Experimental Biology and Medicine

Acetylene dicarboxylate (ADC) is a competitive inhibitor of succinoxidase. The apparent dissociation constant of the ADC-enzyme complex is about 1/5 that of the succinate-enzyme complex when the inhibitor is added to the enzyme after or along with the substrate. However, when the enzyme, inhibitor, and substrate are incubated 18 hours before assay, the value of K_I is about 1/25 that of K_S.

A search was made for presence of infection due to adenovirus type 4 in dormitory freshmen of Case Inst. of Technology, Western Reserve Univ., and U.S. Military and Naval Academies. Antibody analyses revealed no instance of such infection in 126 students. Three students had rises in serum titers of complement-fixing antibodies against adenovirus type 4; however, none of the 3 had rises in serum titers of neutralizing antibodies against adenovirus types 1, 2, 3, 4, 5, 6, and 7. One student from the Naval Academy who had only a 1-tube rise in complement-fixing antibody developed a significant rise in neutralizing antibody against type 7 adenovirus.

The analgesics codeine, morphine, and l-14-hydroxydihydromorphinone (Numorphan) produced inhibition of the conditioned avoidance-escape response in rats, a widely used tranquilizer test. The potencies in blocking conditioned response correlated roughly with analgesic potencies. The very potent analgesic, l-14-hydroxydihydromorphinone, exhibited by subcutaneous injection slightly more conditioned response blocking activity than perphenazine, one of the most active tranquilizers now in clinical use.

(1) Acquired tolerance of male skin isografts has been produced in females of inbred A and C₅₇Bl (subline 1) strains, over a wide age range, either by injection of viable male spleen cells or by placing females in parabiosis with males. (2) Attention is directed to the importance of age and degree of histocompatibility difference in susceptibility to induction of acquired tolerance. (3) The implications of these observations are discussed.

Several preparations of highly purified α-MSH were studied for their adrenocorticotropic activity in vivo and in vitro using USP Reference Corticotropin as a Standard. They showed corticotrophic activity by either assay procedure. β-MSH did not demonstrate similar activity. In view of data obtained, it is suggested that the “active core” of ACTH molecule may be no longer than the tridecapeptide from the N-terminus.

The D.C. specific resistances of dog and beef ventricular muscle strips were compared with those of cat intestine circular smooth muscle, which is a non-syncytial tissue, and of frog sartorius muscle where individual cells extend almost the full length of muscle. Resistances were measured in the direction of fiber orientation before and after the interspace ion concentration was reduced by soaking in isotonic 10% Tyrode's-sucrose solution. Cardiac and smooth muscle resistances were much more sensitive to interspace ion depletion than was skeletal muscle resistance. It is suggested that the current flow path through the cells of cardiac muscle is of high resistance due to large number of high resistance cellular membranes involved, as in smooth muscle. It was concluded that cardiac muscle cells are separated from each other by high resistance membranes and do not form a functional syncytium.

1) RNA-containing fractions have been prepared from crude tissue culture virus suspensions of 6 varieties of enterovirus by the method of Gierer and Schramm. Each RNA preparation has produced plaques on human cell monolayers. The plaque-forming agent is completely inactivated by RNAase but not by DNAase. RNA is, therefore, an essential component for the infectivity of the preparation. 2) The plaques formed by RNA fraction contain intact virus from which RNA is prepared. The RNA directs the cell to replicate not only the specific RNA but also a new highly specific protein needed for synthesis of intact original virus.

An assay is described for measurement of a serum inhibitor of an esterase derived from preparations of the first component of complement (C'1-esterase). Esterolysis of N-acetyl-L-tyrosine ethyl ester by C'1-esterase is inhibited instantaneously and stoichiometrically by fresh human serum. Units of C'1-esterase and of serum inhibitor are defined. The inhibitor in human serum is stable at 45 for at least 4 months, at 37° for at least 24 hr. and at 48° for at least 30 min. It is completely inactivated during 30 min. incubation at 60° and is labile at 0° at pH values below 6. The inhibitor is inactivated at —5 in the presence of methanol concentrations of 15° or greater but is recovered quantitatively in a 40° ammonium sulfate supernatant fraction. These data are being applied to purification of the inhibitor from human serum. Purification procedures and properties of the purified inhibitor will be reported elsewhere.

Monkey and guinea pig sera inhibit human C'1-esterase to about the same extent as human serum, while rabbit serum is about one-third as active. No marked species specificity exists in the interaction of inhibitor in human or rabbit serum with human or rabbit C'1-esterase. These observations are being employed on possible role of inhibitor in experimental hypersensitivity.

1) The virus of infectious canine hepatitis was shown to share the 4 main properties of the adenovirus group, including common complement-fixing antigen. 2) The relationship between the disease of the dog and human adenovirus infections is discussed.

Blood group A activity was demonstrated in purified Vi antigen preparations and blood group B activity in whole cells of Escherichia coli 086. The immunological implications of these findings were investigated. The antigenicity of Vi antigen was essentially as great in group A or AB individuals as in those of other groups. Anti-B exerted a bactericidal effect upon E. coli 086, but further studies are obviously needed to determine the protective effect, if any, of anti-B against E. coli 086 or, indeed, of either isoantibody against any microbial agent which may possess blood group antigens. Nevertheless, cross-reactivity between blood group substance B and E. coli 086 affords a model for the possible influence of blood groups in resistance to infection against those microbial agents which may possess blood group antigens.

1. Fourteen patients with congestive heart failure and 14 control patients without heart failure were studied with the tryptophan load test. 2. Urinary xanthurenic acid excretion following tryptophan load was significantly greater in the cardiac group than in controls. Mean XA excretion of the group with congestive failure was 35.6 mg/24 hours with standard deviation of 22.8. Mean excretion of the control group was 12.9 mg/24 hours with standard deviation of 5.2 mg/24 hours. 3. The heart failure group, which attained a higher level of XA excretion than the control group in the post tryptophan period, showed a greater decline in XA excretion after pyridoxine than the controls. 4. Since pyridoxine is required for orderly catabolism of tryptophan and pyridoxine corrects this metabolic aberration of tryptophan, it is not unreasonable to state that in congestive heart failure availability of Vit. B₆ is limited.

Induction of polycythemia by repeated intravenous injections of homologous erythrocytes produced gastric ulceration in 26 of 29 rats, including normal, adrenalectomized, hypophysectomized, and sham-operated animals. An experimental system has therefore been developed for the study of etiologic factors which may parallel those leading to development of peptic ulcers in patients with polycythemia rubra vera.

Concentrations of nonesterified fatty acids and of glucose in plasma were determined before and after infusion of epinephrine and norepinephrine into dogs. At the same concentration and rate of infusion, norepinephrine produced a greater increase in nonesterified fatty acids.

1. A single subcutaneous injection of 2.14 mg of 4′-fluoro-10-methyl-1,2-benzanthracene in albino rats produced a severe and fatal periarteritis in 4 to 8 weeks. Similar lesions were seen after injection of one-half as much of this hydrocarbon, but they developed more slowly. Equivalent levels of 10-methyl-1,2-benzanthracene or the solvent alone were ineffective. 2. The most severe lesions were observed in pulmonary arteries; this finding is in accord with the cyanotic conditions of some rats. Hyaline necrotic lesions were observed in renal arterioles and glomerular capillaries. 3. A possible immunological basis for these lesions is discussed.

1. A reliable assay procedure has been presented for rapidly quantitating the anti-inflammatory potency of steroids on human skin. 2. Estimated steroid potency values can be used to predict reliably the proper steroid concentration to be studied in topical clinical trials. 3. Data have been adapted to the Stetten theory which concerns relationship of hormone dosage to physiological response. This permitted comparison of steroids with respect to capacity of target organ to bind hormone and affinity of hormone for binding sites. 4. The target organ has increased capacity to bind fluorometholone relative to hydrocortisone. This means that some optimum concentration of fluorometholone is capable of producing a greater anti-inflammatory response than that possible even with very high doses of hydrocortisone. 5. That fluorometholone is approximately 40 times as potent as hydrocortisone topically yet only about equal to hydrocortisone in systemic activity suggests that fluorometholone is a unique compound for topical anti-inflammatory therapy, since even if absorption from the skin occurred, the systemic effect would be negligible.

Continuous phase contrast observation and microcinematographic recording of living rabbit kidney cell cultures infected with herpes simplex virus, established the following phenomena: 1. Progressive dissolution and dispersion of nucleoli, loss of nucleo-plasmic homogeneity, and appearance of intranuclear densification zone but not of a well delineated inclusion body. 2. Persistent activity of cytoplasmic organelles in cells with severe nuclear lesions. 3. Rapid (2-3 hours) formation of giant cells by cell confluence but not by cell division mechanism, accompanied by signs of cytoplasmic liquefaction. The significance of these data is discussed.

Administration by stomach tube of a suspension containing 2-acetylaminofluorene to suckling albino mice was followed by development of high incidence of hepatomas in males (60%) but not in females (1%). A similarly low incidence of hepatomas (0%-4%) was observed in untreated mice from same strain or mice treated with suspension alone. The low dose of 2-acetylaminofluorene represents smallest quantity of this compound effective to date in liver tumorigenesis. Also, results indicate that suckling male mouse is especially susceptible to hepatoma induction with 2-acetylaminofluorene.

Whole cells and cell-free extracts of Leptospira biflexa have been used to demonstrate a series of transamination reactions. Dialysis against versene was used to show that pyridoxal phosphate served as a coenzyme. The possible importance of transamination in leptospiral metabolism is discussed.

Accumulation of ribotide of 5-amino-4-imidazole carboxamide (AICAR), excreted in the riboside form by a purine-requiring mutant of Escherichia coli, was inhibited by a number of structural analogs of purines. The most potent inhibitors were 6-thioguanine, 6-mercaptopurine, and 2,6-diaminopurine. The type of inhibition obtained suggests that the antimetabolites sufficiently resemble natural purines in structure to act as feedback inhibitors in biosynthetic control.

1. Marked elevations of plasma LDH activity in experimental hemorrhagic shock are described. An initial lag period is followed by a sharp rise to 15 to 50 times the original value. 2. Alterations of plasma LDH activity in shock bear a consistent relationship to such parameters of biological deterioration as rate of spontaneous return of blood from the reservoir to the animal (“taking up”) and reversibility to replacement transfusion. 3. Catheterization experiments in which consistent arterio-venous differences were demonstrated indicate that the sources of elevated plasma LDH activity are widespread and include liver, kidney and tissues of an extremity. 4. Electrophoretic separation of normal dog plasma reveals 4 enzymes with LDH activity. All 4 are elevated in plasma of dogs in shock. 5. Marked elevations of plasma GO-T, MDH and alkaline phosphatase were observed in dog plasma during hemorrhagic hypotension. No significant elevations in beta-glucuronidase, alpha-glucosidase, leucine aminopeptidase and ceruloplasmin occurred. 6. These experiments suggest that serial plasma LDH determinations may serve as useful indicators of the extent of biological deterioration in hemorrhagic shock.

We found that the vitelline membrane of hen's egg was ruptured on contact with semen. Also that the destructive action was stronger in spermatozoa than in seminal fluid. Experiments were conducted to determine the substance(s) effecting destruction of the membrane. Trypsin and hyaluronidase are responsible for this phenomenon, but another major factor may exist which determines the destruction.

1. Both muscularis and mucosa of rat show significant changes in amounts of energy-rich phosphate compounds when animal is fasted or fasted and briefly fed. 2. With increasing periods of starvation to 72 hours, the easily hydrolyzable phosphate compounds, Cr.P.-P. and A.T.P.P., diminish in both tissues in a predictable manner. 3. The mucosa appears to be more labile of the 2 tissues, although the muscularis shifts in the same direction and to nearly the same magnitude. 4. The assay of either Cr.P.-P. or A.T.P.P. on whole intestinal segments will provide sufficient data as would the independent assay of each, in the major tissues comprising the intestine, to follow the nature of the mechanism involved.

Fluorometholone is an unusual synthetic corticoid in the rat because its anti-inflammatory potency (131 X hydrocortisone) is much greater than its glucocorticoid potency (26 X hydrocortisone). Its potency in promoting development of ulcers of the pyloric portion of rat stomach is between its glucocorticoid potency and its anti-inflammatory potency. It does not cause sodium retention at doses in which a similar compound (differing only by presence of C-21 hydroxyl) does produce measurable sodium retention.

Insulin in doses that are not hypoglycemic increases the rat's sensitivity to dextran reaction, characterized by edema. A comparable effect was observed in adrenalectomized fasted rats and following treatment with tolbutamide.

1. C3H mice made tolerant by injection of A spleen cells at birth are tolerant of skin homografts from A strain and (A x C3H) F₁ donors. 2. C3H mice made tolerant by injection of (A x C3H) F₁ spleen cells at birth are tolerant of skin homotransplants from both A strain and (A x C3H) F₁ donors. These observations are interpreted as evidence indicating that new histocompatibility antigens do not derive from genie interaction during hybridization in the 2 inbred strains of mice in these studies.

Thrombokinase prepared from bovine plasma was further purified by continuous flow paper electrophoresis. Thrombokinase was assayed by its capacity to activate prothrombin in the presence of oxalate, and also by production of thrombin in a system containing prothrombin, cephalin, calcium and bovine “barium carbonate serum.” Curves of these 2 assays were similar and formed a peak ahead of the thrombin peak. TAMe esterase activity appeared in 2 peaks which corresponded respectively to thrombin and thrombokinase peaks.

A cloned wild-type population (strain D98S) derived from human bone marrow cells, which is inhibited by 8-azaguanine (AG) in concentrations in excess of 0.1 μg/ml, contains approximately 1 to 2% cells resistant to AG in 100-fold higher concentrations. A single-step mutational process (in broad meaning of the word) appears to be involved. The AG-resistant lines are stable upon prolonged subculture either in presence or absence of drug. Mutation rate from sensitivity to resistance is of the order of 5 × 10^-4cell/generation. AG resistance is a useful genetic marker, since at selective AG concentrations (a wide plateau at 1-10 μg/ml) colony formation by resistant cells is not impaired and sensitive cells are destroyed. A method for protein determination is described, based on measurement of eluted bromphenol blue from HgCl₂-fixed, glass-attached cells.

Gastric secretory activity (volume and free HCl concentration) in the pylorus ligated rat is inhibited by bile duct ligation. This is not due to removal of bile from the intestine but appears to be due to increase in level of circulating bile constituents, particularly bile salts, resulting from obstruction to the outflow of bile from the liver.

Metabolism of 1-(p-chlorobenzenesulfonyl)-3-n-propylurea was investigated in dog, rabbit and man. The rabbit excreted more than three-fourths of the drug without chemical alteration. Less than half of orally ingested drug was excreted unchanged by the dog, most of the remainder appeared as p-chlorobenzenesulfonylurea and p-chlorobenzenesulfonamide. As the result of a limited investigation in man, unchanged chlorpropamide and p-chlorobenzenesulfonamide were found in the urine. One other possible metabolite was indicated by paper chromatograms.

Distribution of D-xylose in the uncut rat diaphragm was studied in vitro under a variety of conditions. It was found that epinephrine, triiodothyronine or Cortisol had no effect on distribution of this sugar when administered in vitro. Triiodothyronine or Cortisol, administered parenterally, increased the space occupied by D-xylose in absence of insulin, and in presence of insulin it resulted in comparatively small but consistent addition to the space occupied when insulin alone was used. A 24-hour starvation period was without effect on sugar distribution whereas a 56-hour period increased the xylose space in absence of insulin and was quantitatively additive in its presence.

Carbonic anhydrase was found in the caiman (C. laitrostris) erythrocytes, kidney, gastric mucosa, pancreas and hind-brain. No activity, however, was found in the lens. The enzyme was also found in the alligator (A. mississipiensis) choroid plexus and ciliary processes. Carbonic anhydrase inhibition in the alligator caused a long-lasting rise of plasma total CO₂ but no certain change of anterior aqueous total CO₂ was observed.

An instrument has been described which permits accurate control of degree of constriction of the main renal artery in small animals and facilitates production of hypertension in a large percentage of such animals.

The metrotropic activity of a series of C-21 haloprogestins was assayed in the Clauberg, uterine carbonic anhydrase, and intrauterine (McGinty) assays. C-21 Fluorination of both 17α-acetoxy-progesterone and 6α-methyl-17α-acetoxyprogesterone resulted in a marked increase in oral potency of both substances; subcutaneously, potency was unaltered by this structural modification. Upon local instillation of these 21-halogenated progesterone derivatives, decreased potency was observed as atomic weight of substituted halogen increased. 21-Fluoro-17-acetoxyprogesterone failed to increase progestational potency in the intrauterine assay thus differing from other 21-fluoroprogesterones. The most potent metrotropic agent tested was 6α-methyl-17α-acetoxy-21-fluoroprogesterone. Orally, it was twice as potent as its non-fluorinated analogue and 10 times more potent than subcutaneously administered progesterone. It was 50 times more potent than progesterone when both were given subcutaneously and about 50 times more potent than progesterone when given locally.

The data obtained do not indicate that biotin concentration of the medium in excess of that required for optimum growth of organism exerts any positive effect on protein formation in either L. casei or L. arabinosus. On the contrary, a slight negative effect was noted, particularly when L. casei was used as test organism. The results appear to support the observations that lipide formation is preferentially affected by biotin concentration.

A toxic factor present in our distilled water found lethal to Paramecium was analyzed. The results show that it is not related to pH, can be duplicated by 5 metallic salts at specific concentrations (M) as follows: cupric chloride, 10^-6; cupric sulfate and cupric nitrate, 20^-6; zinc acetate, 10^-4; lead acetate, 25^-4; and can be neutralized by 0.02 M CaCl₂. Calcium chloride is likewise capable of neutralizing cupric chloride and lead acetate, but not cupric sulfate, cupric nitrate or zinc acetate. These results coupled with the demonstration that the presence of SO₄ ⁻⁻ions actually destroys the neutralizing power of calcium indicate strongly that the toxic factor under consideration could possibly be copper chloride and/or lead chloride, not likely sulfate or nitrate of copper or zinc acetate. Generally the results also confirm the protective action of Ca⁺⁺ against ionic imbalance or unknown impurities in water as reported by other workers. Because of the demonstrated sensitivity of Paramecium to metallic ions we commend its use as an indicator for low-level toxicity due to such ions in solutions including distilled water not made and stored in pyrex glass.

1) Electron microscope studies have been carried out on ultrathin sections of lymph nodes from 6 patients with acute lymphatic, 1 with acute and 1 with chronic monocytic, 1 with acute myeloid leukemia, 2 with lymphosarcoma and 1 with Hodgkin's disease. Virus particles of approximately 900 Å in diameter have been observed in cells of lymph nodes from 3 cases of acute lymphatic, 1 of acute myeloid leukemia and 1 of lymphosarcoma. Similar studies of sections of lymph nodes from 5 non-leukemic patients have failed to reveal presence of virus particles. 2) Cytopathogenic changes have been observed in cells of lymph nodes grown in vitro from 15 out of 24 patients with leukemia and lymphosarcoma. Cells of lymph nodes grown in vitro from 4 non-leukemic patients have not shown changes observed in lymph nodes from leukemic patients during similar number of passages. Cell-free extracts of lymph nodes from 8 patients tested have induced changes in monkey kidney cell cultures which could be serially transmitted. These changes have not been seen in similar cultures treated with extract of a non-leukemic lymph node. Extracts of leukemic lymph nodes which induced changes in monkey kidney cell cultures did not have a similar effect in human embryo cultures. 3) Cell-free extracts of biopsy material from leukemic and lymphomatous lymph nodes and fluids from monkey kidney cell cultures treated with these extracts have not shown specific leukemia-inducing activity in Swiss (Holtzman), C₃HZb/Bi and C₃H/Bi strain mice. 4) It is of particular interest that in one patient with lymphosarcoma, not only were virus particles observed in ultrathin sections of the biopsy specimens, but also similar virus particles have been found in cells from the same specimen, grown in vitro for 6 sequential passages during which cytopathogenic changes had been observed.

Excretion of magnesium has been studied in dogs utilizing the “stop-flow” technic and Mg²⁸. The evidence indicates that magnesium is secreted by the distal tubule.

D-alanine, but not L-alanine, competitively reverses the inhibitory action of cycloserine on S. faecalis cells that are synthesizing cell wall substance. Both cycloserine and penicillin can inhibit cells that are not growing but that are making cell wall substance. However, they differ in mode of action.

1) Our studies suggest that neither exhaustion of humoral or cellular factors necessary for inflammation, nor presence of possible inhibitors of the reaction play a dominant role in diminution of inflammation and healing of a hypersensitivity vasculitis of the Arthus type. 2) This study supports the importance of ridding the diseased vessel of antigen and antibody in bringing about a decrease in reaction and healing.

A technic is described for determination of amount of lactogenic hormone required for milk secretion in intact lactating rats. Rats were isolated from their litters for 12 hours on day 14 postpartum then deeply anaesthetized with 4.5 mg/100 g Nembutal and all milk withdrawn by 9 young with aid of oxytocin. With lactogenic hormone discharge blocked and mammary glands empty, significantly less milk (.85 g/100 g maternal body weight) was obtained following a 2nd 12-hour isolation period in comparison with 2.92 g/100 g for unanaesthetized controls. Injection of .5, 1, 1.5 or 2 mg lactogenic hormone during 2nd isolation period significantly restored milk yield approximately 70%. Yields were, nevertheless, significantly less than controls. No greater milk yield resulted from injection of more than 1 mg lactogen. These data indicate that lactogen is necessary for maintenance of milk secretion in intact rats and strongly imply that since lactogen alone was incapable of restoring milk secretion 100%, other pituitary hormones are necessary for full milk secretion and perhaps might be susceptible to discharge by nursing stimuli.

1) After parenteral administration of radiovit. B₁₂, radioactivity reaches the gut mainly with the bile, but other components of duodenal juice also contain radioactivity. 2) The calculated daily biliary excretion of radioactivity in controls is invariably larger than daily fecal excretion, suggesting enterohepatic circulation. In cases with defective B₁₂-absorption, there are indications of an enterohepatic circulation in only about half of the cases. 3) A higher percentage of radio-vitamin administered is retained in serum, and a numerically lower percentage is excreted/day in urine, bile, and feces of patients with B₁₂-avitaminosis than in controls. These facts suggest that more B₁₂ is retained in states of deficiency.

Intact, ovariectomized, and ovariectomized-hysterectomized mice were treated with estrogen, estrogen and relaxin, and relaxin alone for periods of 10 days. It was shown that estrogen and higher levels of relaxin synergize in the promotion of lobule-alveolar growth as indicated by total DNA of the glands and by histological examination of whole mounts. Estrogen or relaxin alone did not stimulate such growth. Mean total DNA at optimal levels of relaxin was equal to that observed in mice pregnant 6 days. In the operated groups, reduced total DNA may be due to gland involution of 12 days.

A simple, accurate method for determination of the 17,21-dihydroxy-20-ketosteroids in plasma and urine has been described. The chloroform extract is dissolved in chloroform and chromatographed on silica gel in a 1 ml serologic pipette. The 17,21-dihydroxy-20-ketosteroids are eluted with ethanol-chloroform mixtures and assayed by a micro Porter-Silber procedure.

Movements of P³²O₄ in and out of fresh and cold-stored human erythrocytes were measured in presence and absence of inosine. I. Small amounts of hemolysis when inosine was present caused a decrease in plasma inorganic phosphate and incorporation of P³² into plasma organic phosphate. This formation of plasma organic phosphate decreased movement of P³² into the cell. II. When P³² was originally present in the red cell, inosine decreased the net movement from cells to plasma. We attribute this effect to utilization of inosine as substrate in the cell. III. Cells containing P³² during 6-day cold-storage lost 3 to 4 times as much P³² as cells having P³² for a short time. Movements from cells to plasma are described as the result of at least 2 phosphate compartments within the cell.

It has been found by the technic of isotope dilution using acetate-1-C¹⁴ that the normal sheep on an all-hay ration produced acetic acid in its rumen by bacterial synthesis at the rate of 0.126 millimole per ml of rumen liquor for the 7-hour period following feeding. Radioactivity was found in the other rumen volatile fatty acid (propionic, butyric and higher acids) demonstrating their formation from acetic acid in the rumen.

Methylphenidate alters the pressor responses in the dog to a series of phenylalkylamines. The amines affected by this central nervous system stimulant are influenced in such a way as to fall into 3 distinct categories, i.e., 1) potentiated or not altered, 2) reversibly blocked and 3) irreversibly blocked. The differences in structure of pressor phenylalkylamines comprising the 3 groups are reviewed along with reference to other compounds which affect pressor action of these amines in a similar manner.

Oxygen at 8 to 12 atmospheres depresses the transport of sodium ion across the isolated frog skin to 57.4%. Addition of 8 to 10 atmospheres of nitrogen without oxygen deprivation had no such effect.

Rabbits and guinea pigs were immunized with untreated crystalline beef and pork insulin in modified Freund adjuvant. Disappearance of insulin-I¹³¹ from subcutaneous sites of injection was markedly reduced by the adjuvant. Fourteen of 17 guinea pigs and all 5 rabbits survived otherwise lethal doses of insulin. Insulin-binding antibodies were produced within 11-36 days in all rabbits and within 10-63 days in all surviving guinea pigs. Insulin-antibody complexes in rabbit plasma migrate with γ globulins and in guinea pig plasma with fibrinogen, on paper electrophoresis. Total insulin-binding capacities of antisera ranged from less than 10 to 5000 mμg/ml in guinea pigs and from 14 to 550 mμg/ml serum in rabbits.

1. Pretreatment of fatty acid with human plasma, plasma albumin or globulin, unesterified cholesterol, or cyanide retards or prevents hemolytic activity. Once lytic quantities of fatty acid are bound by human erythrocytes, the above substances reverse their protective activity and accelerate hemolysis. 2. Plasma and plasma factors appear to accelerate intrinsic oleate hemolytic activity rather than act via a secondary superimposed lytic mechanism; the mode of cyanide action is unknown. 3. The oleate hemolysis protective and accelerating properties of human plasma can be inhibited progressively and simultaneously by pretreatment with oleate. Both mechanisms appear to depend upon plasma ability to bind fatty acid. 4. The intensity of oleate hemolysis acceleration by human plasma or plasma fractions is not uniform. Variations can be attributed to differences in fatty acid binding capacities and to inhibitors which possess unesterified fatty acid activity. 5. A hypothesis is suggested for linking increases in mammalian plasma unesterified fatty acid with in vivo hemolysis.

Synthesis of aldosterone by human adrenal slices from corticosterone-4-C¹⁴ has been demonstrated in vitro.

1. A method is described for determination of magnesium in blood serum using the azo dye Chrome Fast Blue BG. 2. Use of this dye affords immediate determination of serum magnesium without removal of calcium and with no addition of stabilizers. 3. The dye as used here is sensitive to as little as .01 microequivalent of magnesium. 4. Thirty-seven sera were analyzed. These had an over-all magnesium-content of 1.73 meq/l of sera.

1) A study of levels of several electrolytes and various other substances concerned with protein, lipid and carbohydrate metabolism has been made in blood and urine of dogs subjected to removal of alpha cell containing part of pancreas by selective, partial pancreatectomy. 2) Despite previous suggestions by others, there was no definite evidence in our work of a relationship between functional activity of alpha cells and any of the substances studied, with the possible exception of blood non-protein nitrogen. Since this was significantly elevated in all experiments and was not associated with a significant increase in urinary nitrogen excretion nor loss of weight, it is possible that absence of alpha cells exerts a slight inhibitory effect on protein synthesis.

The amount of glycoprotein, expressed in terms of hexosamine concentration, was determined in lipoprotein fractions of 4 human sera, separated in ultracentrifuge at various densities. On an average, no more than approximately 5% of total serum hexosamine was detected in lipoprotein fractions.

Using an eight-day method for screening compounds against experimental visceral leishmaniasis, the following compounds showed activity in hamster or mouse infection or in both: amphotericin B (and Fungizone), fumagillin, nystatin, Sb dimercaptosuccinate and xerosin. The activity of Fungizone is especially noteworthy.

Skin homografts were made between individuals of a single line of random-bred and between 3 strains of inbred rats. Graft survival was determined from histological sections of graft epidermis, and presence or absence of circulation. Survival time of skin homografts was reduced in individuals that had received a prior inoculation of packed erythrocytes.

Continuous flow paper electrophoresis has been applied to isolation of blood clotting factors. Stuart factor has been separated from proconvertin and shown to migrate in the albumin and pre-albumin area. Data presented show factor specificity and freedom from other clotting factors in the case of proconvertin, Stuart, and Hageman preparations.

Ability of several antispasmodic and ganglionic blocking substances to produce immediate death following maximal electro-shock convulsions in mice has been demonstrated. With most agents studied (atropine, atropine methyl nitrate, benactyzine, mepiperphenidol, methantheline, propantheline, scopolamine, scopolamine methiodide, mecamylamine, chlorisondamine, pentolinium and hexamethonium), the post seizure lethal effect was produced in doses approximating that required to produce a peripheral anticholinergic effect in the same species (pupil dilatation). Neither atropine nor mecamylamine altered the electroconvulsive threshold.

Respiratory failure appeared to be the cause of drug-induced post seizure deaths, but the mechanism of action was not revealed.

Genetically different mouse hemoglobins differ in solubility and crystal formation. Differences in hemoglobin solubility and crystal formation can therefore be used to identify homologous erythrocytes in irradiated mouse chimeras if the hemoglobin types of donor and recipient mice are distinguishable by these properties to begin with.

Hepatic cells of chicks fed a purified basal diet consisting of isolated soybean protein (Drackett Assay Protein C-1) with no supplemental zinc, exhibited nucleoli 78% larger than nucleoli of hepatic cells in equal number of chicks fed the same diet supplemented with 40 ppm of zinc. No other histopathologic changes were observed in sections of heart, spleen, kidney and small intestine.

When polygalactosamine (APS) produced by Aspergillus parasiticus was mixed with cells of Cryptococcus neoformans it produced a “capsular reaction” and agglutination similar to that caused by specific antibody. The positively charged APS molecule also caused precipitation of various negatively charged (acidic) polysaccharides including gum acacia and capsular polysaccharides of C. neoformans and pneumococcus. Dextran was not precipitated by APS.

1) Tissue and serum aldolase of normal and tumor-bearing (primary hepatoma and transplanted sarcoma) Osborne-Mendel rats were studied. Blood for aldolase assay was drawn from portal vein and inferior vena cava of hepatoma-bearing and control rats, from tumor vein and heart of sarcoma-bearing rats and also from heart of control rats. 2) Control rats had an average serum aldolase value of 70 units each for portal vein, vena cava and heart blood. Serum aldolase values of sarcoma-bearing rats were about 3 times and those of hepatoma-bearing rats about 8 times the values for control rats. These values averaged 375 units for blood entering tumorous liver and 540 units for blood leaving it. Serum aldolase values for heart blood averaged 176 while that for sarcoma-vein blood of the same rats averaged 194 units. Average aldolase value for normal liver, hepatoma, hepatoma-free liver, sarcoma and sarcoma-rat liver were 14,400, 12,400, 9,600, 7,700 and 9,100 units, respectively. Liver aldolase levels for tumor-bearing rats were about 35% lower than those for control rats. Aldolase values for necrotic areas of tumors were similar to serum aldolase values for respective rats.

1. 6-methylmercaptopurine was identified as a urinary metabolite of 6-mercaptopurine in normal Sprague-Dawley rats. 2. In in vitro experiments, 6-MeMP exerted greater inhibition of both formate and glycine incorporation into the combined nucleic acid and protein fractions of Sarcoma 180 ascites tumor cells than did 6-MP.

Successful implantation, without x-irradiation or other pretreatment of host, of isologous blood forming tissue from normal (ww) mouse fetal liver in viable severely anemic recipients (20 of 24 adult WW ^v mice, 9 of 14 juvenile WW ^v mice) and in lethally anemic recipients (4 of 15 juvenile WW mice) has been demonstrated. Criteria of success were permanent increase in rbc/mm³ to near-normal levels, and decrease of mean cell volume from macrocytic to normocytic levels. These changes appeared in WW ^v adult anemic hosts longer after cell injection than in similar transplant experiments with WW ^v hosts subjected to 200-r whole-body irradiation. These findings fit well with the hypothesis of competition between slow-acting indigenous blood forming tissue of genetically anemic hosts and initially small implants of rapidly functioning blood forming tissue from genetically normal donors. If methods for circumventing homografts can be found, cell implantation may be generally useful for therapy of genetic defects in erythropoiesis.

Thinly encapsulated Cryptococcus neoformans cells were equally as virulent for white Swiss mice as thickly encapsulated cells of the same strain. Yeast cells initially possessing thin capsules promptly developed thick ones upon intracerebral injection into mice, and upon subculture to synthetic capsule medium. Both types of cells produced hydrocephalus to an equal degree. Degree of encapsulation is not a factor in virulence of Cryptococcus neoformans for white Swiss mice.

In both extrahepatic biliary obstruction and intrahepatic cholestasis, alteration of the bile canalicular wall of liver cells and distortion of normally present microvilli can be demonstrated by electron microscopy. The functional defect is localized into membrane of liver cells, the more so since cytoplasm of liver cells does not necessarily show significant alterations. It is assumed that the lesion in extrahepatic biliary obstruction results from increased hydrostatic pressure while it cannot be decided whether in intrahepatic cholestasis it is primary or also secondary to increased pressure. Microvillous changes are not found in chronic idiopathic jaundice and only focally in viral hepatitis with liver cell injury possibly as the result of intrahepatic cholestatic component. In both types of cholestasis, communication between dilated bile canaliculi and perisinusoidal tissue spaces may account by regurgitation for at least part of the jaundice.

A positive result with glyoxylic acid test was obtained in 62.5% of schizophrenic patients, while only 8% of non-schizophrenics gave a positive test. Though non-specific the test affords a statistically significant distinction which cannot be explained by the non-experimental variables. Because of the chance that the test has a significant association with the disease, schizophrenia, further investigation seems warranted.

Succinic dehydrogenase and cytochrome oxidase undergo maturation in respect to distribution and staining intensity in the neonatal kidney of the rat. Recognition of this event provides cytochemical evidence of functional immaturity in the neonatal kidney. Proteinuria or altered tubular reabsorption of protein was not observed in newborns of this species.

Human low density S_f 3-9 lipoproteins can be frozen and thawed, stored at —20°C, and heated to 56°C for 45 minutes without altering their immunological characteristics in precipitin ring and agar diffusion tests. Lipoprotein fractions stored at 4°C are decomposed by bacterial action. Aqueous merthiolate added to final dilution of 1:10,000 prevents bacterial contamination in lipoprotein fractions stored at 4°C without altering immunological characteristics of the lipoproteins. Freezing and thawing increases turbidity of lipoprotein solutions but does not alter lipid and protein patterns obtained with paper electrophoresis.

Survival-mortality data were obtained for tourniquet traumatized, adult Carworth Farms male mice for 120 hours after zero hour (time of removal of both hind leg ligatures). Higher percent survivals were noted throughout for test mice injected intraperitoneally with chlorisondamine dimethochloride, 5 mg/kg, than for control traumatized mice injected with water. The p (probability) values for test vs. control mice were: (A) for mice injected at zero hour, 0.01 or less at 6, 24, 48 and 120 hours thereafter, and (B) for mice injected 15 minutes after zero hour, 0.01 or less ONLY at 6 hours thereafter.

1) Daily administration of 1 μg estradiol benzoate (EB) and 2 mg progesterone (P) to mature ovariectomized rats for 19 days resulted in mammary gland growth (mean total DNA/100 g) comparable to that of rats pregnant 18-20 days. Increasing the progesterone level to 10 mg daily has little further beneficial effect on mammary gland growth. 2) Variability in growth of experimentally developed gland was less than that of pregnant controls. 3) Total DNA appears to be a more reliable index of mammary gland growth when expressed as unit body weight.

Nephrosis was induced in immature rats by subcutaneous administration of aminonucleoside. Subjects were divided in 3 groups: I, hypophysectomized, II, intact and III, adrenalectomized. All 3 groups developed proteinuria and ascites. Examination of stained sections, however, showed that kidneys of intact rats had undergone marked deterioration. Pathological changes were less marked in the adrenalectomized group, and absent in the hypophysectomized group.

1) Time sequence for inhibition of yeast respiration and glycolysis by nystatin is described and its significance is discussed. 2) A series of nystatin-resistant : sensitive pairs of Candida strains was examined. With various pairs, resistance of growth to nystatin was accompanied by similar resistance of metabolic activity to the agent. Resistant strains absorbed less nystatin than did sensitive organisms under same conditions, but there was no quantitative correlation between sensitivity and absorption. Reduced uptake of nystatin is probably only one of the ways by which resistance can be achieved. 3) Addition of serum albumin, zymosan, uranyl ions, arsenate, or dinitrophenol could protect yeast metabolism in varying degrees against the action of nystatin. Inhibition, once obtained, could not be reversed. The protective action of various agents approximated that expected from their effect on absorption of nystatin by yeast. 4) The methyl ester and N-acetyl derivatives of nystatin, which do not inhibit growth or metabolism of fungi, were not absorbed to a significant extent by either fresh yeast or zymosan. Since absorption occurs primarily below pH 5, the ionic species HOOC… NH₃ ⁺ is probably the material absorbed by yeast. 5) The results furnish additional evidence for the critical nature of uptake of nystatin by microorganisms in determining sensitivity to its action.

Intravenous urea has been administered in cats. The flow rate of CSF is radically reduced but the activity of 3 enzymes of the choroid plexus (alkaline phosphatase, succinic dehydrogenase, and carbonic anhydrase) is unaltered.

A reagent with activity resembling that of blood thromboplastin was sedimented on crude cephalin. Activity was lost if washing of sediment was undertaken in absence of calcium ion. The thromboplastic reagent normally clotted plasmas deficient in Stuart factor or factor V; activity was irreversibly destroyed following incubation with serum. Certain properties similar to those of tissue thromboplastin are indicated.

Treatment of rabbits with DE rabies vaccine causes them to withstand otherwise fatal doses of street virus administered intracerebrally. These results with 1 year old DE vaccine parallel Semple's original experience with freshy prepared phenolyzed vaccine. Furthermore, human serum following treatment with these vaccines neutralizes different fixed viruses and also street virus as tested in white mice.

A deficiency of biotin, superimposed over a simple PGA deficiency in rats resulted in further reductions in liver levels of PGA and CF, blood hemoglobin and, additionally, blood erythrocyte count. Administration of biotin to doubly-deficient animals caused almost complete restoration of liver levels of PGA and CF and of blood hemoglobin; under these conditions there was marked excretion of PGA in feces, but not in urine, the amount excreted exceeding by far the tissue rise in level of vitamin. It is suggested that biotin administration may influence synthesis of PGA by intestinal microflora and, as a consequence, improve tissue levels of the vitamin.

An enzyme preparation from mammalian pancreas catalyzes the synthesis of creatine from guanidinoacetate and S-adenosylmethionine. Inasmuch as pancreas, in contrast to liver, can also synthesize the creatine precursor, guanidinoacetate, from arginine plus glycine, it would appear that pancreas may play a unique role in biosynthesis of body creatine.

Hydrocortisone, given to anuric dogs, prolonged survival time with lower rates of accumulation for urea, creatinine and phosphorus in the serum. Testosterone in a second group of animals failed to influence survival time or urea accumulation.

1. Intraperitoneal injection of hydroxylamine resulted in elevation of levels of γ-aminobutyric acid in 8 brain areas of the rat. 2. These elevated levels of γ-aminobutyric acid were attained even after rats were pretreated with methylene blue to reduce methemoglobin formation. 3. The effect of a single injection of hydroxylamine persisted for at least 5 hours. 4. The potential value of this procedure for physiological study has been discussed.

Hydroxylamine when administered to immobilized cats in acute experiments in doses of 10-20 mg/kg markedly reduced duration and spread of electrically-induced afterdischarges while increasing cerebral levels of γ-aminobutyric acid. These findings increase the probability that a causal relationship may exist between metabolism of γ-aminobutyric acid in a particular cerebral area and its excitability.

(1) Hesperidin, hesperetin, naringenin, morin and luteolin were fed to young rats to determine their ability to induce thymus involution. Only luteolin was effective, and in this respect resembles quercetin, dihydroquercetin, and eriodictyol. (2) A commercially available preparation, “Calcium Flavonate Glycoside, Lemon,” caused thymus involution. Some relationships between structure and thymolytic action of flavonoids are discussed.

Glucose-6-phosphatase has been demonstrated in pancreatic B cells of rabbit. guinea pig and dog. and in canine ductular epithelium. Short fixation of fresh frozen cryostat sections in cold neutral 6% calcium formol does not inhibit its activity or alter its distribution in liver or pancreas. Furthermore, specificity of histochemical reaction for glucose-6-phosphatase was demonstrated by differences in staining when other substrates were incubated at same pH for equal time. In view of known relationship of blood sugar concentration to rate of insulin secretion, a hypothesis is advanced relating presence of this enzyme in B cells to control of insulin secretion. It is suggested that net rate of glucose-6-phosphate formation is the controlling factor and that this would be influenced by any increase or decrease of glucose-6-phosphatase activity.

(1) The plasma disappearance and soft tissue uptake of a single i.v. dose of Mg²⁸ was studied in normal rats over a 2 hour period. These results were compared to identical studies in rats given large doses of cortisol and rats made adrenal insufficient. (2) No deviation from normal could be detected in cortisol treated or adrenal insufficient rats.

Typical pancreatic changes were induced by ethionine, which were not altered by addition of cortisone. The high incidence of gastric ulceration and hemorrhage found with ethionine alone was doubled in rats receiving cortisone also.

The influence of phenol concentration, time, and pH on phenolic inactivation of poliovirus at 0° was studied. A minimal phenol concentration of about 0.5 M was required for removal of all the original ribonuclease-stable poliovirus infectivity. This minimal requirement was influenced slightly by pH; it was lower at pH 4.0 than at pH 6.2 and lower at pH 6.2 than pH 7.4. Amount of inactivation was independent of time of exposure to phenol over range of 0.5 to 240 minutes. Poliovirus populations studied were heterogeneous with respect to phenol-stability, some particles requiring higher phenol concentrations than others for inactivation. Under conditions giving phenolic inactivation of all ribonuclease-stable infectivity, the ribonuclease-labile infectivity was present 0.5 minute after start of reaction and its titer remained essentially constant for at least 4 hours.

Nialamide was a potent monoamine oxidase inhibitor by both in vitro and in vivo test. Its potency relative to that of iproniazid varies from 3:1 to 12:1 depending on test and species used. Nialamide was also shown to be qualitatively different from other MAO inhibitors in some of its pharmacological actions, factors which may explain absence of both liver toxicity in dog and postural hypotension in man.

Left-to-right shunts were constructed in 8 mongrel dogs by anastomosis of left subclavian artery to left pulmonary artery. When solutions of krypton⁸⁵ were injected into the root of the aorta proximal to a functioning shunt, the krypton gas could be detected in the airway by a Geiger-Müller tube in less than 5 seconds. When similar injections were made in the absence of a shunt, the appearance time was usually 10 seconds or more. Thus, by monitoring expired gas it is possible to detect the presence of a shunt resulting in early appearance of Kr⁸⁵ in pulmonary arterial blood following injection into the left side of heart or the aorta.

1) Positive binding of fluorescent antimyosin solution to myofibrils of the Purkinje cell demonstrates presence of myosin in myofibrils of the conduction bundle of beef heart. 2) Antibody to myosin whether prepared from skeletal muscle or heart tissue is capable of binding heart muscle antigen.

Fluid production by fetal lung of guinea pigs and goats has been demonstrated through cannulation of the fetal trachea in the last part of the pregnancy. This fluid is probably produced by an ultrafiltration process because its physico-chemical features in the last third of pregnancy are very similar to those of the interstitial fluid. It is suggested that the fetal lung is an important source of amniotic fluid during this period of fetal life.

1. Aggregated human gamma globulin, obtained by heating, gave immediate skin reactions in normal guinea pigs and inactivated complement, whereas aggregated bovine gamma globulin did not have either activity. 2. Aggregated human gamma globulin and antigen-antibody complexes are comparable on weight basis with respect to skin reactivity and complement inactivation. 3. Divalent cation(s) are required for inactivation of complement by aggregated HGG. Inactivation of C'3 by aggregated HGG is markedly dependent upon temperature. 4. A tentative hypothesis concerning the mechanism of complement fixation by antigen-antibody reaction is presented.

Effect of salt replacement on secretion of adrenocortical hormones by rats which had been deprived of sodium for 1 month was investigated. Results demonstrated that total cortical hormone production, which had been depressed, increased to equal that of control animals; that elevated aldosterone synthesis declined to previous levels; and that cortisterone secretion, which had been undetectable, increased rapidly. Thus, within 48 hours after resumption of salt feeding secretion of adrenocortical hormones returned to normal.

1. A tissue culture color test has been developed for titration of influenza virus and antibody using secondary monkey kidney cell cultures. 2. Virus titrations with the color test are comparable in accuracy with those done in roller tube cultures, mean titer being 0.56 logs lower in the color test. 3. Antibody content of immune sera (animal or human) can readily be determined and slopes of neutralization curves determined by color test are comparable to those found in ovo. 4. Color test neutralization titers and HI titers of the same sera show a significant association.

1. Oxidation of linoleic and linolenic acids, both randomly labeled with C¹⁴, to CO₂ was compared in a) fasted and glucose-fed normal rats; b) normal and diabetic fed rats; and c) insulin-treated and untreated, fed diabetic rats. 2. Administration of glucose spared oxidation of both linoleic and linolenic acids. 3. Oxidation of linoleic acid by ad lib.-fed diabetic rat exceeded that by similarly fed normal rat. 4. Insulin spared oxidation of polyunsaturated fatty acids in the diabetic rat.

1. Incorporation of S³⁵ and C¹⁴ from injected doubly labeled rat serum albumin or globulin into the tissue proteins of rats was investigated. The ratio S³⁵/C¹⁴ in tissue proteins varies very little during 9 days after injection of the doubly labeled serum proteins. 2. The ratio S³⁵/C¹⁴ in tisues is approximately equal to (S³⁵ _P/C¹⁴ _P) X (S_T/S_P) × Q_c where the first term is the ratio of the 2 isotopes in the injected plasma protein, S_T and S_P the per cent of total sulfur in the analyzed tissue protein and the injected plasma protein, respectively, and Q_c is approximately 1.5. 3. The increase in S³⁵/C¹⁴ ratio (Q_c > 1) indicates impoverishment of C¹⁴ relative to S³⁵ and is attributed to utilization of C¹⁴-amino acids for other metabolic pathways. 4. In rats injected with serum albumin-S³⁵, I¹³¹ the ratio S³⁵/I¹³¹ increases during 9 days from 50 to 100 times. 5. Our results indicate that conversion of plasma proteins into tissue proteins involves breakdown to amino acids or very small peptide fragments. There is no indication for the transfer of large peptide fragments from plasma protein to tissue protein molecules.

1. Prolonged and sustained elevated phenylalanine plasma values have been observed in monkeys fed 3 g of L-phenylalanine 3 times daily. 2. Phenylpyruvic acid was excreted in the urine of these animals. 3. The biochemical findings are comparable to those found in phenylketonuria.

Ribonuclease-treated homogenates of liver that do not incorporate mevalonic acid into non-saponifiable material or cholesterol were devoid of ATP. ATP was present and mevalonic acid incorporation occurred when ribonuclease pretreatment was carried out in presence of an autoclaved extract of liver. The results are interpreted as indicating existence of a polynucleotide factor essential for maintenance of a positive balance with respect to ATP in liver homogenates.