Experimental Biology and Medicine

Administration of pyridoxine to the four children with homocystinuria who had been on a diet low in pyridoxine resulted in ablation of a general but slight aminoaciduria and a striking reduction in homocystine excretion. Further studies on two of the children showed increases in serum oxalacetic-glutamic transaminase and reduction in xanthurenic acid excretion after tryptophane loading. The marked decrease in homocystine excretion after pyridoxine administration was correlated with increased excretion of inorganic sulfate. The total lack of activity for cystathionine synthetase in liver biopsy tissue after pyridoxine administration to the children even when pyridoxal phosphate was added to the enzyme assay system, lead to the conclusion that the metabolism of methionine sulfur to inorganic sulfate must have been increased through one of the alternate pathways of metabolism. Administration of vitamin B₁₂ and folic acid resulted in a decrease of excretion of homocystine though not as marked as the effects of pyridoxine.

Respiratory fluctuations in ventricular outflows were measured in unanesthetized dogs by simultaneously recording aortic and pulmonary artery flows and intrathoracic pressure. The respective stroke volumes were computed and the beat by beat values were plotted over a series of respiratory cycles. The data showed that in the early postoperative period there was an imbalance between the ventricular outflows during normal breathing. This imbalance was characterized by an increase in right ventricular stroke output and a decrease in left ventricular stroke output during inspiration with a reversal during expiration, that is, they were out of phase. However, this temporal imbalance diminished with recovery from the surgical procedure, so that by the fifth postoperative day the right and left ventricular stroke outputs were essentially in phase. Therefore, our data indicate that the principle of restoration to correct a temporal imbalance between the two ventricular outflows with normal spontaneous breathing is of minor importance.

The daily rhythm of plasma tyrosine that occurs in rats fed ad libitum was altered by controlling the time of food intake. The experimental data suggested that (1) absorption of tyrosine from dietary protein and (2) metabolism of tyrosine by liver tyrosine transaminase may be important mechanisms in the daily rhythm of tyrosine. A normal daily rhythm in serum tyrosine occurred in human phenylketonuric subjects lacking phenylalanine hydroxylase, so variations in the hydroxylation of phenylalanine to tyrosine probably do not play a part in the tyrosine rhythm. In rats fed ad libitum, there was little or no daily variation in plasma phenylalanine, but some changes were produced by restricting the period of food intake. Control human subjects exhibited a phenylalanine rhythm identical to that for tyrosine in serum.

The present study, with conscious dogs, indicates that both pancreatic flow and pancreatic protein output can be affected by the nature of the taste stimuli. Water and a sucrose solution mixed with a basal diet produced a similar and significantly greater stimulation of flow than did citric acid or quinine, which in turn were similar. Although it appeared that protein secretion was more strongly enhanced by sucrose solution than by water and solutions of citric acid and quinine, it was not clearly established that sucrose solution and water were really different. The flow responses for the four taste stimuli were highly correlated with the readiness with which the various food mixtures were accepted by the dogs.

Although connective tissue and collagen induced the formation of huge platelet clumps, they had different effects on lactate production. With collagen, platelet lactate production was linear for 60 min, as compared with 45 min for control platelet suspensions. The rate of lactate production increased during actual aggregation and was similar to that of the control thereafter. Collagenase-treated collagen behaved like the control samples. In contrast to collagen, CT caused an arrest of lactate production within 15 min. Prior to this time, more lactate was produced than in the controls. Collagenase-treated CT also arrested lactate production within 15 min but caused no aggregation. After the addition of hyaluronidase-treated CT or CT with added Mg²⁺, however, aggregation occurred, and lactate production proceeded linearly for 60 min, similar to the rate for control or collagen-treated platelets. These findings appear to show that the arrest of lactate production by CT was due to the binding of divalent cations by acid mucopolysaccharides.

Industrial workers who grind raw sugar cane fiber from which the sucrose content has been extracted and which has been stored in bales under high environmental temperature develop a characteristic respiratory illness called bagassosis. Precipitins against unknown antigens in crude bagasse have been detected in the sera of many patients with this disorder, and thermophilic microorganisms are suspected as likely sources of bagasse antigen. This report describes the most common thermophilic actinomycetes and fungi isolated from the “bagasse” fiber. These include M. vulgaris, S. thermoviolaceus, S. griseoflavus, S. fradiae, S. thermovulgaris, S. olivaceus, and H. lanuginosa. The sera of many patients with bagassosis contained precipitins against cellular extracts of M. vulgaris. Since bagassosis results almost exclusively from inhalation of dried stored sugar cane fiber and M. vulgaris has been the most common isolate from this type of bagasse, it is postulated that M. vulgaris is the main source of “moldy bagasse” antigens.

Earlier studies (1) showed the fluorescein-conjugated antisera to human growth hormone (HGH) localized in acidophils of human, rat, and bovine pituitary glands, and that the fluorescence, which was inhibited when the specific antigen or the unconjugated antisera was applied before staining, was specific.

During inhibition studies, one batch of anti-HGH (titer, 1/12,000: batch HGH-5) inhibited fluorescence caused by another batch of fluorescein-conjugated anti-HGH (titer 1/25,000; Hg-1) in the human but not the rat pituitary gland. Inhibition was repeated with anti-HGH batch HGH-5 and followed by 1:2 and 1:3 dilution of fluorescein-conjugated anti-HGH Hg-1 (which still produced fluorescence); again, fluorescence was not inhibited in the rat pituitary. However, when conjugated and unconjugated anti-HGH Hg-1 from the same batch were used, staining was completely inhibited in both rat and human pituitaries. The present studies were carried out to determine the cause of this discrepancy.

Materials and Methods. The immunofluorescent-staining method used was described previously (2). Preparation of antisera to HGH from 9 rabbits and 1 guinea pig were used for inhibition experiments. Fluorescein-conjugated anti-HGH Hg-1 and Hgh-5, which were used as staining antisera, produced bright fluorescence in rat, human, and bovine pituitaries. The titers were measured by the bis-diazotized benzidine (BDB)-hemagglutination method.

Results. Two antisera did not inhibit staining in any experiment (Table I and II). In human pituitaries, all others produced inhibition of variable degree. In rat, complete inhibition occurred with only two antisera (Table I). In bovine pituitaries, complete inhibition occurred with only one antiserum. Six antisera were conjugated with fluorescein (Table III); since the titer of antisera that failed to stain was unchanged after conjugation, activity was not destroyed during this procedure. Indirect staining, using fluorescein-conjugated sheep anti-rabbit gamma globulin, showed some degree of fluorescence with all eight antisera (Table IV).

These experiments indicate that different rabbits may produce different antisera to a single antigen.

The serum cholesterol ester transferase enzyme is more active in the serum of fasted than fed rats. This increase in activity is associated with principally one cholesterol ester, namely, cholesterol arachidonate. The serum cholesterol esters and phosphatidyl choline of fasted rats contain a higher proportion of arachidonic acid and less linoleic acid than those of fed animals. The findings indicate that the types of cholesterol esters synthesized by the serum enzyme are dependent, in part, on the nutritional state of the animal.

Chimpanzees and certain other nonhuman primates have been implicated in recent years in the transmission of viral hepatitis to their human caretakers and to other contacts (1–6). Mounting epidemiologic evidence for such transmission is documented by more than 100 human cases of the diseas. This evidence supports the postulate that certain nonhuman primates act as carriers of the etiologic agent of human viral hepatitis or of a disease so closely related as to be indistinguishable from it by clinical, biochemical, and histologic findings.

Liver lesions compatible with those of viral hepatitis have been found in several such “carrier” animals and also in other primates spontaneously or experimentally ill with the disease but not implicated in human transmission (4, 7). More recently, inoculation of infective material from human patients with viral hepatitis into certain species of small New World primates has produced a serially transmissible disease highly suggestive of hepatitis (8).

Hillis (4) reported the isolation of viral agents from a group of 25 chimpanzees, 9 of which were specifically associated with either spontaneous or experimental hepatitis. The investigation here reported was conducted in an attempt to relate the agents isolated from these nine hepatitis-associated primates to known viruses. The study presents evidence that seven of the agents are members of the adenovirus group, each of which is serologically related to a specific serologic type of human adenovirus.

Methods. Viral agents, after 2–3 serial tissue-culture passages in primary chimpanzee kidney cells (ChKC) were inoculated into monolayer tube cultures of primary human embryonic kidney (HEK) and rhesus monkey kidney cells (MKC) a 1:10 final dilutions of the previous ChKC passage in maintenance medium (MM). The latter consisted of Medium 199 with 10% fetal bovine serum and standard tissue-culture dosages of penicillin, streptomycin, and amphoterocin B.

The effect of excess ioide in the drinking water on the thyroid function of chicks has been studied. There was no evidence of goiter production in four breeds of chicks fed up to 0.5% (w/v) KI in water for periods of up to six months. There was some evidence in acute experiments to suggest a temporary Wolff-Chaikoff effect with excess iodides. Components of thyroid hydrolysates were not different in iodide-treated chicks as compared to chicks not given excess iodide.

Mycoplasma flora of the pharynx and anterior oral pool of 16 patients with early infectious mononucleosis did not differ significantly from that of 12 controls. Patient-derived strains of M. pharyngis, M. salivarium, M. hominis, and T-strain mycoplasma were employed in metabolic inhibition antibody tests of paired and single sera from patients and controls. The only difference observed was the presence of MI antibody titers of ≥1:4 to M. pharyngis in 40% of 42 patientsapos; sera compared to 14% to 22 sera from controls. Antibody responses to mycoplasmas were not observed during the course of illness.

A sensitive and rapid isotopic microassay method for cholinesterase determination in biological fluid and tissue has been described. The method employed acetyl-1-¹⁴C choline as a specific substrate. Free acetic acid labeled with ¹⁴C liberated enzymatically is selectively extracted in toluene isoamyl alcohol and measured by simple counting technique. The potential of the method is illustrated by determination of the level of the enzyme in brain homogenates and its subcellular fractions as well as in human whole blood. The method has the advantage that it can be applied to samples from 10 μg to 2 mg at constant pH. A wide range of technique is demonstrated.

Considerable amounts of protein and insoluble nitrogen containing compounds are found in the cecal contents of germfree rats after 8 days of ingesting 0 or 10% casein diets. This implied that the majority of the proteins found in the cecal contents was derived from the host and not from the diet. Additional experiments showed that conventional animals reabsorb from 80 to 90% of the nitrogen containing compounds found in the contents of the distal half of the small intestine whereas these compounds were not reabsorbed in the distal half of the germfree small intestine. Thus it appears that the germfree rat cannot efficiently degrade and reabsorb the endogenous proteins which are shed into his gastrointestinal tract. The significance of these findings for the germfree animal are discussed.

Biopsy preparations of pectoral and gastrocnemius muscles from normal and genetic dystrophic chicks, and from chickens developing myopathy following diets deficient in vitamin E and sulfuramino acids, were examined histologically; serial sections were prepared to demonstrate activity of several glycolytic and oxidative enzymes. Histological and histochemical characteristics of genetic and nutritional myopathies are similar in pectoral and gastrocnemius muscles, but more intense in the former. Coagulation necrosis attacks individual fibers or portions of fibers in the earlier stages of the disease. Before other histological alterations appear, white fibers enlarge and manifest the oxidative enzyme characteristics of red muscle fibers. Apparently, muscle fibers early in the myopathic process contain more enzymes, both oxidative and glycolytic, than do the normal muscle fiber. With the development within a muscle fiber of coagulation necrosis, reactions for both oxidative and glycolytic enzymes are diminished or completely absent. The early and extensive vulnerability of the pectoral muscle of chickens for dystrophic change is an index of its high white fiber content. With reference to enzyme activity in the muscle fibers undergoing myopathic changes, apparently two processes occur during the process: at first some enzymes are increased in amount; then when necrosis occurs, various enzymes examined in this study are lost from the damaged fibers.

Purified suspensions of both mature infectious elementary bodies and intermediate reproductive reticulate bodies of meningopneumonitis organisms were tested for NADH-cytochrome C reductase activity. Both intact and disrupted suspensions of reticulate bodies showed a relatively high enzyme activity. Intact elementary body preparations showed very low activity, but a 12-fold increase occurred when the organisms were disrupted by sonic treatment. The results indicate difference in envelope or membrane permeability at different developmental stages.

Ethanol administration to fasted rats increased the hepatic concentration of glutamate, malate, and α-glycerophosphate. The concentration of lactate was unchanged. The concentrations of asparate, 2-phosphoglycerate, 3-phosphoglycerate, fructose-1-6-P, fructose-6-P, and glucose-6-P were decreased by ethanol. Although these data are consistent with the hypothesis that ethanol decreases hepatic glucose production as a result of shunting substrates to lactate and α-glycerophosphate, the amount of α-glycerophosphate, lactate, and malate which accumulated after ethanol feeding was small in comparison to reported estimates of glucose production by the rat liver. It is concluded that the primary mechanism for ethanol-induced inhibition of hepatic glucose production is that ethanol blocks the entry of intermediates into the gluconeogenic pathways.

The development of positive direct Coombs' reactions (DCR) in humans during administration of drugs has attracted increasing attention in recent years. This association can be of practical importance since in some instances the Coombs' reactivity may also be associated with hemolytic anemia; notable examples have included methyldopa and penicillin (1–3). Preliminary studies indicate that 40–70% of patients given cephalothin may develop positive DCR (4,5). This unusually high incidence emphasizes the need to define possible mechanisms of red blood cell alteration or sensitization, and to establish any association with red cell damage that might influence their survival. Although structurally similar to cephalothin (6), cephaloridine has been reported to have caused a positive DCR in only one patient (7).

The present study was conducted to determine (1) the incidence of positive DCR, (2) relationships to serum antibiotic concentrations, and (3) possible associated red blood cell abnormalities in patients and rhesus monkeys treated with cephalothin or cephaloridine.

Materials and Methods. During the period January through June, 1967, 143 patients at University Hospital were observed during cephalothin therapy. Serial DCR were performed by washing 2% suspensions of red blood cells three times in normal saline in 10 × 75-mm tubes, decanting the supernatant fluid; 2 drops of Coombs' anti-human serum (Pfizer) were added and the contents centrifuged for 15 sec in a Clay-Adams serofuge. DCR were interpreted as 4+ to 1+ depending upon the degree of macroscopic agglutination; microscopic agglutination was recorded as ± and considered insignificant. Serial hemoglobin (cyanmet-hemoglobin) and hematocrit (micromethod) determinations were obtained twice weekly during cephalothin treatment (8).

The comparative effect in vitro on DCR of human and monkey erythrocytes exposed to graded concentrations of cephalothin or cephaloridine was also studied; 0.5 ml of blood was added to 0.5-ml dilutions of the antibiotics in normal saline and incubated for 4 hr at 37°.

antibodies in human sera are detectable by double diffusion gel precipitation. These antibodies correlate with growth inhibiting antibody measured by the tetrazolium reduction inhibition test. All positive sera share a common precipitation band which appears to contain lipid.

Plaques of rubella virus infected cells demonstrating granular, cytopathic fluorescence were counted in RK₁₃ cell cultures fixed 72 hr after infection. There was a linear relationship between virus concentration and fluorescent plaque count. Counts on replicate cultures have demonstrated that the technique is very precise. The sensitivity of the fluorescent plaque assay was equal to that of the conventional GMK-interference titration method.

Experimental thyroiditis and thyroglobulin autoantibodies have been produced in both complement intact and deficient mice by injections of a mixture of heterologous (cross-reacting) thyroglobulins without adjuvant. The severity of the immune response and incidence of thyroid lesions in mice varied from strain to strain. The Swiss Webster and A/JAX strains had the highest mean hemagglutinating antibody titer to homologous thyroglobulin in addition to being susceptible to thyroid lesions, whereas the complement intact and deficient strains of B10-D2/SN new line, DBA/1J, B10-D2/SN old line and DBA/2J showed autoantibody production without any definite thyroid lesions. Present data do not substantiate a necessary role for components of complement which are activated after the third component of complement in the production of either thyroiditis or autoantibodies in mice.

Living parental thymus cells injected into heavily irradiated F₁ hybrid recipients augmented erythropoiesis resulting from transplanted bone marrow from the same, but not the second, parental strain. The augmentation increased as parental thymus cell dose was increased. Thymus cells alone, regardless of origin, gave rise to no erythropoiesis. When recipient spleen weight, 8 days after marrow transplantation, was used as a criterion of marrow growth, the data for parental cell doses 4 × 10⁷ and above paralleled those from ⁵⁹Fe-uptake studies in the same mice. Parental thymus cells were effective even when administered 1 to 2 days before or after the marrow transplant.

3, 4-Dihydro-1-isoquinolineacetamide hydrochloride (DIQA) is a novel antiviral agent active in vivo against Columbia SK virus, herpesvirus, ECHO 9 virus (Coxsackie virus A23) and the PR8 and Japan 305/57 strains of influenza A virus. The substance was without effect in vivo against Coxsackie virus B1, Newcastle disease virus and influenza B virus. The compound was inactive when tested in tissue cultures against the viruses for which it had an antiviral effect in vivo. It was also inactive in tissue cultures against poliovirus type I and measles virus. Activity in vitro could be detected against Coxsackie virus B1 and vaccinia virus but, in the latter case, only if a plaque-reduction method was employed. The antiviral agent did not display anti-influenza activity in ovo.

Homogenates prepared from the hearts of hibernating and cold-exposed nonhibernating ground squirrels (Citellus trede-cemlineatus) have been studied with respect to the metabolism of both oxidized and reduced forms of pyridine nucleotide coenzymes in the presence of fluoride and glucose-6-phosphate. The rates of oxygen comsumption by preparations from nonhibernating animals were highest with the oxidized forms of the coenzymes. By contrast, the rates of oxygen consumption by heart homogenates from hibernating animals were highest with the reduced forms. It is postulated that these findings indicate the operation of a cyclic lipid metabolism in the heart of hibernating animals that does not operate or is depressed in the aroused state.

The kinetics of inactivation at 56° and at pH 2 were different for the high-and low-molecular weight components of virus-induced and endotoxin-induced rabbit interferons. The two types of virus-induced interferons were not affected by benzoylchloride or potassium cyanate, suggesting that amino or sulfhydryl groups are not essential for their interferon activity. The integrity of disulfide bonds also seems unnecessary as indicated by the lack of effect of 2-mercaptoethanol and 100 mM thioglycolate, although 1000 mM of the latter compound partially inactivated the interferons. The effect of cyanogen bromide indicated a requirement for the γ-sulfmethyl group of methionine. Sodium metaperiodate in low concentrations rapidly inactivated purified rabbit interferons as well as crude and purified chick interferon. Although the action of periodate may suggest an essential carbohydrate moiety in the interferon molecule, this action may be equally due to an effect on protein.

The action of methandrostenolone on phenylbutazone and its major metabolite, oxyphenbutazone, has been studied in man by determining the half-life and plateau plasma levels of the antirheumatic drugs. The anabolic steroid caused an increase in the plasma level of oxyphenbutazone, confirming previous studies. This effect was previously attributed to a change in volume of distribution. In the present study, in addition to the effect on volume of distribution, a prolongation of half-life of oxyphenbutazone was observed in one subject. In contrast, in six patients receiving continuous therapy with phenylbutazone, plasma levels were unchanged when methandrostenolone was superimposed. The plasma levels of oxyphenbutazone, determined in the same samples, were unaffected. The half-life of phenylbutazone in two patients was not changed upon the administration of methandrostenolone, showing that no alteration had occurred in the metabolism of phenylbutazone. Equilibrium dialysis experiments indicated competition between phenylbutazone and oxyphenbutazone, and that the former has a relatively greater affinity for albumin. These findings suggest that phenylbutazone masks the influence of methadrostenolone on plasma levels of oxyphenbutazone, presumably by displacing oxyphenbutazone from plasma protein binding sites. Comparable in vitro studies with phenobarbital and p-hydroxyphenobarbital indicate the absence of significant competition for albumin binding sites.

In cats, pralidoxime in doses of 30-75 mg/kg produces sedation and an EEG pattern like that seen after administration of CNS depressants. These doses also prevent convulsions induced by pentylenetetrazol or physostigmine. Apparently significant amounts of pralidoxime cross the blood-brain barrier in cats.

Human seminal plasma contains decapacitation factor (DF) as measured by the ability to render rabbit sperm incapable of fertilizing rabbit ova. The presence of DF suggests that capacitation of sperm is required for fertilization in the human. Compared to several other species human seminal plasma has low DF potency. The DF activity sediments in the ultracentrifuge similar to that of the rabbit.

A relationship between iron and manganese metabolism has been suggested by the observation of the incorporation of manganese into the porphyrin molecule of red cells and the increased gastrointestinal absorption of manganese in iron deficiency (1, 2). The present studies were performed to determine variations in the metabolism of each of these elements induced by deficiency or overload of the other.

Methods. Male albino rats (Wistar strain) weighing 200-250 g were used. The animals were fed a standard rat diet (General Biochemicals, Chagrin Falls, Ohio) except that the iron and manganese content was modified from test to test as stated. Iron absorption was measured following a test dose of 0.5 _μCi of ferrous-⁵⁹ Fe citrate per 0.25 mg of ferrous sulfate per 0.5 ml of distilled water, administered through a 17-gauge endoesophageal tube to rats fasted for 16 hr. Whole body radioactivity (0.8 MeV —→ ∞) was measured in a small-animal, whole-body liquid scintillation detector (Packard Armac, LaGrange, Illinois), 3 hr and 7 days after dosing to determine the percentage of the test dose absorbed by the rats.

An isolated intestinal loop technique was utilized to measure manganese absorption because of the significant hepatobiliary excretion of this element. The rat was anesthetized with sodium pentobarbital and the peritoneal cavity was exposed through a midline incision. A 6-cm closed loop of small intestine just distal to the ligament of Treitz was isolated with umbilical tape. One-half μCi of ⁵⁴Mn per 1.0 mg of MnCl₂ in 0.5 ml of saline was injected into the lumen of the loop and the abdominal wall was sutured together. One hr after the injection the animal was placed in the counter and the total radioactivity was measured.

Three out of four cycling ewes followed for two full cycles showed a marked increase in radioimmunoassayable circulating LH levels shortly after the onset of estrus; although samples were taken every 8 hr, only one sample showed the increase in each cycle. The same four ewes were used to determine the half-life and distribution volume of exogenous ovine LH; an average half-life of 28 min and an uncorrected distribution volume of 9.14% of body weight were found. The results have been interpreted as indicating that almost all the LH was released from the pituitary in a surge lasting not more than 3 hr, and that the normal cycling ewe pituitary synthesizes approximately 0.5 mg of NIH-LH-S1 equivalent per day.

Histological evaluation of isolated rat heart preparations has shown that: Incubated isolated right ventricular strips and atria undergo observable degeneration after relatively short periods of time and at temperatures of 27° or higher. The degree of degeneration is a function of: (a) duration of incubation; (b) incubation temperature: and (c) thickness of the soaked preparation. Cardiac degeneration is not prevented by penicillin, streptomycin, polymyxin, chlorte-tracycline, 9-alpha-fluorohydrocortisone, or ouabain in the doses employed. The perfused whole heart is superior to the incubated isolated tissue preparation. The perfused heart shows few signs of degeneration at 37° for prolonged periods.

A dietary level of 20 ppm of dieldrin increased growth in rats raised on rations containing low levels of thiamine, or conversely, the low dietary intake of thiamine produced a smaller growth depression in rats ingesting dieldrin. There was a marked variation among litters in their response to the nutritional stress and the dieldrin produced a more pronounced response in those litters which were more susceptible to the thiamine deficiency stress.

The effect of βAPN on bone collagen was studied in tissue culture. It was found that the lathryrogen modified the solubility characteristics of only that fraction of collagen which was synthesized in the presence of βAPN, and did not affect the bone collagen which was synthesized prior to the exposure of the tissue to βAPN.

A replicative type DNA polymerase has been partially purified from the nonhistone chromosomal proteins of Walker 256 carcinosarcoma. The enzymic polymerization of deoxyribotides into DNA-like product depends on the presence of a complete supplement of all four deoxyriboside-5′-triphosphates and native DNA as template. The enzyme requires Mg²⁺ for its optimal reaction and is inhibited by Na⁺ and K⁺. The DNA in isolated chromatin exhibits 40% of its templating activity. The tumor chromosomal DNA polymerase shows only marginal activity with reconstituted DNA-histone as template.

In searching for an animal model for human adenovirus studies we have attempted to infect rhesus monkeys with a human type 4 adenovirus. The results were inconclusive but we have some evidence to suggest that infection and replication might have occurred in several animals. Communicability among cagemates was not demonstrated. The difficulty encountered in infecting rhesus monkeys despite the large virus dose employed indicates that this animal would not serve as a useful model for vaccine testing.

The cells from fetuses infected in utero with rubella virus were analyzed for the presence or absence of virus. Cells that were virus-free were examined for evidence of prior experience with virus. By infectious center assay of fetal cells in GMK tubes, and by growing fetal cells in culture tubes, it was established that not more than 0.1% of the fetal cells contained infectious virus. No evidence of experience with rubella virus was found in virus-free cells, for such cells were able to support the replication of rubella virus, Newcastle disease virus, and vesicular stomatitis virus. The production of interferon and sensitivity to interferon was normal in virus-free cells, but not in virus-containing cells. The data obtained demonstrate that congenital rubella differs from congenital lymphocytic choriomeningitis virus infection of mice where all of the cells contain viral antigen or are resistant to superinfection by the virus.

The cytotoxic antibody response to skin allografts was examined in calves which received continuous ECIL for up to 28 days. In 3 calves, which maintained posterior skin grafts until ECIL was discontinued, the antibody response was markedly depressed. Following the termination of ECIL cytotoxicity of the sera increased. It was concluded that the antibody response is propagated throughout the body by radiosensitive cells entering the blood via efferent lymphatics.

Our findings support the principle that radiation produces its major effect on hemopoiesis and not on the circulating RBC. The changes found in the DF³²P curves can be explained on the basis of bone marrow depression after irradiation.

Ten strains of Mycoplasma were isolated from brain tissues of a scrapie infected sheep and mouse, joint exudate of an arthritic goat, and cell cultures established from human, chimpanzee, and dog tissues. These strains were related to each other, but were unrelated to known species of Mycoplasma. The present report characterizes the new strains, proposes that they be classified as Mycoplasma ariginini and discusses their ecological significance.

Materials and Methods. Media for isolation. Hayflick (1), BYE (2), and BBL (3) broth and agar media were used under aerobic and anaerobic (5% CO₂ in nitrogen) conditions (2). See Table I for media used for isolation of individual strains.

Media for metabolic reactions. Arginine utilization reaction: Hayflick and BYE broth media were supplemented with 10 mM arginine, 10 mM glutamine, vitamins (4), 0.002% phenol red, and adjusted to pH 7.1.

Glucose fermentation reaction: Hayflick broth medium was supplemented with 0.5% glucose, 10 mM glutamine, vitamins, 0.004% cresol red, and adjusted to pH 7.5.

Urea metabolic reaction: Hayflick broth medium was supplemented with 0.5% urea, 0.002% phenol red, and adjusted to pH 6.0. Utilization of arginine and urea was indicated by an alkaline shift in pH while fermentation was indicated by an acid shift in pH.

Isolation of strains. Source and origin of specimens from which 10 strains were isolated are given in Table I. Two strains were isolated from brain tissues of two animals infected with scrapie, 1 from a sheep (C506) with naturally acquired scrapie and the other from a mouse (G230) with scrapie experimentally induced by the intracerebral injection of a brain suspension from another sheep with naturally acquired scrapie. The latter suspension was not available for direct culturing for Mycoplasma. These 2 Mycoplasma strains were isolated by direct culture in both BYE and Hayflick broth and agar media aerobically and anaerobically.

The activities of the acid hydrolases, cathepsin, β-glucuronidase, β-galactosidase, β-glucosaminidase, and acid phosphatase have been shown to increase significantly in granulomatous tissue of lathyritic rats. These enzymes are of the type normally found in lysosomal particles. An apparent correlation between the increase in acid hydrolase activity and the increase in soluble forms of collagen was demonstrated.

Ethanol administration, in either chronic feeding or acute experiments, increased the level of methionine-activating enzyme in rat liver. The increase in methionine-activating enzyme did not occur in animals treated with puromycin or actinomycin D. The simultaneous administration of methionine or choline failed to inhibit this effect of ethanol. Prolonged ethanol feeding also led to an increase in hepatic cystathionase while acute intoxication resulted in a balanced increment in cystathionine synthase and betaine-homocysteine methyltransferase. We discussed the possible relationship of these results to the increased lipotrope requirement which attends alcohol consumption in rats.

Blood and various organs from MTV infected mice were administered separately to histocompatible MTV-free test mice. With tissues from BALB/cfC3H donors MTV activity was found associated with the blood and with each organ tested. When various organs from MTV-inoculated BALB c mice were assayed prior to the appearance of MTV activity in their blood, activity was localized primarily in the spleen.

The relative stability of cytomegalovirus stored in liquid nitrogen for a period of 3 years is reported.

Experiments were undertaken in marmosets (Callithrix jacchus) to determine the tissue distribution of E. coli after intravenous inoculation and to investigate the susceptibility of the urinary tract of these animals to hematogenous or ascending infection. After intravenous inoculation of 10⁷ E. coli, bacteria were rapidly cleared from the blood. High titers of bacteria were observed initially in liver and spleen but there was a rapid and progressive decline in number of organisms to complete sterility after 4 days. Pyelonephritis or bacteriuria did not result from hematogenous challenge. However, after inoculation of similar numbers of either E. coli or P. mirabilis into the lumen of the bladder, the majority of animals developed bacteriuria and 6 of 20 animals acquired pyelonephritis.

Buffalo strain rats were given a single s.c. injection of 4 mg of MCA in corn oil. Thyroiditis was observed in 10 of 13 females and 3 of 14 males. The glands were larger in the females. The thyroiditis could not be correlated with the presence of s.c. tumors.

Secretion of sodium, chloride, and water was most rapid in the proximal small intestine and showed a progressive distal decline when osmotic and concentration gradients were imposed by intraluminal 300 mM mannitol. Like secretion, absorption of sodium, chloride, and water from 152 mM sodium chloride solution was highest proximally and showed a progressive distal decrease. The pattern of transmucosal potential differences showed no correlation with absorption and secretion rates.

Administration of d-Penicillamine to weanling rats as 1% of the diet was found to produce marked osteolathyrism, with concomitant increases in urinary hydroxyproline and salt-soluble dermal collagen and decreases in dermal monoamine oxidase and proline hydroxylase activities. Supplementation of the diet with copper either partially or completely reversed the osteolathyritic and biochemical changes. Thus, an important role for copper in collagen synthesis is suggested.

Long-term suspension cultures of lymphoid cell lines were studied for phagocytic activity and pyrogen production. These cell lines were shown to actively phagocytize both polystyrene particles and Staphylococcus epidermidis, a function generally not attributed to lymphoid cells. We have observed that release of leukocytic pyrogen, normally associated with phagocytic activity of granulocytes and monocytes, did not accompany phagocytosis by these lymphoid cell lines.

Data are presented which demonstrate that atropine blockade of cholinergic effects in vascular beds is reversible. The reversibility of the blockade is evident in the vasodilatation and blood pressure fall which result from postatropine injections of ACh. It appears to be related not to time, size of dose, or interval between doses, but to the number of times ACh is allowed to compete for the receptor site.

The hemolytic activity of 8 human and 3 animal species of mycoplasmas was studied. With the exception of T-strains of human origin, each of the mycoplasmas tested hemolyzed guinea pig red blood cells.

. The present study was performed to help determine the interaction of some of the factors which determine the development and maintenance of the antiviral state in IF-treated mouse embryo (ME) cell culture. Actinomycin D timing experiments were used to estimate the rate of formation of the hypothesized mRNA for the antiviral protein (AVP) of the IF system. Earliest formation is detectable by 1–2 hr and the amount of mRNA required for the production of the maximum resistance at each dose level is synthesized by 5–7 hr. Inhibition by actinomycin D of RNA production at the time of viral challenge of IF-treated ME cells, indicated that no new mRNA for AVP is transcribed and then translated after removal of IF and simultaneous challenge. Arrest of RNA synthesis in ME cells pretreated with IF was followed by an unchanged level of resistance over 6 hr, thereby suggesting that translation of the hypothesized mRNA into AVP is completed very rapidly and that the mRNA is unstable relative to AVP. Removal of IF from cultures in the presence or absence of inhibition of protein synthesis was also followed by an unchanged level of resistance. This finding was interpreted to mean that removal of IF from ME cultures rapidly results in the cessation of production of the hypothesized mRNA for the AVP.

Staphylococcal enterotoxin B (SEB) may be assayed by a simple and rapid latex-fixation test, with sensitivity improved by several orders of magnitude over conventional methods. Latex particles, coated with specific antitoxin under carefully controlled conditions, were exquisitely sensitive indicators of toxin. The minimal detection limit, 2 × 10^-4 μg of SEB/ml of sample, equivalent to 1 × 10^-5 μg/well in the assay plate, can apparently be extended to an even lower value with instrumented reading of end points. Titrations require only 100 μl of sample.

The relative concentrations of all free amino acids which can be part of protein molecules of animal tissue probably influence the rate of protein synthesis at the cellular level. The other ninhydrin positive compounds in the cell detected during free amino acid analysis should indicate the metabolic activity of these low-molecular weight compounds.

In this report the free amino acid levels in the brain and the liver of fetal monkeys at different stages of gestation and shortly after birth are given. These data are part of a study of fetal development in a subhuman primate, whose placenta is structurally comparable to the human placenta. Other parts of this developmental study referring to free amino acids in the serum during pregnancy and fetal life of the M. mulatta have been reported by Kerr (1).

Although several studies can be found in the literature on free brain amino acids in young animals, fragmentary data on only a small number of fetal brains are available, and then mostly as part of another study. Carver et al. (2) compared free amino acids in fetal rat brain at 19 and 21 days gestation. Dravid et al. (3) determined some free amino acids in dog brain during development, but only a part of this study was concerned with the analysis of a whole fetal brain. No investigation into the free amino acid content of fetal liver of any animal or human has come to our attention.

Materials and Methods. The design of the fetal study and information on fetal specimens of known gestational age obtained by cesarean section have been described in detail by Kerr (1). Surgery was performed under local anesthesia 8–12 hr after the last feeding.

Antibodies against conjugates of DDA and Malathion half ester with bovine fibrinogen were produced by sensitizing rabbits with these conjugates. No direct reaction was found against DDA or Malathion, but these compounds inhibited the hemagglutination of their respective conjugate. The antihapten antibodies were transitory, and their activity was lost during storage, unless frozen and kept at very low temperatures. Due to the hydrophobic nature of DDA, organic solvents were used in the precipitin and hemagglutination tests involving this compound.

The effects of 3-amino-1,2,-4-triazole (ATZ) on weight, content of iodine, and histology of the thyroid in rats were determined and compared to the effects of propylthiouracil (PTU), and KCIO₄. In an 83-day period of treatment, ATZ and PTU produced goiters of comparable size. The KCIO₄ treatment resulted in slightly greater depletion of iodine in the thyroid, smaller goiters, and less thyroxine depletion of the thyroid. The effect of ATZ treatment on intrathyroid metabolism of iodine in rats was studied and compared to similar studies in rats without treatment. In the ATZ-treated group, there was a progressive decrease in T3 and T4 and a slight increase in the proportion of MIT. The biologic half-life of ¹³¹I in the thyroid of the control and treated groups was 4.9 and 1.3 days, respectively. These findings are similar to those found previously for PTU and show that ATZ, like PTU, not only interferes with organification of iodine but also inhibits the coupling of iodotyrosines to form iodothyronines.

Tumor development and CF antibody responses of monkeys inoculated s.c. and i.v. with various dilutions of Yaba tumor virus were investigated. Tumors developed at different times and at different sites with CF antibodies appearing soon after the development of tumors and attaining final CF titers comparable in all inoculated animals. No antigenic relationship between Yaba virus and monkey pox viruses was found by in vivo challenge, or between Yaba, monkey pox, and vaccinia virus by cross CF and HI tests. The development of viral antigen(s) in tumor cells removed from infected monkeys at weekly intervals was monitored by the FA technique and CF test. Although the FA technique detected the viral antigen earlier, both procedures were equally effective for detection of antigen in tumors removed 4 or more weeks postinfection.

The velocity of electrical slow waves recorded from the upper jejunum in unanesthetized dogs was markedly reduced below a transection. This observation suggests that intrinsic nerves are involved in propagation of slow waves.

Selective tritiation of the C-terminal amino acids in polypeptide chains has recently been proposed by one of the present authors (1–5) as a new method for identifying the C-terminal amino acids in proteins.

Proteins are selectively tritiated at their C-terminal amino acids through racemization via oxazolone formation by the action of acetic anhydride and pyridine in a medium containing ³H₂O. The present paper describes the enzymatic digestion of C-terminal tritiated peptides which were readily obtained by the above method. This facilitates the detection of C-terminal fragments in enzymatic digests by radioactivity.

Our investigations were carried out with Scenedesmus ferredoxin and peptides derived from it during the course of structural studies of Scenedesmus ferredoxin, whose primary structure has recently been established [(6); also Sugeno and Matsubara, in preparation]. The results indicate the usefulness of tryptic and chrymotryptic digestion of the tritiated peptides in identifying C-termini.

Tryptic digestion of C-terminal tritiated peptide. As shown in Fig. 1, the peptide (C-V), one of the C-terminal fragments obtained by chymotryptic digestion of carboxymethylated Scenedesmus ferredoxin, had one lysine residue whose peptide linkage is susceptible to trypsin.

To protect the free amino group of lysine from acetylation which might occur during subsequent tritiation reaction, 0.337 μmole of peptide C-V was treated with F₃CCOSEt and 1 N NaOH at pH 9.8 as described by Goldberger and Anfinsen (7). This yielded the corresponding N-trifluoroacetyl peptide (TFA-C-V). Purification was carried out on a small Dowex 1 × 2 column equilibrated with a buffer containing 0.124 M pyridine and 0.003 M acetic acid by successive elutions with the same buffer and 30% acetic acid. Lyophilization of the fraction eluted by acetic acid gave a pure peptide, TFA-C-V (ninhydrin reaction: -, Pauly reaction: +), which was subjected to tritiation reaction by the method described by Matsuo et al. (2) as follows: Peptide TFA-C-V was dissolved in 0.1 ml (10 mCi) of ³H₂O and pyridine (0.2 ml), and acetic anhydride (0.05 ml) was added.

Pectoral muscle mitochondria were isolated from normal and genetically dystrophic year-old New Hampshire cockerels. The mitochondria from the dystrophic muscle showed an elevated rate of oxygen uptake when incubated in a medium containing Mg²⁺ and ATP over a 2-hr respiration period. It is suggested that accelerated loss of ATP from mitochondria in dystrophic muscle is responsible for the abnormal permeability of the mitochondrial membrane and the altered microscopic appearance.

Repeated injections of gold sodium thiomalate in guinea pigs resulted in significant weight gain. Retroperitoneal fat deposits and extractable omental fat were increased in treated animals. Neutron activation analysis and autoradiography localized gold within brain macrophages of treated animals. However, specific concentration of gold in hypothalamic nuclei and tissue destruction were not observed. The absence of lesions and the failure to demonstrate hyper-phagia suggest that gold thiomalate induces a metabolic obesity rather than a regulatory obesity in guinea pigs.

After an oral dose of Na¹³¹ I to rats, sacrificed after 48 hr, liver lecithin had maximum radioactivity compared to other phospholipid components; neutral lipids were devoid of any activity. Liver lipids had more activity than thyroid or plasma lipids. Plasma lipids had negligible activity after intravenous injection of Na¹³¹I after periods ranging from 15 min to 6 hr. Plasma lecithin does not seem to be a carrier of iodide in the blood. Liver slice or homogenate experiments failed to show any enzymatic process connected with association of lecithin with iodide.

Increasing the litter size of rats from 0 to 2, 6, or 12 pups progressively decreased (p<0.05) pituitary growth hormone (GH) content, whereas mammary development (DNA) and metabolic activity (RNA and litter weight gain) progressively increased (p<0.01). Nursing intensity did not significantly alter (p > 0.05) LH content of the pituitary, although LH tended to decrease in the lactating rats with increasing nursing intensity. All non-nursed and one-half of the 2-pup nursing intensity group began their estrous cycles before day 16 of lactation, but rats nursing 6 or 12 pups did not.

Bilirubin production was found to increase twofold during the first 24 hr after administering 100 μg endotoxin (intraperitoneally) to rats. A 50% increase in bilirubin production was observed in host rats 7 days after transplant of the Walker carcinoma 256 (intramuscularly). Ability of the liver to conjugate bilirubin was normal in both types of animals. The results obtained are consistent with the view that the concomitant block in re-utilization of iron occurs at some point after its release from heme.

Proliferation and hyperplasia of cells are phenomena observed widely in various biologic systems. In such effects of overgrowth, it is obvious that the dimensions of the involved tissue exceed those established by normal controls on growth. Very little, however, is understood about this, even as to initiating circumstances (1–3).

One phenomenon that might be explored for possible significance in proliferative overgrowth is the biologic reaction of repair, that is, regeneration after loss of part of a given tissue. In other words, it may be asked whether hyperplasia could be a quite regular result of the regenerative repair response, a result brought out even more when the damage is chronic or repeated.

More specifically, it may be asked whether one or more adequately harmful stimuli to a group of cells would be followed regularly by a rise above normal in the quantity and size of the cells and subsequently by a return to normal. It was felt that the answer to this was desirable first in simple histologic terms as preparation for a biochemical exploration of the mechanisms and effects of regenerative response to damage. Histologic evidence presented here indicates that any of a large number of harmful substances regularly produce a proliferative response in the skin of mouse and guinea pig. Various characteristics of the effect are reported, together with certain related biologic principles.

The constancy of a hyperplastic response to harmful substances reported here raises a second, early question: Is the repletiveproliferation response, after application of a harmful agent, associated with the development of increased resistance in the involved tissue to later invasion by the originally inciting agent? Histologic evidence for the presence of such resistance is given below.

Subacute sclerosing panencephalitis (SSPE) is a progressive degenerative, neurologic disease of children that begins with mental and motor deterioration. After an interval of weeks or months, a state of plastic rigidity, interrupted by myoclonic jerks, intervenes. Coma, with signs of decortication and often of hypothalamic dysfunction, occurs during the terminal stage of this fatal illness.

Observation of cytoplasmic and nuclear inclusion bodies in cortical neurons of patients with SSPE led Dawson to propose a viral etiology for this disease (1). Evidence for an association between SSPE and measles virus has been reviewed and includes: a history of measles infection prior to the onset of symptoms; demonstration by electron microscopy of structures in the diseased brain that are morphologically similar to the nucleocapsids of paramyxoviruses; detection of measles, or a related antigen by immunofluorescence in the brain of patients with SSPE; and presence of exceptionally high levels of antibody against measles virus in both the serum and cerebrospinal fluid of these patients (2). Nevertheless, there has been a recurring failure to isolate or transmit an infectious agent in animals or tissue cultures inoculated with material from SSPE patients.

The present report describes the occurrence of measles antigen and syncytia formation in cells propagated in vitro from the brain of a patient with SSPE, and the production of syncytia in several kinds of cultured cells when exposed to these brain cells.

Materials and Methods. Tissue specimen. Open biopsy was performed by Dr. F. C. Kriss of the Department of Neurosurgery on a patient whose diagnosis of SSPE was based upon clinical and serologic evidence. Bits of cortex in Hanks' balanced salt solution (BSS) containing antibiotics were received in each author's laboratory within 20 min from the time of biopsy.

The immunological competence of embryonic mouse liver cells was studied using the in vitro graft-versus-host system for assay. Embryonic liver cells did not have the capacity to induce splenomegaly when taken from embryos directly, nor did they acquire this capacity when cultivated as organ cultures for four to six days. The capacity to induce splenomegaly was acquired, however, when liver explants were cultivated for several days in combination with thymus tissue.

Subcutaneous nodules were induced at sensitization sites in guinea pigs injected subcutaneously with BCG organisms. When the organisms were suspended in water-in-oil emulsions, the size of the nodule was inversely related to the dose: 1.0 mg induced a larger lesion than 10 mg or 100 mg. Lesions induced by 1.0 mg of BCG were due primarily to delayed hypersensitivity, while lesions induced by 10 or more mg were due initially to toxicity and later to delayed sensitivity.

Steroids labeled with ³H in positions 1 and 2 were incubated with rat ovary slices, and the formation of tritiated water determined. During three hr at 37°, approximately 3.5, 10.0, and 15% of the total tritium present in progesterone-1,2-³H, androstenedione-1,2-³H, and testosterone-1,2-³H respectively was converted to ³H₂O by 100 mg of tissue. The addition of FSH had no effect upon the aromatization of any of the substrates tested, although one of the preparations tested contained a contaminant that significantly inhibited the aromatization of androstenedione.

The mechanisms of virus-host cell interaction resulting in the transformation of normal cells to neoplastic cells has been examined in several systems. Elegant experiments with the SV40-3T3 mouse fibroblast system reported by Todaro and Green (1) demonstrated that active cell replication and DNA synthesis are required to fix the transforming event between virus and target cell. Stoker (2) observed that the frequency of transformation of polyoma virus was increased among BHK-21 hamster cells which have survived X-irradiation. Exposure of mouse fibroblast cells to radiomimetic thymidine analogues enhanced the efficiency of SV40 transformation fivefold (3). Borek and Sachs observed that 300 R of X-irradiation induced the in vitro transformation of hamster embryo cells when cultured on mouse cell feeder layers (4). Processes associated with cell replication were essential soon after irradiation to fix the transformed state (5). A hereditary and a physiological state of susceptibility to transformation appeared to exist in X-irradiated cells and mechanisms for repair of radiation damage (“error correction”) directly controlled the development of the transformed state (6).

Recent studies in this laboratory demonstrated that exposure of primary hamster embryonic cells to 150 R of X-irradiation prior to infection produced a 15- to 50-fold increase in the number of transformed clones when compared with unirradiated controls (7). Radiation alone under these conditions did not produce transformed cells. Characterization of the radiation responses of normal embryonal cells in culture is fundamental to understanding the role of irradiation in sensitizing these cells to transformation by SV40 virus. The present report describes results obtained in the investigation of several parameters of radiation induced changes in normal hamster cells in culture. A parallel study was conducted on the radiation response of SV40 transformed isogenic hamster cells to define potential viral induced changes that may exist.

Effects were studied of insulin and of varying the concentration of glucose upon L-leucine-¹⁴C (UL) metabolism by isolated rat epididymal adipose tissue cells. At lower glucose concentrations, insulin increased CO₂ formation and lipid synthesis from labelled L-leucine. At higher glucose concentrations insulin decreased apparent CO₂ formation and lipid synthesis. Insulin stimulated protein synthesis at all levels of glucose concentration studied, but the increment was less at higher glucose concentrations.

Methylprednisolone (Medrol) was administered to normal and cholesterolfed rabbits for a period of 2 months, and its effects upon plasma cholesterol and triglyceride concentrations and upon the content of aortic acid mucopolysaccharides (AMPS) were studied. Rabbits fed a chow diet developed both a hypercholesterolemia and a hypertriglyceridemia in response to Medrol, and the already elevated cholesterol and triglyceride levels induced by cholesterol feeding were further elevated by Medrol. Despite the hyperlipemia, none of the Medrol-treated animals developed aortic atherosclerosis. Medrol had no significant effect upon the total aortic AMPS content in control animals but it did alter the aortic AMPS content and pattern of cholesterol-fed rabbits. The most significant effect was an increase in an AMPS of low sulfate content. Since all animals fed cholesterol developed some degree of pulmonary atherosclerosis, it is postulated that the antiatherogenic action of the hormone on the aorta is more closely related to alterations in the AMPS pattern than to changes in the pattern of circulating lipids.

The effect of adding dextran T 80 (av mol. wt. 80,000) in certain concentrations to the mixture of antigen, complement (C) and serum in the diagnostic CF test for RS and parainfluenza (P) virus infections has been investigated. Preliminary experiments showed that the diluent employed (Veronal buffered saline, VBS) had to be used with hypertonic salt concentrations to prevent C consumption from increasing when dextran was added to the test system. Sera from infants and children with respiratory infections were titrated in parallel CF tests using either regular VBS without dextran (CF-v) or hypertonic VBS supplemented with dextran (CF-d). In the majority of sera examined the CF-d titers were significantly higher than the corresponding CF-v titers. Furthermore, using dextran, a rise in titer in the second of a pair of sera was obtained, which was not found in regular CF tests without dextran. Addition of dextran was also able to increase the CF titers in tests with LCM antigen and immune sera from mice.

When West Nile virus was added to cultures of L-929 mouse fibroblasts, characteristic morphological changes were produced. The infected cultures were not destroyed although infectious virus was consistently demonstrated in the growth medium. Infected cells were readily subcultured and the virus-cell relationship was continued without any alteration in cultural procedures.

The administration of ferric ammonium citrate to newborn mice enhanced their susceptibility to MHV. Intraperitoneal inoculation of a chelating agent, EDTA, protected a significant proportion of newborn animals against MHV but only when administered shortly after birth.

The effect of endotoxin on mouse renal function was studied. The following dose-related changes were noted 18 hr after the intraperitoneal injection of endotoxin to mice: (i) Elevation of the blood, brain, and liver levels of urea nitrogen; (ii) decreased excretion of urinary urea nitrogen; (iii) diminished output of urine; and (iv) inhibition of the renal clearances of inulin and PAH. These findings clearly indicate that renal function is impaired in mice treated with relatively large doses of endotoxin. It is noteworthy that these doses exceed those usually required to demonstrate nonspecific resistance to experimental microbial infections in mice. Mice were rendered tolerant to the renal inhibitory effects and to the lethal effects of endotoxin by injecting them with a relatively low dose of endotoxin for 8 consecutive days prior to the administration of a high dose of endotoxin, which would ordinarily reduce renal function and produce 85% mortality in 72 hr.

Serum lipid components were measured in eight male subjects exposed to an altitude of 14,000 feet for 14 days. Concentrations of total lipids rapidly decreased after the third day of exposure and a similar, although less pronounced decrease was observed in cholesterol levels. By way of contrast, phospholipid and FFA levels progressively increased during the period of altitude exposure. High altitude had no effect on serum glucose concentration but caused a slight increase in serum water content. The data indicate that high altitude has a marked effect on lipid metabolism.

The serum neutralization test utilizing the continuous line of rabbit kidney-cell strain LLC-RK₁ has been found to be quite sensitive and reproducible. Some differences in results were found when neutralization tests were compared to HI tests. Performance of the HI test at an initial 1:4 dilution of serum appeared to be an important step in decreasing the possibility of false negative results. Even with this change, of 37 sera with no detectable HI antibody, 9 had antibody in the neutralization test. Clearly, there is a need to not only document serological results but to determine the true significance of these conflicting findings. These differences are important for the interpretation of serological tests for all clinical and vaccine studies.

Sequential incubation studies using everted sacs of guinea pig small intestine and vitamin B12 labeled with two different isotopes of cobalt, ⁶⁰Co and ⁵⁷Co, showed that IF-saturated with B12, could effect specific attachment of the complex to distal sacs. In a subsequent incubation IF could: (i) mediate in specific fashion (not exchange) the uptake of additional B12, (ii) interact with IF-“blocking” antibody, and (iii) be partially dissociated by EDTA from the sac (in a form biologically active for this system and retaining specificity of activity) leaving its B12 attached to the mucosa. These results were interpreted to indicate that, in this experimental system, IF is not absorbed into the intestinal cell during the B12 uptake that is mediated by intrinsic factor.

It has been repeatedly demonstrated that a large variety of animals develop ketosis when their diet contains a high proportion of lipid relative to carbohydrate (1). However, the chick embryo (2) as well as growing chicks (3, 4) can utilize diets essentially devoid of carbohydrates without developing ketosis. Since the enzymes necessary for acetoacetate biosynthesis are demonstrable in chick liver (5) and ketosis can be produced if fatty acids furnish all nonprotein calories (4), the lack of ketosis in the chicken can not be attributed to an inability to synthesize ketone bodies.

Ketosis could be avoided, however, if the chick could perform a net synthesis of carbohydrate from acetate via some pathway which is not physiologically important in other animals (6) but may become important in the developing chick where lipid is the only available energy source. Three possible pathways which could account for a net synthesis of carbohydrate from acetate are: (a) initial synthesis of acetoacetate, which then is converted to a glycolytic intermediate via either acetone (7) or pyruvaldehyde (8), (b) synthesis of butyrate which is converted to succinate by ω-oxidation (9), or (c) synthesis of malate via the glyoxalate bypass (10, 11). The first of these would not only decrease ketone body synthesis by making carbohydrate available, but would also reduce circulating ketone bodies by utilization.

Utilization of acetate by any of these pathways can be distinguished from its oxidation by the classical citric acid cycle by injecting acetate-1-¹⁴C and measuring incorporation of radioactivity into amino acids (12). Using this technique, it was found that acetate is metabolized primarily via the citric acid cycle and that, if any of these special pathways are present in the chick, they are of limited importance.

Control and hypothalamic-hyperphagic rats were injected daily with pyridoxine hydrochloride at dosage levels of 10, 40, 80, 120, 160, and 200 μg/day for 32 days. Body weights and food intakes were measured throughout the experimental period; erythrocyte glutamic-pyruvic transaminase activities (GPT) were measured at the termination of the experiment. Based on body weight changes, it is suggested that vitamin B₆ requirement is in the range 80-120 μg/day for both control and hyperphagic rats. However, a daily dose of at least 200 μg is required to elicit maximum erythrocyte GPT activity. A significant correlation between vitamin intake and enzyme activity was observed thus indicating a direct relationship. There appeared to be no evidence of an increased requirement for vitamin B₆ as a consequence of hyperphagia and therefore, it is concluded that in the rat, requirement for this vitamin is not related to calorie intake.

Rhinovirus strains representative of the 55 numbered serotypes and one subtype were tested in HeLa and WI-38 cell culture tubes for inhibition by 223 and 447 μM concentrations of 2-(α-hydroxybenzyl)-benzimidazole (HBB) and 77, 115, 192, 383, and 574 μM concentrations of D - (-) − 2 - (α - hydroxybenzyl) - benzimidazole · HCl (D-HBB · HCl). Half of strains tested showed some inhibition at a 447 μM concentration of HBB and strains of all types except 1A and subtype 1B showed some degree of inhibition at a D-HBB · HCl concentration of 574 μM or less. An increasing proportion of strains were inhibited as test concentrations of the compounds were increased. Multiple strains of rhinovirus types 7 and 24 showed similar degrees of sensitivity to a 192 μM concentration of D-HBB · HCl. Comparative testing of strains of 36 types vs a 192 μM concentration of D-HBB · HCl in WI-38 and HeLa cell culture tubes gave similar but not identical results. D-HBB · HCl concentrations of 574 and 383 μM were toxic for cells. The behavior of viruses which are insensitive to HBB or D-HBB · HCl at non-toxic concentrations, but show some sensitivity at higher concentrations, is difficult to interpret because of the problem in separating virus-specific from toxic actions of the compounds. Indirect evidence for a virus-specific inhibitory effect is provided by the linear increase in the proportion of sensitive strains with increasing compound concentrations and by the growth of some rhinovirus strains in cells exposed to cytotoxic concentrations. Experiments now in progress with a more sensitive gradient plate plaque reduction technique, using D-HBB · HCl concentrations below cytotoxic levels, support this hypothesis. These results suggest that rhino-viruses as a group show a wide range of sensitivity to these compounds but that most types share to some degree the characteristic of D-HBB · HCl sensitivity.