Experimental Biology and Medicine

The intestinal mucosal cells have been shown to contain specific phosphatases along with a group of nonspecific phosphomonoesterases. Ockerman (1) has shown the presence of a glucose 6-phosphatase in the microsomal fraction of the jejunal mucosal cells. A Na⁺, K⁺, activated adenosine triphosphatase activity has been shown to reside in the cell membrane fraction (2, 3). Whereas the adenosine triphosphatase has been implicated in the action of the intestinal sugar pump (4), the metabolic function of glucose-6-phosphatase awaits elucidation. While the precise roles of the intestinal phosphatases remain unknown, their involvement in the processes of mucosal transport and metabolism is a good possibility.

The present report is concerned with changes in the intestinal phosphomonoesterase activity in relation to the age of the animal. This study was undertaken in view of the possibility that the process of aging may have an influence on the behavior of the enzyme apparatus which in turn may suggest changes in the intestinal metabolic and absorptive functions.

Materials and Methods. The homogenate and the cell membrane fraction used in this study as the sources of enzyme were prepared from two groups of female mice of B6D2F₁/J strain (The Jackson Laboratory, Bar Harbor, Maine). One of these, consisting of 11-month-old mice weighing 34.0 + 2.0 g, is referred to in this paper as the adult group. The other group was comprised of 34-month-old animals weighing 26 + 1.5 g and is referred to as the aged group. 1

The animals were sacrificed after a 24-hr fast. The entire small intestine was removed, everted, and scraped on a chilled glass surface by means of a glass slide. Two-tenths percent (w/v) homogenates of mucosal scraping were prepared, in a diluent of one part Krebs-Ringer-bicarbonate and three parts tris (hydroxymethyl) aminomethane-HCl (Tris) buffer adjusted to pH 8.00. The homogenate was centrifuged at 1000g for 40 min, and the resulting sediment was suspended in the Krebs-Ringer-bicarbonate and Tris mixture to a final volume equal to that of the homogenate used in obtaining that amount of sediment.

Dogs with experimental myocardial infarction due to coronary artery ligation, when studied 3-4 days following infarction, displayed reduction in plasma clearance of indocyanine green. This was not fully explained by the mild hemodynamic abnormalities present at the time of study, but may be related to more severe cardiac impairment in the immediate postinfarction period.

The serum lipoprotein levels in partially hepatectomized rats were determined by ultracentrifugal, disc electrophoretic and chemical methods and were compared with corresponding values obtained with sham operated and nonoperated rats. The results indicated a sharp decline in serum high density lipoproteins and an elevation in low density lipoproteins in rats undergoing liver regeneration. The decrease in high density lipoproteins in partially hepatectomized rats was probably due to its utilization by the liver during regeneration.

An overlay employing tragacanth gum as a substitute for agar in the plaque assay of animal viruses is described. The tragacanth overlay was successfully employed with vesicular stomatitis virus (a rhabdovirus), foot and mouth disease virus (a picornavirus), pseudorabies virus (a herpes virus), African horse sickness virus, and measles virus (a paramyxovirus). Plaques under tragacanth were generally smaller but usually equalled or slightly exceeded counts obtained under the agar. The number of plaques developing with measles virus in BSC-1 cells was shown to be directly proportional to the virus concentration and the two variables were shown to have a linear relationship. Tragacanth is easy to prepare, the cells used tolerated long exposure to the material, and it was easier to remove than previously described overlays.

The effects of changes in blood volume by blood transfusion and hemorrhage on the cardiovascular dynamics were studied in dogs with a large A-V fistula. It was found that the increased blood volume by continuous blood transfusion progressively increased mean systemic arterial pressure, pulmonary arterial pressure, left and right atrial pressures, total cardiac output and systemic blood flow, and decreased heart rate, and systemic and pulmonary peripheral resistances. In contrast, the decreased blood volume by hemorrhage increased heart rate, and total, systemic and pulmonary peripheral resistances, and decreased systemic, and pulmonary arterial pressures and left and right atrial pressures. In either instance, the magnitude of the changes in A-V fistula flow was markedly smaller than that in total cardiac output and systemic blood flow. It is concluded that the magnitude of A-V fistula flow is not modified directly by the blood volume or systemic blood flow, but mainly by the A-V pressure gradient across a given A-V fistula if the blood viscosity remains unchanged.

Glutethimide, in relatively high concentrations inhibits corticosterone synthesis by the rat adrenal in vitro. This effect is demonstrable regardless of whether glutethimide is administered to the rat prior to removal of the adrenals or is added directly to the incubation fluid. The conversion of pregnenolone and of deoxycorticosterone to corticosterone by incubated adrenals is inhibited by glutethimide, indicating a block of 11β-hydroxylation. Glutethimide also suppresses 21-hydroxylation as evidenced by a reduced conversion of 11β-hydroxyprogesterone to corticosterone. Chronic treatment of the intact rat with glutethimide significantly lowers plasma corticosterone concentration but does not affect adrenal weight or cholesterol concentration.

A method is described for detecting an aldosterone antagonist in unanesthetized adrenalectomized dogs maintained on long-acting replacement therapy. Using this method, spironolactone blocked mineralocorticoid effects on both Na and K excretion. The characteristics of the aldosterone antagonist were readily differentiated from those of a diuretic, chlorothiazide, by demonstration of qualitatively different effects on K excretion.

Using the ultramicro technique, the authors showed that on day 14 after unilateral nephrectomy there is a significant increase in the activity of the enzymes investigated. Specific activity of glutamic dehydrogenase increased from 6.1 μmoles/1 mg of protein/1 hr to 13.4 μmoles in proximal convoluted tubules and from 3.8 μmoles to 7.3 μmoles in distal tubules. Alkaline phosphatase activity increased from 32.6 to 56.5 μmoles in the proximal and from 14.9 to 25.3 μmoles in the distal ones. In discussing the results, the authors suggest that this increase may be due to the synthesis of the enzymes via the operon.

Because previous reports have demonstrated that intestinal bacterial suppression retards the development of cirrhosis in rats on choline deficient diets, the sensitivity of these animals to the lethal and sublethal effects of E. coli lipopolysaccharide was studied. Rats with fatty livers of choline deficiency had a marked reduction in the LD 50 to endotoxin. In addition, serum transaminase levels of these animals were raised by doses of the endotoxin that were too small to affect the enzyme levels in controls. It is suggested that rats on a choline-deficient cirrhotogenic diet, experience tissue necrosis from amounts of endotoxin that are ordinarily rendered harmless.

The demonstration of dose-dependent increase in plasma deoxycytidine (CdR) in rats after their exposure to relatively low doses of X-rays, suggests that the quantitative determination of this compound as early as 4-hours postirradiation might be of value as a biologic dosimeter or indicator of radiation injury at sublethal doses. Significant elevations in plasma deoxycytidine levels were detected at X-ray doses as low as 25 or 50 R, with a positive dose-response relationship evident at doses up to 100 R. The rapid sampling possible with blood plasma, as well as the absence of significant interference by other UV absorbing substances would suggest that determination of levels of CdR and of other pyrimidines in man after therapeutic irradiation would be feasible and useful. Such studies are presently being pursued.

Influence of octanoate and palmitate on the uilization of glucose by rat aortic tissue was examined. Octanoate but not palmitate depressed the oxidation of glucose-U-¹⁴C to CO_2. No effect was exerted by these fatty acids on glucose uptake, conversion of glucose to lipids, or oxygen uptake. Commercial bovine serum albumin, used as a carrier for these acids, depressed glucose uptake, increased CO₂ production from radioactive glucose, and increased oxygen uptake. These effects were abolished with purification of albumin by dialysis and charcoal treatment. The purified albumin did, however, decrease incorporation of glucose radioactivity into tissue lipid. In a short-term experiment (18-min incubation) octanoate was oxidized to water-soluble products and CO₂ much more rapidly than was palmitate. These results suggest that oxidation of free fatty acid, at the more rapid rate observed with octanoate, has a sparing effect on the oxidation of glucose to CO₂ in aortic tissue.

Osteocyte lacunae were measured in normal rats and in rats in which periosteal hypertrophy had been induced at the adductor longus-pectineus insertion. Lacunae in the cortex beneath the periosteal hypertrophy were significantly larger than lacunae in the cortex of the opposite side of the femur from the hypertrophy. Lacunae in both sides of the femoral cortex of experimental rats were significantly larger than lacunae in the cortex of normal femur. This supports the view that the osteocyte is capable of resorbing bone.

The effect of BAPN on traumatized and nontraumatized periosteum in rats of 1, 3, or 15 months of age was studied. The BAPN caused marked hypertrophy of nontraumatized periosteum in 1-month old rats but not in 3- or 15-month old rats. Response of traumatized periosteum to BAPN was similar in all 3 age groups. It is concluded that BAPN acts on newly formed collagen irrespective of age, but does not act significantly on mature collagen.

Density gradient ultracentrifugation of plasma from estrogenized roosters injected with ⁴⁵Ca or ³²P shows that most of the calcium is complexed to a phosphoprotein of uniform ³²P specific activity and on a molar calcium to phosphorus basis very near 1:1. The bound calcium is also completely exchangeable. Since no phosphoprotein other than phosvitin has been identified in the plasma of laying hens or estrogen treated birds, it is suggested that the multiple serine-phosphates of this unique protein complex calcium directly and are primarily responsible for both the magnitude and the nonfiltrable nature of estrogen induced hypercalcemia.

Studies were continued in SV₄₀ and adenovirus 7 hamster tumor systems to develop means for isolating tumor-specific transplant antigen, essentially free of normal tissue antigens, preparatory to consideration for vaccination experiments employing autochthonous tumor vaccines in man. Consistent with previous experiments, SV₄₀ virus-induced tumor cells lost their immunizing ability for the host when treated with formalin or disrupted by a French pressure cell or nitrogen decompression. By contrast, adenovirus 7 tumor cells treated with formalin or disrupted by freeze-thaw, by use of a French pressure cell, or by nitrogen decompression retained the ability to immunize against the same cell tumor challenge. The latter findings are regarded as a significant step toward eventual development of tumor cell vaccines employing fractionated and purified antigens.

The level of normal PFC to SRC and the immune response to 4 × 10⁸ SRC was determined in germfree mice and compared to that of CFW conventional mice. All three strains of germfree mice had normal PFC in the spleen but the CFW mice had fewer than either the Balb/c or ICR mice. The normal PFC in germfree CFW mice was approximately one half the level determined in conventional CFW mice. There was however no correlation between the level of normal PFC and the immune response to SRC. The direct and indirect technique for detecting cells producing antibody to SRC demonstrated no difference in the immune response of germfree and conventional mice. The number of PFC detected by the direct technique on any day after the injection of SRC was slightly lower in the germfree mice but otherwise the kinetics of the immune response were the same as those observed in conventional mice. The indirect technique demonstrates that the PFC synthesizing antibody other than IgM may occur sooner in germfree but otherwise is similar to that of the conventional mice. Antibody titers in the serum of CFW mice were determined with and without treatment with BME and the titers agree with the results of the PFC assays.

The isolated perfused rabbit liver does not inactivate exogenous interferon either from serum or urine preparations. Such a liver has been found capable of actively removing and then breaking down γ-globulin-¹³¹I aggregates. It is unlikely that absence of breakdown observed with interferon is due to an impairment of the protein catabolic function of the perfused liver.

Insulin secretion in mice with hereditary diabetes was measured in vitro by incubation of pancreatic tissue in the presence of glucose and theophylline. When compared with normal mice of the same age, the absolute secretory capacity of the pancreas from young diabetic mice is either normal or increased; in all cases, however, the pancreas from these young diabetic mice mobilizes a greater fraction of its stores of insulin in response to glucose or the combination of glucose and theophylline. Older mice with more severe diabetes are characterized by depletion of the pancreatic stores of insulin and impairment of the secretory ability. However, maintenance of the hyperactive state is seen occasionally in older diabetic mice that had past histories of consistently mild hyperglycemia.

The intravenous injection of SK in cats results in a decrease of inhibitors of trypsin, plasmin and UK in the plasma. There is also an increase of urinary trypsin and plasmin inhibitors.

Further purification of propionin, an antiviral agent active against Columbia SK virus infections in mice, from cellular extracts of Propionibacterium freudenreichii by dialysis, paper chromatography and gel filtration on Sephadex is described. The active agent is shown to be a mixture of two substances, referred to as propionins B and C and a method of obtaining a fraction rich in these two substances, essentially by gel filtration on Sephadex is also described. Some preliminary data on the probable molecular size and chemical nature is also presented.

Rat-into-mouse chimeras were produced by injection of rat bone marrow into irradiated or CY treated animals. Both groups of animals were given interval injections of CY during the first 2 weeks after the initial procedure. The degree of chimerism in the CY induced chimeras was limited and confined only to hematopoietic tissues. Donor type serum proteins were never detected in cultures of spleens or peritoneal macrophages. In the X-irradiated group, the only serum protein of donor origin produced by the chimeric tissues in vitro was Tr, which correlated with its presence in the sera of these animals.

Isohemagglutinins directed against fetal H−2 antigens inherited from the father are induced by multiparity in inbred mice. It is demonstrated that the hemagglutinins are transmitted in utero to the fetus and to the nursling via milk.

The distribution of ¹⁴C-labeled cortisol was determined in the fetuses of AJAX and CBA strains of mice following a single injection of the radioisotope combined with a teratogenic amount of ‘cold’ cortisol on day 11 of pregnancy. Uptake of the radioisotope was similar but the cleft-palate sensitive strain (AJAX) retained a significantly greater amount of the drug during the 120 min following administration. This suggests that genetic differences in metabolism and/or tissue binding are responsible for the observed differences in retention.

Mice were exposed to 23 cycles each consisting of compression at 45 lb/in.² in an air-oxygen mixture containing 27% oxygen for 72 hr followed by 4 days of recovery. The skin was analyzed for salt-soluble, acid-soluble and insoluble collagen, hyaluronic acid, chondroitin sulfate, and fluorogenic material. Growth was less than that of the controls. There was 25% less salt-soluble collagen and 20% more fluorogenic material but the other values did not differ significantly from that expected on the basis of size.

Multiple injections of isoproterenol (ISO) (5 mg/kg, s.c.) administered twice daily to rats increased wet tissue weight, total RNA content, and the incorporation of ¹⁴C-labeled amino acids into protein of heart and submaxillary glands. The activities of aspartic transcarbamylase and orotidine-5′-phosphate pyrophosphorylase-decarboxylase (measured together) were enhanced in submaxillary glands and in hearts. Dihydroorotase activity could not be detected in heart but was enhanced in the salivary glands taken from rats treated with ISO. These enzymes are considered to be involved in pyrimidine biosynthesis. Incorporation of uridine-³H into salivary gland and heart RNA was enhanced after 2 doses of ISO but then decreased to control (salivary gland) or to below control (heart) with more prolonged ISO treatment

Selective staining was used to determine whether a mutant sex-limited dominant character manifested by sterility in male rats is reflected in the number of primordial germ cells in animals so affected. The number of primordial germ cells in 23.94% of 11-day rat embryos from mothers capable of producing sterile males falls below the least number of such cells found in 11 day embryos from normal mothers.

Small numbers of transplanted marrow cells generate both differentiating hemic cell progeny and new undifferentiated progenitor cells. Comparable grafts of spleen cells generate only the former. Thus, hemopoietic progenitor cells consist of at least two compartments: “precursor cells” which do not self-replicate and their more primitive precursors called “stem cells.” The pool size of stem cells relative to that of precursor cells is much greater in bone marrow than in spleen of adult mice.

An intrauterine device in one rat uterine horn causes an increased incidence of embryonic death in the contralateral horn.

The swelling rate of human red blood cells has been studied by spectrophotometric measurements of the absorbance of erythrocytes dispersed in water or sucrose solutions buffered with sodium phosphate buffer, pH 7.4. The rate of swelling of red cells, prepared from blood stored at 2° in acid-citrate-dextrose solution, increases with increased time of storage. The addition of physiological amounts of progesterone to red blood cells stored in the cold under sterile conditions greatly depresses the increase of the swelling rate; red blood cells preserved for 40 days with addition of progesterone manifest the same rate of swelling as that of red blood cells stored for 10 days without progesterone.

The activity of pyridoxal phosphate-dependent enzymes was investigated in pyridoxine deficiency. Depression of the activity was most pronounced in serine and homoserine dehydratase, whereas the level of serine hydroxymethylase was not changed. Regeneration of liver after partial hepatectomy in pyridoxine deficiency proceeded at the normal rate as measured by the increase in weight and the amounts of protein, RNA and DNA of the regenerating tissue. The rate of incorporation of thymidine-³H was also unchanged. In pyridoxine deficiency, the incorporation of radioactivity from serine-3-¹⁴C into DNA of the regenerating tissue was essentially the same as in the control. This finding together with the resistance of serine hydroxymethylase to the deficiency indicate that the formation of C₁ units from serine is not impaired.

A series of antibiotics have been tested for in vitro activity in various media. A component in routine bacteriologic agar, not present in agarose and which is blocked by addition of protamine or toluidine blue has been found to interfere with diffusion of the polymyxins, aminoglycoside and some other antibiotics. This is thought to be due to acidic sulfate groups present in bacteriologic agar. The relation of this effect to possible in vivo binding to tissue components and their significance for antimicrobial sensitivity tests has been briefly discussed.

Addendum An enhancement of infectivity of respiratory syncytial virus by diethylaminoethyl dextran was recently described by Nomura (Nomura, S., Proc. Soc. Exptl. Biol. Med. 128, 163, 1968). This agent (mol. wt. 2 × 10⁶, Pharmacia, Uppsala, Sweden) was tested in bacteriologic agar to determine whether or not it would increase the size of zones of inhibition of polymyxin B. A marked enhancing effect was found with as little as 0.03 mg per ml. Concentrations in medium 199 and 2% agar of 1 mg per ml of DEAE dextran increased the zone size of polymyxin disks against E. coli from 10.7 mm to 16.3 mm, corresponding to the zone size found with agarose gel using medium 199 and the same test organism. Thus, DEAE dextran has been shown to have an effect in agar as well as on virus infectivity.

Spleen explants from hamsters kept at room temperature responded strongly when immunized with sheep red blood cells in vitro. Spleens from cold-adapted animals behaved essentially like those obtained from normal animals. In contrast, spleen explants obtained from hibernating animals tended to be less capable or incapable of giving a detectable response to the same antigen under identical conditons of cultivation. Restoration of competence was obtained by cultivation of spleen explants in combination with a number of other tissues.

Evidence is presented that treatment of C'2 with C'la yields fragments which are dissimilar with respect to size and/or shape, as well as antigenic determinants.

Anaerobic glycolysis rate was significantly higher in the thymuses of high-incidence leukemic mice (AKR) than in the thymuses of low-incidence leukemic mice (Ha/ICR). In addition, there was a marked decrease of anaerobic glycolysis rate in the thymuses of AKR mice at 1-6 days after inoculation of tumor (BW5147), in the absence of either hematologic or histologic evidence of tumor. From the sixth day after the inoculation, there was a gradual increase in rate. It is postulated that this alteration of anaerobic glycolysis in the thymus following inoculation of tumor might represent one of the earliest preleukemic changes in the AKR mouse.

The incubation of choroid plexus tissue with ³⁵SO₄ ^2- reveals that sulfate is accumulated in the tissue against a concentration gradient. The magnitude of tissue accumulation appears to depend on cellular metabolism as evidenced by the adverse effect of metabolic inhibitors. The inhibition of sulfate accumulation by thiosulfate and thiocyanate implies that these anions share a common carrier. These findings support the contention that the low concentration of sulfate in CSF is due to the active transport of this ion out of the CSF across the choroid plexus.

A requirement of calcium ion for normal insulin release has been demonstrated in vivo. It was shown that subnormal amounts of immunoreactive insulin were released in response to infusions of glucose during the severe postparturient hypocalcemia that occurs in dairy cows (postparturient paresis). A diabetic glucose tolerance curve was also observed with this condition. In the presence of hyperglycemia following an infusion of glucose or that occurring naturally in postparturient paresis, a prompt and marked release of immunoreactive insulin was observed in response to an infusion of calcium borogluconate.

The radioprotective compounds studied do not alter the wound healing pattern in the nonirradiated rat when wounding follows drug administration by 4 days. These compounds also do not demonstrate any deleterious interaction with wounding under these circumstances. The radioprotective compounds studied promoted the wound healing pattern of irradiated, wounded rats toward the pattern of nonirradiated wounded rats. The increased mortality observed with the combination of a nonlethal surgical wound and radiation was not observed in animals treated prior to radiation with the radioprotective agents studied (MEA, serotonin, and WR 2721-C).

The DL-kynurenine-keto-¹⁴C was resolved by chromatography on powdered paper columns. Rats were injected intraperitoneally with aqueous solutions of a ¹⁴C-isomer either alone, or together with an equimolar amount of unlabeled enantiomer, or 30 min after a loading dose of the enantiomer. Respiratory and urinary radioactivities were measured. Urinary quinolinic acid, nicotinic acid, kynurenic acid, xanthurenic acid, kynurenine, hydroxykynurenine, and N'-methylnicotinamide were isolated by carrier techniques and their radioactivity was measured. The L-kynurenine-¹⁴C was more rapidly metabolized to ¹⁴CO₂ than was the D-isomer and was also a better precursor of quinolinic acid. Only traces of label appeared in nicotinic acid and there seen in approximately equal amounts in each isomer. A load of D-isomer inhibited the metabolism of the L-¹⁴C-isomer to ¹⁴CO₂ and on the basis of changes in urinary metabolites, it is suggested that D-kynurenine inhibits L-kynurenine hydroxylation. A load of the L-isomer increased the metabolism of the D-¹⁴C-isomer very slightly. Kynurenic acid was the main product of D-kynurenine-¹⁴C metabolism identified in the urine. This could arise by deamination and cyclization as a result of the action of a transaminase or D-amino acid oxidase. Other urinary metabolites were also labeled from D-kynurenine-¹⁴C, indicating further metabolism of this isomer without conversion to the L-form. The appearance of label from D-kynurenine-¹⁴C in niacin metabolites indicates the presence of metabolic pathways in the rat capable of converting D-kynurenine to this vitamin.

Effects of thiocyanate on iodide concentration by the calf abomasum, thyroid, and salivary glands were investigated. Five calves were anesthetized 20 hr after intravenous dosing with 200 μCi ¹²⁵I and the left gastroepiploic vein, inferior thyroid vein, and carotid artery cannulated. Saliva flowing from the mouth and blood from each cannula were collected simultaneously at 5-min intervals for 40 min. A second 40-min collection was made after intrajugular injection of 0.33 mM NaSCN/kg of live wt. Thiocyanate increased venous/arterial ¹²⁵I ratios by 22% for the abomasum and 26% for the thyroid, indicating a comparable degree of inhibition of iodide uptake in both concentrating mechanisms. Secretion of ¹²⁵I into mixed saliva was reduced by 29%. In two additional calves, fitted with parotid duct cannulas, secretion of radioiodine into parotid saliva decreased by 75% after thiocyanate administration.

Insulin-induced hypoglycemia produced a marked increase in adrenal catecholamine output in intact dogs as well as in dogs with midbrain transection at the level of the inferior colliculus. An increase in plasma FFA also occurred in these groups of animals with an increase in ACO. In 10 animals with cervical (C₁) spinal cord transection in which the pH remained above 7.35 there was no increase in ACO or FFA following the production of severe hypoglycemia. In an animal with cervical spinal cord transection that developed an acidosis to pH 7.12, a rise in ACO occurred which returned to normal with correction of the acidosis. In a previous similar study pH was not followed and it seems likely that acidosis may have caused increased ACO in dogs with C₁ transection. Our findings suggest that the locus at which hypoglycemia acts to initiate an increase in ACO and FFA is within the brainstem, below the level of the inferior colliculus.

The effects of gonadectomy and the administration of male or female hormones on the hepatic level of methionine adenosyltransferase have been studied in the rats of both sexes. It was found that the sex difference in the level of this enzyme can be attributed to the action of both estrogens and androgenic-anabolic hormones. Ovariectomy decreased the enzyme level, whereas the administration of 17β-estradiol or diethylstilbestrol, but not progesterone, reversed the effect of ovariectomy. Estradiol also raised the enzyme level in intact male rats to that of the female. Adrenalectomy had no effect on the response of the enzyme to estradiol. Castration of the male resulted in an increase in the activity of methionine adenosyltransferase, and the administration of androgenic-anabolic hormones brought the elevated activity down to the normal level of intact males or even lower. Adrenalectomy did not abolish the effect of castration. 17α-Ethyl-19-nortestosterone and 1-methyl-δ¹-androstenolone were more effective in this respect than testosterone, 17α-methyl-δ⁵-androstene-3β,17/β-diol and 5α-androstan-17β-ol-3-one. The effect of the steroids in decreasing methionine adenosyltransferase activity seems to be associated with their anabolic rather than their androgenic action.

Melanin catalyzes the nonenzymatic oxidation of NADH and PPD. One atom of oxygen is consumed for 1 mole of NADH oxidized and H₂O₂ is formed. The NADH reduces the concentration of free radicals in melanin indicating the formation of reduced melanin during the reaction. Melanin's oxidative activity is inhibited by pharmacologic compounds that combine with melanin such as phenothiazines and chloroquine.

Aerobic and anaerobic activity measured in heart homogenates were generally lower in trained rats than in controls. This depressed activity was not seen in non-trained animals subjected to a single bout of exercise or to chronic aortic coarctation of a degree to simulate “training” hypertrophy. The mildly trained heart also did not spare its endogenous glycogen, as might be expected, during acute exercise.

Acid phosphatase activity in the peripheral blood cells of BALB/c/jax mice infected with Rauscher leukemogenic virus was demonstrated, using unfixed smears and azo dye coupling techniques. Enhanced enzyme activity was noted in neutrophils and lymphocytes with an increased percentage of cells staining and augmented numbers of granules per cell. A number of factors which could account for the rise in acid phosphatase are discussed. This activation of lysosomal enzymes may play a significant role in malignant cell transformation to the leukemic state.

Sperm cells from rat, rabbit, and man are fibrinolytically active. Studied histochemically, an activator of plasminogen is localized to the sperm cell head, while the tail is inactive. The presence of inhibitory compounds, removable by washing, prevents the direct demonstration of fibrinolytically active sperm cells in frozen sections of testis or epididymis of rat and rabbit, and smears prepared from unwashed material require a prolonged incubation period. Human semen liquefies the fibrin layer after only brief incubation.

Many of the properties of the so-called postheparin plasma phospholipase were found to be similar to those of postheparin plasma lipase. Dissimilarities were mainly in the effects of various cations, suggesting that the differences observed were due to the electrolytic effects on the substrate micelles rather than differences in enzyme species. The site of attack by postheparin plasma on the α'-C of the phospholipid molecule was confirmed with synthetic phospholipids. The similar properties of the two enzymic activities and the inability to separate the two enzyme species physically suggest that a single enzyme is capable of hydrolyzing both glyceride and phospholipid molecules. That this enzyme is a lipase and not a phospholipase is indicated by the preferential attack on the α'-C of the phospholipid molecule and by the heat lability of the enzyme.

Striking differences in capacity of the Kupffer cells of the isolated perfused rat liver to recognize and clear B. abortus on the one hand and B. melitensis and S. typhosa on the other have been demonstrated. B. abortus organisms not removed from the perfusate in absence of antibody are promptly engulfed in the presence of antibodies. It is suggested from in vivo experiments involving clearance and distribution of tagged bacteria that striking compartmentalization exists in the RE system.

An antitumor substance has been isolated and fractionated from the bulb of the African onion. Antitumor activity of this material against Walker 256 (intramuscular) carcinosarcoma was demonstrated.

Nodamura virus, an ether and chloroform resistant arbovirus, was likewise resistant to sodium deoxycholate by either of two SDC test methods when virus was tested in suspensions of infected suckling mouse brain, but by only one of two methods when tested in suspensions of infected suckling mouse muscle. By the method in which muscle suspensions of virus were incubated with 0.5% final concentration of SDC at 37° for 1 hr, and then diluted and tested in mice, inactivation occurred irregularly and unpredictably. These variable results were unexplained, but were not due to brand of SDC, duration of incubation, individual muscle suspensions, concentration of muscle tissue in virus-SDC mixtures, passage history of virus in mice or arthropods, dates of experiments or methods of sterilizing SDC. However, the results indicated that tissue source of virus and method of performing SDC tests can influence results.

The cholinesterase activities of pectoral, biceps, and adductor muscles from selected lines of normal and genetically dystrophic chickens were investigated using spectrophotometric assays of acetylthiocholine and butyrylthiocholine hydrolysis and acryl-amide disk gel electrophoresis. It was found that predominately white-fibered muscles such as the pectoral or biceps of dystrophic chickens has 30-40 times the acetylcholinesterase activity of their normal counterparts when the data were expressed on a per protein basis. In contrast, the cholinesterase activity of the predominately red-fibered adductor muscle was little different in samples from dystrophic and normal chickens. Acryl-amide gel electrophoresis revealed that the high acetylcholinesterase activity of dystrophic pectoral and biceps muscles was associated with isoenzyme bands of cholinesterase activity characteristic of embryonic skeletal muscle. In the normal these bands disappeared from the gels before the chicks were 2 weeks of age; in dystrophic lines many of the embryonic isoenzyme bands were retained until the chickens were at least 1 year of age. The possible significance of these findings with respect to the mechanism of action of the gene causing muscular dystrophy of the chicken was discussed.

Samples of human liver, obtained at autopsy from 54 patients were analyzed for 16 amino acids, and adjacent areas were evaluated histologically for several morphological features. No correlation between amino acid levels of hepatic parenchyma and histological findings was obtained.

Chronic intrapituitary implantation of a mixture of estradiol (1 part) and progesterone (10 parts) significantly prolonged the duration of pseudopregnancy. Similar implantation of estradiol (1 part) and cholesterol (10 parts) or of pure progesterone were without effect.

The renal mechanism for the increased sodium excretion due to dopamine infusion was investigated. Infusion of 10 μg/kg/min of dopamine into the dog caused a mean increase in sodium excretion of 43 μeq/min. There was no change in glomerular filtration rate. Proximal tubule sodium reabsorption, as measured by tubule fluid to plasma inulin ratios and the transit time of lissamine green through the proximal tubule, was likewise unchanged. It is concluded that dopamine increases sodium excretion by a direct or indirect tubule effect at a site distal to the proximal tubule.

Urinary estrogen excretion was followed throughout pregnancy (9-35 weeks) in a lowland gorilla (Gorilla gorilla). During this period, estrogen levels increased, with estriol showing a marked elevation similar to that reported in pregnant women, with a sharp decline after parturition. Estriol was not found 5 months after parturition.

Quinidine given in small intravenous doses appears to act at the prejunctional area with no apparent action on the postjunctional membrane as judged by a lack of effect on the ACh-evoked twitch. However, larger intra-arterial doses of quinidine produce a neuromuscular blockade which has the characteristics of the postjunctional depolarizing type. No evidence of muscle membrane depression is present since quinidine does not depress twitch tension elicited from direct muscle stimulation of a curarized cat. Edrophonium potentiated the neuromuscular blockade produced by intra-arterial quinidine, while calcium was an inconsistent antagonist.

Transplantation techniques in irradiated mice were used to demonstrate that the size of the population of colony-forming cells increases during the first 96 hr after injection of bone marrow cells into lethally irradiated animals. The doubling time in the population in the spleen was not constant, but was about 48 hr between 1 and 3 days decreasing to 24 hr between 3 and 4 days. Transplantation techniques probably grossly underestimate the size and growth-rate of the population of colony-forming cells in lethally irradiated mice injected with isogeneic bone marrow cells.

Passive transfers of allergic adrenalitis and encephalomyelitis have been achieved in 24 hr by direct transfusion of whole blood from 1 or 2 specifically immunized donors. It was necessary that recipients were isogenic or hybrids and had nonspecific injuries of the target organ which lowered the threshold for disease. It is likely that the active components of the blood were the agranular leukocytes because these autoimmune diseases have been transferred previously by lymph node, spleen, or isolated blood leukocytes but not by serum (2-5). However, a contributory role of plasma has not been excluded. This new method will facilitate the study of cell migration between lymphoid and target organs in autoimmune diseases.

The effect of metaraminol on lipolysis was studied in isolated rat fat cells. It was found that metaraminol directly stimulates FFA release from isolated fat cells. However, the lipolytic activity of metaraminol was approximately 1/1000 that of NE. The lipolytic effect of metaraminol was blocked by both propranolol and phenoxybenzamine. In addition, metaraminol reduced NE-induced lipolysis significantly.

On the basis of preliminary observations, it appears that plasma lipoproteins may be partially separated into four groups by chromatography on columns of 2% agarose gel: “chylomicrons”endogenous VLD; LD; and HD. Reproducibility and recovery of lipid components are satisfactory. The four groups are not clearly separated on the basis of molecular size, the spectrum of particles being continuous but with peaks of concentration corresponding to the four groups. There is overlap with plasma proteins containing little or no lipid, especially in the HD area.

By the combined determination of LDH and isoenzymes in rat plasma, the degree and extent of training required to prevent the marked rise of enzymatic activity associated with exercise was investigated. Training by swimming 1 hr daily for 1-10 weeks provided little or partial protection against severe exercise. Training 4 hr daily for 6-8 weeks prevented the enzymatic changes observed after severe exercise. By isoenzyme analysis the rise of plasma LDH activity was associaed with the kidney, heart, liver, and muscle; they further showed that adaptation to exercise by training varied in the different organs.

Cutaneous herpes simplex infection was produced experimentally in baby rabbits and hairless mice. Infection in rabbits progressed to give a flaccid hind limb paralysis, whereas massive skin lesions without the flaccid paralysis occurred in hairless mice. Progression of infection was readily scored in both baby rabbits and hairless mice so that these provided good systems for evaluation of antiviral agents. Kethoxal (α-keto-β-ethoxybutyraldehyde hydrate), a potent virucidal compound, showed marked antiherpes activity in both baby rabbits and hairless mice when applied topically. It is suggested that kethoxal may have potential utility in treatment of cutaneous herpes in man.

Radiation can damage human chromosomes, both in vivo (1-5) and in vitro (6-8). This report describes more fully the results of an experiment devised to distinguish between the direct effects of X-rays on leukocyte chromosomes and a possible indirect effect mediated by way of blood plasma. Blood plasma from patients who had received large doses of X-ray enhanced the degree and frequency of chromosomal aberrations of normal nonirradiated lymphocytes in short-term cell culture (9).

Materials and Methods. Six patients with a variety of tumors were chosen because they had received large doses of X-rays through several different ports (see Table I). X-ray integral doses varying from 2.88 to 25.1 × 10⁶ gR were given with a 2 meV van de Graaf generator. Six other patients with similar tumors, studied prior to X-ray, served as controls for the first group. Three of the control patients with tumors were studied before and after X-ray. None of the patients received chemotherapeutic agents or had symptoms of viral infections during the studies. Blood for the studies was drawn from the irradiated patients 24−72 hr after irradiation. Subjects who had not received therapeutic X-ray furnished the normal lymphocytes and plasma.

Chromosomes were studied by a modified method of Moorhead et al. (10). Plasma from the following sources was added to normal nonirradiated lymphocytes in short-term culture: (i) plasma from irradiated tumor patients “irradiated tumor plasma”, (ii) plasma from tumor patients before irradiation “nonirradiated tumor plasma”, (iii) homologous “normal plasma,” or (iv) autologous “normal plasma.”

The cells for culture were obtained from leukocyte-rich plasma after sedimentation of heparinized blood. The leukocyte-containing plasma remaining after removal of erythrocytes, was centrifuged at 2400 rpm (IEC Universal model UV, head no. 240) for 20 min to obtain leukocyte-free plasma.

We aspirated peritoneal fluid from newborn, weanling, and adult mice of both sexes. From the time the mice were born and up to their maturity, mesothelial cells gradually increased in numbers in females but not in males. Lymphocytes also increased with advancing age from birth, however, the adult male count was lower than the mature female distribution. Polymorphonuclear leukocytes and mast cells were lower in mature animals than day-old mice. We observed a 4-fold difference in histiocyte counts between samples from adult males and females. Barr bodies were seen only in newborn females, and daisy cells were seen only at weaning.

Four experiments were conducted with growing lambs to study the influence of diethylstilbestrol administration upon plasma urea-N levels. A reduction was observed within 24 hr that magnified until maximum depression was observed 12-19 days after administration of the compound at which time plasma urea-N levels tended to return toward those observed in control lambs. This response was similar with either oral or implant administration of the compound. With oral administration, the response appeared to be dose-dependent; only one level was tested via implantation. Several possible explanations exist for this response. It is felt that growth hormone release could account for this effect, especially the early (24-hr) response that was observed.

The conditions for eliciting PCA reactions in chickens were described. Optimal reactions were obtained in 3-week-old chickens with an antigen dose of 0.06 mg of BSA/g of body weight administered after a latent period of 3 hr. As determined by the high-salt precipitin test, 0.28 μg of chicken anti-BSA N gave minimal reactions in 3-week-old chickens. Older chickens required larger amounts of antibody to yield minimal reactions. Skin sensitizing antibody resided in the electrophoretically fast-moving globulin fractions.

Antithymocyte globulin was administered to 33 NZB-NZW F₁ hybrid mice ranging from 36 to 47 weeks of age at the start of treatment. Thirty-one age matched female controls were studied. The ATG had a deleterious effect on survival, but no marked effect on renal histopathologic changes. The ATG did appear to prevent the development of fibrinoid arteritis. We speculate that the decrease in survival rate in the treated animals was related to an exacerbation of an underlying viral disorder.

The ability to attack and destroy allogeneic kidney cells in tissue culture was transferred to previously unsensitized rabbit lymphoid cells by incubation in vitro with RNA extracted from the lymph nodes of a specifically sensitized animal. Brief incubation with small amounts of ribonuclease destroyed the ability of the RNA to mediate the apparent transfer of immune capability. The RNA active in the transfer of immunity was located within the 8-18S fraction after sucrose density-gradient ultracentrifugation.

In comparison with the very limited or not detectable reactivity of the versatile carcinogen N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) (1-3) with proteins and nucleic acids (4-7), its esters have shown a strong nonenzymatic reactivity at pH 7 with these tissue constituents and certain of their components (4, 8-13). The sulfonium derivatives formed by reaction of esters of N-hydroxy-AAF with methionine decompose spontaneously to yield 1- and 3-methylmercapto-AAF (0-CH₃S-AAF) (8, 11), and the same compounds can be liberated by alkali at room temperature from the liver protein of rats fed N-hydroxy-AAF (11, 14). Similarly, a product obtained by enzymatic digestion of liver RNA from rats administered N-hydroxy-AAF appears to be identical with N-(guanosin-8-yl)-AAF, formed by reaction of esters of N-hydroxy-AAF with guanosine in vitro (11, 14). These findings indicate that esters of N-hydroxy-AAF are involved in the formation in vivo of protein- and nucleic acid-bound derivatives of N-hydroxy-AAF in the rat (11, 15-17).

In a search for enzymatic esterification of N-hydroxy-AAF we have noted the enzymatic activation of N-hydroxy-AAF with 3′-phosphoadenosine-5′-phosphosulfate (PAPS) by the soluble fraction of male rat liver for reaction with endogenous protein or added methionine, sRNA, or DNA. These studies confirm and extend the recent findings of King and Phillips (18) for sRNA, which appeared while our studies were being prepared for publication. In addition, we have found that the soluble fractions from the livers of female rats and four male rodent species resistant to hepatocarcinogenesis by N-hydroxy-AAF have lower capacities to transfer sulfate from PAPS to N-hydroxy-AAF than do the soluble fractions of male rat livers. Except for rabbit liver these levels parallel the susceptibility of the livers of these species to carcinogenesis by N-hydroxy-AAF.

Materials and Methods. Animals. Adult animals were obtained from the following dealers: male and female albino rats (Holtzman Rat Co., Madison, Wis.); male guinea pigs (O'Brien Co., Madison, Wis.); male Syrian golden hamsters (Con Olson Co., Madison, Wis.); male New Zealand rabbits (Willard Voss, Madison, Wis.); and male albino mice (CD-1 stock, Charles River Breeding Laboratory, Wilmington, Mass.).

Data is presented and reviewed which demonstrates that rat virus is highly sensitive to the antiviral action of interferon in rat cells. However, it is also shown that the virus does not stimulate detectable interferon in primary rat embryo cultures or in hamsters. One of the three strains of rat virus tested induced circulating interferon and lung interferon in rats. It was noted that the interferon-stimulating strain was a wild isolate passed only once in the laboratory. These findings indicate that in rat virus infection of rats and hamsters the interferon system may not play the decisive role during recovery although it may contribute to the host defense.

Transverse sections of human kidney were obtained at autopsy, frozen, and subsequently analyzed for uric acid. In 15 of 18 kidneys, 14 of which were free of renal pathology, tissue urate concentrations increased progressively from the renal cortex to papillary tip. Similarly in 5 antidiuretic rhesus monkeys infused with a sodium urate solution, increasing corticomedullary urate gradients were found in frozen kidney slices. In hydrated monkeys similar infusions were not associated with the occurrence of a urate gradient. The data indicate that urate may undergo stratification in human and antidiuretic primate kidney with higher concentrations occurring in the renal medulla. Although the lack of microscopic evidence does not exclude urate microcrystallization in medullary interstitial tissue, the possibilities that urate may undergo either countercurrent multiplication as a consequence of reabsorption beyond proximal tubules or countercurrent exchange by the vasa recta after passive reabsorption in the collecting ducts are entertained.

Following our observation that dimethylcysteine (DMC) detoxifies without inactivating arsenicals and antimonials, DMC chelates formed with antimonyl tartrates were investigated for toxicity and activity against S. mansoni, T. equiperdum, T. venezuelense. On basis of Sb content the chelates maintained the higher order of activity of the antimonyl tartrates, while their toxicity was considerably reduced. It is suggested that besides the magnitude of the relative safety margin, the absolute (minimal) amount of Sb required for effectiveness may be a factor reflecting clinical usefulness.

The present experiments indicate that urethane is a satisfactory anesthetic agent for electrophysiological studies on hypothalamic ovulatory mechanisms, provided that it is administered early in the morning of proestrus. However, if treatment is delayed until 11/2 hr prior to the beginning of the critical period, this drug will block ovulation.

Further studies demonstrated that progesterone would advance ovulation by 24 hr in 5-day cyclic rats when administered either subcutaneously or intravenously. The minimal intravenous dose for this effect was 50 μg in propylene glycol. In contrast, a dosage of 20-α-hydroxypregn-4-en-3-one, sixteenfold greater than the minimal effective dose of progesterone, failed to advance ovulation. Treatment of urethanized rats with progesterone did not depress hypothalamic ovulatory mechanisms. In these animals, intravenous administration of the steroid was more effective in advancing ovulation than was subcutaneous treatment.

Continuous cell lines derived from embryo bovine trachea (EBTr), pig kidney (PK(15)) and calf kidney (MDBK) were tested for their ability to support the multiplication of visna virus. Visna virus multiplied to a significant degree in EBTr and PK(15) cells and produced distinctive cytopathic changes in EBTr cells, but not in PK(15) cells. Serial passage was successful in EBTr cells, and virus from a late passage was immunologically similar to visna virus propagated in cell cultures prepared from sheep choroid plexus cells. Inoculation of EBTr monolayers with concentrated visna virus at high virus/cell multiplicities resulted in widespread polykaryocyte formation within 6 hr. EBTr cells may furnish a more convenient in vitro system for the study of visna virus than the cell culture systems now in use.

The effect of serum from normal humans and cancer patients on the kinetics of the in vitro hydrolysis of hyaluronic acid by testicular hyaluronidase (E.C. 3.2.1.35) was studied. In cancerous serum the presence of an inhibitory factor, distinct from the physiological hyaluronidase inhibitor, was demonstrated. This factor was absent from normal sera. It is thought to originate in the peritumoral connective tissue.

Serum arginase activity in newborn calf, adult human, and adult horse sera was 0-1 μmoles of urea formed/hr/ml. In fetal calf serum, it is much higher and was observed to be 2-12 μmoles of urea formed/hr/ml. Thus in long term cultures in which fetal calf serum is used, arginine depletion may occur unless the media is changed frequently.

The pressor effect of angiotensin II has been demonstrated in vivo in the fetal as well as the maternal circulations of five pregnant rhesus monkeys. The pressor effect was not transmitted in either direction across the placenta. A possible role for the renin-angiotensin system in the pathogenesis of fetal growth retardation and mortality in human pregnancies complicated by maternal hypertension is suggested.

Hepatic microsomes from the homozygous Gunn rat have been tested for their ability to effect the conjugation of THE and p-nitrophenol with glucuronic acid. Para-nitrophenol conjugation was impaired in these animals but THE was not. Neither aglycone inhibited conjugation of the other in normal animals. It is concluded that the glucuronyltransferases active for each of these substrates in the rat are separate entities.

Rates of filtration and urinary excretion of water, sodium, potassium, and total osmols were determined in renal clearance studies with nonpregnant and 20-day pregnant albino rats. Excretion of radiosodium-22 (²²Na) was determined as the percentage of dose excreted in the clearance urine sample begun 1 hr after a single intravenous dose of 100,000 cpm. Twenty-three rats were given no drugs and 25 were injected intramuscularly with 20 mg of spironolactone (Aldactone) just prior to initial hydration with saline solution. Significant reductions in the rates of excretion and in the percentage excretion of filtered loads of water, sodium, and total osmols were seen in pregnant rats as compared to nonpregnant females, and excretion of ²²Na was reduced by approximately 50%. Aldactone did not prevent these decreases in excretion of water, sodium, total osmols, and ²²Na. Under the conditions of this experiment water, sodium, osmols, and labeled sodium were retained by pregnant rats despite unchanged or increased GFR and a diminished concentration of effective salt-saving mineralocorticoids. These findings suggest increased proximal reabsorption of sodium as a mechanism for reduced urinary excretion of sodium late in gestation.

Mice subjected to femoral trauma in order to induce periosteal proliferation were exposed to atmospheres of either air, 15% O₂ or 10% O_2. TDR-³H autoradiographs revealed that 15% ambient O₂ concentrations did not markedly affect the periosteal response. Ambient gas concentrations of 10% O₂ reduced the labeling index of intact and traumatized femurs only slightly. The decreased ambient concentrations of O₂ do not appear to affect the trauma site sufficiently to enhance a local tissue factor capable of stimulating cell proliferation.

Cell cultures derived from various kinds of simians (African green, rhesus, marmoset, baboon) were found to respond in different ways to adenovirus infection. Some permitted only early steps in replicative cycle, including the production of T antigen. Others were as susceptible as human cells, producing similar amounts of viral antigen and approaching human embryonic kidney cells in assay sensitivity.