Experimental Biology and Medicine

To determine whether the dilute locus was associated with a greater behavioral need for pyridoxine and to ascertain the effects of this locus per se on audiogenic seizure severity in mice, the dilute gene was varied over several backgrounds. C57Bl/6J, DBA/2J, first and second filial generations and backcrosses were made sensitive to audiogenic seizures by acoustic priming at age 21 days, placed on regimens of 0, 15, or 30 mg pyridoxine HCl/kg diet for 14 days, and then tested for audiogenic seizures. It was concluded that: 1) lack of pyridoxine enhanced audiogenic seizure severity; 2) the dilute locus per se was not associated with a difference of audiogenic seizure severity; 3) the dilute gene was not associated with a greater behavioral need for pyridoxine, as measured by audiogenic seizures; and 4) the DBA/2J was recessive to the C57Bl/6J for audiogenic seizure severity under the conditions of this experiment.

Under laboratory conditions the percentage of ovulations in rabbits may be increased significantly (p < 0.01) by an intravenous injection of human chorionic gonadotrophin in addition to the natural stimulus of copulation. In particular this treatment is useful for does of poor reproductive performance. It is suggested that such procedures only be used for short-term experimental problems, since the effects of repeated usage of HCG are as yet unknown. The data show that failure to ovulate following copulation is a major factor in rabbit infertility.

The norepinephrine and epinephrine content of ventricular cardiac muscle and spleen were analyzed in 2 groups of anesthetized white rats. One group served as controls, while the other group was subjected to burn shock induced by immersing the lower 1/2 of the body for 90 sec in water heated to 90°C. Myocardial norepinephrine and epinephrine levels of control rats averaged 0.74 ± 0.15 and 0.07 ± 0.06 μg/gm, respectively. In burn shock, norepinephrine decreased markedly to 0.26 ± 0.20 μg/gm (p <.003), while epinephrine rose to 0.24 ± 0.07 μg/gm (p<.003). On the other hand, splenic norepinephrine rose from 0.55 ± 0.15 to 1.02 ± 0.25 μg/gm (p<.05), while epinephrine content declined from 0.36 ± 0.12 to 0.09 ± 0.06 μg/gm (p<.003) in burn shock. Excessive sympathetic nervous system stimulation, or the formation of toxic substances may be involved in reducing norepinephrine stores of the myocardium in burn shock. Unlike the depletion of splenic norepinephrine in hemorrhagic shock, the norepinephrine content of the spleen rose. This finding together with postmortem observations of a soft and relaxed spleen would suggest a different role than that observed for this organ in hemorrhagic shock.

In a previous study it was reported that in the chronic cardiac denervated dog the natriuretic and diuretic responses to acute isotonic, iso-oncotic intravascular volume expansion are significantly attenuated (1). Subsequently it was reported by Knox and associates (2) that the natriuretic response to an acute isotonic sodium chloride load is also attenuated in the same preparation. These studies suggest that cardiac nerves influence both sodium and water excretion. The present report describes experiments which were carried out on three dogs at least 3 years after surgical denervation of the heart to determine if reinnervation is associated with a return of the normal diuretic and natriuretic response to intravascular expansion.

Methods. The methods employed in these experiments are essentially those reported previously (1). All experiments were carried out under pentobarbital sodium anesthesia (30 mg/kg). A small branch of the femoral artery was cannulated for blood pressure recording and for obtaining blood samples. Drugs were administered through a catheter introduced through a needle into a foreleg vein. Urine samples were obtained through a catheter inserted into the bladder. Following these preparations urine was collected in graduated cylinders until at least 2 ml of urine were obtained. The bladder was then flushed with water and air. A blood sample was obtained between two such collection periods that showed similar urine flows. Dextran (6% clinical dextran and isotonic saline) was then infused in an amount equal to 3% body weight at a rate of approximately 30 ml/min. When urine flow achieved a new steady level, blood and urine samples were again obtained and the experiment continued until urine volume declined. At the end of the experiment the bladder was flushed with Furacin solution, antibiotics were administered, catheters were removed and the animals were returned to the kennels for recovery.

DMSO was found to inhibit DNA synthesis in S-180 ascites tumor cells in vitro. This inhibition was a linear function of DMSO concentration from 5 to 15% (v/v). Removal of the DMSO resulted in an immediate restoration of the capacity for TdR-³H incorporation.

Synthesis of DNA in human kidney cell cultures was prevented by X-irradiation. Adenovirus type 12 replicated in irradiated cells as well as unirradiated cells. Virus production was preceded and accompanied by marked increase in cells showing DNA synthesis in the nucleus as monitored by autoradiography, which may be virus and/ or virus-stimulated cell DNA.

The h and m antigenic components of histoplasmin previously described by Heiner have been separated by anion-exchange column chromatography and fraction collection using a phosphate buffer system. The separation of the two antigens was demonstrated by the agar gel, double-diffusion precipitin method. The m antigen was found in earlier fractions at higher pH ranges, and the h was found in later fractions at lower pH ranges. A distinct separation occurred with no detectable overlap. The use of specific antigens should give more reliable experimental and diagnostic results.

Six out of 10 local anesthetics inhibited clot retraction, platelet aggregation, and ATPase activity; and produced platelet potassium loss. Another anesthetic, dibucaine, inhibited clot retraction but did not inhibit platelet aggregation. Thus these two phenomena were separable. Dibucaine, however, produced platelet potassium loss and inhibited ATPase activity. These studies have shown that platelet aggregation may be dissociated from clot retraction, changes in platelet permeability to potassium and ATPase activity.

Mitochondria were isolated from the pectoralis major muscle of normal and genetically dystrophic New Hampshire cockerels. The mitochondria were incubated in Tris-buffered KCl solution and the rate of efflux of soluble protein was measured and compared with the amount of protein released from mitochondria of the same isolate by Triton X-100. The efflux of protein from the mitochondria of dystrophic muscle was significantly greater than that from normal muscle.

Cystaselenonine, the selenium analog of cystathionine, causes a hair loss in mice that is limited to the older growing hairs which have aged 16 days after mechanical epilation. The hair does not regrow for 20 days if the mice are treated with cystaselenonine for 12 days. The mice lose weight during treatment, but eventually recover. Cystaselenonine may act by interfering with sulfhydryl metabolism in the keratogenous zone of the hair follicle and not by interfering with mitosis.

The fatty acid compostions of the sterol ester and phospholipid fractions of mammary carcinomas and a derived, transplantable sarcoma were determined; the composition of the sarcoma differed from that of the carcinoma. Hormonal modification (ovariectomy plus 50 μg estradiol-17β with or without progesterone) of hosts bearing mammary carcinomas did not per se result in changes in the fatty acid composition of the phospholipid fraction of the carcinomas, except in those carcinomas which became laden with fat. The phospholipid and sterol ester fractions of fat-laden carcinomas had increased proportions of the shorter chain (C₁₀-₁₄) fatty acids and decreased proportions of polyunsaturated fatty acids but differences were not as great as in the triglycerides.

The AAV partially inhibits oncogenesis of adenovirus 12 when the two are inoculated together. Sixty percent of newborn hamsters inoculated with adenovirus 12 alone developed subcutaneous undifferentiated malignant tumors. Only 19% of animals inoculated with identical amounts of adenovirus 12 with AAV developed tumors.

The synthesis and secretion of progestins from cholesterol-7_a-³H and acetate-1-¹⁴C has been investigated in the rat with luteinized ovaries by retrograde infusion of precursors into vena cava. In this system “solubilized” plasma cholesterol-7_a-³H but not acetate-1-¹⁴C derived from the circulation was a major immediate precursor which could easily enter and label the “available” cholesterol pool(s) for the biosynthesis of progestins of the rat luteinized ovary. The LH and the NADPH system increased the mass and tritium activity of progesterone converted from cholesterol-7_a-³H, whereas progesterone-¹⁴C transformed from acetate-1-¹⁴C was not increased. The higher specific activities of secreted progesterone-³H derived from cholesterol-7_a-³H was found after the addition of the NADPH when compared with LH stimulated and control groups.

The rate of Ca uptake was studied in grana isolated from skeletal muscle of normal rats and rats with myotonia induced by 20,25-diazacholesterol. No differences were found. It was concluded that the slow relaxation phase of contraction in skeletal muscle seen in this drug induced myotonia can not be explained by a slow uptake of calcium by the sarcoplasmic reticulum in vivo.

A monovalent type 5 soluble antigen vaccine was prepared by methanol concentration, alkaline treatment, and brushite purification from virus propagated in WI-38 (diploid) cells. The vaccine was water-clear, contained minimal protein, and evoked high levels of neutralizing antibody in guinea pigs, suggesting a high degree of purity and effectiveness. There is no U. S. standard of potency with which to compare the results obtained, but indications are that the results are comparable, if not superior, to those reported for killed adenovirus vaccines types 3, 4, and 7. The procedures described show promise of meeting the present urgent requirements for the preparation of safe, effective adenovirus vaccines for the prevention of acute upper respiratory diseases in children and selected adult populations.

Concerning the cross reactivity of anti-ECHO virus 1 immune serum with ECHO virus 12, previous publications (1,2) as well as our data on the domestic reference serum prepared in monkeys showed no evidence for the cross neutralization. However, Berg et al. (4) reported that these two serotypes crossed each other in rabbit immune sera with very low heterologous titers. They described that absorption of these sera with packed monkey kidney cells did not significantly reduce the neutralizing capacity of ECHO virus 1 antiserum against ECHO virus 12, but the efficiency of absorption of anti monkey kidney cell antibody, as described by Habel et al. (6), was not specified.

The present data are considered to substantiate that one way cross neutralization between ECHO virus types 1 and 12 so far reported had been due to the contamination of the antigen used to immunize animals. It is thus concluded that ECHO virus types 1 and 12 are unrelated serologically.

The tensile strength of a healing surgical incision in the integument of the zinc-deficient rat has been found to be significantly decreased 12 days after operation, compared to pair fed control rats.

Dihydrosphingosine-4,5-³H, injected intracranially into nursing rats, was converted to sphingosine-³H, utilized for the synthesis of sphingolipids and dihydrosphingolipids, and catabolized with the incorporation of isotope into cholesterol and fatty acids from phospholipids and sphingolipids. Pathways of long-chain base metabolism are discussed.

Two species of Muridae neonata are more resistant to the lung edema produced by inhalation of nitrogen dioxide and to the lung edema produced by intraperitoneal injections of thiourea than comparable weight-adjusted adults. These results are in direct contrast to both original data and data from the literature on the ordinarily increased sensitivity of neonata to most drugs and toxic agents. The normal lung-body weight percentage in rats decreases from 1.7% at birth to 0.5% at 90 days.

In rats starved for periods up to 10 days or fed a protein-free diet for periods up to 11 weeks, the number of cells per crypt of the small intestine declined steadily. During this time, however, with the exception of the rats kept on a protein-free diet for 11 weeks, the number of cells per crypt synthesizing DNA and dividing remained the same as in the control crypts, thus resulting in an increase in the labeling and mitotic indices in the experimental animals. The hypoplasia in the crypts, despite the fact that there were a constant number of cells per crypt undergoing proliferative activity was due to the inhibitory effect of the dietary regimes on the generation cycle time. The synthetic and the mitotic phases of the cycle are inhibited to the greatest extent.

Long-term observations up to 24 months in guinea pigs and 39 months in monkeys given multiple doses of emulsified preparations of adjuvant 65 or their aqueous counterparts were carried out. Extensive gross and microscopic pathologic observations of the major organs and tissues were made. Aside from local reactions at the injection site, there was no evidence for immunopathologic lesions, disseminated granulomatous lesions, tumor formation, or other pathologic processes referable to the vaccine. The local reactions observed were those to be expected following the injection of nonirritating and metabolizable fatty materials, viz., lipoid granulomata with gradual disappearance of oil cysts, residual collection of phagocytic cells, and final disappearance of reactions after some months. There was no evidence that adjuvant 65 would be likely to cause abscess or nodule formation. A test for teratogenicity in rabbits given adjuvant 65 emulsified preparations, or their aqueous counterparts on the eighth day of pregnancy revealed no apparent effects on fetal development.

Interferon production and antiviral activity by L cells treated with double-stranded synthetic polynucleotide complex I:C is increased up to one hundred times by addition of DEAE-dextran. Highest interferon production (300 units) was observed when I:C (10 μg/ml) and DEAE-dx (200-400 μg/ml) were held in contact with the cells for a 12-hour period. However increased amounts of interferon were also produced when the inducer and DEAE-dx were removed 2 hours after their addition and the cultures were reincubated for 10 additional hours. The inhibitor produced was shown to have properties consistent with those of interferon.

Administration of a single large sublethal dose of CCl₄ in the rabbit results in severe centrilobular hepatic necrosis and acute derangement of liver function, followed by rapid healing. At the height of the lesion (48 hours) animals are approximately 120 times more susceptible than normal animals to the lethal effect of bacterial endotoxin, whereas animals tested 7 days after CCl₄ are only 4 times as susceptible as normals. Measurement of the 5-hour fever index following small doses of endotoxin (10^-4 to 10^-5 LD₅₀) reveals that animals tested 48 hours after CCl₄ are approximately 40 times more susceptible than normal rabbits to the pyrogenic action of endotoxin. Animals tested 5 days after CCl₄ show only a normal febrile response. Normal rabbit liver is capable of detoxifying endotoxin in vitro, whereas liver from CCl₄-treated rabbits is not. Results of these studies indicate that acute CCl₄-induced liver injury in the rabbit is associated with enhanced susceptibility to both lethal and pyrogenic effects of bacterial endotoxin.

Serum creatine phosphokinase (SCPK) activity was determined in mongrel dogs before and after 45-60 min of treadmill exercise (268 m/min; 10° grade). The ability of prednisolone to prevent the elevation of SCPK levels observed after exercise was investigated. This exercise load, in untreated animals, resulted in a large increase in SCPK activity. Serum creatine phosphokinase activity did not increase to the extent observed in the untreated dogs if prednisolone was administered. The cellular permeability change and/or necrosis occurring during exercise may be reduced by the action of prednisolone.

Comparative tests with 4,5-dimethyl- and 4,5,10-trimethylbenz(a)anthracine show that both compounds were highly carcinogenic for Fischer line 344 rats. Both compounds were more effective than the 3 most active previously reported monomethyl derivatives with methyl groups in the 4,5, and 10 position. Tumors induced by the trimethyl compound occurred in a significantly shorter average latent period than tumors induced by the dimethyl derivative and were probably more malignant.

The 76 subcutaneous sarcomas induced by the trimethyl compound were observed in an average latent period of 170 ± 5.9 days compared with 186 ± 4.1 days observed for the 77 similar tumors induced by the dimethyl compound. Metastatic dissemination was observed in 20% of the rats with tumors induced by the trimethyl compound compared with only 6% of the rats with tumors induced by the dimethyl compound.

Natural antibodies specific for Escherichia coli serotype 0126:B16:H were demonstrated in both the sera and secretions of normal volunteers by means of an indirect fluorescent antibody test (IFA). Each immunoglobulin class of bacterial specific antibody was identified by using fluorescein conjugated antisera for human γA, γG, and γM in the IFA test system. Both serum and concentrated saliva from normal donors contained bacterial specific antibodies representative of all three classes of immunoglobulin. The predominant bacterial specific antibody in the serum of seven donors was of the γG class. Concentrated saliva from five donors contained bacterial specific γA, γG and γM antibodies. The concentrated urine of five donors contained bacterial specific γA and γG antibodies, however, bacterial specific γM antibodies were detectable in only one urine specimen. Significant bacterial hemagglutination titers were obtained from all seven sera, three salivas, and one urine. Hemolysis titers paralleled hemagglutination titers in every case. Secretory γA from both saliva and urine was shown to be reactive with E. coli cells with specific antisera. E. coli cells sensitized with salivary and urinary γA preparations showed specific fluorescence when treated with antisera specific for either serum γA or secretory “piece.” However, cells sensitized with serum reacted only with γA specific antisera.

Phosphorylase activity was measured at intervals during the first 8 days of pregnancy in the ovarian interstitial tissue of New Zealand white rabbits. Levels of phosphorylase a remained constant during the period studied while a rapid transformation of phosphorylase b (37% at estrus) to phosphorylase a (95% phosphorylase a 1.5 hours after mating) was noted.

The oxidizing agent, iodoacetate, used to stabilize cysteine in blood and urine had an adverse effect on the absolute amounts of the S-containing amino acids in blood plasma, in both in vitro and in vivo studies. Methionine concentrations were decreased, while taurine, methionine sulfoxides, cystathionine, and cystine increased over those of the nontreated control samples. The use of the qualitative relationship among methionine and its catabolites, however, seemed to be valid for the in vivo investigation of the metabolism of these amino acids.

Erythrocytes of 63 persons were examined for the presence of copper. Copper-containing granules were demonstrated in all erythrocytes of 50 patients containing hemoglobin S in homozygous (sickle cell anemia) or heterozygous (sickle cell trait) forms. No copper was found in the erythrocytes of the 13 control subjects with normal hemaglobin (Hb^{1 A} Hb₁ ^A). Good correlation was obtained between the presence and amount of copper and the hemoglobin type.

An experimental model of anemia (decrease in venous hematocrit) in association with chronic inflammation has been described. It was readily induced in adult female C57B1/6 mice by the intraperitoneal injection of 25% allogeneic C3H or syngeneic C57B1/6 erythrocytes emulsified in complete Freund's adjuvant. Direct Coombs tests were negative but hemolysis was a significant factor. Increased plasma volume appeared to be a second important component. Splenomegaly, peritoneal inflammation and fibrosis, hyperplasia of the reticuloendothelial system, plasmocytosis, and miliary hepatic granulomata occurred when the adjuvant was intraperitoneally injected. The intensity of these reactions bore a close relationship to the anemia. Splenectomy prevented hemolysis and modified the anemia but it did not totally abolish the events producing the increased plasma volume and decreased venous hematocrit.

Injections of purified bovine LH (NIH-LH-B3) in rabbits produced circulating antibodies which were associated with degenerative changes in uterine, ovarian, and adenohypophyseal structures and affected mating behavior and ovulation. Final body weight; gain in body weight; adrenal, thyroid, and thymus weights; and histology were unaffected. Freund's complete adjuvant caused microscopic changes in the ovaries and adenohypophysis. Bovine LH and crude rabbit anterior pituitary extract contained a similar antigen which cross-reacted with anti-BLH serum. The cross-reacting antigen was not bovine gamma globulin or bovine serum albumin. Results were attributed to an autoimmune phenomenon with circulating antibodies common to NIH-LH-B3 and endogenous gonadotropin.

Lysozyme, a naturally occurring antimicrobial enzyme, has been shown to be present in the renal cortex in a concentration 7 times that of the renal medulla. Exogenous lysozyme given intraperitoneally accumulated in the kidney and raised substantially the concentration of lysozyme in renal cortex and medulla. Repeated intraperitoneal injections of lysozyme, however, did not influence the growth of Streptococcus fecalis in the renal cortex or medulla nor did administration of this enzyme enhance the formation of protoplasts in the hypertonic tissue fluid of the renal medulla.

The fact that the selenium content of tissues and the active transport function of the intestine are the same in the dystrophic and normal hamsters supports the conclusion that selenium is not involved in the disease. In the genetically conditioned muscular dystrophic hamster, intestinal absorption of selenium is not significantly different from that of the normal animal.

The effects of acute and chronically administered morphine on the threshold voltage required to elicit a response to tooth pulp stimulation in the dog and cat were quantitatively compared. A single dose of morphine elevated tooth pulp thresholds to a greater extent in dogs than in cats. After chronic administration, a decrease in the threshold occurred in cats but not in dogs. In the latter, a tolerance to (but not a reversal) of the threshold elevating effect was observed. Tolerance was seen to the overt depressant effects of morphine in dogs but not to the overt excitatory effects in cats. Studies concerned with attempts to delineate the reason (s) for these differences may be useful in advancing our knowledge concerning the mechanisms responsible for the analgesic effect of morphine and the development of tolerance to this drug.

An apparatus has been devised to perfuse livers under conditions which simulate those existing in the body. It consists of two identical perfusion sets so the metabolism of two livers can be compared under the same conditions. The perfused liver continues to produce plasma protein for 10 hours. After this time there is no vacuolation or necrotic change in any part of the liver; only slight atrophic changes in hepatic cells are present. A replacement of the blood plasma of the perfusate by Ringer-Locke solution caused an increase in plasma protein production and a decrease in the synthesis of liver tissue protein. When PVP was added to Ringer-Locke solution the production of plasma protein decreased. In both perfused liver and in vivo globulin appears to have a higher rate of synthesis than albumin. Gamma-globulin shows a faster turnover rate than albumin and alpha-globulin shows a still faster rate.

Cell lines and strains were studied with respect to possession of G-6-PD isoenzymes, using cellulose acetate strips and sucrose-agar gel electrophoretic media. The latter provided higher resolution of A and B bands. The A and B types of G-6-PD were found to occur in human, diploid cultured cell populations in accordance with racial origin of the donor; while certain other heteroploid cell lines were found to possess A type G-6-PD irrespective of donor race.

The penicillin G- and nafcillin-mediated lysis of growing cells of S. aureus by lysozyme and trypsin was used as a test to determine the influence of sublethal concentrations of other antibiotics on this response. Seventeen antibiotics were tested with two strains of S. aureus (G50 and CHP). Rifamycin, novobiocin, tetracycline HCl, actinomycin D, erythromycin, and streptomycin interfered with or blocked lysis whereas methicillin, cephalothin, cloxacillin, dicloxacillin, cycloserine, and vancomycin either enhanced it or had no effect depending on the concentrations of these antibiotics. Rifamycin, chloramphenicol, and novobiocin were found to be the most active antagonists of penicillin G and nafcillin. Under the experimental conditions, none of the bactericidal antibiotics acted synergistically with either penicillin G or nafcillin. The results suggest that the change in lytic activity when certain antibiotics are combined with penicillin depends on some specific biologic activity against S. aureus by which these antibiotics directly or indirectly reduce the ability of the penicillin to block cell wall synthesis.

The present studies were on the concentrations of progesterone and 20-α-hydroxypregn-4-en-3-one (20-α-OHP) in ovarian vein blood of androgen-sterilized rats prior to and following ovulation induced by exogenous HCG or LH. Neither steroid was present prior to ovulation and following HCG treatment the dominant steroid secreted was 20-α-OHP. Reserpine or prolactin (i.v. and s.c.) administration resulted in secretion of progesterone in high concentrations whereas the hydroxy steroid was essentially undetectable.

The level of plasma erythropoietin (ESF) from untreated baboon renal allotransplants, compatibly or incompatibly matched in terms of their major human ABO blood groups, was assayed in polycythemic mice. Postoperative rises in ESF occur within 7 days if graft thrombosis ensues. Other grafts exhibit progressive elevations in ESF activity, which correlate directly with the reduction in measured renal blood flow due to renal rejection. Azotemia does not evidently influence this response in this species. Compatibly matched animals demonstrate continued ESF responses despite restoration of peripheral hematocrit values to preoperative ranges.

Male rats were subjected to stress by graded reduction of available food or an environmental temperature of 4°C with food ad libitum. The stressed animals exhibited increased adrenal and thyroid weights and atrophy of the thymus. Isometric tail tendon contraction carried out in 40% KI solution at room temperature showed significant increases, indicative of aging, as food was reduced 60-80% of control consumption. However, the environmental stress of 4°C (without food restriction) did not significantly change the isometric tension of the tail tendon.

The oral administration of digestible lipids was followed by prolonged depression of reticuloendothelial system phagocytosis as measured by blood clearance of colloidal carbon. In contrast to the effects of intravenously administered lipids oral trioleate was as effective a depressant as ethyl stearate.

Insulinase activity was determined by quantitating the degradation of insulin ¹³¹I substrate by rat liver homogenates. Plasma insulin was determined by a two-antibody radioimmunoassy procedure. In rats fasted for 1-4 days, insulin levels were 30% at 1 day and 10% at 4 days. Insulinase activity decreased less rapidly, being 80% at 1 day, 60% at 2 days, 35% at 3 days, and 25% at 4 days. On refeeding, insulin levels rose to normal promptly; whereas, insulinase activity did not return to normal until the third day. Refed animals treated with actinomycin D had only 48% of normal insulinase activity after 3 days of refeeding. Actinomycin D also inhibited the renewal of insulinase activity in insulin treated alloxan diabetic rats. These results are compatible with the hypothesis that insulin is an inducer of insulinase biosynthesis.

The NE and E content of the SA node region and right atrium of the dog was determined fluorometrically. The NE concentration of the SA node region was found to be significantly greater than in the right atrium. No difference in the epinephrine content could be demonstrated between the two regions.

The possible toxic effects of a condensed tannin isolated from sericea, which has pectinase and cellulase inhibitor properties, was investigated by feeding this polymeric proanthocyanidin to rats for as long as 150 days at a dietary level of 2%. No effects were observed on growth, feed intake, hematology, reproductive performance, metabolic fate, gross pathology, organ weights, or histopathology. Also included is a summary of the results of feeding a phlobatannin isolated from grape pomace to rats continuously for 700 days. When compared to control rats, there were no toxic effects produced that could be attributed to the ingestion of the isolated sericea tannin or the grape phlobatannin.

A reproducible and simple chromatographic method with DEAE-Sephadex was used for fractionation of GOT isoenzymes. In the sera of health individuals three distinct isoenzymes were found. In patients with liver diseases, the SGOT isoenzyme distribution was distorted. Two isoenzymes, (A₁ and B), were substantially increased, whereas the third one (A₂) remained the same. More striking changes were noticed in acute viral hepatitis. Isoenzyme A₂ was not detectable, and the isoenzyme pattern was similar to that in liver homogenates. A possible explanation of this altered isoenzyme distribution was discussed. The change of isoenzymes in the sera reflect more precisely the extent of the hepatocellular damage, rather than the total GOT activity.

A highly sensitive and specific radioimmunoassay for rat LH has been developed utilizing antiovine LH serum and ovine LH for iodination. Estimates of the LH potency of pituitary preparations with widely varying LH to FSH and LH to TSH potencies obtained with this assay agree with estimates obtained with bioassay and with those obtained with a radioimmunoassay utilizing antirat LH serum and rat LH for iodination. With this technique it has been possible to measure the LH in serum obtained from females throughout the estrus cycle and in serum from normal and castrated males.

Intraperitoneal injection of a navy bean fraction (Fraction 4) into young rats reduced the incorporation of leucine-¹⁴C into protein of skeletal muscle; increased the incorporation into liver protein; and had no effect on the level of incorporation into tissue protein of the small intestine. Decreased protein synthesis in skeletal muscle appears to be a specific phenomenon related to a toxic factor present in Fraction 4.

Sodium octyl, decyl, and dodecyl sulfate, in the concentrations used enhance the hypoglycemic effect of insulin in fasted and glucose-loaded rats. The molar relationships among these surface active agents indicate that higher concentrations of the shorter alkyl chain substances are required to produce the hypoglycemic effect. The adherence or penetration of insulin into rat liver cells has been measured by the hypoglycemic response of rats to injections of insulin-treated cells or the wash fluids of these cells. Detergents do not alter the adherence of the insulin to cells, but do decrease the amount of insulin present in the supernatant. This decrease is attributed to the penetration of insulin into the cells in the presence of detergent.

Oxygen consumption and aerobic and anaerobic lactate production of testes from prepuberal and adult rats in the presence and in the absence of different substrates were studied. Oxygen consumption was greater at the prepuberal age. Aerobic lactate production was similar in both groups but anaerobic lactate production was greater in prepuberal animals. Only the testes from adult animals were able to oxidize exogenous substrates (glucose, pyruvate, or glutamate). The Pasteur effect was observed in the adult gland whereas the Crabtree effect appeared only in immature testes.

In eight studies, the plasma renin activity (PRA) was determined in blood drawn consecutively from the hepatic vein, a peripheral vessel, and renal veins. The renal venous PRA was consistently higher than the arterial PRA, indicating renal secretion of renin. In seven of the eight studies, hepatic venous PRA was, on the average, 26% lower than the PRA of blood entering the liver indicating hepatic extraction of circulating renin. That the elevated PRA seen in cirrhosis of the liver may in part be related to impared hepatic inactivation of renin is an hypothesis that merits further study.

It is concluded that reaction products obtained by acetaldehyde treatment of certain biologically active amines manifest cerebrocortical EEG stimulant activity significantly greater than the parent compounds. We postulate that such products may indeed be formed endogenously, perhaps especially in pathologic conditions, to the extent of producing enhanced CNS stimulation. This further supports the hypothesis that prolonged CNS hyperstimulation induced by biochemical errors, may be etiological in some schizophrenic states.

Reserpine-induced hypothermia occurs in the presence of shivering, piloerection, and hunched posture in contrast to the absence of these defenses following CPZ. Serum FFA and glucose levels remain normal in the reserpinized animal down to a body temperature of about 25°C below which they fall markedly. The CPZ induced hypothermia is associated with increases in serum FFA and glucose concentration proportional to the degree of hypothermia. Corticosterone levels are elevated with CPZ-induced hypothermia, but this increase is blocked by reserpine pretreatment. Serum insulin is unaffected by CPZ hypothermia, but the reserpinized animals have insulin levels elevated at 22°C and depressed at 3°C ambient temperature. In essence CPZ-induced hypothermia is associated with effective mobilization, but decreased utilization, of chlorigenic substrates. On the other hand, reserpine-induced hypothermia is associated with decreased mobilization of these substrates and their eventual depletion resulting from effective utilization.

Carbohydrate protein complexes of costal cartilage of rats, aged 3-4, 6, 10-13, and 20-27 months, were extracted with water and precipitated with EtOH in the presence of potassium acetate. The PPL was extracted with 0.15 M NaCl and separated into CMP and GP by the addition of (NH₄)₂SO₄ to 55% saturation. Ultracentrifugal analysis revealed that the CMP fraction consisted of approximately 90% 1.4 S and 10% 3.0 S components and the GP fraction was made up of equal amounts of 2.8 S and 3.8 S components. The protein: polysaccharide ratios are approximately 1:3 and 1:0.5 for CMP and GP, respectively. With increasing age, there were highly significant decreases of protein, uronic acid, hexose, and hexosamine in the PPL fraction. After separation of CMP and GP, this change is seen only in the CMP fraction.

Thirteen, male and female sheep were electrically anesthesized (using a variable voltage-frequency, bitemporal electrode technique) during whole body exposure to 1 of 3 ambient temperature ranges, —10 to 5°C (“cold stressing”), 18 to 23°C (“thermoneutral”) and 27 to 29°C (“heat stressing”). Thermoregulatory mechanisms were examined at each temperature range, without drug treatment, during mild tranquilization (1.1 mg Propiopromazine HCl, i.v.), during 2 hours of electro-anesthesia and for a subsequent 3-hour recovery period. Except during “cold stressing” exposures, rectal temperature rose as hyperthermic polypneic reflexes were inhibited (15 breaths/°C change in rectal temperature) during electro-anesthesia but near-normal respiratory responses were reinstated after transcranial current flow ceased (120 breaths/°C change in rectal temperature) with rectal temperature returning to a normal range. Internal temperatures did not increase with electro-anesthesia during “cold stress.” A concept is proposed wherein it is suggested that the effect of electro-anesthesia is to inhibit partially normal thermoregulatory reflexes, resulting in transient heterothermia (similar to but more profound than chemical tranquilizer effects) with an additional shifting of a “thermoneutral zone” to a lower environmental temperature range due to increased somatic muscle tone.

Four consecutive daily doses of cortisone on days 11 through 14 of pregnancy were injected into several strains of mice and their hybrids and the incidence of cleft palate noted in the offspring. In Strong A mice the incidence of induced cleft palate was 100% compared to 9% of spontaneous cleft palate. In C3H mice, the incidence of induced cleft palate was 36% compared to less than 1% of spontaneous cleft palate. In the BALB/c the incidence of induced cleft palate was 62% and no cases of spontaneous cleft palate were noticed. The incidence varied greatly in the hybrids and backcrossings according to the genetic pattern of the fetuses. Of particular interest is the backcrossing of A × BALB/c females to A males with an incidence of 92% of cortisone-induced cleft palate, which may be useful as a pharmacogenetic tool in studying drug-induced orofacial malformations.

An experimental model system of virus-induced polycythemia in Ha/ICR Swiss mice is shown to represent an example of erythropoietin-independent erythropoiesis. This conclusion is based on the following findings: (i) No ESF activity was detected in the plasma or urine of mice at any time after virus infection, nor was there an increased level of REF, and (ii) multiple injections of antierythropoietin did not inhibit the re-initiation of erythropoiesis in plethorized, infected mice or the formation of macroscopic spleen foci in nonplethorized infected mice.

A bovine rhinovirus was isolated from an apparently normal calf in BEK cell cultures. The virus was found to contain RNA, was 25-27 mμ in diameter, acid labile, chloroform resistant, and was stabilized against thermal inactivation by molar MgCl_2. The virus grew slightly better in 33°C than at 37°C. The virus was serologically related to 2 German isolates. Antibody to the bovine rhinovirus isolate was highly prevalent in the cattle population of the State of Maryland.

Net inward calcium flux occurs across isolated short-circuited frog skin in the absence of a calcium concentration gradient. Calcium influx and calcium outflux are inversely related to the intermittent open-circuit skin PD but are not a function of short-circuit current. The changes in calcium flux in the hour after PTE administration suggest that PTE increases influx and decreases outflux. PTE and PTH do not alter short-circuit current or skin PD.

A series of experiments was conducted with Black Minorcas and partially inbred White Leghorn chicks of 1, 12, 14, and 16 days of age. Six-mercaptopurine and a platinum complex of this compound were administered orally to chicks of various ages at 612 mg/kg/day, 306 mg/kg/day, and equimolar dosage levels for various treatment periods. At equal dosage levels the capacity of the two compounds to induce leukopenia in the chick was essentially the same, however, the platinum complex was less toxic, as evidenced by greater weight gain, lower mortality, and LD₅₀ determinations. When the 6-MP complex was compared with 6-MP at twice the dosage level of the latter, and at equimolar dosage levels, results were similar to those obtained when equal dosage levels of the two compounds were evaluated.

Turkeys treated with diethylstilbestrol (DES) were fed various levels of iodinated casein (Protomone). Mortality was lowered and body weights were elevated by increasing amounts of Protomone in the feed of DES-treated poults. Systolic blood pressure and plasma composition of DES-treated poults fed a high level of Protomone, most nearly approximated the corresponding values for control birds, but did not attain the values for control poults. The histology of the thyroid glands of DES-treated turkeys was altered by the feeding of a high level of Protomone.

Complement binding antibodies were induced readily in rabbits by multiple injections of dextran or DEAE dextran. Antisera to DEAE dextran cross reacted with dextran, and preliminary evidence indicates that antisera to dextran also cross reacts with DEAE dextran and DEAE Sephadex. In contrast, complement binding antibodies to DEAE Sephadex developed very slowly. Low titers were observed only after multiple subcutaneous and intravenous doses of the antigen with complete Freund's adjuvant and in saline over a 3-month period.

Food deprivation was found to significantly impair the functional activity of the reticuloendothelial system as evaluated by the vascular clearance of the gelatinized “RE test lipid emulsion.” Depression was associated with an impairment in hepatic phagocytosis and an increased lung uptake. Opsonization of the gelatinized “RE test lipid emulsion” prior to injection completely eliminated the RE depression observed during the starved state. Determinations of the relative opsonic activities of sera from normal and starved rats suggested that the RE depression induced by starvation is mediated by a derangement in opsonic activity. Opsonin depression and subsequent impairment in RE phagocytosis due to a failure in the “recognition” mechanism may be the basis for the lower resistance to infection during profound malnutrition.

Skeletal muscle FFA contents are increased during starvation in the rat. An attempt has been made to determine the source of these FFA by incubating the intact rat diaphragm preparations of fed and 72-hour starved animals in palmitate-albumin solutions containing palmitate-1-¹⁴C and by assaying the composition of the plasma and tissue FFA and tissue TGFA of fed and starved animals. The 1-min uptake of label was minimally influenced by starvation and not at all by incubation in ouabain, Amytal or 100% nitrogen. Steady-state levels of label were minimally increased by starvation but the increases were insufficient to account for the enhanced tissue FFA levels. Tissue FFA composition differed from both plasma FFA and tissue TGFA. Starvation from 0 to 96 hours did not affect tissue FFA composition. These results are compatible with the following conclusions: The initial association between the albumin-FFA complex and the diaphragm is not affected by metabolic inhibitors; intracellular FFA do not approach the composition of either the plasma or muscle TGFA during progressive starvation suggesting selectivity in uptake or utilization or both, the selectivity of the processes responsible for tissue FFA size and composition is unaltered by starvation, and tissue FFA are increased during starvation primarily from intracellular sources.

The hemoglobin of 24 rabbits was analyzed by the “finger-printing” technique and amino acid analyses. The data revealed the presence of dimorphism in rabbit hemoglobin. The difference in composition and/or structure appears to be located in the β-chain.

The activites of the hepatic glycolytic enzymes, phosphorylase, phosphoglucomutase, and glucose-6-phosphatase were determined in normal and scorbutic guinea pigs administered acetoacetate. Scurvy caused an increase in the total available activity of glucose-6-phosphatase and a decrease in phosphorylase and phosphoglucomutase activities. Acetoacetate caused an increase in the activites of phosphorylase and glucose-6-phosphatase.

An examination of hexosemonophosphate shunt dehydrogenase activity in the rat exudate polymorphonuclear leukocyte reveals an exceedingly high NADPH generating capacity which is not modified by changes in composition of diet or alterations in thyroid status of the animal. This is in marked contrast to the hepatic shunt dehydrogenases which show marked alterations in response to the above stimuli. Thus, leukocyte HMPS dehydrogenase activity cannot be used as a peripheral index of hepatic shunt dehydrogenase activity.

The degree of ingestion of opsonized heterologous red cells by mouse peritoneal macrophages was greatly increased if the red cells were treated with neuraminidase before opsonization. An opsonin specific for the enzyme-treated red cells was shown to be present in normal mouse serum.

Thoracic duct lymph diversion decreased the gastric secretory response to feeding by 59%. When lymph was subsequently able to flow per via naturalis, the secretory response was restored to normal levels. Preliminary fractionation of lymph suggests that a secretagogue may be carried in the lymph, perhaps gastrin.

Following eradication of erythroid marrow by single injection of 5-FU, spontaneous repopulation began on day 6 and resulted in a sharp rise in ⁵⁹Fe incorporation on day 9/10. Mice which received a large dose of erythropoietin within 32 hours after 5-FU, repopulated their erythroid marrow 72 hours after 5-FU and showed an early peak in ⁵⁹Fe incorporation on day 6/7. Injections of erythropoietin earlier or later than between 8 and 32 hours after 5-FU had little or no effect, indicating that the induced erythropoiesis was not due to a carry-over of injected erythropoietin into the phase of spontaneous stem cell recovery. A delayed action on erythropoietin-sensitive stem cells present at the time of injection, or an action of erythropoietin on their precursors are considered as possible mechanism.

The growth of Sindbis, pseudorabies, and vaccinia viruses in primary chick embryo cultures was notably inhibited by 10^-2 M SHA but this is probably due to a nonspecific toxic effect. At 10^-3 M SHA selectively inhibited DNA synthesis in uninfected cells and impaired the growth of only the DNA viruses.

Diphenylhydantoin, 50 mg/kg, s.c., was administered to primigravid Swiss-Webster and A/J mice during early (days 7, 8, 9) and late (days 11, 12, 13) embryogenesis. Litters were delivered by caesarean section on day 18 of gestation, weighed, examined, and measured. Resorption rates were not altered in either strain treated during early embryogenesis. The A/J, but not Swiss-Webster, mice treated during late embryogenesis had a significant (p<0.05) increase in resorption. Diphenylhydantoin resulted in reduced fetal weight in both strains when given during late embryogenesis. Fetal long bone length, measured in alizarin red stained fetuses, was shortened only in late treated Swiss-Webster mice. The incidence of cleft palate was significantly elevated in late, but not early, treated progeny of both strains. Cleft palate with cleft lip occurred only in A/J mice; the rate was not different from vehicle treated controls. No other soft tissue malformations were found in either strain following Bouin's fixation and freehand cross-section examination.

The distribution of ceramic, glass, and carbonized microspheres within the gastric micorcirculation has been compared following simultaneous left ventricular injection. No significant differences between the extraction of the 3 types of microspheres by the vessels of the stomach were apparent. This similar distribution of the different microspheres within the stomach suggests that physical properties of the microspheres do not affect their distribution.

Soybean trypsin inhibitor (SBTI) can act to reduce early radiation lethality (<24 hours) in the 3-day chick as well as in the 14-day chick embryo. Plasma acid phosphatase activity was increased more than 2-fold in lethally irradiated chicks, but only small increases were demonstrated in groups that received near-lethal exposures or that were protected by SBTI before a lethal exposure.

The isolation of rat Kupffer cells was effected by the use of collagenase and trypsin digestion of liver tissue. The isolated cells demonstrated the ability to phagocytize, but required both serum and heparin. Metabolic studies demonstrated that the cells might possess a significant fatty acid metabolism and that the phagocytic process of the isolated Kupffer cell was both cyanide and iodoacetate sensitive. Microelectrophoretic studies suggested that the surface integrity of the isolated cells, as measured by surface charge, remained intact during the isolation procedure.

High titered adenovirus type 12 T antigen preparations were produced in LLC-RK-1 cells; this was achieved with little or no virus replication. The T antigens which cross-reacted with B group tumor antibody developed in the same cultures. Infection of the LLC-RK-1 cells with adenovirus type 7 and other representatives of group B failed to yield appreciable amounts of T antigen.