Experimental Biology and Medicine

Erythropoietin activity has been studied in eight intact and renoprival baboons. These studies demonstrate that renoprival baboons revealed elevations of plasma ESF activity in response to bleeding stimulus as intact baboons subjected to similar repeated bleeding stimuli. It appears that humoral regulation of erythropoiesis persists in anephric baboons.

Anesthetized dogs were hyperventilated via a respirator pump to increase blood pH to high levels. Canine erythrocytes hemolyze in vivo when blood pH is maintained at a high level (7.7 or higher) for 30 min or longer. Hemolysis occurs whether body temperature of the dog is rising or falling.

It was demonstrated that dietary amino acids appear in the mesenteric lymph and are transported from the intestinal lumen both as free amino acids and newly synthesized protein via the lymphatic system. The amino acid profiles in intestinal lymph are compared to those in plasma and the intestinal mucosa. There seems to be a depression of lymph amino acid levels during the early phase of intestinal absorption which returns to normal in the later phase. This depression occurs at the same time the newly synthesized protein appears in the lymph.

The effects of the protein antagonists cycloheximide and puromycin upon the transport of amino acids and newly synthesized protein into the mesenteric lymph of the intact rat was investigated. Upon feeding a fatty meal (bovine cream) fortified with leucine-¹⁴C these antagonists depressed the formation of lymph protein with a concurrent increase of amino acids, as measured chemically and by tracing radioactivity of these fractions. The influence of cycloheximide was more lasting than puromycin. These two antagonists affect protein synthesis differently as evidenced by the rates of new protein synthesis and the nature of the amino acids transported by the lymph leaving the upper small intestine.

Comparative viral susceptibility tests using human diploid lung fibroblasts (Led-130) and human lymphoblasts (RPMI 7466) have been made. Lymphoblasts 7466 were found resistant to the cytopathic effect of most viruses tested, but were capable of supporting persistent viral infections with many of these. Exceptions were herpes simplex and Coxsackie type A-15 viruses which were highly cytopathic for lymphoblasts 7466; viral replication in these instances was accompanied by total destruction of the culture. Concomitant virus replication and cell multiplication occurred in lymphoblasts 7466 persistently infected with poliovirus type 1 during 7 months of continuous culture. No recognizable cell destruction, reduction in growth rate, or interference to infection with two other viruses could be measured in these cultures. The mechanisms which operate to control and maintain persistent virus infections in cultures of human lymphoblasts has been studied and discussed.

Intravenous injection of insulin (2.0 U/kg) did not have any effect on the elevation of plasma FFA concentration produced by the injection of 20.0 μg/kg of glucagon in geese, ducks, and roosters. Intravenous injection of insulin (2.0 U/kg) in geese caused marked hypoglycemia, but no decrease of plasma FFA concentration.

Ethylenedinitramine (EDNA), a plant growth stimulator (1) and high impact explosive, ameliorates the symptoms of dietary and hereditary muscular dystrophy in laboratory animals (2). These beneficial effects have been correlated with enhanced muscle regeneration. In mechanically injured muscle, EDNA increases the regenerative yield most dramatically when its administration is timed to coincide with a certain critical period (3) before the new fibers appear. We speculated that su banes t he tic concentrations of ethylene, too, might increase muscle regeneration.

Materials and Methods. Male mice, Re 129 dydy, 3 months of age, were paired in two groups, littermates being represented in each group. One group was exposed to ambient air and the other to USP grade ethylene. The cage containing the latter was enclosed in a gas-proof Saran bag. Ethylene-air mixtures were delivered at 1.8 liters/min. Animals inhaled ethylene continuously for 7 days, three at a concentration of 5.7% and the remaining four at 0.2%. Ethylene concentrations were determined at the Ethylene Analysis facilities of The Dow Chemical Company. Upon termination of exposure, animals were sacrificed and tibialis anterior muscle was dissected out, fixed in formalin, and paraffin sectioned serially and tangential to the anterior surface of the muscle belly. Sections were mounted in even rows convenient for random sampling. Slides were stained with Ehrlich's hematoxylin and eosin.

Among several infections in mice, both lethal and nonlethal, it was found that an antiglobulin plaque neutralization test was superior to a standard plaque neutralization test for the detection of antibody. In most cases, the antiglobulin plaque neutralization test detected antibody sooner and to higher titer than the standard plaque neutralization test. In one of the nonlethal infections, the antiglobulin plaque neutralization test detected antibody concentrations ranging from 1:80 and 1:1280 where it was otherwise undetectable by a standard plaque neutralization test, complement-fixation test or hemagglutination-inhibition test. In certain hyperimmune sera there was sometimes little difference in the results of the two tests, but absorption of the antisera with viruses revealed significantly higher titers by the antiglobulin plaque neutralization test than the standard plaque neutralization test in the remaining antibody population in at least 2 of 4 sera examined. In addition, infectious virus-antibody complexes which remained after absorption of antibody could be neutralized by the addition of antimouse gamma globulin but not by control fluids.

In an attempt to identify the mycobacterial cell wall components which enhance resistance in mice to airborne infection with Mycobacterium tuberculosis H37Rv, fractions of BCG were prepared by organic solvent extraction and by alkali or lipase treatment. Although certain of these fractions, when tested individually, were impotent in our protection test, vaccines containing combinations of solvent-extracted, alkali-treated, or lipase-treated cell walls and of an inactive chloroform or methyl alcohol extract were significantly effective. The wax D fraction, but not the wax B or C fractions, of the chloroform extract in combination with “inactive” cell walls was highly protective. Since combinations of either the “inactive” BCG cell wall residue or the BCG chloroform extract with heterologous substances failed to enhance resistance of mice, two or more specific and distinct mycobacterial components may be required in an effective vaccine.

By the techniques of zygote transfer and allotype suppression, b⁵ homozygous rabbits were produced with an altered expression of immunoglobulin light chain allotypes; i.e., at 24 weeks of age, only 27% of the total γG was b5γG compared to 90% in nonsuppressed homozygotes. Of the 73% b-negative molecules, 44% were c7γG and 29% were both b- and c-negative. These results suggest that there may be at least three “loci” which control the synthesis of light chains.

Fatty infiltration of the liver following i.p. administration of partially-purified E. coli l-asparaginase was investigated using mice as experimental animals. Normal male C3H mice given varying dosage of low specific activity l-asparaginase over a 5-day period developed a marked hepatic lipidosis. Quantitative analyses and cytological studies confirmed this abnormal fat accumulation, in the form of triglycerides, compared with control animals given physiologic saline. By contrast, enzyme of high specific activity was not steatogenic to mice at the highest dose levels administered. Therefore, pure l-asparaginase per se does not appear to be steatogenic to mice. Quantitative analyses of liver tissues of a small group of l-asparaginase-treated leukemic patients showed marked hepatic lipidosis, regardless of amount or specific activity of enzyme administered. In fact, the severity of lipidosis seemed quite unrelated to the dose or purity of enzyme used. The limitations and possible implications of these findings are briefly discussed.

Human plasminogen was treated with either DFP, TLCK, or SBTI under conditions which completely inhibited the plasmin contaminant in our preparation. Each treated plasminogen preparation could be completely activated with low molar ratios of streptokinase to plasminogen in 24 hr, in 25% glycerol. Activation of DFP-treated plasminogen with either streptokinase or a DFP-treated equimolar human plasmin-streptokinase complex could also be carried out in 10-² M DFP, in 25% glycerol; however, much longer periods of time were required to obtain nearly complete activation. Under these conditions for activation, the plasmin contaminant in our human plasminogen preparation is therefore not essential for streptokinase-activation of the proenzyme. These data indicate that streptokinase, alone or in the complex, acts directly on human plasminogen. A new electrophoretic method for differentiating human plasminogen from human plasmin is described.

The effects of passively coating, with specific isoantibodies, erythrocyte isoantigens determined by the B blood-group locus in chickens on the immune response to A blood-group system isoantigens were investigated. Antibody synthesis to the B isoantigens was suppressed while the immunological response to A isoantigens, present on the same erythrocytes, was significantly enhanced. Since B isoantigens act as an adjuvant to A isoantigens, these results suggest that passive anti-B antibodies diminish competition between B and A antigens or, alternatively, enhance the adjuvant activity of the B antigens.

The preparation of a dog renal cell suspension suitable for the study of renin production is described. This suspension was utilized to study the in vitro effects of agents that have previously been shown to affect renin production in vivo. Since production and destruction of renin could not be separately quantified, data were limited to “net production” of renin. Incubated controls had only slightly higher renin content than did the nonincubated controls, but the addition of epinephrine, norepinephrine or cyclic AMP caused striking increases in “net production” of renin. The effects of these agents on “net renin production” were abolished by cycloheximide, an inhibitor of protein synthesis. It is suggested that the reninstimulating effect of catecholamines in the intact animal might be due, at least in part, to a direct chemical action on the renal cells. Cyclic AMP may play a role as an intracellular mediator of the action of catecholamines.

The activity product of Ca²⁺ and HPO₄ ²⁻ was calculated for the urine before and after incubation with brushite (CaHPO₄ · 2H₂O). This permitted an estimation of the degree of saturation of urine in terms of Ca²⁺ and HPO₄ ²⁻. The urine of patients with idiopathic hypercalciuria was supersaturated with respect to brushite at all pH. However, the urine of normocalciuric subjects with stone was supersaturated only at high urinary pH.

The rate of intestinal transit affects the absorption of dietary iron. The parenteral administration of alpha adrenergic blocking agents or parasympathetic blocking agents significantly increases the absorption of iron by decreasing intestinal motility. That the intestinal transit of iron can play an important role in iron absorption is suggested by effects observed in both normal animals and animals with a decreased capability to absorb iron.

An experiment was conducted with weanling mice to evaluate the protein quality of two samples of gourd seeds (Cucurbita digitata gray and C. foetidissima HBK). These materials were compared with whole egg protein. The gourd seeds were fed after fat extraction and after autoclaving (30 min at 120°) in addition to the feeding of the untreated ground material. Amino acid analyses showed that the C. digitata seeds were deficient in phenylalanine plus tyrosine, methionine, and threonine; while, the C. foetidissima seeds were deficient in methionine and threonine. Nitrogen retention data, liver transaminase activities and amino acid retention estimates all indicated that C. foetidissima seeds were superior to C. digitata in protein quality and that C. foetidissima seeds could be improved by autoclaving but was still of lower protein quality than whole egg.

Water-soluble fraction IV (component 15; R_f 0.73-0.74) purified by electrophoresis of sonicated extracts of strain 2 guinea pig spleen cells, was shown to induce specific allograft immunity in inbred strain 13 animals, thus establishing this material as a transplantation antigen, i.e., a substance which specifically effects the fate of grafts.

Nonvirion T antigen was isolated from adenovirus-type-31 solid tumors by a series of differential extractions, including overnight digestion with papain, centrifugation, acetone and ammonium sulfate precipitation, and acrylamide-gel electrophoresis on a vertical flat-bed apparatus which enabled us to isolate individual bands of protein. The antigen is membrane-associated, has a sedimentation coefficient of 2.93, is partially heat labile, stable to freeze-thaw, and travels rapidly in acrylamide gel. It is probably not the only T antigen moiety present in these tumors.

Absorption of micellar lipid into thoracic duct lymph of unanesthetized rats was studied after administration of actinomycin D or cycloheximide, agents known to inhibit protein synthesis. Micellar lipid was infused steadily into the upper small intestine to avoid complications by defective gastric emptying or defective lipolysis. Actinomycin D, 50 μg/rat, inhibited mucosal protein synthesis, but absorption of fat into the lymph was unimpaired after this dosage. Malabsorption produced by larger doses of actinomycin D or cycloheximide was accompanied by a marked decline in lymph flow. The change in lymph flow was closely related to decreased recoveries of lipid in the lymph, and reduced flow appeared to be more important than suppression of mucosal protein synthesis. The results indicate that chylomicron formation is not dependent on normal mucosal protein synthesis.

The effect of EFA deficiency and chronic ethanol ingestion on liver mitochondrial fragility and fatty acid composition was studied using young growing male rats. The results indicated that EFA deficiency (fat-free diet) partially protected the mitochondria from developing the increased fragility caused by ethanol ingestion. The mitochondrial fragility induced by chronic ethanol feeding was not related to changes in the proportions of arachidonic acid or other mitochondrial fatty acids. Additionally, it was found that chronic ethanol feeding partially prevented the changes in the mitochondrial fatty acid composition induced by EFA deficiency.

Host tissue reaction was a stumbling block in the progress of immunodiffusion and immunoelectrophoretic studies of arboviruses in this laboratory. Partial purification by flurocarbon or by protamine sulfate, of the infected rodent brain tissue homogenates used as test antigens or as inocula into rabbits to prepare antisera failed to remove the tissue antigen reactions. Antisera prepared against infected mouse brain or normal mouse brain reacted with normal hamster brain and vice versa. However, two methods were successful in eliminating the reaction: (i) Pellets obtained from clarified infected mouse brain homogenates following ultracentrifugation at 106,000g was free from rodent brain antigen but contained reacting viral antigens. (ii) Absorption of antisera either by 50% normal mouse brain or normal hamster brain eliminated the antibodies to the rodent brain tissue antigens. Two methods accomplished this latter—conventional tube absorption or absorption-in-gel. The latter is described in detail and found to be highly effective, simple, easy, time saving, and sparing of reagents. It is now used routinely in this laboratory.

The acute toxicity (death within 24 hr) of 10 drugs representing four drug classes (anticonvulsants, hypnotics, hypoglycemics, and psychopharmacologics) was studied in irradiated male CF₁ mice 2 hr, 1 and 6 days after 500 rad; 2 hr and 1 day after 1000 rad; and 2 hr after 10,000 rad whole-body doses of mixed gamma-neutron radiations. The LD₃₀ value for each drug in irradiated mice was calculated and compared to the LD₅₀ value obtained in unirradiated controls. The LD₅₀ values for most drugs studied were altered significantly in some of the groups of irradiated mice. All drugs tended to be more toxic in irradiated mice on the sixth day following 500 rads. In general, the anticonvultants were more toxic in mice receiving 1000 and 10,000-rad doses, while the psychopharmacologics were less toxic in the mice receiving 1000 rads but more toxic in those receiving 10,000-rad doses.

The most commonly used method for measuring prolactin in biological samples has been the pigeon crop bioassay of Lyons (1). It is possible with this method to estimate the prolactin content of pituitary preparations and of other tissues containing relatively high concentrations of this hormone (a minimum of about 0.03 IU or 2 μg of NIH-P-B1, 13 IU/mg); however, it was not possible to assay the prolactin present in serum from normal animals. Recently, double antibody radioimmunoassay techniques have been developed for ovine (2) and bovine prolactin (3) and a radioimmunoassay for rat prolactin (4) which uses a chromatoelectrophoresis step to separate free prolactin-¹²⁵ I from that bound to the antibody has also been reported. These techniques are sufficiently sensitive to quantitate serum levels of prolactin. This communication describes the development of a double antibody radioimmunoassay for rat prolactin capable of measuring the amount of prolactin in 100 μl of serum from immature female rats or in 25 μl of serum from normal females during diestrus.

Methods and Materials. Purified rat prolactin (HIV-8-C) for radioiodination and for immunization was prepared from whole, frozen rat pituitary glands by Ellis et al. (5). This preparation is designated B329 in the remainder of the present study. Other preparations with differing concentrations of rat prolactin were obtained by physiologic manipulation of three groups of mature, female Sprague-Dawley rats. Rats in the first group (B350) were ovariectomized and injected subcutaneously with 10 μg of estradiol benzoate in corn oil daily for 10 days. Rats in the second group (B349) were ovariectomized and injected with corn oil for 10 days while the third group was composed of untreated intact females during diestrus (B351). At the end of 2 weeks the rats were bled terminally via the abdominal vena cava, and the blood from each group was pooled and serum was obtained by centrifugation.

The relationship between some of the chemotherapeutic effects of N-isopropyl-α-(2-methylhydrazino)-p-toluamide (MIH) and the development of resistance in Ehrlich ascites cells was explored. Whereas DNA and protein synthesis is moderately inhibited in these cells after the in vivo administration of the drug, the synthesis of RNA is stimulated. Cell division is markedly suppressed by MIH for periods of time beyond its effects on nucleic acid metabolism. Energy metabolism of the cells is not significantly affected by the drug. It is concluded that the primary chemotherapeutic effect of MIH is on some process associated with cell division other than nucleic acid or energy producing metabolism. A line of cells resistant to MIH was developed. Resistance was induced much more rapidly when MIH was given to coincide with periods of normal DNA synthesis. Resistance developed more slowly when the drug was given over a period in which DNA synthesis was depressed. In both cases, the resistant line was characterized by the appearance of two new metacentric chromosomes in each cell in mitosis.

Weanling rats were used in 4 experiments to determine the effect of Zn and Fe added to the diet alone and in combination in preventing Cd toxicity. Cd added to the diet at a level of 100 ppm depressed weight gain and blood hemoglobin in all 4 experiments. Supplemental Fe at 300 ppm in the diet offset the effect of Cd on weight gain and hemoglobin. Liver Cu was reduced and liver Zn was increased by Cd. Liver Fe was reduced by Cd at 100 ppm dietary Fe but not at 400 ppm. Depression of weight gain by Cd in one experiment was prevented by 68 ppm of Fe and 200 ppm of Zn added to a low Fe-low Zn diet, but not when either was added singly. Weight gain and hemoglobin of rats fed Cd were increased when rats were kept in galvanized cages as compared to stainless steel cages in one of two experiments. A Cd X Fe X Zn interaction appears to exist in the growing rat and environmental contamination with Zn is an important factor in determining the response to variable dietary Cd and Fe levels.

Cycloheximide, at a dose (0.5 mg/kg, i.m.) which is known to inhibit incorporation of amino acids in the proteins of the stomach was found to decrease gastric secretory volume and acid and pepsin production in rats with pylorus ligation. When various stimuli to gastric secretion (alcohol, insulin, gastrin pentapeptide, and 2-deoxyglucose) were given, there were smaller absolute responses in the cycloheximide-treated rats than in controls.

Inhibition of porcine tissue plasminogen activator by ∊-aminocaproic acid (EACA), trans-4-aminomethylcyclohexancarboxylic acid (trans-AMCHA) and p-axmnomethylbenzoic acid (PAMBA) increased uniformly with increasing concentrations of inhibitor. In contrast, with human urokinase an early phase of inhibition at low inhibitor concentrations was followed by a phase of enhancement of fibrinolysis turning into a second phase of inhibition at inhibitor concentrations 300 times higher than required in the first phase. This biphasic inhibition of urokinase distinguishes it chemically from tissue plasminogen activator.

Radioimmunoelectrophoresis was used to detect antibodies to myelin basic protein (BP) in sera from guinea pigs given a single encephalitogenic challenge containing homologous BP or CNS. Antibodies which were found in only 13% of sensitized guinea pigs were detected late in the course of disease and were found only in the 7S-_γ2 class of immunoglobulins. With such a low incidence, it is unlikely that this antibody has any pathogenetic significance. Although the incidence of antibodies is higher in guinea pigs with mild clinical and histologic evidence of allergic encephalomyelitis (EAE) than in the entire group, the increased incidence is not sufficient to suggest a protective function for the antibody.

A low casein diet decreases the incidence and delays the appearance of Leiskmania donovani-induced amyloidosis in the golden hamster. In contrast, 75% of the animals on the normal casein diet developed extensive amyloid infiltration in the liver, spleen, adrenal, and kidney.

The intestinal absorption of radioiron was determined following X-irradiation of the exteriorized small intestine in the rat. The morphologic lesion resulting was discontinuous, with areas of intestinal radiation injury alternating with areas which were essentially intact or hypertrophic. The results indicate that there was an increase in radioiron absorption following irradiation and suggest that this increase was due to augmented absorption in those areas of the small intestine which did not develop the morphologic characteristics of severe radiation injury. Possible mechanisms are discussed.

A leukemogenic treatment with urethan in infant mice induced a prolonged depression of primary hemagglutinin response against sheep red blood cells. The development of lymphosarcomas was found to be correlated with the degree of immunological impairment.

A procedure was established whereby a microbial indicator was incorporated in an animal system to identify and characterize mutagenic agents. The organisms, histidine auxotrophs of Salmonella typhimurium, were injected into mice intraperitoneally, and the test compound was administered intramuscularly. Mutagenesis, indicated by an increase in the incidence of prototrophs in the population, was induced by dimethylnitrosamine, N-methyl-N′-nitro-N-nitrosoguanidine, and streptozotocin. Such a system appears capable of detecting microbial mutagenic activity in compounds which remain or become physiologically active after administration.

Lake trout raised in a hatchery environment develop corneal lesions and opacities with a gross configuration similar to that observed in human keratoconus. Ultrastructural examination of this lesion shows that in the early stages of disease, the endothelial cells become distorted in shape and vacuolization occurs within the cytoplasm. As the corneal changes progress, the kerato-cytes enlarge and develop extensive endoplasmic reticulum, increasing the stromal thickness and displacing the parallel alignment of the collagen lamellae. There are no observable changes in epithelial thickness or morphology.

The respiratory burst associated with phagocytosis can be significantly inhibited by high concentrations of chloramphenicol without impairment of phagocytosis perse. The impaired respiratory burst reflects decreased activation of the HMP shunt probably through the mechanism of enzyme inhibition.

The experiment investigated the effect of a high ambient temperature (35.6°) of varying duration on the hypothalamic content of growth hormone releasing activity (GHRA). These experiments consisted of five treatment groups: one group of normals at 24°, one sham-treated group receiving only diluent in which hypothalami were extracted, and three groups, each heatexposed for 1, 24, or 168 hr, respectively. The GHRA was measured by in vivo depletion of pituitary growth hormone (GH) content in 30-day-old rats. Pituitaries of the recipient rats were assayed by the “tibia test” method for GH content. The standard assay was based on three doses of bovine growth hormone (NIH-GH-B12) with a threefold difference between consecutive levels. Growth hormone was evaluated by two parameters: (a) epiphyseal cartilage width, and (b) GH content of the unknown estimated from the response in terms of the reference standard. An exposure to heat for 1 or 24 hr resulted in an enhanced hypothalamic GHRA with a concomitant marked (significant at 1% level) depletion of pituitary GH content and a return to near normal values by 168 hr post-exposure.

Bone marrow cells were ineffective in producing significant graft-versus-host reactions as measured by a spleen assay test. No synergism between bone marrow and thymus was observed in the graft-versus-host reactions.

The purine analog, 6-mercaptopurine, failed to inhibit the synthesis and the action of interferon in primary chicken fetal cells. Actinomycin D, included as a positive control, completely blocked the synthesis and the action of interferon. Several aspects of host resistance to viral infection other than interferon are impaired by 6-mercaptopurine and delayed hypersensitivity may be the more important component.

The metabolism of calcium was studied in manganese-deficient and zinc-deficient rats by a method combining a kinetic study using ⁴⁵Ca with a short-term classical balance study. There were no differences in managanese-deficient rats as compared with controls in any of the parameters of calcium metabolism, including pool size, endogenous fecal calcium, urinary clacium, or in the rates of calcium entering or leaving bone. The zinc-deficient animals, however, showed significant decreases, as compared with both ad libitum and restricted-intake controls, in a number of parameters of calcium metabolism. These included the rates of calcium entering and leaving bone, pool size, the rate of slow exhange, and the slowly exchangeable calcium in bone. The results suggest that the skeletal abnormals are not the result of abnormal calcium metabolism. In zinc deficiency, on the other hand, specific effects on calcium metabolism were noted. Thus it appears that zinc, but not manganese, is required for normal metabolism of calcium.

Cycloleucine administration to rats produces a generalized aminoaciduria which is largely the result of failure of renal tubular reabsorption. The greatest competition for renal tubular transport appears to be with the site for the dibasic amino acids.

The testicles from hypophysectomized rats respond to small doses of HCG, with the maximal concentration found at 45 min after injection of HCG. A method is reported for measuring testicular testosterone in the rat, using gas-liquid chromatography with a hydrogen flame ionization detector.

Acute and long-term changes in pituitary prolactin and mammary nucleic acids of rats following induction of pseudopregnancy by cervical vibration were investigated. After maintaining a constant level (0.045-0.047 IU/mg) between 0 and 16 hr of stimulation, prolactin concentration declined progressively to the lowest level (0.012 IU/mg) on day 10 of pseudopregnancy. This decrease was followed by substantial increases in DNA and RNA contents of the mammary gland. Pituitary prolactin and mammary nucleic acid content were highly but negatively correlated. The data suggest that prolactin in rats may be involved in mammogenesis and luteal function during pseudopregnancy.

7-Chloro-3,4-dihydro-2-(3-pyridyl)-1 (2H)-napthalenone (Su-10603), a compound known to inhibit particularly 17α-hydroxylation in the adrenal of the dog, was studied for its effects on gonad and adrenal function in rats. Administration to immature animals for 30 days (100-400 mg/kg/day) inhibited growth of seminal vesicles, ventral prostates, levatores ani and uteri without other obvious effects. It was similarly active in adrenalectomized rats. These results are consistent with previous demonstrations that the drug interferes with testosterone synthesis in vitro. The Su-10603 also inhibited the response of immature males but not females to chorionic gonadotropin as judged by weights of sex accessories. It interfered with the normal processes of pregnancy by means as yet undetermined. Unlike the situation in the dog, it was without effect on adrenal morphology or the corticosterone secretion rate in rats.

Parotid fluid was collected without exogenous stimulation from 309 healthy young adult males. Subjects with rates of flow less than 0.018 ml/min were excluded due to sample size requirements. The mean rate of flow for all participants was 0.049 ml/min. Other means were: total solids, 0.72%; pH 5.92, viscosity, 1.08 cP; calcium 1.63 meq/liter, and magnesium 0.25 meq/liter. Rate of flow exerted a significant negative effect on total solids, viscoscity, calcium, and magnesium. The positive correlation between flow and pH was explained as reflecting a parallel relationship between bicarbonate and flow rate. These changes are compatible with the premise that, at these very low levels of gland function, decreases in flow rate bring into dominance the process of passive diffusion of water from the saliva down an osmotic gradient produced by sodium reabsorption. This tentative explanation is offered in the absence of definitive data regarding ductal secretion of the variables under study.

The intraduodenal intubation of heparin^-35S in solution with sulfated or sulfonated surfactants in both the rat and dog has been shown to result in the appearance of circulating clearing factor (lipoprotein lipase). The increase in enzymic activity was paralleled by a proportional increase in serum radioactivity. The clearing factor response occurred within 15 min in both species and reached a maximum in the dog within 30–40 minutes. The relatively slow appearance of both clearing factor and radioactivity in the thoracic duct lymph of the dog indicated that absorption was proceeding via the portal circulation. Approximately 2% of the total dose was absorbed, 50% of the absorbed heparin accumulating in the liver. Negligible radioactivity was found in the muscle and perirenal fat.

The comparative effect of sucrose and complex carbohydrates in the form of corn starch and four different modified food starches was studied in the cholesterol-fed rat. Two separate experiments were conducted, one in which the starch was fed in the cooked form, the second in which the starch was fed raw. Sucrose-fed rats had significantly lower serum and hepatic cholesterol levels (p<0.01) and total hepatic lipid than any of the animals fed modified food starches. This finding held true regardless of whether the starch was cooked or raw; however, cholesterol and lipid levels were markedly higher when raw starch was fed. Feeding unmodified corn starch resulted in serum and hepatic cholesterol levels which were significantly lower than those observed with modified food starch, but not significantly higher than the sucrose-fed controls with the exception of hepatic cholesterol which was significantly elevated when raw corn starch was fed.

Nariva virus induced syncytium formation in BHK-21 and Vero cultures. Such infected cultures showed positive hemadsorption for guinea pig red blood cells (RBC). A hemagglutinin for guinea pig RBC was extracted from infected Vero cells by sonication. The hemagglutinin did not act on 1-day chick or goose RBC, and it showed an enhancement in titer upon treatment with Tween-ether. The hemagglutination reaction was highly temperature-dependent, with optimal titers being obtained at 4° and no activity manifested at 37°. Serological studies by hemagglutination-inhibition and complement-fixation indicated no antigenic relation to the better known myxoviruses or to a number of other viruses. The nucleic acid component of Nariva virus is probably RNA.

Evidence has been presented that potassium depletion occurs in the skeletal muscle, heart, kidney, and plasma of pregnant rats and in the skeletal muscle and plasma of the nonpregnant rat following chronic administration of 0.6% acetazolamide in the diet for 20 days. A similar pattern of depletion followed two injections of drug spaced 18 hr apart. Sodium recompensation was observed in both pregnant and nonpregnant rat tissue that was partially depleted of potassium. A rise in liver potassium was observed in the pregnant and nonpregnant animal following the injection of 500 mg/kg of acetazolamide on days 10 and 11 of gestation. A similar rise was observed only in the pregnant rat on the 0.6% diet for 20 days.

Analyses of rabbit myometrial longitudinal and circular muscle activity indicate that these two muscle layers contract simultaneously in vitro. Simultaneous contractions of the longitudinal fibers were recorded by the Grass tranducer and the longitudinal ECFT (Fig. 4). Tone levels of bursts recorded by the longitudinal ECFT have been observed to be a function of the tension set on the underlying muscle at the time of ECFT attachment. If this tension was large, a decreased tone was recorded; if small, an increased tone was recorded. This apparent decreased tone was likely induced by contraction of adjacent longitudinal muscle above and below the ECFT.

Homogenates of BHK21-F cells which fused rapidly after inoculation with a high concentration of the parainfluenza virus SV5, or which fused more slowly with SV5 infection and virus multiplication, showed no greater breakdown of lecithin than uninoculated control cells. Cellular lecithin did not undergo appreciable hydrolysis in intact cells under conditions in which extensive cell fusion occurred. BHK21-F cells possess lysolecithinase activity, and the ability to convert lysolecithin to lecithin. The cells thus possess the pathways for elimination of the membrane-lytic agent lysolecithin. No phospholipase activities were found associated with purified SVS virions. We conclude that the cell fusion induced by this virus is not due to an effect on a major structural lipid such as lecithin.

After preincubation with dexamethasone (0.016 μg/ml) and growth hormone (1 μg/ml), segments of epididymal fat were tested for sensitivity to the lipolytic effects of epinephrine. When the data from the entire group of 36 rats were pooled and analyzed, it appeared that dexamethasone and growth hormone significantly increased basal lipolysis, but had no significant effect on epinephrine-induced lipolysis. Tissues from only 13 of the 36 rats had increased basal lipolysis of the order of twofold or greater. When the data from these rats were pooled, it appeared that pretreatment with dexamethasone and growth hormone actually reduced lipolysis in response to 0.001 μg/ml of epinephrine and had no effect on higher concentrations of epinephrine. Pooling the data from the remaining 23 rats indicated that the preincubation procedure did not increase basal lipolysis, but significantly increased lipolysis in response to 0.001 and 0.01 μg/ml of epinephrine. The difference in the response of the two groups of rats cannot be attributed to inhibitors present in the albumin solution.

When renal mass is reduced, sodium excretion per remaining nephron increases so that normal sodium balance is maintained. Since this adjustment might be expected to involve changes in the secretion of sodium-retaining hormones, the rate of production of aldosterone was estimated in rats with azotemia and in control rats. The rate of production of aldosterone, in vitro, was 0.85 ± 0.05 mg/100 g of adrenal/hour in rats with azotemia and 0.96 ± 0.08 mg/100 g of adrenal/hr in their paired controls. The similar rate of production in both groups indicates that changes in aldosterone secretion are not primarily responsible for the adjustments in sodium excretion in renal failure.

With the in vitro kidney slice technique, the in vivo steady state and stopflow technique, we have studied the sites and the interrelationship of tubular secretion and biosynthesis of organic acids and found that both occur in the cortex, in the same proximal segment and probably in the same cells. Addition of transported substance, such as PAH or Diodrast, in the medium does not alter the rate of biosynthesis. Simultaneous administration of aminobenzoic acid does not augment, but exhibits either no effect or a slight decrease of tubular transport maximum of the aminohippurate. However, administration of Diodrast would markedly depress the urinary excretion of aminohippurate either by administration or by biosynthesis. Our results indicate that the second step of the transport process, from cell to lumen, is carrier-mediated, and that this carrier can be saturated at a Tm level.

The tissue iron levels and tissue distribution of an intravenous dose of radioiron were studied in germfree and conventionalized mice. The germfree animals demonstrated similar plasma iron levels, diminished plasma radioiron clearance rate, diminished iron concentration in the liver and spleen, a slower rate of accumulation of radioiron in the liver and spleen, and no difference in the percentage of radioiron found in the red blood cell at 18 hr when compared to conventionalized mice.

Human skeletal muscle extracts at acid pH brought about a loss of myoglobin's antigenic precipitating activity. This property was also present in cardiac muscle, uterine smooth muscle, spleen, liver, and urine, but was absent in serum. The “myoglobinolytic” activity of human skeletal muscle was soluble, nondialyzable, inactive near neutral pH, most active at pH 3.4, stable at 56° for 0.5 hr, and precipitable by ammonium sulfate. It was not possible to obtain adequate separation of this activity using DEAE-cellulose chromatography. Partially purified preparations of this “myoglobinolytic” activity caused the loss of the precipitating ability of myoglobin with antiserum, as well as the loss of its electrophoretic character, and the appearance of new electrophoretic species. The substrate for this reaction appeared to be a complex molecular form of acid myoglobin. The “myoglobinolytic” activity of tissue extracts is presumed to be due to the presence of one or more acid activated proteolytic enzymes. The substrate specificity is not known. However, active fractions did not seem to attack human serum albumin.

Isoantibodies prepared in C57BL/6 mice against lymphocytic leukemia cells of DBA/2 mice (L1210) exhibited in vivo localization in the brain of DBA/2 mice. Rabbit antisera prepared against the same cells exhibited a lower localization in DBA/2 brain, while rabbit antibodies prepared against normal lymphoid organs of DBA/2 mice exhibited a much lower localization. The localization of both the isoantibodies and the heteroantibodies was very rapid. Maximal amounts of antibodies were fixed to the brain 2 hr after i.v. injection.

The transport of 3-O-methyl-D-glucose was studied in 20-cm segments of upper jejunum removed with an intact circulation from the animal and perfused extracorporeally. Intestinal preparations from alloxanized animals had a significantly higher percentage minute absorption of the substrate. Insulin failed to modify the transport rate in both the control and the alloxantreated animals. However, insulin stimulation of transport was observed in experiments carried out in a group of rats where diabetes was induced by anti-insulin serum.

Hydrocortisone, a compound capable of prolonging postmitotic life span of primary human amnion cells in vitro was also capable of abolishing the age-dependent increase in lipid/CO₂ ratio of cultures fed with palmitate or oleate, and of delaying the age-dependent increase in incorporation of cholesterol into cell lipids.

The study of the quantity of immunoglobulins in adult axenic C3H/J mice thymectomized at birth, reveals a significant increase in the IgG₂ and IgG₁ immunoglobulins until the age of 8 months, in comparison to the control axenic mice. These results point out the role of infection and/or the wasting syndrome in the variation of the quantities of immunoglobulins in thymectomized holoxenic mice. The existence of an homeostatic inhibition effect on the immunoglobulins is considered.

The correlation between the suppression of thyroidal uptake of radioactive iodide and the serum percent free thyroxine (PFT) in rats with hormone-secreting pituitary tumors was studied. Thyroid glands in intact male or female rats bearing the pituitary tumor 7315a, which secretes prolactin and ACTH, took up 30 and 34% of Na¹³¹ I as compared with nontumor intact controls. The serum PFT of these tumor-bearing rats was 72% higher than controls. Adrenalectomy resulted in complete restoration of thyroidal function to normal levels and was accompanied by a fall in serum PFT. Serum PFT was significantly decreased in the sera of adrenalectomized nontumor-bearing rats compared with intact controls, and this decrease was prevented by predniso-lone treatment. The PFT in sera of predniso-lone-treated adrenalectomized rats was not significantly different from intact controls. It is suggested that in the 7315a tumor-bearing animals the decreased thyroid function is related to the increase in serum PFT. This increase in PFT may reflect a decrease in total binding of thyroxine to serum proteins and this effect may be mediated by the elevated serum corticosteroids caused by the ACTH secreted by the pituitary tumor.

Two new cyclic nitrosamines, nitrosoheptamethyleneimine and nitrosooctamethyleneimine, have been prepared and tested biologically by feeding to rats in drinking water, at two concentrations, 50 and 200 mg/liter. Both nitrosamines produced a high incidence of squamous carcinomas in the lung and of squamous tumors in the esophagus. The lower dose of the compounds did not seem less effective than the 200 mg/liter concentration. Nitrosooctamethyleneimine appeared to be a less potent tumorigen than nitrosoheptamethyleneimine, the latter giving rise to lung tumors after less than 16 weeks treatment, and to esophageal tumors after 7 weeks. Although large single doses of both nitrosamines caused rapid death of the animals with severe necrosis of the liver, there were no tumors or other lesions of the liver in animals treated chronically.

The appearance of the REF in the light mitochondrial and microsomal fractions of rat kidney was studied during a continuous 22.5 hr exposure to hypoxia. Within 15 min a depletion of the REF occurred in the microsomal fraction (105,000g) but ESF-generating activity was detectable at this time in the light mitochondrial fraction (21,000g). Beginning with 1 hr of hypoxia, REF activity reappeared in the microsomal but decreased in the light mitochondrial fraction. At 10 hrs, REF activity was again present in the light mitochondrial but not in the microsomal fraction. With further exposure, the REF was again evident in both fractions up to 22.5 hr. Plasma ESF values rose steadily from hours 2 to 10 followed by a decrease at hour 15 and a secondary rise at 22.5 hours. It is suggested that the REF is generated in the microsomal fraction from which the factor is transported to the peroxisomes contained within the light mitochondrial fraction.

The individual and additive effects upon the elimination of ¹³⁷cesium from the body of supplementation of the diet with 1% ferric ferrocyanide (Prussian blue), 20% beet pulp, and 0.007% sodium fluoride was determined in rat muscle, femur, and carcass 30 days following a single intraperitoneal injection of the isotope. Ferric ferrocyanide showed a significant effect in reducing ¹³⁷Cs retention in each of the examined tissues. Beet pulp showed a significant effect in reducing ¹³⁷Cs retention in muscle tissue when used separately, and a greater effect when used in combination with ferric ferrocyanide and fluoride. Fluoride significantly reduced ¹³⁷Cs retention in the femur when used separately. The combination of all dietary regimens resulted in the removal of 98.7 and 97.0% of the ¹³⁷Cs in muscle and femur, respectively, and in the complete elimination of ¹³⁷Cs from the remaining carcass of the rat.

Results of the ¹⁴C-distribution studies suggest that equilibria (steady state conditions) are maintained between the various constituents of the cells, at least during the early stages of endogenous respiration and before death becomes a major factor. The loss of nitrogen from the cells as NH₃ would result in some shift in internal conditions. The results indicate that the oxidative assimilatory and endogenous respiratory activities of the oxidative yeast R. graminis are similar to those reported for fermentative yeasts and for bacteria. The results of the RQ studies and those on the amino acid pool and on NH₃ production indicate that amino acids are a major endogenous substrate for this organism. Carbohydrate, which is an endogenous substrate for various species of yeast, does not appear to act as such to a marked extent for R. graminis.

Interferon-treated cells yielded less murine sarcoma virus and were less susceptible to focus formation than were untreated cells or cells treated with control mouse serum. The protective effect of interferon appeared to be lost or at least significantly reduced 24 hr after treatment.

Results are presented for a group of beagle dogs given single, short inhalation exposures to a CsCl aerosol contaminated with ¹³⁷Cs. The mean biological half-life derived from the long-term component of whole-body retention was 42 days before, and 36 days after, a colony-wide diet change. For times greater than about 10 days post exposure, approximately constant percentages of the body burden were excreted each day in the urine and feces. The U/F ratio normally fell between 3 and 7. Highest tissue concentrations of ¹³⁷Cs were observed in skeletal muscle, mandibular salivary glands, and kidneys while the lowest were in lung, skin, fat, and bone. These results indicate that since ¹³⁷Cs is very soluble in body fluids, the route of administration has very little effect on its subsequent distribution and extion.

Tumor tissues from 12 neuro-blastomas, one benign pheochromocytoma, one malignant pheochromocytoma, and one ganglioneuroma were analyzed for HVA. Autopsy specimens of heart, lung, liver, spleen, kidney, and adrenal gland were analyzed also. The compound was detected in 9 of the neuroblastomas and in the malignant pheochromocytoma. A small amount was detected in adrenal tissue.

Using a chelating agent which binds calcium ions 100,000-fold more avidly than magnesium ions (EGTA) it was demonstrated that PMN adhesion to glass or to other leukocytes was not a calcium-dependent process and that a slight excess of magnesium ion actually caused increased PMN adhesiveness in the virtual absence of calcium.

Tracer study showed that 25% dietary triolein caused better absorption of calcium than 25% tripalmitin, 80% as compared to 50%. Including 4% L-phenylalanine in the diet, however, did not influence calcium absorption significantly, regardless of the nature of triglyceride.

A comparative study was made of the fatty acid compositions of serum glycerides, phospholipids, and sterol esters of White Carneau pigeons (WC) which are susceptible to naturally-occurring aortic atherosclerosis and Show Racer pigeons (SR) which are resistant. The significantly lower proportion of arachidonic acid in the serum sterol esters of 8-month-old WC compared to SR, was not observed in 5-year-old pigeons. No significant difference was observed in the fatty acid compositions of the three lipid fractions between 5-year-old WC and SR. Significant differences in the fatty acid compositions of all three serum lipid fractions were found between F₁ generation of SR X WC and either one of their parent breeds. Significant changes in the fatty acid composition of serum sterol esters were noted between 8-month-old and 5-year-old pigeons of both breeds.

The membrane-associated enzyme adenyl cyclase gives rise to 3′5′ cyclic adenosine monophosphate (cyclic A) within the hepatocyte; and the availability of intra-hepatocytic glucose is determined to a considerable extent by the mobilization of glycogen via cyclic A. Following 70% hepatectomy, a significant turnover of membrane structures occurs concurrently with loss of cellular glycogen. The present study revealed that glucagon and epinephrine activated adenyl cyclase remains at a normal level throughout the regenerative process. These findings suggest that the regenerating hepatocyte is capable of producing adenyl cyclase and inserting it into newly formed membranes.

A single i.v. dose of hydroxyurea (0.9 g/kg) produced transient marrow erythroid depopulation and reticulocytopenia in the mouse. Within minutes after drug administration there was a virtually complete erradication of marrow DNA synthesis which persisted for approximately 2 hr. Erythroid cell cycle kinetics, estimated with colchicine, revealed an entry of erythroid cells into mitosis for 80 min followed by no further accumulation of metaphase forms until the seventh hour. Thereafter erythroid cells accumulated in mitosis at a normal rate. These findings are consistent with the hypothesis that hydroxyurea kills erythroid cells in DNA synthesis; the initial 80-min accumulation of erythroid mitoses represents the progression of G2 cells into mitosis. Unaffected cells in G1 at the time of drug administration require 7-8 hr to progress through DNA synthesis, G2, and enter mitosis. From these data it would appear that hydroxyurea may prove a useful agent in the study of erythropoiesis because of its selective effect on DNA synthesis in the erythroid series.

An electron microscope study of the shell of living schistosome eggs was made in an effort to elucidate further the mechanism of their nutrition and excretion. Stenger et al. (1) studied the fine structure of murine he-patosplenic Schistosomiasis mansoni and also studied the fine structure of the schistosome egg. These authors described a fenestrated shell and also observed microspicules. Extracellular degradation of these shells in host tissues was observed. They also credit J. H. Smith and F. Von Lichtenberg for observing the fenestra and microspicules independently. The published photographs by Stenger et al. show their fenestrations to be irregular in size, of random distribution, and to be unlined by a plasma or unit membrane. They suggested that these fenestrations might facilitate diffusion of antigenic material from S. mansoni eggs, since Andrade et al. (2) and Lichtenberg (3) demonstrated schistosomal antigens in granuloma tissue.

In our study, living eggs grown in vitro by the method of Lee and Michaels (4) were isolated, fixed in collidine buffered paraformaldehyde, and embedded in epon according to the method of Lynn et al. (5). Thin sections were made on an ultramicrotome, stained with lead citrate (Reynolds, 6) and uranyl acetate (Watson, 7), and viewed with an RCA EMU 3. A shell with microbarbs was observed (Fig. 1). The shell also had discrete pores that traversed the shell in a direct manner. These pores were located principally in the anterior portion of the eggs. No pores could be demonstrated in the spurs, suggesting that the function of the spur is essentially mechanical. Each pore was lined by a trilaminar unit membrane, in contradistinction to the unlined, randomly oriented, fenestrated spaces described in the presumed degenerating eggs of other studies.

Animals given nicotinamide either as a pretreatment or as long as 2 hr (but not 4 hr) after streptozotocin were protected from diabetes. Nicotinic acid and NAD pretreatment were ineffective in preventing streptozotocin diabetes. It was postualted that streptozotocin may interfere with NAD formation in the beta cell and that treatment with nicotinamide reverses the streptozotocin effect by reversing its effect on levels of NAD within beta cells.

Arthritis could be induced in rats by the oral and subcutaneous administration of 6-sulfanilamidoindazole (6-SAI). M. arthritidis could be occasionally isolated from involved joints, but the results obtained from tetracycline therapy, histopathology, and serologic studies, in comparison with mycoplasma-induced arthritis, indicated that these organisms were not the etiologic agent of the disease. It was concluded that mycoplasma could appear in 6-SAI arthritis as secondary invaders.

The effect of ultimobranchialectomy upon the maintenance of normal skeletal structure in the young chicken has been examined. Mortality, body growth, serum calcium, phosphorus, alkaline phosphatase, X-Ray appearance and microradio-graphs of the tibia of calcitonin-deficient animals was not different from sham-operated birds. It was demonstrated that the fall of serum calcium to hypocalcemic levels following parathyroid hormone induced hypercalcemia is calcitonin dependent. Similar observations were made after the infusion of calcium studies indicate that calcitonin is essential in in thyroidectomized animals. (20). These studies indicate that calcitonin is essential in the prevention of hypercalcemia but has no major role in the growth of the skeleton under normal conditions.

The D-C potential at the surface of human skin has been measured in sweating and nonsweating subjects. In non-sweating skin a steady D-C potential was present which changed to a rapidly fluctuating pattern when the skin began to sweat actively. These fluctuating potentials occurred synchronously in the skin of both arms. The potential changes were independent of local hemodynamic events and could be abolished by atropine; which suggests they are a manifestation of sweat gland activity. An influence of breathing on skin potential and the rate of sweating was also found. In response to deep breathing, skin potential transiently became more positive while sweat output increased. These respiration-induced changes were not caused by a transient hypocapnia and could be observed only in skin that was already sweating actively.

The plaque-reduction, serum-dilution neutralization method satisfied most of the important criteria for efficient measurement of antibodies against Tacaribe-group viruses. This technique is type specific, economical to perform both in time and expenditure of reagents (0.025 ml of serum provides quantitative data), does not require special additives such as complement, is highly reproducible and easy to read. In practice, a team of two technicians easily tested 600 sera/week. By reversing the basic procedure and diluting virus rather than serum, it is simple to identify Tacaribe-group isolates.

The number of nucleoli in myocardial nuclei was determined for human hearts in the weight range of 300-640 g. The variation of nucleolar abundance was significant, but slight, with larger hearts tending to show more nucleoli per nucleus. The maximum number of nucleoli per nucleus was six, regardless of the heart weight. It is concluded that even if the amount of DNA per nucleus increases during the development of myocardial hypertrophy, as suggested by microspectrophotometric studies, the number of potential nucleolar organizers remains fixed at six per nucleus.

Studies on protective agents in oxygen toxicity experiments led us to believe that a glutathione (GSH)-glutamate-γ-aminobutyrate (GABA)-succinate pathway may serve as a secondary support system in the maintenance of brain energy levels (adenosine triphosphate [ATP] concentration). This “shunt” is shown in Fig. 1. The glutamate-GABA-succinicsemialdehyde-succinate shunt is a well established pathway (1-8) to which no major physiological significance has been attached. The GABA-succinate shunt has been suggested as a means of metabolizing GABA (9, 10). It has also been reported to function as a means of bypassing inhibition of the alpha-ketoglutarate dehydrogenase system of the citric acid cycle by withdrawal of alpha-ketoglutarate from the cycle by transamination with GABA to yield glutamate and reentry of the carbon chain of GABA into the cycle at the succinate level (9, 10). The possible physiological importance of the shunt is seen if one recognizes that succinate markedly stimulates respiration and oxidative phosphorylation.

Krebs et al. (11) reported that succinate oxidation can monopolize the respiratory-electron transport chain which is the major source of ATP production. Sanders et al. (12, 13) observed significantly higher respiration rates with succinate in brain, liver, and kidney of rats when compared with alpha-ketoglutarate and glutamate. Data shown later indicate that succinate stimulates brain respiration and oxidative phosphorylation in the mouse, rat, guinea pig, rabbit, cat and dog.

The factor limiting the amount of succinate available for metabolism has generally been considered to be the rate at which alpha-ketoglutarate is converted to succinate by enzymes of the Krebs cycle. Roberts (8) showed that conversion of GABA to succinate by GABA transaminase is rapid, and that both glutamic acid decarboxylase and GABA transaminase have pH optima (6.5 and 8.2, respectively) such that small changes in intracellular pH—within the physiological range—could result in large changes in these enzyme activities in situ (8, 14, 15).