Experimental Biology and Medicine

In the attempted isolation from mammalian tissues of polysaccharides with endotoxic properties, only materials devoid of such activity were obtained under conditions in which exclusion of bacteria was demonstrated.

Rabbits which had been exposed to whole body x-irradiation showed a significant mortality rate following injection of 1.5 mg/kg body weight nitrogen mustard in contrast to the control group where no deaths occurred. This difference was noted even though bone marrows of the x-irradiated group were functionally normal when nitrogen mustard was administered.

Studies on the interactions between brucellaphage and Br. abortus, either under extracellular conditions or when the bacteria resided intracellularly within guinea pig mononuclear phagocytes maintained in vitro, showed: 1) inhibitory effects of untreated or UV-treated phage on the multiplication of brucellae, and 2) a lack of such effects in the case of heat-treated phage. These findings suggest an association between brucellacidal, or possibly brucellastatic, effects and phage protein. It was demonstrated that the effects on intracellular bacteria can occur even when exposure to phage is delayed until 2 hours after phagocytosis of the bacteria.

Previously, it was demonstrated that nephrectomy causes a 90% reduction in creatine synthesis in rats. In the present study, nephrectomy was found to cause a 37.5% decrease in methylation of guanidinoacetic acid (GAA)-2-C¹⁴. Rats loaded with creatinine by intraperitoneal injection demonstrated a 30% reduction in GAA methylation and a 45% reduction in total synthesis of creatine from glycine. It is concluded that chronic renal disease causes reduced creatine (and creatinine) synthesis both by loss of the renal mechanism and by interference with methylation. It is suggested that extra-renal (pancreatic?) synthesis may be relatively increased to partially compensate for these 2 inhibitory factors.

1) Hageman factor and plasma thromboplastin antecedent have been separated from human plasma and each other by ion exchange chromatography. 2) The defect in “exhausted plasma” is not corrected by purified PTA, purified HF, or a combination of the two.

Human growth hormone, 8 mg, has been administered intravenously to 6 normal volunteers, and blood samples have been obtained before and from 10 to 240 minutes after injection of the hormone. The growth hormone preparation used exhibited a definite early insulin-like effect both in terms of blood glucose and plasma free fatty acid levels. This was not associated with a change in serum insulin-like activity, as measured with rat epididymal adipose tissue. Four hours after growth hormone had been given, there was a mild hyperglycemic effect, and a marked increase in plasma free fatty acids, together with a small but significant elevation in insulin-like activity of serum. These observations are interpreted as compatible with an intrinsic in vivo insulin-like activity of the growth hormone preparation used, which could be either a property of the growth hormone molecule itself, or represent the activity of unrelated substances present in the preparation.

The newborn rabbit is limited in its capacity to accept the passive transfer of delayed hypersensitivity, as is the case in the human newborn. The ability to accept passively transferred delayed sensitivity develops by 4 weeks of age in newborn rabbits.

Preputial glands from rats, when incubated with C¹⁴ acetate show a marked incorporation of C¹⁴ into squalene, a sterol precursor, and little into sterols. In this regard, the preputial glands differ from many other mammalian tissues which show incorporation of C¹⁴ mainly into sterols and little into squalene. Human dermis, which contains sebaceous glands, is another mammalian tissue which shows preferential accumulation of the C¹⁴ into squalene. The mechanism of the difference is unknown, though it seems likely that factors necessary for conversion of squalene to sterols are missing in these tissues, or that an inhibitor of this reaction is present.

Differences in flocculating characteristics of erythrocytic stromata prepared from blood of uninfected calves and calves infected with Anaplasma marginale are described. The differences in flocculating properties are due to a change in isoelectric point brought about by the altered total phospholipid content.

Oxytocin failed to initiate lactation when injected 4 times daily into 45 mature female New Zealand White rabbits initially primed with estradiol or made pseudopregnant to develop their mammary glands. Prolactin injections initiated copious milk production in pseudopregnant rabbits. 2. Neither oxytocin nor vasopressin inhibited mammary involution or induced secretion in estrogen-primed rats, whereas injections of prolactin alone retarded mammary involution and addition of ACTH initiated mammary secretion. 3. It is concluded that oxytocin does not induce prolactin release in rabbits or rats.

A series of analogues of 1-methyl-3-nitro-1-nitrosoguanidines (NG), differing in the alkyl substituent, was tested against 2 mutants of E. coli strain S, selected for successively increasing resistance to NG. In general, increasing resistance to NG was observed to be accompanied by increasing resistance to all the nitrosoguanidines. When N-methyl-N-nitrosourea and N-methyl-N-nitroso - p - toluenesulfonamide were tested against NG-resistant strains of E. coli, it was found that the first step resistant mutant was cross-resistant. However, the second step mutant, derived from the first, was more sensitive to both of these compounds than was the first step mutant. A first step mutant of E. coli strain S, selected for resistance to N-methyl-N-nitrosourea, was resistant to NG. S-methyl-N-nitropseudothiourea did not inhibit E. coli strain S.

Survival of Cr⁵¹ tagged erythrocytes, autologously transfused, is severely shortened in monkeys maintained on Vit. E-deficient diets. Maximal survival times of 35, 45, and 49 days were obtained after monkeys had been on the diet for 349 or more days, as compared with about 100 days for replete controls. The shortened survival time, associated with anemia, could be reversed upon addition of Vit. E. The mechanism is discussed.

Cytochemical changes associated with psittacosis virus infection of tissue cultures were studied by acridine orange staining and fluorescence microscopy. Normal virus development was interrupted by the addition of 5-fluorouracil and 5-fluoroorotic acid which caused the accumulation of “fraudulent” RNA. It is suggested that the substituted pyrimidines interfered with development of intact virus through inhibition of protein synthesis.

Adenine enters Ehrlich ascites cells by simple permeability. The permeability constant for adenine is in the range 10^-4 to 2.10^-4 cm/min at 37°C and 2.10^-5 cm/min at 4°C.

The effect of D₂O on growth of 3 stable mammalian cell lines (HeLa, L and L-5178Y) has been investigated. As D₂O concentration was increased all cells showed increased water content and dry weight and a slower growth rate. Cytological investigations showed an increase in number of multinucleated cells and a moderate increase in sudanophilic material. No significant changes in nucleic acid content were demonstrable with the staining technics used.

Parenteral administration of 5 mg/mouse/day of diethyl-1, 4-dihydro-2, 4,6-trimethylpyridine-3,5-dicarboxylate (“collidine derivative”) in corn oil causes increased hepatic and biliary proto- and coproporphyrin levels. Hepatic coproporphyrin was found to be entirely Type III isomer. No green porphyrins are found in livers of animals receiving “collidine derivative.” Oxidation products of “collidine derivative” did not produce abnormalities in porphyrin metabolism within a 10-day observation period.

Glucose-6-phosphatase activity was estimated in various sheep tissues. It was appreciable in liver and kidney, detectable in intestine and salivary gland, but negligible in brain, muscle, stomach, rumen epithelium and mammary gland. The results suggest the relative importance of these tissues in contributing glucose to the blood in sheep. They indicate particularly the relatively small potential role of sheep intestinal wall as a source of blood glucose.

An ultra-sensitive method of assay for ribonuclease (RNase) has been devised using a biologically active ribonucleic acid (RNA) as its substrate. Infectious RNA isolated from foot-and-mouth disease virus was exposed to suitable dilutions of RNase, and the resulting loss in RNA infectivity was measured by plaque assay in calf-kidney cultures. Concentrations of enzyme as low as 0.1 mμg per ml were readily detected. The RNase level in sera from man and several animal species was determined by this method. Most sera contained 0.02 to 0.1 μg/ml of RNase, but guinea pig serum had a 20- to 30-fold higher content of this enzyme.

1. Highly hormone dependent mammary tumors were induced in rats by a combined treatment of a subthreshold dose of 3-methylcholanthrene and mammotropic hormone(s) (administered by grafts of a functional mammotropic tumor). 2. Growth of these mammary tumors was dependent upon continuation of stimulation by mammotropic hormone(s). 3. Grafts of the mammary tumors remained latent up to 262 days, growing only after mammotropic hormone(s) was administered. 4. Upon resection of mammotropic tumor, a primary mammary tumor promptly and completely regressed; 5 other primary tumors in rats carrying functional mammotropic tumors grew progressively.

The authors acknowledge the technical assistance of Mary Campbell.

1. An improved method for demonstration of lactic dehydrogenase after starch gel electrophoresis is described. This method employs a more sensitive tetrazolium and phenazine methosulfate. 2. Rat kidney contained 5 lactic dehydrogenase fractions which varied in cofactor requirements. DPN diaphorase was not demonstrable in any of these fractions, when reduced DPN was employed as the substrate, but was demonstrated in 2 additional bands. 3. The possibility is discussed that at least certain of the lactic dehydrogenase fractions from rat kidney exhibit electrophoretic heterogenity because of their association with various components of the respiratory system.

We are deeply indebted to Mrs. Julia Ternak for technical assistance.

Colistin sulfate, neomycin sulfate, polymyxin A sulfate, polymyxin B sulfate, and viomycin sulfate produced neuromuscular blockade when tested on sciatic nerve-gastrocnemius muscle preparations of the rabbit. Data obtained from the neuromuscular preparations were statistically analyzed and the relative potencies of the antibiotics in relation to colistin sulfate were determined. Polymyxin B sulfate was the most active antibiotic, approximately 1.5 times as active as colistin sulfate. Neostigmine methylsulfate antagonized the neuromuscular blockade produced by neomycin sulfate and viomycin sulfate, but was not an effective antagonist of the blockade produced by colistin sulfate, polymyxin A sulfate, and polymyxin B sulfate. Colistin sulfate, polymyxin A sulfate and polymyxin B sulfate do not produce contracture when administered to the chicken. The amino acids and other constituents comprising the various peptide antibiotics failed to show neuromuscular blocking activity.

Ten fractions have been isolated from abalone juice by ion exchange chromatography (Cellex-D) and tested for in vitro antibacterial activity against Staphylococcus aureus. Four fractions of effluent showed high activity. One active fraction was tested and found effective against Staphylococcus aureus, (including a penicillin-resistant strain), Streptococcus pyogenes, beta-hemolytic strain, and Salmonella typhi, and paratyphi A and B. The action for Staphylococcus aureus seemingly was bactericidal.

The authors express thanks to Dr. C. W. Hiatt for valuable suggestions and to Dr. R. C. Woodworth, W. C. Jahnes, E. C. Martino and J. L. Rogers for technical assistance.

Data were presented revealing that the paucity of growth of certain strains of yeast at 38°C is somehow related to a reduced ability to carry out the biosynthesis of pantothenic acid. Results were discussed in the light of the accepted pathway of pantothenate biosynthesis. Since the organisms are genetically endowed with the enzymic make up to synthesize pantothenate, this provides another example of the fact that environment plays an important role in genetic expression.

A method of purifying and crystallizing human myoglobin is described. This method depends on the observation that myoglobin is soluble in saturated ammonium sulfate solutions at pH 8.

Administration of diethylstilbestrol to intact rabbits, or to rabbits made cirrhotic with carbon tetrachloride, produced in all animals a marked bile duct proliferation and in one animal changes suggestive of neoplasia of the bile ducts. The possibility that hyperestrogenism may be responsible for the increased incidence of carcinoma of the liver in cases of liver cirrhosis is suggested.

A technic is described for avoiding loss of lecithin at point of origin during quantitative separation of phospholipids on silicic acid impregnated paper.

Methyl prednisolone and fluorometholone prevented the appearance of CxRP in rabbit serum following subcutaneous injection of mineral oil-Aquaphor emulsion. Fluorometholone was unable to block CxRP responses to 250 r whole body irradiation or to intravenous injection of Thorotrast or Varidase. It did block CxRP responses to intravenous injection of post irradiation seromucoid or TMV and the intravascular formation of antigen-antibody complexes. Under appropriate time dose conditions fluorometholone inhibited both antibody production and CxRP appearance while under other time dose conditions CxRP appearance was prevented without alteration of antibody production.

The results clarify a previous report(2) in which the possibility of a direct action of PABA on the adrenal gland was suggested as a more likely mechanism for induction of adrenal atrophy by this goitrogen. These data indicate that under conditions where administration of PABA produced adrenal atrophy, inhibition of the thyroid gland occurred as indicated by reduced thyroidal I¹³¹ uptake. Thus further evidence is presented to associate goitrogenic activity with ability to induce adrenal atrophy, though the possibility of a direct action of PABA on the adrenal gland coupled with thyroid inhibition can not be excluded.

Hydroxocobalamin, when injected intramuscularly into dogs, produces more prolonged elevation of serum Vit. B₁₂ than does injection of equal quantities of cyanocobalamin. The possible physiological significance of this observation is discussed.

Using an in vivo, acute loop technic it has been shown in the rat that absorptive activity for conjugated cholate increases nearly 4-fold as the small intestine is descended from duodenum to terminal ileum.

The occurrence of pyridoxine deficiency was determined in 14 patients with hyperthyroidism and 14 control euthyroid patients by the tryptophan load test. Urinary xanthurenic acid excretion following the tryptophan load test was significantly greater in hyperthyroid patients than in controls. Mean excretion of xanthurenic acid in hyperthyroid cases was 71.9 mg/24 hr with standard deviation of 56.0. Mean xanthurenic acid excretion of control cases was 15.4 with standard deviation of 6.1. After pyridoxine administration, mean xanthurenic acid excretion was 13.2 with standard deviation of 8.7 in the hyperthyroid cases, while in the control group, xanthurenic acid excretion was 12.5 with standard deviation of 5.2. Since pyridoxine is required for orderly catabolism of tryptophan and pyridoxine corrected this metabolic aberration, it is not unreasonable to state that in hyperthyroidism, the availability of pyridoxine is limited. Until the actual significance of these findings becomes more clearly defined, it would seem reasonable to suggest that use of pyridoxine in addition to anti-thyroid medication may prove of distinct benefit in treatment of the hyperthyroid patients.

Spinal cord section results in early adrenal ascorbic acid depletion. Recovery of normal levels occurs after 48 hours and is maintained for at least one week. Cord-sectioned rats (2- or 7-day), exhibiting normal initial adrenal ascorbic acid levels, respond to injected ACTH with ascorbic acid depletion similar to that observed in 24-hour hypophysectomized rats. Adrenal ascorbic acid depletion response was employed as an index of blood ACTH levels. Intact rats respond to electric current flow through either forepaws or hindpaws with equivalent ACTH release. Cord-sectioned rats respond to forepaw stimulation with ACTH release, but do not respond to hindpaw stimulation. Sham stimulation of intact or cord-sectioned rats does not release ACTH.

Marion D. Lipscomb rendered valuable technical assistance.

1. A pharmacological procedure is described which has clearly demonstrated the oral analgesic activity of carisoprodol (350 mg), acetylsalicylic acid (1200 mg), and codeine phosphate (30 mg) in man. Drugs were given with placebos in a “double-blind” investigation. Pain was induced by a high-frequency electronic stimulator applied to normal intact teeth. 2. Since the pain threshold end-point did not require activation of skeletal muscle, carisoprodol must have induced analgesia independently of its muscle relaxant action. 3. By this method, carisoprodol was 5 times more potent than acetylsalicylic acid in raising tooth pain threshold in man.

Systemic administration of ThioTEPA caused the development of hyperpigmentation of the ears, tail, and extremities of brown or black mice, but not of mice of lighter coat color or albinos. These changes resulted from increased numbers of dermal and epidermal melanocytes and increased deposition of epidermal melanin. Similar microscopic changes were seen to a lesser degree in the lighter-colored mice, but not in albinos. Gross or microscopic evidence of hyperpigmentation of the general body skin was not found.

In guinea pigs the effect of mycobacteria in adjuvant on production of precipitating antibody to a constant amount of antigen depends upon the immunization scheme employed. With multiple large injections of adjuvant, mycobacteria had no effect on antibody formation; with a single large injection, mycobacteria had a slight enhancing effect; and, with a single small injection, mycobacteria had an extreme enhancing effect. Mycobacteria had no effect on persistence of antibody production. There was a persistence of antibody production independent of the level of the antibody, with all the immunization schemes employed. Adjuvants containing mycobacteria produced considerably more extensive granulomata at injection sites than adjuvants lacking mycobacteria. However, there was no relationship between size of the granuloma produced at the injection site and antibody production. In guinea pigs injected with a small dose-of adjuvant containing mycobacteria, there was both a greater persistence of antigen int he local adjuvant depot and in the whole body and a larger antibody production than when mycobacteria were omitted.

The hydroxamate of anthranilic acid injected intraperitoneally into rats in a dose of 1.0 mg/g causes anesthesia lasting 10 hours or more. Recovery is apparently complete. One half this dose causes tranquilization and suppression of the conditioned avoidance response. The compound protects mice against twice the lethal dose of strychnine. It merely prolongs the life of animals given metrazol or picrotoxin. A dose of 1.0 g/kg injected intravenously in a dog has no effect on heart rate, EKG or blood pressure and only minor effects on respiration. Equivalent amounts of the hydroxamates of salicylic and benzoic acids have none of the pharmacological effects.

Our thanks are due to Mr. W. J. Novick and Dr. E. M. Scarpelli for assistance in this study.

Under continuous exposure to arsenicals in vitro, mammalian cells showed a remarkably wide range of tolerance to arsenic. To suppress growth of cells by 50% the absolute quantity of arsenic administered as atoxyl was up to 5000 times that in the form of arsenosobenzene. Pentavalent organic arsenicals with nitrogen groups were comparatively well tolerated in cell culture. In cells originating from different tissues and cultured in the same medium, degree of growth inhibition by a particular arsenical was fairly uniform. An adaptation of a DNA assay, useful in estimating cell populations in tissue culture, is described.

Dogs inoculated with live measles virus readily developed neutralizing and complement-fixing antibodies to measles. The dogs showed no signs of illness and did not spread the infection to unit contact litter mates. Following measles inoculation, 7 of 11 dogs developed low titers of canine distemper neutralizing antibody. All measles-inoculated dogs, including those with no demonstrable canine distemper neutralizing antibody, were protected against intravenous or intracerebral challenge with virulent canine distemper virus, while the contact controls, not receiving measles virus, became ill, and some died. The dogs which had received distemper virus alone developed distemper neutralizing antibody, but developed no measles neutralizing antibody.

Niacinamide in large doses (3-5 g) administered intravenously increased significantly cerebral blood flow and cerebral oxygen consumption, but decreased mean arterial blood pressure and cerebral vascular resistance in dogs. This drug in small doses had no such effects. The increase in cerebral blood flow was attributed to an increase in cardiac output associated with a decrease in cerebral vascular resistance.

A tissue culture adapted strain of Coxsackie virus A-10 has been obtained from primary human amnion cells inoculated with virus from a highly purified lot of suckling mouse grown material. The adapted virus (A-10TC) was neutralized by Coxsackie virus A-10 antiserum. It was no longer virulent in mice and caused gross cytopathic changes in primary human amnion cells indistinguishable from those changes caused by the polio viruses. Plaque assays of the virus were obtained on monolayer cultures of primary human amnion cells.

The authors wish to thank Mr. A. N. Koutsoukis for excellent technical assistance.

Tritiated thymidine, when given to young male rats 20 hours following partial hepatectomy in doses of 1.0 μc g body weight or greater, causes a transient inhibition of regeneration as mirrored in rate of increase of residual liver mass, mitotic rate, and incidence of abnormal mitotic figures. The importance of this effect in tracer studies is obvious.

1. Atropine and morphine effectively block angiotensin and nicotine-induced spasm on the isolated guinea pig ileum. 2. Mecamylamine, pempidine, pentolinium and hexamethonium blocked nicotine-induced spasms; however, only mecamylamine and pempidine blocked angiotensin spasms, but very high concentrations were required. 3. It is proposed that angiotensin acts on the post-ganglionic cholinergic mechanism of the ileum and that its site of action is probably peripheral to the ganglion.

In an in vitro test system at pH 7.4 it was found that degradation of serotonin by ceruloplasmin was markedly inhibited by sodium metabisulfite, ascorbic acid, glutathione, BAL, isoniazid, iproniazid, hydralazine, an analog of hydralazine (Ba-20523), isocarboxazid, thiocarbohydrazide, hydrazine hydrate, syrosingopine (Su-3118) and its isomer, Su-5171. Lesser effects were produced by reserpine, tetraethylammonium chloride, and a thiazide diuretic (Su-7075). A number of other sulfhydryl containing, chelating, anti-hypertension, and psychopharmacologic agents were without effect in the test system employed. It was not possible to correlate reaction inhibition with simple presence of sulfhydryl groups, the hydrazine nucleus, or with monoamine oxidase and diamine oxidase inhibition.

The authors wish to express their appreciation to Mrs. Claire Graff and Miss Faye Baldwin for technical assistance during these studies.

The amount of coenzymatically active Vit. B₆ was determined in plasma and leukocytes in patients with various diseases. Values for plasma and leukocytes varied widely in all groups examined. A statistically significant decrease of plasma B₆ was found only in patients with nutritional anemia and various forms of leukemia. However, the amount of pyridoxal phosphate was very low in individual patients with various other diseases. This may indicate a decreased intake of vitamin in some hospitalized patients or an increased need for it. B₆ values in leukocytes were consistently decreased in patients with various forms of leukemia. In contrast, cells from normal bone marrow and from tonsils showed significantly higher amounts of pyridoxal phosphate. The findings are discussed with reference to a specific disturbance in B₆ metabolism in some neoplastic conditions.

High fat feeding increases blood sugar, liver fats and tissue weights (liver and kidneys). It increases acetoacetate production by liver slices and utilization by kidney slices, but this high rate of utilization is not maintained on prolonged feeding (19 weeks). High fat feeding augments hypothyroidism and suppresses hyperthyroidism to some extent. A beneficial effect of hydrolyzed GCA feeding with high fat diets to hyper- and hypothyroid rats is indicated.

Forearm blood flow and venomotor responses to intravenous injection of mephentermine were studied in normal men. In each experiment blood flow decreased while arterial pressure increased. Venous pressure increased while forearm venous distensibility and venous volume decreased in each subject. In each case blood shifted from the forearm. These experiments indicate that mephentermine administration increases both peripheral resistance and venous tone in man.

The occurrence and distribution of serum enzymes and proteins in 7 mammalian species have been studied by means of starch gel electrophoresis and appropriate staining technics. Species differences were observed with respect to the presence, number, mobility, and concentration of components exhibiting various enzymatic activities. Similar observations were also made with respect to bands staining for protein.

The technical assistance of Carl Howell and John A. Cogswell is gratefully acknowledged.

The food intake and urinary excretion of glucose and NPN were measured daily in alloxan diabetic rats under the conditions of free access to food. The animals were then adapted to force feeding and fed in this manner twice daily the same quantity of diet they had consumed ad libitum. Insulin dosage was kept constant throughout. Under conditions of forced feeding, glycosuria increased by 215% and NPN excretion by 23%. These findings were reproducible when animals were recycled 2 or 3 times, demonstrating that eating patterns, not ebb and flow of severity of diabetes, were responsible for the findings.

Myotis lucifugus, simultaneously exhibits enlarged accessory sex glands and atrophied testis during hibernation. Evidence is presented for presence of an unidentified androgenic substance in the interscapular brown fat obtained from the bat during the hibernation season. The active substance isolated from the nonsaponifiable matter of one gram of brown fat elicits a growth response (rat seminal vesicle assay) equivalent to that produced by 676 μg of testosterone. The presence of an androgenic factor in brown fat suggests a unique role for this tissue in the sexual cycle of the bat.

We wish to acknowledge the valuable assistance of Messrs. Charles R. Cook and Robert Kolek.

Urinary excretion of 5-hydroxyindolacetic acid (5-HIAA) has been evaluated in rats treated with various histamine liberators. Excretion of 5-HIAA was unchanged, even when the liberator was administered repeatedly. When rats were given Compound 48/80 in increasing dosage during 8 days, they were entirely resistant to edema normally produced by other liberators. These animals, however, excreted as much 5-HIAA after injection of reserpine as did the untreated controls. These observations do not militate in favor of a serotonin mediation in development of anaphylactoid edema in rats.

Data suggest that major pathway of glucose oxidation in growing cultures of A. niger was via gluconate. The citric acid cycle played a minor role in young mycelium and inhibitory action of DNB appeared to involve further depression of formation of citric acid cycle intermediates with consequent decrease in rate of amino acid biosynthesis.

The effects of androsterone and testosterone propionate on atheroma and plasma lipid shifts induced by cholesterol feeding in cockerels were compared. Both compounds produced significant inhibition of coronary arterial atheroma. Aortic atherogenesis was not affected. The effects on plasma lipids were similar; non-significant reduction in cholesterol (C), significant decrease in phospholipid (P) with elevated C/P ratios. Testosterone propionate, but not androsterone, caused a significant reduction in testicular weight; both compounds stimulated comb growth. Further experimentation with androgens should provide useful information on the relationship between plasma lipid changes and inhibition of coronary atherogenesis.

The technical assistance of Astrida Kalnmalis and Margot Turner is gratefully acknowledged.

Intraperitoneal and intravenous LD₅₀ values of 12 strains of staphylococci, grouped according to free coagulase and the so-called clumping factor or bound coagulase, indicate that mouse virulence of S. aureus is influenced less by route of injection than by nature of the organisms injected. Mucin markedly enhanced mouse virulence for intraperitoneally injected coagulase positive, clumping factor negative staphylococci but not that of other strains. Neither free nor bound coagulase per se appeared to be a critical factor of virulence for mice.

The sequence of development of hypoproteinemia and hypercholesterolemia was studied in 15 aminonucleoside nephrotic animals. Hypercholesterolemia succeeds rather than precedes hypoproteinemia.

ACTH produces hypergranulation of the renal juxtaglomerular cells even in absence of pituitary and adrenal glands, and the chronic absence of ACTH depletes juxtaglomerular granules. The renal effect of vasopressin and of hydralazine, substances which produce hypergranulation of juxtaglomerular cells, is blocked by hypophysectomy. These findings support the concept that ACTH mediates many of the responses of the juxtaglomerular cells, and that the juxtaglomerular apparatus is an extra-adrenal site for the direct action of ACTH.

A highly reproducible and precise method for determination of “insulinlike” activity using glucose uptake by the epididymal fat body has been presented. It permits the assay of “insulin-like” activity in as little as 3 ml of serum with a calculation of relative potency with confidence limits as well as a test for assay validity.

The authors are indebted to J. L. Ciminera and A. G. Itkin for their assistance with statistical evaluation of data.

1. The properdin content of sera from germfree rats is lower than that of conventional animals. 2. Properdin titers of germfree animals gradually reach normal values after exposure to a conventional environment. 3. It is concluded that properdin level, like gamma-globulin level, is influenced by the normal bacterial flora.

Bovine kidney culture cells were found to be resistant to high concentrations of poisons of the glycolytic cycle, the citric acid cycle, and the cytochrome oxidation chain, as measured by respiration and cell loosening from the glass. Infection with FMDV had no influence on the results, and it may be that respiration of the cells is more dependent on other metabolic cycles. Iodo-acetate and fluoride, present in concentrations which caused large decreases in glycolysis, decreased virus production more than any other poisons. Malonate had an insignificant effect on virus production, while the larger effect of fluoroacetate was probably due to its action in removing viral precursors. The fact that maintenance of infected cells in an anaerobic environment did not affect virus production was a further indication that glycolytic sequences of the host cell are more essential to viral replication than oxidative sequences.

The presence of an inhibitor of the electron transport system of a number of mycobacteria and of lung homogenates of normal mice was found in normal mice and in those infected with M. tuberculosis H₃₇Rv. This inhibitor was previously found in tubercle bacilli grown in vivo and in lung extracts from infected mice. This work shows that the amount of inhibitor is greatly increased in animals infected with H₃₇Rv. The significance of these findings is discussed.

It was shown that in vivo grown tubercle bacilli are readily permeable to glucose and oxidize it to an even greater extent than those grown in vitro. It was therefore concluded that the apparent lack of respiratory response to glucose by in vivo grown tubercle bacilli was not due to either permeability factor or to inability of these cells to oxidize exogenous glucose. Possible causes for the apparent lack of respiratory response of LH₃₇Rv cells to glucose are discussed.

Two killer stocks of Paramecium aurelia, 51 and 299, have been cultivated axenically in a medium consisting of vitamins, a salt mixture, stigmasterol, proteose-peptone, an enzymatic digest of lactalbumin and the nondialysable fraction of a hot water extract of yeast. All components are neeessaty for growth of the ciliates and for maintenance of the kappa character. Differences in nutritional requirements of the two stocks have been noted. As far as is known, this is the first instance of cultivation of a kappa-bearing strain of Paramecium in bacteria-free medium.

Since this paper was submitted for publication, stocks 138 and 139 of P. aurelia, containing respectively μ and π particles, have been placed in axenic culture (medium No. 8 as given in Table II). The organisms have been maintained in tube culture for two months without loss of particles.

There appears to be no report regarding the influence of dietary protein on biosynthesis of ascorbic acid in the rat. Prompted by a chance observation that rats receiving a high protein diet excreted relatively large amounts of ascorbic acid in urine, we investigated the influence of dietary protein on the Vit. C status of the rat. In the first instance, the effect of variations in dietary protein levels was examined.

Materials and methods. Adult male albino rats (Wistar strain) with body weights in the range 180-200 g were employed in all experiments. Animals were distributed according to the randomized block design for the various test treatments. Diets fed had the following composition: Protein (Polson's lactic casein)—according to choice; fat (refined peanut oil)—10%; vitaminized starch—1%; salt mixture‡—2%; and corn starch—to 100.

The vitaminized starch contained thiamine hydrochloride 40 mg, riboflavin 80 mg. niacin 100 mg. pyridoxine hydrochloride 40 mg, calcium pantothenate 100 mg and folic acid 5 mg per 100 g. The rats were given necessary amounts of diluted shark liver oil twice a week to provide the requirements of Yit. A and D.

Cages with wide mesh wire bottoms which were well covered with resistant paint were used to house the animals individually. Urine was collected in brown glass bottles containing 10% trichloracetic acid (TCA) over 3-day periods. Collecting funnels and bottoms of cages were rinsed with 10% TCA solution at least thrice a day during the collection period. Urine collections were made up to known volume and aliquots used for estimation of total Vit. C (ascorbic acid. dehydroascorbic acid and diketogulonic acid) according to the dinitrophenylhydrazine (DNPH) method of Roe and Keuther(1). The same procedure was employed for estimation of ascorbic acid in tissues and blood.

The incidence of deaths due to arteriosclerotic heart disease is being compared in men recovered from myocardial infarction and treated 75 days or more with either Premarin or no estrogen (control), allocation of treatments being randomized. Survival rate in those receiving Premarin is significantly higher than in controls.

Normal control and fowlpox-infected chick scalp were separated by trypsin into connective tissue and epithelium. By appropriate technics the infected epithelial tissue was separated into surface epithelium and follicles. Comparison was made between normal epithelium and connective tissue and between normal and infected tissues with respect to certain lipids and incorporation of P³² into phospholipid. Distinct differences in phospholipid and total, free, and esterified cholesterol were found between normal epithelium and connective tissue. The above components were increased 2- to 3-fold at some interval of time in both infected epithelium and connective tissue. The specific activities of phospholipid P from all tissues were approximate at 3 days but that of normal epithelium and connective tissue fell approximately 50% at 5 and 7 days. In contrast the incorporation of P³² in infected tissues was 1.5 to 1.7 times that of normal controls.

Simultaneous dialysis of 2 diluted plasma samples in the same system at 37°C is an acceptable method for measurement of the relative amounts of corticosteroid binding protein present. Dissociation of the steroid-protein complex in undiluted samples is slow even at 37°. The kinetics of binding do not permit attainment of equilibrium at 4°, even when the plasma is diluted. The differences noted by previous investigators in comparison of pregnant and non-pregnant samples are explainable by this incomplete equilibrium.

A study has been conducted on factors which affect the “readily extractable” cholesterol concentrations in serum. The results showed that (a) in general, REC values increased as triglyceride and cholesterol contents of the serum and chylomicra increased, but a notable exception was high REC values in nephrotic subjects in whom chylomicron contents of cholesterol and triglyceride were low, (b) addition of the sodium salts of long chain fatty acids to serum prior to lyophilization markedly increased cholesterol extractability, (c) addition of crystalline human serum albumin to sera with high REC concentrations markedly reduced REC values; the albumin also reduced REC concentration in most normal sera but in no case did it completely disappear, (d) traces of moisture in the chloroform used for the extraction had little or no effect on most sera with low REC concentration but markedly increased it in sera with high REC concentration. It is suggested that the concentration of non-albumin bound free fatty acids may play a major role in regulating REC concentrations in various sera.

Material obtained from sonically disrupted HeLa cells by extraction of trichloracetic-acid precipitate with buffered saline was used to sensitize tanned sheep erythrocytes. Sensitized erythrocytes were agglutinated to high titer by anti-HeLa rabbit serum, but minimally by normal rabbit serum. Reactivity of the sensitizing extract was considerably reduced by treatment of HeLa cells with tannic, acid prior to disruption and extraction.

A simple hemagglutination-inhibition (HI) technic is presented which enables detection of inhibitors in patients' sera during diarrhea. These inhibitors thus far appear to be antigenically related to bacteria isolated from the patients' intestine at the same time and may well be substances derived from them.

The authors express their gratitude to Major D. H. Hunter for his help in the initial stages of this work.

An investigation was undertaken to determine the immunologic sequence of events during attacks of diarrhea associated with the presence of “normal” fecal flora, E. coli serotype 01 through 025, in the intestinal tract. It revealed that 12 of 30 or almost half of those infants from whom acute and convalescent phase sera were available exhibited a rise in hemagglutinin titer. At some time during their illness a greater number, two-thirds, had inhibitors present in their sera which were antigenically related to the E. coli serotype isolated from their fecal flora.

The authors wish to express gratitude to Dr. Antonio Ortiz, Chief, Dept. of Pediatrics, Municipal Hospital, San Juan, P. R., and to his staff, as well as to the staff of Children's Hospital, Washington, D. C., for providing the samples studied.

1. A 2-stage operation for production of pure complete hepatic venous outflow obstruction has been devised. Ten of 18 dogs survived from 18 to 270 days after operation. Crimped Dacron prostheses function satisfactorily in the low pressure venous system. 2. This preparation is presented as a satisfactory means of studying hemodynamic. hormonal, and chemical factors affecting development of ascites.

At low ionic strength, atabrine inhibition of myofibrillar ATPase activity was potentiated by Mg++. Ca++ counteracted atabrine inhibition in absence but not in presence of Mg++. The assumption of the occurrence of an Mg-atabrine coordination complex as the strong inhibitor is proposed as the best explanation of the experimental results. At high ionic strength, atabrine failed to inhibit Ca++ activated myofibrillar ATPase (myosin ATPase) with or without Mg++ being present.

Studies demonstrate that isopregnenolone-21-carboxylates are potent inhibitors of the renal electrolyte effects of deoxycorticosterone in adrenalectomized rats. Compound III, the most active in the series, was also effective against aldosterone. Available evidence indicates that the compounds affect electrolyte excretion by competition with aldosterone-like steroids at receptor sites in target tissue.

The authors are indebted to Drs. R. R. Burtner, V. A., Drill and B. Riegel for generous advice and helpful criticism in preparation of the manuscript.

A method for producing a high titer, specific anti-P. tularensis globulin is presented. Roosters immunized with living P. tularensis organisms produced an antiserum that is more specific and has a higher working dilution than rabbit and monkey sera. The suspending medium for the organisms does not affect degree and intensity of specific staining or working dilution of serum. Cross-reactions obtained suggest that a specific antigenic relationship exists between P. tularensis and the Pseudomonas used in this study. This cross-reaction was eliminated by specific adsorption with heterologous living organisms.

A relatively simple method for separation of fluorescein isothiocyanate-labeled gamma globulin from serum has been presented. The procedure has been applied to a number of labeled anti-bacterial and anti-viral sera, and the labeled gamma globulin obtained gave definite specific fluorescence when used in the staining procedure with markedly low background fluorescence.

The authors wish to acknowledge the technical assistance of Mrs. F. Malone.

1. Gentle stretching of the rabbit aortic strip produces rapid relaxation, and single doses can be applied at intervals as short as every 7 minutes. 2. Use of this preparation for a variety of purposes is described, including a 4-point bioassay of catecholamines and pharmacological studies, as exemplified by potentiation and inactivation.

The cholesterol ester, triglyceride and phospholipid fatty acid composition (gas-liquid chromatography) of aortic media, thickened intima and plaque material were determined in 6 human subjects. For comparison, the fatty acid composition of those fractions in serum was determined in 6 subjects (aged 55-70 years) with occlusive atherosclerosis in a good nutritional status. The triglyceride fraction of those tissues and serum were similar in their fatty acid composition. Some slight differences were noted in the fatty acid composition of the phospholipid fractions, most notably in the arachidonic and long chain fatty acids. The cholesterol ester fraction of the tissues studied showed the greatest differences in fatty acid composition. Both early and advanced plaques had significantly less linoleic and more oleic acid in that fraction than serum or media. Media and serum CEFA were similar in their fatty acid composition. The significance of these findings in relation to atherogenesis is discussed.

Respiratory deficient mutants of yeast, obtained by incorporating acriflavine into the growth medium, as well as S. aureus “mutants” obtained from ATCC were used for testing cancer chemotherapeutic agents. Although there is a feasible theory that supports the use of a respiratory mutant with a change in the mode of utilization of glucose, the procedure described appears to be of limited value. Examination of the S. aureus mutants (ATCC #13680-13681) from Cause lead to doubt of their authenticity.

Injection of complement (guinea pig serum) into the laboratory albino mouse failed to alter capacity of these animals to withstand parenterally induced bacterial infection with K. pneumoniae or S. typhimurium. Addition of complement to mouse serum failed to induce bactericidal activity in vitro.

Total exchangeable potassium, using K⁴², was determined in a group of female control subjects and 2 groups of patients with systemic lupus erythematosus. One of these groups was receiving adrenal cortical steroids including triamcinolone, the other salicylates and antimalarials only. Serial determinations were also done on one patient receiving long term steroid therapy. No significant differences in total exchangeable potassium values could be demonstrated among the 3 groups, and steroid therapy, including triamcinolone, did not appear to have an effect on total exchangeable potassium.

The authors are indebted to Juanita Lestina and Marva Jenkins for their technical assistance.

These results indicate that ESF is produced in BDF1 mice after a 6-hour exposure to 22,000 feet. It is further demonstrated that these mice have augmented response to hypoxia at time of increased endocrine function at puberty.

A persistent hog cholera viremia was produced in pigs from non-immune mothers that were 6 weeks of age, but not in pigs that were 3 months of age or in pigs from immune mothers that were 6 weeks of age. Although a similar condition of immunological tolerance has been reported for other animals, it was produced either in utero or shortly after birth. These pigs, therefore, appear unusual because persistence of virus was established at an older age. Pigs with persistent viremia failed to grow and showed elevated temperatures but survived for periods from 6 to 17 weeks. During this interval, persistent virus reverted to a fully virulent state. When they became moribund, virus titers of spleens from stunted pigs ranged from 10⁷ to 10⁹ in contrast to 10⁵ found in spleens from moribund pigs infected with virulent virus in the usual manner. The ability of hog cholera virus to transmit in utero and to persist in pigs after birth may have epidemiological implications.

A strain of hog cholera virus has been procured that produced CPE in tissue cultures of pig kidney cells. This tissue-cultured virus produced typical hog cholera when inoculated into susceptible swine and it was neutralized by immune sera.

Substitution of L-, DL- and D-valic acids in place of valine in the nutrition of 10 lactic acid bacteria has been investigated. Five of the bacteria were able to utilize either optical form of valic acid efficiently in place of L-valine, even though the requirement for this amino acid is highly L-specific. Three of the latter 5 bacteria, on the other hand, could be made to exhibit a marked preference for D-valic acid by selective antagonism of L-valic acid with the inhibitor, DL-a-hydroxybutyric acid. One other organism showed a similar preference for D-valic acid without use of the inhibitor, but in this case utilization of D-valic acid was relatively inefficient. The a-hydroxy acid-dependent organism, L. casei 280-16A, was D-specific in its utilization of valic acid as an a-hydroxy acid source, but was not optically specific in its utilization of valic acid as a valine substitute.