Experimental Biology and Medicine

The liver, but not the ovary, spleen or diaphragm inactivates homologous rat pituitary gonadotrophin in vitro. Liver tissue damaged by carbon tetrachloride loses this property altogether. Thyroxine prevents such inactivation of gonadotrophin when added to the incubation medium. Liver tissue from thyroidectomized animals causes more potent inactivation of gonadotrophin, which could be prevented by thyroxine both in vitro and in vivo. Liver tissue of intact thyroxine-treated animals does not inactivate gonadotrophin. The possible mechanism of such inactivation is discussed.

Day-old Japanese quail fed a zinc-low purified diet containing soybean protein were severely deficient at 4 weeks of age. The zinc deficiency was characterized by slow growth, abnormal feathering, labored respiration, an incoordinate gait, low ash content of the tibias and low concentrations of zinc in the livers and tibias. Control birds fed the same diet supplemented with zinc grew and developed normally. Successful use of the Japanese quail in this study indicates that the animal has great potential usefulness for nutrition studies.

In vivo lytic sensitivity of mouse erythrocytes to hyperbaric oxygenation paralleled their in vitro sensitivity to H₂O₂. In vitro lysis was associated with lipid peroxide formation. The in vivo and in vitro lysis and in vitro lipid peroxide formation were enhanced by vit. E deficiency and reduced by treatment of animals with a tocopherol acetate. It was suggested that hyperoxic hemolysis in vivo was a direct or indirect result of abnormal lipid peroxidation.

It has been suggested that the protein concentration of peritubular plasma may be an important determinant of sodium and water reabsorption by the renal tubule (1,2 −3). The effect of protein on excretion of water and electrolyte has been the subject of several investigations (4,5,6,7). Infusions of hyperoncotic albumin reduce excretion of sodium in normal man(4,5,7). In the dog, infusion of hyperoncotic albumin may not result in increased excretion of sodium(6)) whereas the infusion of iso-oncotic “plasma-like” solutions is associated with a natriuresis similar to that resulting from the infusion of equal volumes of saline(8). Since the excretion of sodium must be the difference between total filtered load and net tubular reabsorption, changes in filtration rate could mask changes in tubular reabsorption when plasma volume is expanded during the infusion of solutions containing protein. Thus, an increase in filtration rate associated with infusions of protein solutions may result in a natriuresis which could be independent of any effect that plasma protein concentration may have on tubular reabsorption. Despite the demonstration by micropuncture that active reabsorption by the proximal tubule of Necturus is not grossly influenced by the transtubular gradient of oncotic pressure (9), the possibility cannot be excluded that overall net tubular reabsorption by the mammalian kidney is in some way affected by the protein concentration of peritubular plasma. Even though the osmotic gradient for water produced by the protein concentration gradient between tubular lumen and capillary is too small to account for a major fraction of tubular reabsorption(9), changes in protein concentration could conceivably influence net reabsorption indirectly through such mechanisms as a change in interstitial volume or a redistribution of blood within the kidney. The present studies were designed to determine whether increasing the protein concentration of renal arterial blood during the natriuresis of saline loading results in any major change in sodium excretion.

Urethan was found to be eliminated from newborn SWR mice at about one-tenth of the rate found in adults, when injected at a dose of 0.5 mg/g body weight. The longer time during which urethan remains in the body may explain the greater carcinogenic action in newborn mice as compared with adults.

1. A method for analysis of microgram quantities of hippuric acid in plasma with good precision and accuracy is presented. 2. There is no significant difference in the clearance of hippuric acid and p-aminohippuric acid by the dog.

1. Prolonged inhibition of water diuresis occurred after bilateral cervical vagotomy in anesthetized dogs. 2. The urine became hypertonic to plasma, and the decline in urine flow was due solely to increased tubular reabsorption of solute-free water. 3. Glomerular filtration rate, renal plasma flow, and solute excretion did not change significantly.

Administration of ATP with ethionine enhanced, rather than counteracted, the ethionine-induced resorption of the rat litter. ATP decreased the maternal weight loss caused by ethionine, thereby indicating a lack of correlation between maternal and fetal effects of the compounds.

Muramidase activity in kidney increases progressively for 16 days after implantation of Walker-256 carcinosarcoma into rats. A similar progressive increase in rate of iron removal from plasma was found in these rats. At 16 days both muramidase activity and iron removal were increased 4 times above control values. A relation between kidney muramidase and hemopoietic activity is suggested.

Studies were carried out with chickens and rats to relate the ingestion of medium- and long-chain saturated triglycerides to the blood and liver cholesterol level. It was found that MCT significantly elevated the plasma cholesterol level of chickens compared with LCT. The reverse observation was made in rats where MCT was effective in lowering serum cholesterol. On the other hand, MCT-fed chickens and rats had a significantly lower liver cholesterol level than LCT-fed individuals. The significance of these findings with reference to the relationship between dietary fat and blood lipid concentration is discussed.

We woulil like to acknowledge with thanks, excellent technical assistance of Mrs. Olga Donis and Messrs. Neil Boorman and Hans 1, utz. Wc would also like to thank Mr. V. K. Babayan of the E. F. Drew Co. for a supply of the medium- and longchain saturated triglycerides used in these studies.

Concentrations of human C'1 esterase which are below threshold for direct enhancement of vascular permeability in guinea pig skin have been shown capable of generating a partially heat stable (56†C, 30 minutes) permeability factor from normal guinea pig serum in vitro. The action of this factor was blocked by treatment of test animals with an anti-histaminic drug, tripolidine. Generation of the factor from serum was prevented by inactivation of the enzymatic activity of C'l esterase with DFP or human serum inhibitor of C'l esterase. SBTI, without effect on C'l esterase, did not inhibit generation. Concentrations of C'l esterase which elaborated permeability activity in guinea pig serum also produced marked depression of the hemolytic complement titer of the serum. A relationship was also observed between the residual complement titers of heated sera and their capacity to produce a permeability factor upon mixture with C'l esterase. The data are consistent with, but do not prove, the hypothesis that C'l esterase mediates increased vascular permeability by interaction with the complement system.

1) The radioactivity of I¹³¹ labeled insulin injected by tail vein into mice was rapidly concentrated by liver and kidney, and converted by these organs from TCA insoluble into TCA soluble form. 2) Overnight fasting plus bromobenzene administration resulted in significant decreases in: (a) liver and kidney non-protein sulfhydryl (reduced glutathione) concentrations, and (b) abilities of these organs to convert radioactivity of administered labeled insulin from TCA insoluble into TCA soluble form. 3) Rapid repletion of liver and kidney GSH, previously depleted by use of bromobenzene, was obtained by use of L-cysteine. The accompanying bromobenzene induced impairment of mouse tissue abilities to solubilize radioactivity of labeled insulin was not restored by L-cysteine administration. 4) Benzyl chloride administration to overnight fasted mice resulted within 2 hours in marked decrease in liver GSH concentration, not accompanied by measurable change in insulin degradation. 5) It therefore appears unlikely that insulin degradation rate varies significantly as result of physiologic variations in intracellular GSH concentrations.

The approximate requirement of the guinea pig for pyridoxine with a purified diet containing 30% protein is 2.0 to 3.0 mg/kg of diet.

The author gratefully acknowledges the assistance of Woodrow Duvall in care of the animals and that of Esther Hurley for preparation of the vitamin pre-mixes.

The oxidation of intravenously administered carboxyl-labeled octanoic acid was estimated in 2 groups of rats by continuously measuring and recording the expired C¹⁴O₂ in a carbon-14 analyzer. In 5 normal rats an average of 27.4% of the administered dose of radioactivity was recovered in the expired air in one hour. In 5 rats in which the liver had been excluded from the circulation an average of 23.7% was recovered. These results suggest that extra-hepatic tissues of the rat are capable of oxidizing octanoic acid at nearly the same rate as the intact animal.

An agent has been isolated from Ha/ICR Swiss mice which induces lymphocytic leukemia in approximately 80% of Swiss mice injected at birth with cell-free filtrates. This agent retains its infectivity after storage at room temperature for 24 hours, at 4°C for 48 hours, at —20°C for 1 month, and in the CO₂ deep freeze for 1 month. There is some loss of infectivity under all storage conditions, greatest at —20°C for 1 month. Storage in the CO₂ deep freeze for 6 months reduces infectivity to about one-third. In all instances, latency is extended. The agent is inactivated at 56°C for 30 minutes, but is not destroyed by exposure to ether overnight. It can be neutralized by incubation with specific antiserum produced in rabbits. Sprague-Dawley rats and Rf strain mice are susceptible to infection when injected at birth; weanling Swiss mice are resistant to infection. Similarities and differences between this agent and other murine leukemia viruses are discussed.

Punned suspensions of human blood lymphocytes in 50% normal human serum were incubated at 37°C in small test tubes and the number of viable lymphocytes were counted weekly with a phase contrast microscope. The tubes were not shaken and the medium not changed. In suspensions from hematologically normal individuals, 24% of the lymphocytes survived 2 weeks and 5%, 3 weeks. In blood cell suspensions derived from 13 patients with chronic lymphocytic leukemia, 29% of the lymphocytes survived 3 weeks and some of the cells survived 7 weeks. The percentages of leukemic lymphocytes that survived 3 weeks varied considerably for the 13 patients and were directly correlated with the absolute lymphocyte counts of the patients.

Late lesions with the qualities of a delayed hypersensitivity reaction occur in the skin of rats injected intradermally with rabbit anti-rat tissue sera. These lesions are related to the fixation and persistence of antibodies directed against antigens in the epithelial and mesenchymal basement membranes of skin.

The effects on adrenal cortical function of removing the hypothalamus and other neural tissue above the midbrain were determined in 11 dogs in which the pituitary stalk had been sectioned previously and a plastic plate placed over the pituitary. There was a clear-cut response to stress 2 to 10 weeks after stalk section, and 17-hydroxy-corticoid output in the unstressed state 24 hours after adrenal vein cannulation (before brain removal) was low. However, following subsequent brain removal, 17-hydroxycorti-coid output was elevated. All neurohypo-physeal tissue below the plastic plate was atrophic in these dogs. There was no marked adrenocortical atrophy. The results suggest that the stress response in severely stressed dogs is not due entirely to CRF from the hypothalamus, and may be due in part to an ACTH-stimulating humoral agent liberated from traumatized tissues.

Poliovirus and SV40 in tissue culture fluids are neither removed nor inactivated by extraction with diethyl ether, chloroform or fluorocarbon. However, similar treatment with 1-butanol removes large amounts of SV40 from aqueous suspensions. The infectious titer of poliovirus is practically unaffected by the same procedure. That the action of 1-butanol on SV40 is one of extraction into the solvent phase rather than virus inactivation has been demonstrated in two ways. First, an addition of 1-butanol sufficient to saturate the aqueous suspension did not reduce the infectious titer significantly. Second, the virus was recovered quantitatively from an emulsion of equal volumes of 1-butanol and virus fluid. However, when the aqueous and solvent phases were separated, the titer in the aqueous layer was reduced by at least 4 logs (base 10). Extraction of samples of rejected Sabin polio vaccine/high in adventitious SV4Q, with an equal volume of 1-butanol and subsequent Seitz filtration reduced the concentration of the contaminating virus to a very low level. The infectious titer of 11 viruses, other than polio and SV40, was reduced on extraction with 1-butanol. It has not been determined whether the effect on these viruses was due to inactivation or removal from the aqueous layer.

Various surface-active agents were evaluated for their ability to bind heparin to graphite-coated plastic and thus produce a prosthetic surface resistant to thrombus formation. Both in vitro and in vivo studies indicated that only cationic agents bound heparin to graphite. After the positively charged cationic agent is adsorbed to the graphite surface, it can in turn bind the negatively charged heparin and thus present to the blood stream, at least initially, a surface rich in heparin.

1. The resistance of S. aureus to lysis by lysozome and trypsin was abolished by exposing growing cells to subinhibitory concentrations of nafcillin. This observation, together with the results of electron microscopic studies of nafcillin-treated cells exposed to maximal enzymatic action, reflect an actual interference with cell wall synthesis. 2. Although the precise cytological action of the subinhibitory levels of the penicillins on the integrity of the cell wall is not known, the limited lytic action of lysozyme on cells treated with oxacillin and cloxacillin, as well as the resistance of these cells to further lysis by trypsin, suggest at least a quantitative difference between nafcillin and the other penicillins in the disorganization of the cell wall structure. 3. On the basis of the experiments described, evidence is presented which indicates that subinhibitory levels of nafcillin produce an alteration of cell wall structure, thereby increasing permeability and rendering the cell wall and proto-plasmic constituents accessible to the action of lysozyme and trypsin.

Mice of the C57BL strain are normally highly resistant to the oncogenic activity of polyoma virus. Such mice were thymectornized at 3 days and injected with the virus at 4 days. Controls were sham-operated and injected with virus. Most of the thymectomized mice gained weight at the same rate as the controls and produced hemagglutination inhibiting antibodies. Pleomorphic tumors of the salivary glands developed within 2 months in 18 of 20 thymectomized mice and in only 1 of 16 controls at 4 months. It is suggested that the resistance of the C57BL strain to the oncogenic action of polyoma virus has an immunologic basis. Impairment of the maturation of immunologic faculty in mice of this strain allows the

Nuclear and total cell volume changes have been studied as a function of extracellular osmotic gradients in respiring rat liver slices. The nucleus and intact cell respond approximately as osmometers in a manner suggesting that the intracellular water activity is the same in the nucleus and cytoplasm of the cell. A mean increase of 37.2% or a mean decrease of 22.3% of cell water volume did not significantly change the cell O₂ uptake when compared with cell O₂ uptake at isotonicity although the O₂ uptake was significantly increased in cells respiring in hypertonic solutions when compared with cells respiring in hypotonic solutions.

Cardiac stimulating actions of selected sympathomimetic amines have been compared. In the case of the β-phenethanola-mines described here, this effect was increased by substitution of a cyclopentyl or isopropyl group in the N-center. Alkyl (methyl, ethyl or propyl) substitution of the a-carbon of the side chain markedly reduced cardiac stimulation and in the case of the N-substituted catecholamines described here, disclosed differences in potency for effects on force as compared to rate. The structural requirements for initiating sympathomimetic cardio-acceleration and for increases in the force of contraction differ from those which are optimal for bronchodilator action. The receptors responding to sympathomimetic amines may consist of a population differing in sensitivity to structural variation of the agonist (and possibly also to the various antagonists).

The incidence of aortic ruptures in turkeys induced by injection of DES in a liquid form was modified by dietary composition including high NaCl levels. Diethylstil-bestrol injections also caused hyperproteinemia, hypercholesterolemia, hyperlipemia and lowered blood pressure. High NaCl diets did not affect blood pressure.

The technical assistance of J. W. Carlisle and C. J. Miller is acknowledged.

The Na and K concentration of follicular fluid is similar to that of plasma. The K concentrations of diestrus, estrus, and pre-ovulatory rabbit follicles are not measurably different. Average [K] values are slightly above those for normal plasma. Na concentration of diestrous follicles was somewhat higher than that of plasma, whereas the Na content of fluid from estrous and pre-ovulatory follicles was somewhat lower.

A method which appears a practical and easy way to produce cloned cell lines is presented, with some observations and problems to be considered when cloning. Although the method attempts to derive cell lines from single cells by taking many precautions, it cannot be definitely stated that this goal is actually accomplished. It has yet to be demonstrated satisfactorily that a single diploid cell can in fact be cloned. The method, however, definitely produces cell lines which are derived from such a reduced population that morphological differences cannot be demonstrated within a clone, but can be demonstrated between cells from different clones.

The minimum protective dose of ethynyl estradiol (EE) which prevented pregnancy in all female rats treated before mating (MPDioo) was found to be 0.05 mg/kg body weight. This concentration of EE appeared to increase the sexual receptivity of some of the females. In attempting to clarify the mode of contraceptive action at the MPD₁₀₀, EE was found to inhibit pituitary gonadotropin release but not ova transport or ova nidation. This was concluded from the facts that EE inhibited compensatory ovarian hypertrophy of the unilateral ovariectomized rat but did not prevent implantation when given after coitus, although a high rate of resorption was noted. In addition/ the MPD₁₀₀ of EE did not inhibit formation of deciduomata.

Acid hydrolysates of melanoprotein isolated by differential centrifugation from amphiuma liver and from a human liver melanoma show them to be exceedingly similar with 20 amino acids identified. Bleaching of the melanoprotein by H₂O₂ fragmented both the melanin and the protein into a large number of unidentified ninhydrin reactive components. The bleached particle was positive to the PAS reaction.

Plasma chylomicra of fasting subjects with hyperlipemia were separated into α and β types by PVP gradient flocculation. Subjects with diabetes, CHO-induced hyperlipemia and the nephrotic syndrome had predominantly or exclusively β chylomicra (Group 1), while those with fat-induced hyperlipemia and severe hyperlipemia associated with alcoholism and probable pancreatitis had predominantly α chylomicra (Group 2). Consideration of the diet of each subject and of serial observations of a subject from each group during dietary manipulation leads to these conclusions: β chylomicronemia occurs primarily because of increased synthesis. Clearing may be grossly normal or moderately decreased at moderately elevated TG levels, and may become severely impaired at high TG levels, due to these levels per se. α-Chylomicronemia occurs primarily because of impaired clearing but is regularly accompanied by retention of small quantities of β chylomicra.

These studies have shown that the HeLa cell strain in fluid media is a satisfactory host cell system for arbovirus titrations as well as for neutralization tests. Of 94 arbovirus strains tested, 89 grouped as well as ungrouped viruses caused constant and regularly occurring destructive changes in HeLa cells. Partial destruction of the cell sheet was observed with one-third of the viruses tested, subtotal necrosis with another third and total necrosis with the remainder. In one instance, the cytopathic effect progressed only to a stage characterized by presence of multiple focal areas of necrosis. In 5 instances, no CPE was obtained. As far as tested, specific antiserum prevented development of CPE. Fifty-four strains were propagated serially without difficulty.

Fractions were prepared by 0 to 33% saturation with ammonium sulfate from the sera of two patients with lupus erythematosus who had circulating anticoagulants. The fractions prolonged the prothrombin and partial thromboplastin times of human but not bovine or canine plasma and inhibited the conversion of human but not bovine prothrombin to thrombin by “product 2.” The findings suggest that the circulating anticoagulants are species specific inhibitors of prothrombin.

Variants of herpes simplex virus were shown to differ in virulence for both newborn mice and hamsters. Intraperitoneal introduction of all variants led to a higher death rate than subcutaneous inoculation. The virulent variant caused transplacental infection of hamster, but not of mouse, embryos. Inoculation of 10⁵ plaque-forming units of the attenuated variants into newborn hamsters and mice within 24 hours after birth failed to induce tumors within a period of 6 months.

Germfree mice were found to be more susceptible than conventional mice to mouse-adapted influenza A strain PR8, although the difference in susceptibility is small. Work with more precise dosage methods is indicated to ascertain the nature of the difference. At 21 days after infection, the predominant leukocytic response in GF lungs was lymphocytic infiltration, while conventional mice responded with lymphocytes, polymorphonuclear leukocytes, and macro-phages. Gamma₂ globulin was not detected by IE prior to infection either in GF mice or in 20 of 23 GF mice at 3 weeks after infection. HI antibody was not detected in GF mice before and after infection, but was detected in conventional mice after infection. Addendum. Since the submission of this paper, we have been able to elicit HI antibody production in germfree mice after intra-peritoneal inoculation of five LD₅₀ of live PR8 virus.

Mercuric cyanide has a dual mechanism of poisoning. In dogs receiving this poison intramuscularly, the cyanide radicle may be detoxified by intravenous injection of sodium nitrite and sodium thiosul-fate, one after the other; and the mercury radicle, by prompt injection of BAL. The latter can be given first by intravenous injection, and repeated by intramuscular injection. The LD₅₀ of mercuric cyanide in dogs without treatment is 2.71 ± 0.17 mg per kg; and that with nitrite-thiosulfate therapy and BAL injections, 8.66 ± 0.66 mg per kg.

Yolk-sac suspensions of 3 strains of trachoma and 3 strains of inclusion conjunctivitis virus isolated and grown in this laboratory, and the TE 55 strain of trachoma, were tested for toxicity for mice following intravenous injection. All suspensions were equally toxic for mice, requiring between 3.3 × 10⁵ and 8.4 × 10⁶ ELD 50 per MLD 50, and all contained similar numbers of virus particles, an average of 6 × 10⁹/g of yolk sac. The egg-lethal titers of TE 55 virus suspensions were 10-fold higher than those of the other strains tested, but this strain was no more toxic for mice and possessed the same total number of particles per g of yolk sac. Biological differences of the strains, such as virulence for chick embryos and pathogenicity for the conjunctiva, do not appear to depend on the relative mouse toxicity of the strains.

The tissue distribution of Cs¹³⁷ after chronic administration is markedly similar in mice and dogs. The highest concentration is found in muscle, while fat, blood, skin, and bone have lower concentrations. Other tissues tend to conform to the concentration in the whole body, which is less than in muscle and more than in fat. Differences in whole bone concentration are due in part to the state (red or fatty) and quantity of marrow; for example, dog ribs have a higher concentration than dog femurs.

Administration of 20% THAM citrate (pH 8.50) to dogs on a meat diet results in increase of the base content of the blood and in the production of an alkaline urine. Between 60 and 70% of the THAM administered is excreted daily in the urine indicating intestinal absorption. This treatment which was well tolerated over a 3-month period could have useful applications in clinical medicine.

Alkaline extracts of the whole pancreas were found to be capable of rendering about one-half the hydroxyproline content of native soluble collagen into a dialyzable form. These extracts could also solubilize small amounts of dialyzable hydroxyproline from washed insoluble collagen. This apparent collagenolytic activity of pancreas extracts was not inhibited by EDTA, soybean trypsin inhibitor or mercurobenzoate. These fractions did not demonstrate any proteolytic activity upon denatured hemoglobin.

A series of 15 corticoids was assayed for anti-granuloma, thymolytic and liver glycogen deposition activities in adrenalectomized male rats. The order of relative potency for the majority of the steroids was similar in the three bioassays. Exceptions were 6α-chloro-9α-fluoro-16α-hydroxyprednisolone acetonide acetate and 6α-fluoro-16α-methylprednisolone which had greater relative potencies in the anti-granuloma and thymolytic assays, and 9α-fluoro-16α-hydroxyprednisolone acetonide which had a greater relative potency in the liver glycogen deposition assay.

The inactive precursor of a lipolytic enzyme which requires bile salts for activation in vitro has been identified in serum from dogs and cats, and bile from dogs. It is absent from the serum of humans, rabbits, rats and goats. The activated enzyme has been referred to as cholate activated lipase(CAL). Both CAL and its inactive precursor are heat labile. The pH optimum of CAL is 8.0-8.3. CAL rapidly hydrolyzes long and short chain fatty acid triglyceride. Eserine and hexestrol are inhibitors of CAL. Comparison of the effect of various potential inhibitors on CAL with their effects on pancreatic lipase and postheparin lipase suggest that the lipolytic enzymes from these three sources are not identical.

Data on the flow and composition of cardiac lymph have been collected in 9 dogs subjected to various types of increased left ventricular resistance loads. Aortic stenosis, intravenous norepinephrine and intravenous angiotensin II significantly increased the rate of cardiac lymph flow. No significant alterations in lymph composition were found. The data demonstrate that increased resistance to left ventricular emptying causes increased lymph flow. This effect is undoubtedly a reflection of increased capillary hydrostatic pressure and/or increased capillary permeability. The suggestively greater lymph flows with the use of angiotensin II may be due to increased capillary permeability as a result of increased cardiac effort without a proportional increase in coronary blood flow.

The toxicity of several preparations of “hematoporphyrin” has been studied in female ZBC mice and in rabbits. The MLD₁₀₀ in mice ranged from 0.2 to 0.7 mg per gram body weight depending on source of the porphyrin and route of administration. The intraperitoneal route was generally most toxic. A marked fall in total body oxygen uptake associated with severe hypotension and decreased blood flow occurred at lethal or near-lethal dose levels. In vitro measurement of oxygen uptake by mouse liver homogenates showed no effect at porphyrin concentrations up to 3 × 10^-4M (5 mg/gram of liver in a 10% liver homogenate) at pH 7.4 to 8.9 but 50 per cent or more depression at pH 6.5 to 6.6. Associated phenomena and some implications of these results in relation to studies on the modification of radiosensitivity by porphyrins are considered.

Of 18 compounds studied, only protoporphyrin significantly protected mice injected with lethal doses of “hematoporphyrin.” Results of separate experiments with 277 mice showed (a) 14% survival in 76 mice which received “Hp” and vehicle alone, (b) 47% survival in 90 mice injected with protoporphyrin in addition to the “Hp” and kept in the dark, and (c) 30% survival in 109 mice injected with both porphyrins and kept in a room illuminated with fluorescent lights. The marked fall in total body oxygen uptake associated with “Hp” administration was decreased considerably by administration of protoporphyrin.

1. 5-Hydroxytryptophan (5-HTP) given parenterally to cats exerts emesis. This effect is enhanced by various monoamine oxidase inhibitors and blocked by pentobarbital. 2. The emetic effect of 5HTP is antagonized by chlorpromazine and by compounds that deplete brain catecholamines (reserprine, α-methyl DOPA and α-methyl-m-tyrosine) suggesting that 5HTP acts through release of norepinephrine.

The entire population of peritoneal cells maintained in suspension is susceptible to cytolysis by isoantibody and complement in vitro. In tissue culture, the macrophages spread on glass and become resistant to cytolysis by isoantibody and limited quantities of complement. This contrasts with the marked susceptibility of the other peritoneal elements (lymphocytes, eosinophils, neutrophils and mast cells). In the presence of isoimmune serum and additional complement, however, macrophages are also lysed. Peritoneal macrophages are capable of phagocytizing isogenic tumor cells in vitro in the presence of isoimmune serum directed against both macrophages and tumor cells.

The high degree of flocculation of unsensitized particles of bentonite by cancer sera and not by normal or by sera from other disease conditions, may be of help in developing a test for cancer diagnosis. Heating of the sera at 50°C for 30 minutes is required to establish the difference in reactivity between cancer and other sera. The factors affected by such heat treatment are not yet clear.

Although the flocculation reaction occurs in cancer sera in a gradient and at high titers, no other indication could be found to show that this may be an antigen-antibody reaction, as could have been expected if the bentonite particles became coated with either excess antigen or antibody present in the sera. Also, addition of guinea pig complement did not restore the reaction in sera heated to 56OC for 30 minutes. Since in vivo antigen-antibody interaction has been considered to take place in some acute or chronic diseases such as systemic lupus erythematosus and glomerulonephritis, this theory should not be discarded.

Absorption of the α₂ globulin by the bentonite suggests an ionic type of reaction with some component of this fraction. Immunoelectrophoretic studies using antian globulin sera and bentonite may clarify this point. The low percentage of reactivity of normal and other sera as compared with cancer sera suggests the possibility of a test based on the effect of bentonite on specific components of the α₂ globulin.

Direct placental transfer of albumin-bound, labeled palmitate, stearate or linoleate administered to albino rats was very small, whether introduced by intraaortic, intra-amniotic or intra-fetal injection. Time-course experiments indicated a small indirect transfer of radioactivity initially assimilated by the tissues as TG or PL. The distribution of the assimilated FFA between tissue TG and PL was generally similar on fetal and maternal side.

Rats made less resistant to anthrax by means of a gluten diet have been studied as to their ability to elicit an adequate phagocytic response. When followed by the Rebuck technique, the gluten-fed animals do not sustain a total leukocyte response as do the controls. In addition, the relative number of monocytes is reduced by more than 50%. When followed by the ability to mobilize leukocytes in response to an intraperitoneal irritant, the less resistant group responded with a decreased rate as well as decreased numbers of leukocytes.

Intradermal injection of GSH into healing sites of Arthus phenomenon produces a reactivation of the inflammatory process having the same aspect as the original reaction. The interval during which reactivation occurs is correlated with the susceptibility to GSH of a protease extracted from the healing sites, which decreases with the age of the lesion. Cysteine is less active in vivo than GSH but no difference was observed between the in vitro effects of the 2 thiol compounds.

A decrease in total iron-binding capacity of the serum of rats was found to occur after injection of Escherichia coli lipopolysaccharide. The decrease was apparently not due to an increased blood volume and was accompanied by other alterations of the serum proteins.

1. Endogenous lipoprotein lipase activity was found in the plasma in 75% of normal subjects. 2. In vitro clearing of a coconut oil substrate is a much less reliable indicator of the presence of the endogenous enzyme.

3,5 diiodo-4-hydroxybenzene-sulfonamide was found to inhibit the growth of the influenza virus in chick embryo lung tissue culture. The compound appeared to exert a selective direct effect upon the extracellular virus particle.

The activity of phenylalanine hydroxylase from livers of young, male rats fed L-phenylalanine is suppressed by as little as 1% of the amino acid fed for 2 weeks. Larger amounts and a longer period of feeding result in further decreases in measured activities whether animals are pair-fed or fed ad libitum. Growth is depressed also with high levels of phenylalanine in the diet. The lowered activity of the hydroxylase may be attributable, at least partially, to a real suppression of enzyme synthesis.

A series of neutral ammo acids (1-leucine, 1-serine, dl-serine, 1-cysteine, 1-methionine, 1-threonine, glycine) consistently produced positive inotropic and pressor responses when administered intravenously and intra-aortically in vagotomized, open-chest dogs. The ability of DCI to decrease the positive inotropic response to the intra-aortic injection of 1-leucine and 1-methionine indicates that adrenergic receptor sites situated in the myocardium are involved. Acute bilateral adrenalectomy also decreased these positive inotropic and pressor responses, suggesting a close interaction between the adrenal system and the circulatory stimulant effects of these agents. The acidic amino acids, 1-aspartic acid and 1-glutamic acid, and the basic amino acid 1-arginine primarily depressed the myocardial force responses, with the blood pressure exhibiting a biphasic response.

Tetrodotoxin injected into the lateral cerebral ventricle of cats produced a reversible loss of volitional control. This syndrome differs from general anesthesia in that the EEG showed a waking pattern which could be “activated” by sensory stimulation throughout the course of motor dysfunction. The behavioral effects of lateral ventricular procaine mimicked those of tetrodotoxin, but procaine interfered with EEG activation due to sensory stimulation.

Experiments on adult female cats have demonstrated, as measured by roentgenographic appearance, microradio-graphic data, and tetracycline labeling, the influence of dietary levels of calcium on the structure of bone. It is shown that a low-calcium diet will produce osteoporosis in the adult cat and that this can be reversed by a high-calcium diet.

Hyocholic acid effectively prevented accumulation of dietary cholesterol in mice fed diets supplemented with cholesterol but had little or no effect on tissue cholesterol concentrations in mice fed normal diets. Treating mice with antibiotics largely reversed the effect of hyocholic acid, and permitted the accumulation of dietary cholesterol in hyocholic acid-treated mice. It was demonstrated that the hypocholesteremic effect is probably mediated by hyodeoxycholic acid, which arises from the action of intestinal bacteria on hyocholic acid. In animals which received hyocholic or hyodeoxycholic acid, cholic acid disappeared from the bile acid spectrum of the small intestine. Treatment with antibiotics restored cholic acid to the bile acid spectrum of hyocholic acid-treated mice but not to that of hyodeoxycholic acid-treated mice.

1. Millipore filtered tissue culture medium in which dog spleen cells have been cultivated in vitro was found to contain subcellular material with transplantation antigenic activity which could be concentrated by ultracentrifugation. Antigenic activity was demonstrated by the accelerated rejection by antigen recipients of a kidney homotransplant from the original spleen donor. 2. The antigenic material did not induce accelerated rejection of kidney homotransplants when injected back into the original spleen donor. 3. The Millipore filtered culture medium containing antigenic activity was apparently non-toxic when infused intravenously into homologous recipients.

Treatment of a 7 S anti DNP-BGG antibody with 2-ME resulted in a marked diminution of its ability to trigger positive PCA reactions in mice and guinea pigs; 3 to 4 times more ME treated antibody was necessary to obtain a reaction comparable to that produced by native antibody. In the reverse PCA technique, ME treatment was without influence on the skin reaction. Therefore, the diminution of the direct PCA reaction is not the result of an impaired antigen antibody reaction, but rather the consequence of a diminished tissue fixation of ME treated antibody. Complement fixing capacity of the antibody was practically abolished by ME treatment. From this fact and from results of PCA reactions in decomplemented animals, it follows that PCA reactions in mice and guinea pigs require little, if any complement.

Rats fed d1-ethionine for 25 days showed a significant decrease in small intestinal trypsin, chymotrypsin and lipase activity when compared to controls. The results suggest that levels of intestinal enzyme activity may be used as a quantitative indicator of pancreatic exocrine damage.

We wish to express our gratitude to Dr. David C. H. Sun for helpful criticism of the data and manuscript and to Miss Jeanne Chen for technical advice.

Rats with cobalt epileptogenic foci had a reduced convulsive threshold which could be increased toward normal by producing homolateral brain infarction. The alleviation of the epileptic state is assumed to be due to damage to epileptogenic areas or associated tracts in some, but not all, rats.

Encapsulated nodes of lymphoid tissue have been demonstrated in the upper thorax, neck and axilla of the amphibian, Bujo marinus. The nodes phagocytize India ink and form antibody to bovine serum albumin. The anatomical and functional characteristics of these lymph nodes indicate that they are comparable to lymph nodes of mammals.

Fatty livers produced by cerium injection have shown 3 main alterations in the liver phosphclipid pattern separated on silicic acid columns: (1) the appearance of a secondary peak in the polyglycerol phosphatide fraction (eluted with chloroform: methanol, 8:1); (2) a small but consistent increase in the phosphatidic acid-polyglycerol phosphatide fraction; and (3) a decrease in the lecithin fraction for all fatty livers measured in this study.

Nine continuous, 2 diploid and one primary cell line were successfully preserved by freezing in liquid nitrogen vapor at cooling rates of 1.4°C/min to 43°C/min, but poor survival was observed when cells were frozen by immersion directly in liquid nitrogen. The cells were frozen directly in a liquid nitrogen refrigerator by placing the ampuls at different distances below the lip, demonstrating that elaborate and expensive cell preserving equipment is not required. The plating efficiency technique was shown to be the most sensitive indicator of cell damage.

A modification of the Querido bio-assay for thyrotropin is presented along with log dose-response curves for USP Reference Standard Thyrotropin in 2 different strains of mice. The immimobiologic effects of antisera to bovine thyrotropin (TSH) on either endogenous or exogenous TSH were determined in guinea pigs, intact or hypophysectomized rats and mice. In thyroxinized guinea pigs and thyroxinized intact or hypophysectomized rats, passively administered rabbit antiserum to bovine TSH neutralized the biologic activity of exogenous bovine TSH. Rabbit antiserum passively administered to guinea pigs, rats or mice inhibited the effects of propylthiouracil (PTU) on goitrogenesis and on radioactive iodine uptake, indicating biologic neutralization of endogenous TSH in these species. These findings suggest a molecular similarity of guinea pig, rat and mouse thyrotropin.

Actively or passively established tuberculin hypersensitivity in Strain 2 guinea pigs is suppressed by methotrexate treatment for as short a period as 15 days. The suppression of this immune response does not seem to be due to a lymphopenic or anti-inflammatory effect of the drug.

These results indicate that a 7S γ2 rabbit antibody having a low affinity for its antigen was equally as efficient as a 7S γ2 high affinity antibody in producing cutaneous anaphylaxis in guinea pigs when abundant antigen was present. However, when the concentration of antigen in the reaction was reduced the antibody of low affinity was not as potent as the antibody of higher affinity. A possible explanation of the inefficiency, marked by a slow rate of development and at times less severe anaphylactic reaction, was found to lie in a relatively slower rate of association noted between the absorbed antiserum and its antigen (Fig. 1). This slower rate of association may well bear causal significance when considering the finding that when one merely increases the concentration of antigen in the circulation, the rate of anaphylactic response of the slowly associating antiserum was increased to equal that of the control, rapidly associating antiserum (Table I). These latter conditions favored more rapid and complete binding of the lower affinity antiserum by antigen. These findings fail to support the possibility that the absorbed, slower associating antibody was less capable of “fixing” to tissues or for other reasons less capable of reacting, since merely by raising the dosage of antigen it became fully reactive in vivo. Other tests failed to show significant differences between antibodies in the 2 antisera: both were 7S, γ2 globulins as determined by density gradient ultracentrifugation and radioimmunoelectro-phoresis. When mixed with antigen they both fixed abundant hemolytic complement. The antigen-antibody dissociation rates of the 2 antisera were not appreciably different considering the amount of time required to demonstrate as much as 15-20% difference. Further, in other unpublished studies in this laboratory, results similar to those herein reported have been found using anti BSA obtained from individual rabbits after primary immunization and absorbed once with a small amount of antigen. This diminishes the possibility that the results could be explained by some unique quality of the absorbed antiserum.

The role played by low affinity antibody in immunopathologic lesions may be considerable. In serum sickness the antibody responsible is without much doubt of relatively low affinity. In this disease alone such diverse lesions as endothelial proliferation, increased glomerular permeability, vascular necrosis and anaphylactic alterations in vessels take place. Anaphylactic reactions bringing about an increase in vascular permeability may be of special importance in causing circulating antigen-antibody complexes to localize in vessel walls to produce the lesions. Anaphylaxis may thus act as a trigger mechanism in initiating the development of the disease (11-13). The extent to which low affinity antibody may influence the development of related lesions has not been determined. These studies, along with those of Osler(l), indicate that low affinity antibody may be less efficient in precipitating anaphylactic responses under conditions of low concentrations of antigen. However, when an optimum of antigen is present such antibody is fully reactive.

Characterization of the diuresis experienced by a 19-year-old recipient of a renal homotransplant revealed certain features in common with the diuresis seen in experimental osmotic diuresis in normal subjects, and with the diuresis seen following relief of urinary tract obstruction and in other salt-wasting syndromes. The preponderant unreabsorbed solutes were sodium and chloride rather than urea. Since the salt wasting state did not persist, it may have been due to volume expansion and to an enhanced filtration rate, rather than to a tubular reabsorptive defect.

Rat myocardial preparations were stimulated to contract at one per second in an oxygenated Ringer's solution. The effects of 0%, 2% and 5% CO₂ on the magnitude of the isometric developed tension were studied during a 3-hour experimental period. It was noted that muscles aerated with a gas mixture containing 2% CO₂ revealed not only a constant developed tension but also a small variation in contractile response during the experiments. The detrimental effects of the other percentages of CO₂ on the developed tension were not immediately evident.

(EC), a Bartonella-like organism existing as a widespread latent infection in rodents, has been separated from other known infectious contaminants so that its influence on plasma LDH (lactate dehydrogenase) elevation and other pathological properties could be studied and compared with those of an LDH elevating viruslike agent. Distinct differences in their LDH elevating characteristics were demonstrated. The viruslike agent induces a high and sustained elevation while that produced by EC is usually unremarkable in magnitude in normal intact animals, and is invariably transitory. In combination, the two infectious entities act synergistically to produce higher plasma enzyme elevations than their additive separate effects. Recent suggestions that EC may be responsible for the LDH elevations originally reported appear to be in error.

Histochemical studies of 12 strains of diploid human fibroblasts derived from newborn foreskins revealed marked variation of alkaline phosphatase activity within all populations, whether classified as constitutive for the enzyme or inducible by substrate. Variation was also apparent in non-induced populations having low activity and in “self-induced” populations having high activity.

The antibody response measured by HI and CF tests in 7 goats, 2 sheep and a Mexican burro was followed after inoculation with influenza type A, B and C viruses. Volume production of useful hemagglutination-inhibiting antisera in these animals is possible. Nonspecific inhibitor to Asian influenza virus in normal goat and burro sera was eliminated by periodate treatment.

An enzymatically unhydrolyzable ester of cholesterol, cholesterol-4-C¹⁴ α,α-methyl ethyl caproate, has been synthesized. This ester was infused in micellar dispersion into the duodenum of lymph fistula rats. After 12 hours, 98.1% of the administered ester was recovered unaltered from the feces and intestinal contents. There was none of this ester in intestinal tissue or lymph. The data provide convincing evidence that the initial transfer of cholesterol into mucosal cells is specific for free cholesterol, and that cholesterol esters are not absorbed from the intestinal lumen prior to hydrolysis.

Dogs were bled 40% of their blood volume (32 ml/kg). Thereafter, at intervals to 52 hours, the in vitro uptake of tritiated thymidine by erythrocytic precursors of the marrow did not differ from the controls as measured by percent labeling and grain counts in morphologically differentiated cells. Also, no alterations from control values were found with regard to morphological distribution, relative numbers of the marrow normoblasts, and mitotic index. The data suggest that the acute blood loss did not influence detectably the chosen parameters for the time intervals studied.

Several glucocorticoids were administered orally or intravenously to fasted, intact dogs and their effects on circulating eosinophils determined. The calculated eosinopenic potencies were not quantitatively related to those reported in man. The Possible implication of these data with respect to dosage selection of steroids for studies in dogs is briefly discussed.

F₁, F₂, first, second, and third backcross generation mice resulting from crosses of A2G with A, and F₁ F₂ and first backcross generation mice resulting from crosses of A2G with C3H were challenged with neurotropic influenza A virus, strain NWS. The results were compatible with the hypothesis that a single dominant autosomal allele was responsible for resistance to the virus. The symbol Mx (myxovirus-resistant) is tentatively proposed for the allele involved.

Exposure of cultured human amnion cells to some tetracycline compounds produced slight suppression of growth. When drug treated cells were subjected to irradiation with short-wave (2537Å) UV light, those to which demethylchlortetracycline had been added were severely damaged, the effect being moderate at a concentration of 1.6 μg per ml of the drug and marked at a level of 6.4 μg per ml. Methacycline and tetracycline were without significant effect. Exposure to long-wave (3960Ä) UV light did not increase the slight damaging effects of the antibiotics on cells. The phenomenon was observed at concentrations of demethylchlortetracycline commonly present in the serum of patients treated with this agent and appears to be due to cellular injury produced by irradiation on cells which have been sensitized by exposure to drug.

The distribution in pregnant mice of ¹⁴C-labelled thalidomide and glutethimide was investigated autoradiographically. After administration of ¹⁴C-thalidomide the radioactivity was found to penetrate through the placenta and to be distributed very evenly in both mother and fetuses. After administration of ¹⁴C-glutethimide a specific accumulation was seen in fat, brain, myocardium, blood vessel walls and mammary glands.

A new immunologic adjuvant, adjuvant 65, is described and the results of extensive tests of polyvalent influenza virus vaccine in the adjuvant are presented. The vaccine consists of a water-in-oil emulsion in which the adjuvant portion consists of peanut oil, mannide monooleate and aluminum monostearate. The adjuvant effect measured in terms of hemagglutination-inhibition antibody responses 28 days after vaccination in mice or guinea pigs is roughly equal to that obtained with Freund's incomplete mineral oil adjuvant. Adjuvant 65 influenza vaccine showed striking enhancement and long-term persistence of antibody in guinea pigs, rabbits and monkeys compared with the corresponding aqueous vaccines. The findings in histomorphologic-pathologic studies in animals and the clinical and serologic results obtained in large-scale studies in man are presented elsewhere. Adjuvant 65 shows promise of meeting the present urgent need for a safe and effective adjuvant for enhancing immunologic responses to viral and other vaccines and possibly also for treating allergic disease.

A new immunologic adjuvant, called adjuvant 65, was assayed for toxicity, safety and irritancy in several species of animals. Clinical, gross and histomorphologic examinations were made. The reactions which resulted from adjuvant 65 vaccines were mild and inconsequential and far less than were obtained in comparable tests with Freund's incomplete mineral oil adjuvant vaccine. The comparative pathology for the 2 different kinds of adjuvant is presented and the theoretical and practical advantages of adjuvant 65 are discussed.

The effects of estrogen, or pro-lactin and STH, on mammary tumor induction by a single feeding of 20 mg of 7, 12-dimethyl-1,2-benzanthracene (DMBA) in ovariectomized Sprague-Dawley rat was investigated. After DMBA administration, mammary tumor incidence in sham-operated controls was 100%. In ovariectomized rats, DMBA failed to induce any mammary tumors by the end of 40 weeks. However, daily treatment with 1 or 10 μg estradiol, for 7 days before and after (total 14 days) DMBA administration, induced mammary tumors in 33 and 23% of rats, respectively. Similar treatment with 1 mg STH and 1 mg prolactin, twice daily for 14 days, elicited mammary tumors in 44% of the rats, while continuous daily treatment with increasing amounts of prolactin and STH for 75 days, resulted in a 66% incidence of mammary tumors. The average number of tumors per tumor-bearing rat was lower and mean tumor latency was longer in the ovariectomized rats given estrogen or STH and prolactin than in sham-operated controls. All tumors were mammary carcinomas. These results indicate that the 2 pituitary hormones, STH and prolactin, can induce mammary carcinogenesis in DMBA-treated rats in the absence of the ovaries. They also suggest that these 2 hormones, as well as estrogen, participate in the initiating and promoting phases of mammary carcinogenesis; and that the effects of estrogen may be partially mediated through stimulation of STH and prolactin secretion by the pituitary.

Similar increases in urinary excretion of taurine follow drug-induced muscle necrosis and x-irradiation. The excess taurinuria appears to be unrelated to altered renal function but is probably a non-specific manifestation of increased tissue catabolism.

In rat liver homogenates 22,25-diazacholestanol blocked to the same extent the incorporation into cholesterol of 2-C¹⁴-acetate, 2-C¹⁴-mevalonate and of 26,27-C¹⁴-desmosterol. Thus, direct evidence is provided for a triparanol-like interference with hepatic cholesterogenesis in vitro. This is in agreement with the finding of appreciable desmosterol levels in humans and rats treated with 22,25-diazacholestanol per os.

Triparanol suppressed the synthesis of total lipids in 3 strains of Tetrahymena pyriformis. Strain differences were noted when triparanol inhibition was reversed with oleic acid or by prolonged incubation. Supplements of short-chained carboxylic acids influenced the effect of triparanol on the total lipids, the percentage composition of saturated and unsaturated fatty acids and the specific fatty acids synthesized. There was an increase in the content of odd-numbered and branched-chain saturated fatty acids synthesized with triparanol plus pro-pionate or a-methyl-n-butyrate supplements, and an increase in the percentage of oleic acid synthesized with triparanol plus acetate supplements. Triparanol may act by inhibiting the ability of T. pyriformis to demethylate and to dehydrogenate saturated fatty acids in formation of unsaturated fatty acids.

The electrophoretic distribution of serum proteins has been observed in a variety of clinical conditions. Severe malnutrition, wasting diseases and liver cirrhosis result in a decrease in albumin, attributed to impaired synthesis, and usually an elevation of the globulins. These changes occur in so many conditions that they are seldom useful for diagnostic purposes. The more specific hyperglo-bulinemia of multiple myeloma has been found to consist of a series of protein molecules in the γ group(l).

Analysis of the serum protein in human beings with diabetes mellitus shows a decrease in albumin and an increase in α₂ globulin. Less consistent changes occur in the β lipoprotein and γ globulin(2). Particular interest surrounds these changes as they may be related to the abnormalities of lipid and cholesterol metabolism found in these patients. Serum protein changes in alloxan-diabetic rats are not entirely comparable to those changes due to diabetes mellitus in human beings(3), but since insulin is necessary for the protein anabolic effect of growth hormone and has been shown to cause an increased incorporation of amino acids into the protein of alloxan-diabetic rat liver(4), the changes in serum protein may result from the lack of an insulin effect on liver protein synthesis.

Studies of liver function after a portacaval shunt in rats reveal changes in plasma proteins that are quite similar to those found in human diabetics and suggest that the biologic effect of insulin on the liver may be a significant factor in these changes.

Six months after end-to-side portacaval shunt, heparinized capillary blood was taken for protein analysis. Six lambda was applied to Schleicher and Schuell 2043A filter paper. After the run, the strips were stained with bromphenol blue and electrophoretic curves for the components determined on a densitometer (Spinco Analytrol).

Reciprocal tests for genetic recombination by transduction have divided 19 mtl— mutants of Staphylo coccus aureus strain 233 into 3 genetic groups. One group was composed of 4 mutants having a pleiotropic defect in their fermentation of carbohydrates. One group was composed of 2 mutants postulated to be defective in their phosphorylation of mannitol. The remaining mutants, postulated to have defects at specific steps in the utilization of mannitol, were linked genetically into the third group.

Viral inhibitory factors were isolated from several different bacterial species including representatives of both gram negative and positive groups. Two of the factors were found to prevent viral cytopathogenicity even when added as late as 18 hours after viral inoculation. Trypsin sensitivity indicates that these inhibitors are probably proteins. Evidence is presented that viral inhibition appears to occur during the intra-cellular stages of viral replication. Each factor has a sharply specific spectrum of antiviral activity. This specificity of action may reflect blocking of cellular metabolic pathways necessary only for the replication of the virus affected.