skin or vitreous humor.

Elimination of the 7-hydroxysubstituent of the bile acids by intestinal microorganisms is a common reaction occurring in many species (1) and has been reproduced in vitro by a mixture of anaerobic intestinal microorganisms from the rat(2). Samuelsson has demonstrated that the reaction mechanism is a dehydration to a Δ⁶-derivative, followed by reduction (3).

Human bile contains 3 main bile acids, cholic, chenodeoxycholic and deoxycholic acid, with a mean ratio of 1.1:1.0:0.6(4). Cholic and chenodeoxycholic acids are formed directly from cholesterol in the liver (1). Due to the action of microbial enzymes the 7-hydroxyl group of cholic acid is removed and the “secondary” bile acid deoxycholic acid is formed(5). Contrary to the rapid transformation of cholic acid, chenodeoxycholic acid is transformed slowly during the enterohepatic circulation. Three days after administration of chenodeoxycholic acid-24-C¹⁴ only 3% of the isotope in the human bile was recovered as lithocholic acid(6). Although definitive proof is lacking, investigation so far indicates that deoxycholic and lithocholic acids are not metabolized by the human liver to a significant extent(6,7). The different concentration of deoxycholic and lithocholic acids in human bile, therefore, might be explained by a difference in ability of the intestinal flora to remove the 7-hydroxyl group from cholic and chenodeoxycholic acid or by a difference in accessibility for absorption of the “secondary” bile acids.

The aim of the present investigation was to study the formation of deoxycholic and lithocholic acid in subcultures of mixed human intestinal microorganisms.

Experimental. The 24-C¹⁴-labeled cholic, deoxycholic and chenodeoxycholic acids were prepared according to Bergström et al.(8).

Labeled bile acids as sodium salts dissolved in 80% ethanol were added to thick walled centrifuge tubes (inside diameter 13 mm, height 100 mm). After evaporation, the residue was taken up in 4 ml of culture medium. The bile acid concentration was always 0.2 mM/l broth. The tubes were plugged with cotton and autoclaved for 20 minutes. The first subculture was made by inoculating one loop of human feces into each experimental tube and into a tube without bile acid. Subsequent serial stock subcultures of mixed microorganisms were made in medium free of bile acids. One drop from a serial stock subculturc was inoculated into the respective set of experimental tubes containing the bile acids. For aerobic incubation the tubes were cotton-stoppered.

Radioautographs of liver sections from mice given tritiated thymidine were used to identify the cells which were preparing for division and colloidal saccharated iron oxide was used to identify the active phagocytic cells. In livers of mice whose reticulo-endothelial system was stimulated by estradiol, it was established that the cells preparing for division and those which had recently divided were actively phagocytic. In livers of mice whose reticulo-endothelial system had been “blockaded” with saccharated iron oxide, it was established that the cells which had phagocytized colloid were able to divide in the process of recovery from “blockade.” No evidence was found for a stem cell which proliferates and differentiates to provide the active phagocytic population.

The galvanic skin response from a macroelectrode covering the toe pad of the cat was compared with simultaneously recorded potentials arising from individual sweat glands and cells of the surrounding epidermis and dermis of the toe pad skin of the same animal. By direct measurements employing microelectrode technics, the fast negative component of the galvanic skin response was shown to originate in the sweat glands, and to be related to sweat gland activity. The slow positive component of the galvanic skin response was shown to originate in the cells of the epidermal layer of the skin and is therefore unrelated to sweating.

The effect of ITSC, a highly specific antiviral agent, on some of the biochemical parameters of vaccinia virus replication in HeLa cell monolayers has been studied. At concentrations which completely inhibited infectious virus production this compound did not affect the previously observed enhancement of thymidine-H³ incorporation, and the inhibition of valine-C¹⁴ incorporation in infected cells, nor did it affect these reactions in control cells. There was a typical cytoplasmic accumulation of thymidine-H³ at virus-synthesizing sites in presence of the drug. In kinetic experiments, ITSC prevented virus formation only in proportion to the part of the virus replication cycle during which it was present, and independent of the absolute time in the cycle at which it was first added. The implications of these results on the mode of action of ITSC are discussed.

Studies on the kinetics of formation of CxRP in rabbits have been carried out. By measuring relative specific activities of CxRP and serum albumin in rabbits given glycine-1-C¹⁴ and induced with typhoid vaccine to form CxRP, evidence has been obtained to suggest that CxRP is being manufactured constantly and appears in the serum on challenge.

I wish to acknowledge the kind and generous counsel of Dr. Chandler A. Stetson in the design and execution of these experiments.

Tracer amounts of cholesterol-4-C¹⁴ added to hog adrenal homogenates and incubated under conditions favorable for steroid synthesis were rapidly and efficiently converted to labeled steroids. Addition of cholesterol-4-C¹⁴ esters, prepared enzymatically and incubated under the same conditions, did not give C¹⁴-activity in the steroids produced. Conversion of free cholesterol to cholesterol esters or of esters to free cholesterol as measured by C¹⁴-activity of the sterol digitonides did not occur in this system.

Four different methods for determination of “free” vit B₁₂ and the fraction “bound” by the serum proteins have been compared. The serum binding capacity for vit B₁₂. at varying concentrations of total vit B₁₂ has been determined with the same methods. None of the methods showed correlation between original serum vit B₁₂ concentrations and binding of added vit B₁₂. When binding capacity was determined by dialysis and by using uptake of vit B₁₂ by wild type E. coli, there was good agreement. This showed a mean binding capacity of 1350 pg vit B₁₂/ml serum when total vit B₁₂ concentration was below 10,000 pg/ml. Amount of vit B₁₂ unavailable for L. leichmannii increased almost proportionally with total concentration of vit B₁₂. Amount of vit B₁₂ unavailable for E. gracilis was almost constant, irrespective of total concentration of vit B₁₂.

We therefore propose that the term “microbiologically unavailable vit B¹²” should always include the name of the organism and the initial total concentration of vit B₁₂, and that the serum binding capacity for vit B₁₂ should be defined as the amount of vit B₁₂ per ml serum which is non-dialyzable at such initial total concentration of vit B₁₂ that more than one-fourth and less than half of the initial total amount of vit B₁₂ remains undialyzed.

I. Three H-reflexes, 2 of which have not been described previously, have been obtained in 13 normal human neonates when the ulnar nerve was stimulated. These electrically induced reflexes (EIR's) were demonstrated by muscle action potential recordings from the flexor muscles of the forearm (UFEIR), the hypothenar (UHEIR) and the first dorsal interosseus muscles (UIEIR) of the hand. 2. The UFEIR had a lower threshold than either the UIEIR, or the previously described UHEIR. 3. All 3 EIR's were reduced in amplitude in neonates and in those subjects reexamined 2 to 4 months later, as the infants fell asleep. They were restored by spontaneous arousal or by waking induced by sensory stimulation with sound, light, or touch.

The increment in liver cholesterol and liver total lipid induced by cholesterol feeding in the rat was largely counteracted by concurrent feeding of Gum Guar, Locust Bean Gum or carrageenan at a 10% level in the diet. Effects were similar to, although slightly less marked, than that obtained with a comparable amount of pectin N.F.

1. During development fetal chick liver and muscle show sequential alterations in distribution of LDH isozymes. These changes are characterized by a progressive increase in concentration of cathodal bands relative to anodal bands. In chick heart the LDH isozyme pattern remains relatively constant during development. The number of bands depends on the activity applied to the gel, and with high LDH activity chick heart, muscle and liver 10 days after fertilization show the full tissue complement of LDH isozymes. 2. The chick embryo tissues studied in tissue culture reveal under the conditions employed in this study a common pattern characterized by 1 or 2 heavily stained cathodal bands and 1 or 2 lightly stained anodal bands. During prolonged culture the anodal bands decrease in concentration. Isozymes may prove to be a sensitive index of the maintenance of physiological activities of cells grown in tissue culture.

Guinea pigs were given daily intraperitoneal injections of 136,000 I.U. of vit A-alcohol dissolved in sterile mineral oil. Peritoneal leucocytes, harvested from these animals 24-48 hours after the third dose of vitamin, showed a highly significant depression of total acid phosphatase activity when compared with leucocytes obtained from control animals treated with oil alone. Smaller doses of the vitamin (68,000 I.U.) had a similar action. This effect of vit A was not prevented by simultaneous treatment of the animals with modest doses of cortisone or hydrocortisone. The observation that leucocytes of vit A treated animals show a marked loss of acid phosphatase supports the contention that this agent has a disruptive action on lysosomal membranes in vivo.

Hydrocortisone, as the free alcohol, was kindly provided by Dr. H. J. Robinson, Merck Institute of Therapeutic Research. The technical assistance of Miss Nili Kratka is gratefully acknowledged.

Significant increase of cell membrane resting electrical potential (REP) of adipose tissue obtained from young rats occurred in response to 1-1,000 micro-units/ ml of insulin. Decrease of adipose tissue resting potentials occurred following replacement of insulin by control buffer. Resting electrical potentials of adipose tissue from older rats, weighing 330-405 g, showed no such increase in response to 1,000 micro-units/ml of insulin. Presence or absence of glucose in the buffer medium appeared to have no effect upon the response of cell membrane REP.

An ATPase has been demonstrated in water extracts and microsomal suspensions prepared from rabbit ventricles. It has an absolute requirement for Mg and it can be further stimulated by Na and K. The Na + K active component is susceptible to inhibition by G-Strophanthin. Ageing increases the Mg + Na + K/Mg ATPase ratio.

Two kinds of immunological comparisons between several bovine and human preparations of thyrotropin (TSH), including an I¹³¹-labeled bovine preparation, were made with the use of a rabbit antiserum against a purified bovine preparation. Using erythrocytes sensitized with a sample of the immunizing antigen, the inhibition of hemagglutination demonstrated cross-reaction between a crude human TSH preparation and anti-bovine TSH serum, but degree of inhibition was not proportional to biological activity when 3 bovine preparations were compared. Both the in vivo mouse I¹³¹ release and the in vitro bovine thyroid slice bioassay showed that the antiserum neutralized TSH. Results from the 2 assays were in agreement. Crude human TSH preparations required considerably more antiserum per unit of biological activity than did either crude or purified bovine TSH preparations. The labeled bovine preparation retained both biological potency and immunological integrity.

Diphenhydramine hydrochloride was estimated to be about 0.4 as potent as codeine phosphate in terms both of peak and of 2-hour total effect of subcutaneous administrations, in raising tussal thresholds to ammonia in unanesthetized dogs.

Prechallenge properdin levels were not significantly different in 35 vaccinated or non-vaccinated volunteers who did or did not become ill after respiratory challenge with P. tularensis. No change was observed in properdin levels after infection in 4 volunteers so studied. P. tularensis was not sensitive to the properdin system in vitro. It would appear that the properdin system is not involved in immunity of humans to tularemia.

Photodynamic destruction of monkey kidney cells by irradiation with visible light from a fluorescent light source at .20 watt/cm² for 15 minutes occurs 4 hours after staining with acridine orange, neutral red, proflavine, and toluidine blue. Resistance to this photodynamic effect occurs when cells stained with the same dyes are incubated in the dark for 24-48 hours prior to exposure. Lowering the temperature of incubation to 4°C inhibited the resistance forming process. A resistance-producing factor could not be obtained from resistant cells which would convert sensitive cells. Aeration of the cells or addition of fresh media does not reverse their photodynamic resistance. Thus, the depletion of a media constituent is not the cause of photodynamic resistance. Cell lines vary as to their susceptibility to photodynamic action and their ability to develop photodynamic resistance. However, under the experimental conditions outlined in this paper, primary cell cultures display resistance, whereas, continuous cell lines do not.

X-ray diffraction analyses, and Sr⁸⁹ and Ca⁴⁵ uptake studies in tibiae and incisors of control and fluoride treated one-year-old rats were carried out. In the tibia ends and shafts an improvement of bone apatite crystallinity in the direction of the a axis and not the c axis was produced by fluoride ingestion. No changes in incisor apatite crystallinity were observed. Uptake of Ca⁴⁵ and Sr⁸⁹ was decreased in the tibia ends of the 25 and 50 ppm F treated animals. Sr⁸⁹/Ca⁴⁵ ratios in the tibia samples were the same as in the serum specimens. There was a discrimination against Sr in relation to Ca in the incisor bases. Radioactivity in the tibia of one-year-old animals was 1/40 to 1/50 of that found in a group of young animals treated similarly in an earlier experiment. In the young rat the tibia ends were found to incorporate more Sr⁸⁹ and Ca⁴⁵ than the incisor, while the opposite was found in the older rats.

Ouabain in concentrations from 10^-3 to 10^-6 M inhibits acid secretion by frog gastric mucosas in vitro and at 10^-4 M causes tissue Na to rise and K to fall. Increasing K concentration in the medium from 9 to 29 mM largely abolishes the inhibitory effect of 10^-4 M ouabain. We conclude that at least some of the inhibitory effect is the result of alteration of intracellular electrolyte pattern.

Purified sheep erythropoietin was found to enhance iron uptake and heme synthesis when added to a suspension of rabbit bone marrow. The greatest difference between control suspensions and erythropoietintreated suspension was found after 45 hours incubation in suspensions with a low cell count, with a medium enriched with tissue culture fluid and with an atmospheric oxygen tension of 13% oxygen.

The author is grateful for the able assistance of Yolanda Rivero.

The amino acid composition of deproteinized cerebrospinal fluid was quantitatively determined by paper chromatography in 25 healthy adults, 28 patients with hepatic disease, (16 with hepatic coma, and 12 without coma). A significant increase of all free amino acids in the cerebrospinal fluid of those with hepatic coma was found. There was also an increased concentration of 14 free amino acids in the fluid of patients with liver disease without coma. Methionine sulfone and methionine sulfoxide were also found as pathologic breakdown products in the fluid of those with hepatic coma. A decrease in oxidative properties of the liver is suggested as a possible cause.

A variety of respiratory inhibitors, phosphorylation uncouplers, amidine and guanidine derivatives, including the hypoglycemic agents, synthalin and phenethyl-biguanide, appreciably stimulated lactic acid production and glucose utilization of cell cultures. The simplicity and effectiveness of the procedures described suggest their applicability to further studies of respiratory inhibitors, uncouplers of phosphorylation, and the evaluation of certain types of hypoglycemic compounds.

Cultured cells exposed to phenethylbiguanide and other respiratory inhibitors or uncouplers of phosphorylation exhibited an increased susceptibility to cytotoxicity of certain inhibitors of glycolysis.

Mature female Sprague-Dawley-Rolfsmeyer rats with a mean food consumption of 5.26 g/100 g BW of Purina Lab Chow were subjected to 8, 17, 22 and 48% food restriction. Thyroxine secretion rates (TSR) were determined beginning 3 and 17 days later. Only 48% restriction resulted in a significant reduction in TSR during initial period (28%). In second period a 22% restriction resulted in a significant 42% and 48% restriction resulted in a highly significant 54% decrease in TSR.

S. aureus grown on whole blood agar produced a diffusible material which caused changes in the nitrogen, hemin and red cell content of the agar. Experiments using blood cells tagged with Cr⁵¹ indicated that the dense red band around the growth of the organism was due to a concentration of red blood cells which had been moved into the area of the band from the area just outside it.

Injections into normal mice of serum obtained from rabbits previously immunized with human urinary erythropoietin results in a progressive daily decrease in the relative numbers of erythroid cells in the bone marrow. Injections of this serum into polycythemic mice 24 to 44 hours after initiation of a wave of erythropoiesis with erythropoietin has no effect on the magnitude of the erythropoietic response, although injections of the serum one hour before or at the same time that exogenous erythropoietin is administered completely abolish the erythropoietic response. These results support the concepts that erythropoietin is involved in normal regulation of red cell production and that this regulation is the result of the effect of erythropoietin on the differentiation of stem cells. Some speculative interpretations of the effect of injections of the immune serum in polycythemic mice during the first 18 hours after injection of exogenous erythropoietin are also presented.

Rat platelets suspended in a glucose-sodium citrate-citric acid solution plus plasma have been introduced in polyethylene tubing of 0.125 inch in diameter and rapidly frozen by immersion of the whole container in liquid nitrogen. The material was stored in the same environment. The material was thawed by immersing the whole container in water at 45°C. Administration of these platelets to thrombocytopenic animals was associated with average increase of platelet counts from 36,900 to 205,793 in one hour. Eighteen hours after administration, 63.9% of these platelets were detectable in the circulation. Satisfactory correction of the bleeding tendency was observed in 66.6% of the recipients.

The assistance of Dr. Charles H. Burns of the School of Public Health in production of thrombocytopenia in the rats and the technical assistance of Miss Lynne Ballantyne are gratefully acknowledged.

The effect of epinephrine and norepinephrine on the individual free fatty acids of plasma was investigated in anesthetized dogs. The increased arterial FFA after administration of catecholamines was due to a rise in concentration of most major fatty acids. The percentage contribution of oleic and linoleic acids to total arterial FFA was also increased. A direct relationship was found, both in normal and in diabetic dogs, between the resting arterial FFA level and percentage of oleic acid in FFA. An attempt was made to relate these findings to the selective release of FFA from adipose tissue that occurs in the normal animal.

Estrogen induced extension of the duct system of mammary glands of suckling, ovariectomized, hooded Norway rats. Two μg of 17 β-estradiol dipropionate stimulated greater mammary growth than did 1.0 μg but 4.0 μg were not superior to 2 μg. Estrogen (0.5 μg) every other day plus progesterone (1.0 mg) daily stimulated greater mammary duct growth than did 2.0 μg of estrogen alone.

1. Histidine decarboxylase activity in the Walker carcinosarcoma 256, although measurable, is very low. 2. No consistent change occurred in excretion of histamine in the urine of female rats when the tumors were implanted or growing rapidly, or after their removal and subsequent recurrence.