Experimental Biology and Medicine

The steps in the heme biosynthetic pathway were evaluated in normal and in copper deficient swine. As anemia developed, the activity of heme biosynthetic enzymes increased. These data suggest that the anemia of copper deficiency is not the result of defective heme biosynthesis and, therefore, that copper is not a cofactor in any of the reactions studied. Since the morphologic characteristics of the anemia of copper deficiency suggest defective hemoglobin synthesis, it is concluded that copper is essential either for the normal metabolism of iron or for the synthesis of globin.

The centrifugally packed column of human or canine RBC hardened with acetaldehyde consists of only 60% cells, in contrast to the near 100% packing for normal RBC. This incomplete packing of hardened RBC was demonstrated with the isotope dilution method using three different macromolecules (albumin, dextran, and PVP) and by following the hematocrit values during the hardening process, and the results agree with theoretical calculations. The striking difference in the centrifugal packing of hardened vs normal RBC indicates that normal RBC are deformable during centrifugal packing and that the low degree of packing must be considered in any study on hardened RBC.

Liver damage was produced in rats by administration of thioacetamide both by intraperitoneal injection and feeding via a stomach tube. Such damage is accompanied by a diminished liver amylase synthesis and leads to lowering of liver and serum amylase levels to 50% of normal or less with the higher doses of drug.

The incorporation of glucosamine-³H into hyaluronate-³H by cultured mouse fibroblasts and human synovial cells is only partially reduced by cycloheximide or puromycin, under conditions where protein synthesis by these cells is almost completely inhibited.

Three isolates of hemadsorption virus were recovered from children with respiratory infections in January 1966. The three isolates could not be distinguished from the prototype Greer strain of parainfluenza type 2 virus by the complement-fixation test using antisera against both the new isolate and Greer strain. However, the neutralization test and the hemagglutination-inhibition test revealed a difference between the new isolates and the prototype Greer strain. The antiserum against Greer strain was not inhibitory for the new isolates in the hemagglutination-inhibition test of the latter viruses before and after sonication and also in the neutralization test, whereas ether disruption of the hemagglutinin disclosed a common antigen in the new isolates and the Greer strain. In contrast to this, 6 isolates of 1965 were the same as the Greer strain in all serological tests.

Arterial blood pO₂, pCO₂, pH, lactate, and pyruvate concentrations were measured in chickens at normal body temperature and at temperatures of 43, 44, and 45°C. A decrease in pCO₂ and increases in the other components were observed. It was concluded that acute anoxia was not the cause of thermal death in chickens.

Phenol-water purification of TCA extracted Serratia marcescens endotoxin results in an apparent enhancement of toxicity as measured by two methods for testing biological activity, whereas three other methods indicate only an insignificant increase in toxicity. Partial degradation of endotoxin by NaOH shows a rapid loss of chick embryo lethality both by the iv and CAM routes of inoculation. Mouse lethality persists longer, almost complete detoxification occurring between 1 and 6 hours of NaOH treatment. The FI₄₀ figures in rabbits tend to show an increase in pyrogenicity after 5 min of treatment and a progressive decrease between 30 min and 24 hours, although a low level of pyrogenicity is retained at the latter time. Shwartzman reactivity increases up to 1 hour of NaOH treatment, followed by a decrease to approximately the initial level after 24 hours of treatment and thus would appear to be a less reliable indicator of detoxification when compared to the other testing procedures. It is thus seen that one method of testing endotoxin may indicate an active preparation whereas another parameter may indicate otherwise to a greater or lesser extent. As a result, it is important to recognize the inherent danger involved in using a single assay technique for describing the toxic potency of an endotoxin preparation. The observed discrepancies in biological assays also indicate that the mode of endotoxic action is obviously triggered and channeled by a number of different pathways in the various host systems.

An increased excretion of biliary free cholesterol and phospholipid was shown to be produced by infusion of various bile acids into the isolated perfused rat liver system. The derivatives studied were the sodium salts of taurocholic, glycocholic, chenodeoxycholic, deoxycholic, lithocholic, hyodeoxycholic, and dehydrocholic acids. Hence, the major bile acids, normally found in the enterohepatic circulation of the rat, can exert stimulatory effects on bile lipid excretion.

Femurs from normal and vitamin D-deficient rats are equally capable of taking up calcium in vitro. However, normal rat serum contains a vitamin D-dependent substance that stimulates the calcium uptake by bone. This factor which is not present in the serum from vitamin D-deficient rats, is apparently not vitamin D itself or a metabolic product of vitamin D; however, vitamin D may regulate the synthesis of this factor.

The effect of administration of highly purified pig GH-releasing factor (GRF) on the depletion of pituitary growth hormone (GH) content in recipient rats and levels of plasma GH-like activity were investigated using the tibia test method. It was demonstrated that GRF induced a significant increase in plasma GH-like activity which was accompanied by a simultaneous decrease in pituitary GH content. The levels of plasma GH-like activity in recipient rats treated with saline were not detectable (less than 1.25 μg/ml). These results seem to confirm that the “depletion” of pituitary GH content in rats caused by GRF indicates the “release” of GH.

Blood sera from four species of nonhuman primates were analyzed for their protein and protein-bound sugar content. Generally, the levels of total protein, protein-bound hexose, sialic acid, and fucose of Pan troglodytes (chimpanzee), Theropithecus gelada (baboon), and Macaca mulatta (rhesus monkey) are comparable to those of humans. In all species examined except Hylobates lar (gibbon), the protein-bound hexosamine levels are considerably higher than those of humans. In Hylobates lar, the total serum protein concentration is lower than in humans, while the protein-bound hexosamine level is very close to human levels. The electrophoretic patterns of the four species of nonhuman primates examined are very different from that of humans and from each other.

Marine elasmobranchs maintain their body fluids hyperosmotic to sea water, continually taking up water through their gills and integument, while maintaining water balance through the renal excretion of a urine which is hypo-osmotic to both sea water and plasma. An increased uptake of water occurs when these fish migrate to fresh water or are placed in dilute sea water. In these circumstances plasma osmolality becomes reduced, urine flow rate and glomerular filtration rate increase, and the ratio of urine osmolality to plasma osmolality decreases (1). A similar response to dilution is observed in higher vertebrates (2-4) and it has been repeatedly suggested that endocrine control of water excretion exists in the elasimobranchs as it does in higher forms. In teleosts the caudal neurosecretory system of the spinal cord has been implicated in the control of water excretion, and the neurosecretory cells in the spinal cords of elasmobranchs have been considered as having analogous functions (5,6). However, despite many conjectures no experimental evidence has supported the presence of a humoral factor capable of influencing water excretion in elasmobranchs, nor is there evidence directly implicating the caudal neurosecretory cells or any other elasmobranch tissue as the source of these postulated factors. In a search for such fmtors we have examined the effect of injections of extracts of various tissues taken from the smooth dogfish, Mustelus canis, on the renal function of other Mustelus undergoing water diuresis while in dilute sea water.

Methods. Donor fish, (male and female, weighing between 1 and 4 kg) were placed in fresh running dilute sea water (7040%) for 2-3 days, a period sufficient to promote development of the maximum drop in plasma osmolality and the maximum water diuresis abtaimble in this salinifty.

The rate of passage of oxygen through isolated sheets of human stratum corneum was measured in vitro and compared to other materials. There was a significant passage of oxygen through the stratum corneum, comparable to that through cellulose acetate, but less than through teflon, and more than through Kodar or Al-foil. Some limitations and therapeutic applications of these experiments are discussed.

Strains of herpesvirus hominis (HVH) which were recovered primarily from genital sites and which belonged to antigenic type 2 were found to produce large pocks (av diameter > 0.5 mm) on the chorioallantoic membrane (CAM) of embryonated eggs. Type 1 HVH strains isolated primarily from nongenital sites produced small pocks (av diameter < 0.5 mm) on the CAM. Histological examinations of the infected CAM have revealed that type 1 HVH strains produce ectodermal proliferation with little subectodermal involvement. Type 2 HVH strains involve ectodermal, mesodermal and endodermal layers with marked necrosis, hemorrhages and inflammatory cells. Multinucleated giant cells are more readily found in CAM infected with type 2 strains. The conflicting reports of other workers regarding pock size and histological findings after infection with various HVH strains may be explained by their multiple passages in eggs and other selective factors. Also included is a comparison of CAM infection with type 1 and 2 HVH and variola and vaccinia viruses. Evaluation of the pock size of freshly isolated strains of HVH which have had no prior egg passage could serve as a rapid presumptive test for ascertaining the antigenic type of HVH strains.

Adenovirus infection of man is common and some adenoviruses have been implicated as causative factors of cancer in animals. Some types of adenovirus produce sufficiently mild infections, however, so that volunteer studies with these agents appear relatively simple and risk free (1,2).

The testing of drugs on randomly occurring diseases is difficult, since a definite clinical diagnosis may not be possible until the disease is past. In addition, the variability of randomly occurring cases, except in very large epidemics, would require that very large numbers of people be tested with any new drug which is to be evaluated in order for such studies to be meaningful. Volunteer studies of induced infection appeared safer and more practical than the study of random cases as a method of drug evaluation, and adenovirus type III, passed only in human amnion cells, and isolated from a very mild local infection seemed an ideal agent to provide this test system.

Two drugs possess in vitro activity against adenovirus and seem reasonably safe for use in man. Methislazone (N-methylisatin-B-thiosemicarbazone) is of proven value in a variety of pox virus infections of man, clearly preventing smallpox in contacts and probably providing therapeutic benefit in the treatment of vaccinia gangrenosa (3-13). Recent studies of this drug in tissue culture indicate that it has some in vitro effect in suppressing adenovirus multiplication (14), and a broad experience with thousands of subjects provides detailed information as to its safety and toxicity (3-6). Trifluorothymidine (F₃TdR) is a potent and nontoxic antimetabolite of proven benefit in the experimental and clinical treatmcnt of herpes simplex infection in the eye when administered locally as drops (15,16). Since it suppresses and interferes with virus DNA synthesis, and drugs of this class have been shown to have activity against adenovirus in tissue culture (14) the topical administration of trifluorothymidine also seemed of possible clinical value.

The earliest change seen after infection of human embryo fibroblasts with human CMV was a contraction of the cell, followed by the appearance of an area of increased cytoplasmic activity adjacent to one pole of the nucleus. The area of cytoplasmic activity moved to a paranuclear position before it impinged upon and invaginated the nucleus. Slow rotation of the nucleus about the cytoplasmic lesion within the cell was apparent. Intranuclear inclusions, distinct from the nucleoli, appeared and matured before the cell exploded and all activity ceased.

Hemagglutinin of rabies virus was prepared from CVS 11 strain grown in suspension culture of BHK-21 cells maintained in a medium containing 0.4% bovine albumin and no serum. Optimal conditions for demonstration of rabies hemagglutinin included low temperature, pH 6.2, and the use of goose erythrocytes. Normal human, burro, and goat sera contained high titered nonspecific HA inhibitors which were difficult to remove. Specificity of rabies HA antigen was, however, demonstrated with antirabies hyperimmune sera which were treated with a modified kaolin adsorption technique. The same methods were used with minor modifications in the preparation of hemagglutinins of VSV-Indiana, VSV-New Jersey, Cocal, and Kern Canyon viruses.

The influences of dose level and route of administration on fat storage and excretion of ethynylestradiol-3-cyclopentyl ether (EECPE) were studied in rats. The EECPE was tagged with ³H in the steroid nucleus and with ¹⁴C in the cyclopentyl group in order to be able to trace the de-etherification process. Over a range of oral doses of 20 μg to 12.5 mg, there was a proportional increase in the concentration of compound in body fat and brain. The storage increment exceeded the dose increment suggesting that with increasing dose more of the compound escaped metabolic alteration. Nearly twice as much radioactivity was found in fat, brain, and blood of rats in which EECPE had been injected parenterally (iv, ip, sc) rather than administered orally. Evidence was sought but not found that EECPE is metabolized by the gastrointestinal tract; in fact, orally and intravenously administered EECPE were excreted in qualitatively similar patterns in bile and urine. Excretion of radioactivity following treatment of rats with cyclopentanol-¹⁴C led to the conclusion that EECPE follows two basic metabolic pathways: (i) de-etherification with urinary excretion of cyclopentanol derivatives; and (ii) conjugation and biliary excretion of metabolites in which the ether linkage is still intact.

Weanling male rats receiving a diet low in zinc for 2-3 weeks had a significant increase of liver nonprotein SH compounds and glutathione over those fed an adequate zinc diet. Deficiency of this trace element caused an increase in the incorporation rate of glycine-1-¹⁴C into liver glutathione moiety, but it had no effect on protein synthesis.

The dog is shown to have very low blood catalase activity levels, ranging from an immeasurably low blood catalase to a level which in other species would be considered hypocatalasemic. Although some tissue levels of catalase activity are lower than, say, those of the normal mouse, the dog and the duck are not acatalatic.

Feeding antibiotics to chemically bursectomized (CB) birds has demonstrated that immunological incompetence in CB birds is a direct result of embryonic bursectomy and not secondary to growth rate or factors that increase mortality. Mature CB birds appear to produce normal amounts of agglutinin to sheep red blood cells, but are still deficient precipitin producers. Apparently, the chicken is capable of adapting immunologically to the altered internal environment produced by embryonic bursectomy.

These experiments demonstrate that hypothalamic obesity can develop in the absence of hyperphagia and of pituitary involvement, and establish that VMM lesions must have caused additional disturbance contributing to this surgically produced obesity.

An oral hydrophilic colloid (9.6 gm/day), after a 4-week preexperimental control period, was fed for a 6-week experimental period to 2 normally active young adult males on a self-selected mixed diet, with the following results: Total fecal bile acids per 24 hours obtained during the experimental period averaged 3 times (302%) the excretion level of the control period. Serum cholesterol levels during the experimental period averaged 17% lower than during the control period. These differences were statistically highly significant by t test (p < 0.005). Total neutral sterol excretion per 24 hours in the experimental period was unchanged from the values measured during the control period. The mechanism by which the mucilloid increases fecal bile acid excretion is unknown.

Immune guinea pig gamma₂-globulin is cytotoxic in vitro for tumor cells in the presence of guinea pig complement. F(ab')₂ antibody fragments, prepared by pepsin degradation of the gamma₂-globulin, are not cytotoxic. Preincubation of tumor cells with F(ab')₂ fragments blocks the cytotoxic activity of gamma₂-globulin in high F(ab')₂/globulin ratios. The implication of these findings for immunologic enhancement of tumors is discussed.

Red cell G-6-PD, 6-PGD, and GOT were studied in mice during and after prolonged arrest of erythropoiesis by means of repeated subcutaneous injections of actinomycin D. No clear downward trend in the activities of any of these enzymes in the progressively aging cells was found except for GOT, which initially declined as the reticulocytes disappeared and then stabilized at a new lower level. Discontinuation of the drug evoked a marked reticulocytosis accompanied by a sharp increase of G-6-PD and GOT activity. These data suggest that the major factor determining the red blood cell enzymes level is the presence or absence of reticulocytes.

Jejunal fistulas of conscious dogs were perfused with mixtures containing NaCl, and a test sugar at concentrations up to ISO mM and an inhibitor sugar at concentrations between 0 and 75 mM. The absorption rates of glucose, galactose, and xylose increased with increasing bowel lumen concentrations of each sugar, and increased with increasing net rates of fluid absorption. Glucose reduced the absorption rates of galactose and of xylose, and galactose reduced glucose absorption. Xylose did not have a consistent effect on glucose absorption.

The effect of serotonin on oxidative phosphorylation by rat brain mitochondria was studied. It was found that both phosphorus and oxygen uptake were inhibited by the addition of serotonin. However, the uptake of phosphorus was more sensitive to serotonin inhibition. The addition of serotonin caused an uncoupling of phosphorylation from oxidation as evidenced by a reduced P/O ratio. This effect of serotonin was removed by monoamine oxidase blockade. Indication that the effect of serotonin is mediated via its carbonyl derivative was further strengthened by the use of 3-indole-acetaldehyde which gave similar inhibition of phosphorus and oxygen uptake. These findings support the hypothesis that serotonin could impede brain function by inhibition of energy producing reactions.

In three serial experiments, chimpanzees free of specific neutralizing antibody were infected with rhinovirus types 14 or 43. Although infected animals shed virus from 6 to 21 days and produced high levels of specific antibody, clinical illness was not discernible. Providing serum neutralizing antibody was still present, chimpanzees rechallenged with the same agent shed virus for less than 5 hours. To the author's knowledge this is the first report of successful experimental infection by human rhinoviruses of species other than man.

A spectrophotometric method using DTNB for the estimation of nonprotein -SH in the presence of protein -SH groups in rat brain homogenates is described. Details of the optimum conditions for the color development which were found to be dependent on the reaction time and pH are also given. Estimates of nonprotein -SH and total -SH in rat brain reported by a number of investigators using various methods are in good quantitative agreement with those obtained in this study. Thus, findings led us to conclude that the relative specificity, rapidity, and accuracy of this chemical method are comparable with the existent methods requiring specialized instruments or techniques. This spectrophotometric method has a further advantage of not requiring measurement in the region between 250 and 280 mμ where interference by ultraviolet-absorbing substances is often a complication.

Aminoglutethimide was found to be a competitive inhibitor of the enzymatic conversion of cholesterol to pregnenolone by the adrenals. This effect is due wholly or in part to an inhibition of the 20-hydroxylation of cholesterol.

Twelve rats were subjected to hemorrhagic shock. Histochemical study of the liver revealed increased adenosine triphosphatase and glucose-6-phosphatase activity when compared to controls. The reason for these alterations may be either an actual increase in enzymes in the parenchymal cells or altered histochemical reactivity due to cell necrosis, changes in membrane permeability, or deranged intra and extracellular ionic concentrations as a result of hypovolemia. The increased enzyme activities relate logically to altered carbohydrate metabolism and fluid dynamics in shock.

The dose response curves measured 24 hours (“early”) after erythropoietin injection differ significantly from those measured 48 hours after (“late”). The slope of the curve relating increase in iron uptake by total erythroid tissue to EP dose is greater (p < 0.01) in the 48-hour response. Total uptake saturates above 8 units in the “early” response but not in the “late.” Marrow uptake drops at higher doses (16 units) in the “early” response but does not do so in the “late.” The curve for uptake by circulating erythrocytes shows only one slope in the “early” but two distinct slopes in the “late” response: one from 1-4 units the other much steeper from 4-16 units. The results are discussed in relation to the possibility of different mechanisms being involved in the two responses.

The influence of exogenous ascorbic acid on the movement of ⁴⁵Ca from the bone to the blood was measured in the young chick. The isotope was given 6 days prior to the vitamin and was presumably located mainly in mature crystals when the ascorbic acid was given. Blood levels of ⁴⁵Ca were significantly elevated from 4 to 8 hours following administration of ascorbic acid. Twenty-four hours after this injection blood ⁴⁵Ca was approximately 25% less than controls. These results suggest that ascorbic acid, as used in these studies, had a bone salt mobilizing influence. Whether this effect was direct or indirect remains to be elucidated. If the mobilizing effect were direct, it would indicate that ascorbic acid has a more extensive role in skeletal physiology than previously thought.

The protein fractions present in fetal lung fluid were determined by means of cellulose acetate and immunoelectrophoresis. Proteins with identical electrophoretic mobility to that of sheep albumin and α globulins were detected in all; prealbumin, beta and gamma globulins were observed in some. An unidentified γ globulin other than γ G-globulin was shown to be present in fetal lung fluid and absent in fetal serum. The α₂ globulins in fetal lung fluid were found in a concentration which exceeds that of the serum. The local synthesis of both proteins in the lung is suggested.

Lactic acid absorption was studied in anesthetized rats by perfusing isolated segments of small intestine or colon with sodium lactate-1-¹⁴C for 2 hours. The concentration and pH of the perfusate were varied. Absorption was maximum at pH 2.8, near the pK of lactate, and minimum at physiologic pH. Absorption was directly related to concentration of the lactate perfused and it was not affected by a metabolic inhibitor. The jejunum was the site of maximum absorption. Absorbed lactate was metabolized to ¹⁴CO₂. Poor lactate absorption at physiologic pH and in the distal intestine might lead to an osmotic diarrhea in certain patients with malabsorption.

It has been attempted in the present study to compare the malignancy of the two transplantable carcinomas of rabbit, both originating from the cutaneous papillomas induced by Shope papilloma virus some 20 years ago, from somewhat quantitative aspects. The results confirmed the generally accepted impression that the Yx2 carcinoma is more malignant than the Vx7 carcinoma. Comparative studies on the growth pattern and tendency to metastasize to remote organs from the primary site of transplantation have clearly shown that the Vx2 has a more rapid growth rate, and shows a higher frequency of formation of metastatic lesions in retroperitoneal lymph nodes, lungs, and other organs than the Vx7 carcinomas.

Studies were made to determine if raising or lowering the metabolic rate of experimental animals would increase or decrease the incubation period of rabies virus.

The metabolic rate of mice, rats, hamsters, and guinea pigs was increased from 17 to 84% with iodinated casein with no significant change in the incubation period or mortality rate of rabies when compared to control animals. The host metabolic rate was decreased by thyroidectomy, hypophysectomy, or propylthiouracil in the diet. In most cases the incubation period of the treated groups did not differ significantly from the rabies controls. However, in thyroidectomized rats with the metabolic rate decreased 13% there was a significant delay in the onset of rabies symptoms (p = .10). In mice fed PTU the mean metabolic rate ranged from a 2.2% increase to a 4% decrease. An analysis of 216 mice suggests a delaying effect in the onset of rabies symptoms and a higher survival rate for mice with PTU in the diet than controls with a normal diet. It is suggested that the decreased metabolic rate of a host could delay virus propagation and give more time for active and passive treatment and possibly increase the chances of survival for an animal exposed to rabies virus.

An avian leukosis virus (ALV) free primary yellow fever vaccine seed has been developed by ridding the 17D virus vaccine seed of its ALV contaminant. By differential filtration through Millipore membrane filters of different pore sizes, yellow fever virus was separated from its contaminant by physical means. The ALV could not be detected in the new primary and secondary vaccine seeds by RIF, COFAL, and FAB tests.

Cholesteryl ethers of aliphatic alcohols when fed in triolein are absorbed by rat intestinal mucosa and transported in the thoracic duct lymph of rats fed these ethers. The recovery of the ethers in the lymph decreases with the chain length of the aliphatic alcohol moiety of the ether. Around 1/3 of the methyl ether fed being recovered and 4% of the decyl ether. The cholesteryl ethers are recovered in the lymph in general to more than 90% in unchanged form.

The results obtained clearly show that the free form is not the only possible transport form for cholesterol into the intestinal mucosa cells.

Insulin, labeled with ¹³¹I, was injected intracisternally in normal and vagotomized dogs. In both types of experiments a significant fall in plasma glucose concentration was observed. The amount of insulin which passed into the blood from the cerebrospinal fluid, as determined by the amount of insulin-¹³¹I in the plasma, was more than sufficient to account for the observed hypoglycemia. These findings indicate that insulin enters the blood after intracisternal injection, and that the hypoglycemia observed after intracisternal insulin administration is most likely due to a peripheral rather than a central effect of insulin.

A sensitive bioassay for the lactogenic activity of rat chorionic mammotropin, a secretion of the midpregnancy rat placenta, utilizes the synergism of this hormone with prednisolone acetate to induce mammary secretion in the intact, immature, male rat. Activity in locally injected solutions is detected by the appearance of distended alveoli in stained whole mounts of the treated mammary gland. The contralateral, untreated gland serves as a control. Reliable responses to extracts containing RCM have been produced in 2-4 days, depending upon dosage and number of injections.

The effects of dl-ethionine on fetal development in the rat were studied, with particular reference to the formation of the exocrine pancreas. Pregnant rats were injected from day 14 through day 20 of pregnancy, and sacrificed on day 21 of the 22-day gestation period. Controls were injected with equivalent volumes of saline. Ethionine caused significant decreases in litter size and fetal body weight, and had a selectively disruptive effect on the formation of the fetal pancreas as indicated by measures of pancreatic relative weight and histological examination of fetal pancreatic tissue.

Rats having permanent gastric fistulas were infused intravenously for 24 hours with calcium in saline, or with saline alone. Gastric secretion was collected for the period of infusion. In calcium-treated rats significant reduction of gastric acid secretion was found. In these animals blood pepsinogen was increased but gastric tissue pepsinogen was reduced. Reduction of zymogenic granules of chief cells was also found in glandular mucosa of calcium-infused rats. Hypercalcemia appeared to alter the function of both parietal and chief cells of rat stomach under the conditions of this experiment.

Plasma protein bound iodine (PBI) of female rats fed an essential fatty acid (EFA) deficient diet for 15 weeks was higher than that of controls receiving dietary EFA, even though weight of the thyroid gland was less in deficient rats. After refeeding EFA to the deficient rats either as soybean oil or the colony stock diet for 3 weeks, the PBI concentration was decreased to the control values and the weights of the thyroid gland increased. Thiouracil added to the EFA-deficient diet lowered the PBI concentration in plasma and increased the weight of the thyroid gland. These data suggest that the increase in plasma PBI in EFA-deficiency is not dependent on TSH but the control mechanism that regulates the secretion of thyroidal iodine is dependent on essential fatty acids

Peptide map of F(ab')₂ fragments of anti-DNP-PLL antibodies obtained from responder guinea pigs immunized with DNP-PLL and from guinea pigs lacking the PLL gene immunized with DNP-PLL ovalbumin complexes were compared. No structural differences that could be attributed to the “PLL gene” were observed.

Extracts of African lymphomas (Burkitt's tumor) frequently induced morphologic alterations in cultured primary human amnion cells. The changes were associated with resistance to infection by several viruses.

The administration of D-ribose was followed quickly by a sharp rise in plasma insulin levels in 9 awake, alert, unstressed dogs (16 experiments). The elevated insulin levels occurred while blood glucose levels remained constant or dropped. Oral administration of ribose was not as effective as intravenous administration in causing insulin release. Thus the mechanism of action of ribose in these experiments does not appear to be associated with any gastrointestinal factor.

Chloramphenicol pretreatment reduced the lethality of cyclophosphamide in vivo and resulted in an increased time of mean death and a higher mean lethal dose. Chloramphenicol pretreatment decreased the in vivo conversion of cyclophosphamide to a metabolite capable of alkylation. After pretreatment with chloramphenicol, a decreased amount of activated product appeared in the urine during 90 min and blood levels of active metabolite were less when measured after 90 min. In vitro chloramphenicol decreased the metabolism of cyclophospamide 50% at approximately 10^-3 M. It is concluded that the protection afforded animals by chloramphenicol after treatment with lethal levels of cyclophosphamide is due to an inhibition of microsomal drug-metabolizing enzymes which are responsible for the in vivo activation of cyclophosphamide.

Intravenous injection of 10 mg/kg of bretylium produced a sustained elevation of the heart rate in ganglioplegic dogs and in vagotomized spinal dogs and did not have this effect in reserpinized ganglioplegic dogs. At any time after bretylium injection the elevated heart rate could be reduced to the control level by injection of the beta adrenergic receptor-blocking agent, propranolol. The urinary norepinephrine output increased strikingly immediately after bretylium injection and then decreased to control levels, whereas the epinephrine output in the urine was not increased. On the basis of these results the tachycardia throughout the experiments could be attributed largely or entirely to the action of norepinephrine despite the inactivation of the cardioaccelerator pathway.

Sotalol and propranolol enhanced insulin-induced hypoglycemia in the rat. Neither agent displayed any significant hypoglycemic activity per se.

The effects of PGE₁, PGA₁, and PGF_2α on the coronary and peripheral circulations were studied in anesthetized dogs. The ia injection of either PGE₁ or PGA₁ increases blood flows and decreases the peripheral resistances in the coronary, brachial, femoral, carotid, and renal arteries. On the other hand, PGF_2α decreases blood flows and increases peripheral resistance in the brachial, femoral, and renal arteries. The present study indicates that PGE₁, and PGA₁ dilate and PGF_2α constricts directly the regional blood vessels.

The ability of certain tissues from anemic dogs to form copro- and protoporphyrins from glycine and acetate or from delta-aminolevulinic acid (ALA) has been compared with that of tissues from normal control dogs. None of the tissues studied formed porphyrins from glycine and acetate. However, tissues from normal dogs formed porphyrins from ALA, and in decreasing order of activity, liver, spleen, kidney, and heart. The tissues from the phenylhydrazine-treated dogs showed significantly less copro- and protoporphyrin formation than did those of the control dogs. Porphyrin synthesis in the spleen was especially decreased. In contrast, all tissues from the group made anemic by bleeding showed increased porphyrinogenesis as compared with the controls. The same results were obtained in dogs similarly treated but administered added iron. It is suggested that the decrease of porphyrin biosynthesis observed in the tissues from the phenylhy-drazine-treated dogs may result from an in vivo “feedback” inhibition by coordinate repression by heme or possibly a related metabolite. The increased porphyrinogenesis observed in the tissues of the dogs made anemic by bleeding may be related, either directly or indirectly, to erythropoietin or to some other hemopoietic factor.

Transfer of tolerance to the analgesic effects of morphine to mice by injection of cerebral homogenates from morphine-tolerant rats was studied. After such pretreatment the mice were injected with a test dose of morphine and then tested for analgesia using the hot plate method. Neither the dose-response curve nor the mean increase in reaction time was significantly altered by pretreatment with the cerebral homogenate from morphine-tolerant rats. The possible reasons for the lack of confirmation of results obtained by other workers were discussed.

Guamanians eat the fleshy husk of the seeds of the cycad, Cycas circinalis, which is indigenous to Guam. The fresh husk is eaten to relieve thirst. The dried husk is eaten as candy. The kernel of this seed is soaked in water, sun dried, and ground for preparation as an ingredient in foods. This processed kernel was not toxic or carcinogenic in contrast to unprocessed kernels. Since the husk is consumed without cooking or processing other than drying, and since the husk is reported to be eaten primarily by children, it is imperative to evaluate the safety of this food. The acute toxic effects have already been studied whereas the carcinogenic properties of the husk have not. For this reason rats were fed diets containing 0.5-2.0% of the dry husk for prolonged periods. Malignant tumors were found in the liver and kidney of experimental animals. Metastatic tumors were found in the pancreas, spleen, and lung.

Studies demonstrating the effect of low salt intake on the activity of Na⁺-K⁺-activated adenosine triphosphatase in the rat kidney have been described. The results indicated that no detectable changes in the level of this enzyme occurred.

The injection of 3′,5′-AMP elicited an increase in blood glucose concentration of both the CFW and CFI strains of mice and had no effect on the histamine sensitivity of either strain. Administration of 5′-AMP was followed by hyperglycemia and histamine hypersensitivity in the CFW mice; the CFI animal was unresponsive to 5′-AMP in terms of both phenomena.

Mice infected by the i.v. route with P. mirabilis or Ps. aeruginosa were treated with several chemotherapeutics commonly used in human pyelonephritis or recommended for gram-negative infections. Streptomycin, neomycin, and kanamycin were the most effective agents, and with sulfadiazine, the only ones active against both infections. When therapy was delayed as long as 5 days, the 3 antibiotics substantially reduced kidney damage and the number of positive cultures obtained if given in large doses. Sulfadiazine was not beneficial for either infection if treatment was delayed more than one day after inoculation. The same was true for polymyxin (Pseudomonas infection). When one treatment i.v. was given 1 hour after challenge, only those agents found active in the delayed therapy experiments gave good results. The other chemotherapeutics were ineffective by any test method. Gamma globulin as well as specific antisera reduced the incidence of gross kidney damage and infection when administered before or up to 24 hours after i.v. inoculation.

The β-galactosidase of Pseudomonas aeruginosa was isolated and enriched approximately 25-fold. Gel filtration experiments provide an estimated molecular weight of 45,000. The comparatively small size of this enzyme is evidence for the nonrelatedness of pseudomonads and previously characterized microorganisms (e.g., Aeromonas formicans).

The anticonvulsant activity of synthetic antiparkinsonism agents has been studied in mice with experimental seizures and in children with myoclonic spasms refractory to conventional medications. Procyclidine and trihexyphenidyl protected mice from maximal electroshock seizures. Procyclidine was the more potent compound and was also effective against pentylenetetrazol seizures. In a preliminary, short-term clinical trial, procyclidine caused a reduction in the incidence of seizures in 10 of 16 patients treated. The antiparkinsonism agents are free of severe chronic toxicity and are contraindicated only in adults with glaucoma or urethral obstruction. Further laboratory and clinical investigation of the anticonvulsant activity of this class of compounds is recommended.

1-Hydroxybiuret (1-HB) and 3-hydroxybiuret (3-HB) were shown to be inhibitors of DNA synthesis in an in vitro Ehrlich ascites tumor test system in NCTC-109 medium. Inhibition was manifest almost immediately upon addition of either compound to the cells. The rates of synthesis of RNA and protein were not substantially altered by either compound. Removal of each inhibitor by washing the cells did not completely restore the rate of DNA synthesis to its control value. The inhibitory action of each against DNA synthesis was antagonized by a mixture of deoxyribosides, suggesting that inhibition of ribonucleotide bioreduction may be a major mode of action, similar to that of hydroxyurea. Morphological evidence indicated that 3-HB induced unbalanced growth in several strains of Pseudomonas, but 1-HB was devoid of activity in the microbial system.

Rabbits injected with streptococcal extracellular protein (SEP) developed degenerative and infiltrative lesions in the heart and liver, with coagulation necrosis and multinucleated giant cells in the latter organ. The majority of these rabbits also showed elevated levels of glutamic-oxalacetic transaminase, or of sorbitol dehydrogenase, and all showed elevated serum lipids. These biochemical indications of cell injury could be found within 2 to 4 hours after a first injection of SEP. No such biochemical or pathologic effects were found following an injection of heated SEP or a commercially available streptokinase-streptodornase preparation from group C streptococcal culture. In a preliminary fractionation of SEP all the activity was found in a fraction not adsorbed to DEAE cellulose at pH 7.4 (0.05 M P0₄). The active fraction contained at least five antigens and four of the known streptococcal enzymes. Injection of extracts of sonically disrupted streptococci produced similar biochemical and pathologic changes. There was some cross-reacting material between the sonicates and anti-SEP serum.

Protamine zinc insulin (PZI) was administered daily to male rats by subcutaneous injection (2 units/100 gm of body weight) and comparisons were made with saline-injected control animals. As a consequence of PZI administration, the following observations were made: hyperphagia, increased body weight gain, increased lipogenesis from acetate-1-¹⁴C and glucose-U-¹⁴C, and no alteration in oxidation of glucose-U-¹⁴C to ¹⁴CO_2, in lipolysis in vitro, nor in the fat concentration of adipose tissue. These results are compared with those reported previously with hypothalamic-hyperphagic rats and it is concluded that the metabolic mechanisms of increased fat deposition differ between the two forms of hyperphagia.

Spleen cells from Coombs positive 480-500-day-old male NZB mice were incapable of inducing graft-versus-host reactions when injected into (NZBXA) F1 hybrids. This occurred even when the total number of nucleated cells injected was adjusted to contain 15-20 × 10⁶ lymphoid cells. Younger, 45 and 150 day old, NZB mice were capable of inducing graft-versus-host reactions in a normal fashion.

Studies from this laboratory have suggested that there is a decreased availability of insulin in vitamin B₆ deficient rats (1). Although the role of zinc on insulin metabolism has never been determined, associations of zinc and insulin have been reported for more than 30 years. Recent in vitro studies (2,3) using isolated epididymal adipose tissue have clearly shown that zinc in concentrations in the range of those observed in human and rat plasma and higher inhibit insulin activity. Hsu (4) has reported that vitamin B₆ deficiency in rats results in decreased zinc content of plasma, liver, pancreas, and heart tissues. Work in this laboratory using a different experimental protocol appeared in disagreement with Hsu's conclusions. This report contains a study of the effects of vitamin B₆ deficiency on tissue zinc concentrations.

Since some pyridoxal enzymes are magnesium dependent and it has been shown that magnesium supplementation of vitamin B₆ deficient rations for rats prevents at least in part some effects of vitamin B₆ deficiency, [protects against calcium oxalate urolithiasis, increases urinary citrate and decreases xanthurenic acid excretion (5)] the effects of vitamin B₆ deficiency on tissue Mg levels were also investigated. To partially determine how specific the effects observed were, studies of the effects of thiamine deficiency and restricted feeding on zinc tissue levels and the effects of restricted feeding and vitamin B₆ deficiency on potassium tissue levels were also under taken.

Experimental. Weanling male Charles River CD rats were used in these studies. They were fed semipurified diets containing in percent: casein, 25; sucrose, 70.7; corn oil, 9; cod liver oil, 1; salts IV (6), 4; and choline, 0.3. Eight mg of riboflavin, 40 mg of niacin, 20 mg of calcium pantothenate, 1 mg of folic acid, 1 mg of menadione, 0.2 of biotin, 0.05 mg of vitamin B12 and when used 4 mg of thiamine hydrochloride, and 4 mg of pyridoxine hydrochloride were added to each kilogram of diet. The basal diet contained 40 mg of magnesium and 1.4 mg of zinc/100 gm.

The antiviral inhibitor substance produced by reovirus type 2 in a stable human amnion cell line (RA) was found to have several properties similar to interferon. It inhibited the growth of inducing virion as well as of heterologous virions, was not sedimentable at 45,000 rpm for 1.5 hours, was not neutralized by reovirus antiserum, was not toxic to cells, was stable upon treatment at pH 2, was inactivated by trypsin, and was ineffective in cells of unrelated species (mouse). These results indicate that the reovirus can induce interferon formation in cells and, like other virions, are also susceptible to the action of the inhibitor.

The ether, neutral, and phenolic fractions of the urine of normal individuals and of patients with carcinoma of the breast, cervix, gastrointestinal tract, and with various leukemias were analyzed polarographically. Reducible compounds were found in all the fractions. In the urine phenolic fraction the ratio (of the number of polarographically reducible compounds with an E _1/2 more negative than—0.5 V vs SCE to that more positive than—0.5 V) was significantly different for male and female patients with gastrointestinal cancer as compared to normal persons of both sexes. There was no difference in this ratio between the neutral urine fraction of normal persons and cancer patients. The ether fraction of the urine of males with gastrointestinal cancer had a much higher ratio (number of compounds with an E _1/2 more negative than—0.5 V vs SCE/number of compounds with E _1/2 more positive than —0.5 V) than that of the same fraction of normal males. Finally a significant difference was found in the ratio (of the number of reducible compounds with an E _1/2 more negative than—0.76 V vs SCE to that more positive than—0.76 V) of the ether fraction of normal male urine as compared with that of men with gastrointestinal cancer. Patients with various types of cancer did not excrete reducible compounds characteristic of any form of this disease.

The migration of mononuclear cells isolated from the spleens of rats with experimental autoimmune nephrosis was inhibited by extracts of rat kidney in both allogeneic and isogeneic strains. The migration was partially inhibited by the presence in the medium of rat liver but not by other tissue extracts. This effect appeared whether or not circulating precipitating antibody against kidney antigens could be deomonstrated. The specificity of this reaction was further demonstrated in aminonucleoside nephrosis which involves no known immunologic mechanism; splenic monocytes from these animals, as well as from control animals, failed to show any inhibition of migration in the presence of these same antigens. There was no correlation between the degree of inhibition and the severity of disease. The role, if any, played by this specific cellular sensitivity in the pathogenesis of autoimmune nephrosis remains to be elucidated.

A positively graded ovulatory response to human chorionic gonadotropin and pregnant mare's serum exists. Ovulation can be induced using much smaller dosages of HCG than have been commonly used heretofore. The relative ovulation-inducing abilities of HCG:PMS are approximately 5:1.

The “basal” rate of energy utilization, as measured by PC disappearance, in mouse anterior tibial muscles exposed to a N₂-IAA environment is about 0.25 μM/gm per min or 2.5 mcal/gm per min. This rate, as expected, is greater than the “basal” value for amphibian skeletal muscle and less than the “basal” value for mammalian cardiac muscle. By making certain assumptions about active ion transport and by using certain data from other investigators, the conclusion is reached that mammalian skeletal muscle sustains itself with a restricted energy supply much less well than amphibian skeletal muscle.

The buoyant density of rubella virus HA was determined to be 1.23 in Cs₂SO_4. A density of 1.33 was obtained in CsCl. Rubella virus HA, either Tween-80. ether-treated or untreated, and infectivity could not be separated on sucrose by rate zonal or density gradient techniques. These activities also could not be separated on CsCl and Cs₂SO₄ density gradients. The banding density of rubella virus HA preparations was unaffected by the type of cell culture used for antigen production; neither the passage level nor the strain of virus altered the point at which rubella virus HA banded in CsCl.

In separating the various effects of neomycin sulfate which may be exerted in systems designed to measure the activity of lipase, it is apparent that the antibiotic enhances the activity of both porcine and human lipase on an insoluble substrate. Additionally, it has the propensity to unstabilize emulsions. These effects are antagonistic and may account for divergent results previously recorded regarding neomycin-lipase interrelationships. Both the enzyme enchancing and emulsion unstabilizing properties of the antibiotic seem to reside in the presence of its free amino groups.

Treatment of human erythrocytes with tannic acid caused irreversible inactivation of the surface located acetylcholinesterase (ACHE). This effect was dependent on concentration, time, temperature, and pH. The ACHE inactivation was prevented by albumin but not by coating the cells with blood-group specific antibodies. The enzyme was not affected by the other phenolic compounds tested.

Electrolytes and amylase concentrations of rat saliva which is evoked by physalaemin, are virtually identical to those found in saliva evoked by stimulation of postganglionic cholinergic nerve fibers. Mean Na concentrations of parotid and submaxillary saliva were found to be respectively, 139 ± 4 and 32 ± 5, and K, 20 ± 4 and 46 ± 2; submaxillary flow rate was higher than flow rate from parotid, but both exhibited the high rates characteristic of cholinergic nerve stimulation; amylase concentration of parotid saliva was about 20 mg/mg. The secretion was, however, neither blocked nor modified in any way when the cholinergic blocking agent atropine was present. Furthermore, it was not modified by the presence of either α or β adrenergic blocking agents, or all three blocking agents simultaneously. An indiscriminate action on all receptor sites is thus ruled out. These data provide further evidence for the conclusion previously indicated (1-3) that this agent causes secretion by a direct action on gland cells that differs from that usually produced by autonomic transmitter substances.

The effects of sham operation, bilateral ligature of blood vessels joining the uterus to the ovary, and hysterectomy were determined on the length of pseudopregnancy in rats. Hysterectomy or bilateral ligature of blood vessels joining the uterus to the ovary significantly increased the length of pseudo-pregnancy when compared to sham-operated controls. These results indicate that the circulation between the uterus and ovaries is important in local control of luteal lifespan in the rat.

Under certain experimental conditions, a specific antigenic component of pregnant mare's serum gonadotropin (PMSG) reacted with a hyperimmune antiserum to ovine luteinizing hormone (LH) to form an early-appearing heavy precipitin line in agar gel double diffusion preparations. It was demonstrated that it interfered with the free diffusion of some unrelated antibodies in a multiple precipitating antigen/antibody system. In addition, removal of this line by treatment of PMSG with urea unmasked a well-defined line identified in another study as a contaminant of follicle-stimulating hormone (FSH). Failure of the latter line to appear under similar conditions when untreated PMSG was used was ascribed to the interference with diffusion of the FSH contaminant through the strong mechanical barrier formed by the specific component of PMSG.

Ouabain (1.0 μg/kg per min) administered iv for 60 min in dogs caused a significant decrease in glucose and glycerol plasma concentration. The same dose of ouabain significantly inhibited the lipolytic and glycogenolytic effect of epinephrine. These metabolic changes caused by ouabain in intact animals might be explained either by a direct inhibitory effect on the adenyl cyclase system or by an indirect effect mediated through insulin.

Studies of the comparative induction of interference by virus, statolon, and interferon in mouse embryo (ME) and African green monkey kidney (AGMK) cell cultures were performed. The major portion of the interference induced by different viruses in AGMK was characterized as being interferonlike. In both cell systems resistance was observed to develop before appearance of detectable interferon. Decline of resistance after removal from ME cells of unadsorbed statolon and NDV was slower than after removal of interferon. Possible significances of these observations are considered.

The kidneys were removed from 11 euthermic and 8 hypothermic (average rectal temp. 25°C) dogs and four slices of the tissue from papilla to cortex were taken from each kidney. The Na, K, and urea concentrations of the slice were then determined in slice extracts. Although urine osmolality was significantly reduced in hypothermia, the papillary Na and urea concentrations at a given urine osmolality were markedly higher in hypothermic kidneys than in euthermic, suggesting that the impairment of renal concentrating ability in hypothermia is partly attributable to a failure of the collecting duct urine to osmotically equilibrate with the surrounding media. On the basis of the latter finding, it is postulated that the action of ADH is less effective in hypothermia.

A study of 17 day fetal mouse blood revealed that almost all cell types were much lower than even in the newborn (2-3 days later) with the exception of the nucleated erythrocytes which constituted about 50% in both the males and females. The graphs of blood counts represent the averages of 25 mice for each age up to 9 weeks, and at 12 months there were no less than 20 mice, and at 24 months no less than 13 mice examined. Females tended to show a higher red cell and hemoglobin count than did males of the same age, and the males after 2 weeks had the higher leukocytes counts and after 5 weeks showed higher platelet counts. With respect to the neutrophils and lymphocytes there was little difference between the sexes.

Red cell production is progressively increased in male and female mice from birth until a plateau is reached at 8-9 weeks of age, while the hemoglobin shows a drop from birth through 3 weeks. Thereafter the hemoglobin values parallel those of the red cells possibly because of the decrease of red cell size and of polychromatophilia. Peak production in hemoglobin and red cell production was reached at 8 weeks of age, and this was generally a bit higher than the plateau established for the fully matured mouse at 12 months of age or the early senile mouse at 24 months. The average counts from week to week were rather steadily altered except for the leukocytes which curiously showed a decline at 6 weeks for both sexes. The decrease of leukocytes in both sexes from birth to the lowest point at 2 weeks of age, and the slight dip at 6 weeks, is caused by the steep drop in the neutrophils while the lymphocytes showed a steady increment from birth to a plateau at 8-9 weeks of age.

Isoimmunization of rabbits with semen, testis, and conceptus resulted in lowered embryo and fetal survival rates compared with immunization with seminal plasma and physiological saline. In situ recovery of ova from one oviduct of rabbits after insemination revealed a depression of fertilization rates in rabbits immunized with semen and testis and normal rates in rabbits immunized with conceptus, seminal plasma, and saline. Embryo survival rate adjusted for fertilization rate and the fetal survival rate were lowest in the testis group, intermediate in the semen and conceptus groups, and normal in the seminal plasma and saline groups. There was an increase in fertility of the 3 low-fertility groups from first to second breeding. Embryo survival rates of transferred ova were similar to those described above for the immunized groups. Fetal survival, however, was decreased only in rabbits immunized with testis. Isoimmunization with washed epididymal sperm induced antibodies and infertility in rabbits. In general, there was negative association between fertility and the serum titer of sperm agglutinins in rabbits immunized with semen and testis.