Experimental Biology and Medicine

In 3 separate experiments small groups of mice of 2 strains were exposed to infection with Schistosoma mansoni and half of them were treated with cortisone beginning shortly prior to infection and ending on the 29th, 15th and 7th days after infection, respectively. The various regimens of cortisone treatment employed did not increase susceptibility to the parasite, as judged by numbers of adult worms recovered from hepatic portal tracts of the mice 7–8 weeks after infection. The treatments may have enhanced the natural immunity slightly, since significantly fewer worms were recovered from the cortisone-treated mice.

Three pairs of male littermate rabbits were fed a commercial vit. E deficient and a vit. E supplemented diet. When the vit. E deficient member of each pair showed typical vit. E deficient symptoms, the pair of animals was sacrificed and the water soluble muscle proteins extracted from the striated muscle. The electrophoretic pattern of the deficient animals showed a significant change in distribution of water soluble proteins as compared to the control group. This change was an increase in the fast moving peak and a decrease in the slow moving peak.

It has been shown that niacin deficiency in rats has the effect of increasing rate of alcohol metabolism in spite of the fact that DPN is generally conceded to be involved in conversion of alcohol to acetaldehyde.

A simplified coating method for radioautography has been described in which the radioactive slides are dipped in melted photographic emulsion. After exposure, the developed emulsion is protected with a coat of vinylite and mounted under coverslip.

Methylglyoxal has been incubated with rat liver extracts in the presence of kidney antiglyoxalase. During incubation, methylglyoxal and inorganic P decrease, whereas total hydrolyzable P increases. The hydrolysis curve of the Ba insoluble fraction of liver incubated with methylglyoxal resembles that obtained from hexose phosphate. Paper chromatography also gave evidence for the formation of hexose phosphate from methylglyoxal.

Low doses of l-triiodothyronine suppressed the mild forms of aminoacetonitrile poisoning in rats, whereas extremely high doses of thyroxine were necessary to accomplish the same result. On the basis of the prevention of aminonitrile lesions in rats, l-triiodothyronine was estimated to be over 100 times more active than l-thyroxine when administered orally and over 50 times when administered parenterally.

1. Chlorpromazine and reserpine inhibit spinal reflex activity. 2. The inhibition affects predominantly monosynaptic reflex pathways when submaximal stimuli and minimal doses of chlorpromazine or of reserpine are used. 3. The spinal effects of chlorpromazine and reserpine do not result from circulatory changes, unless these be local alterations in the circulation within the spinal cord. That possibility has not been studied.

A procedure is described for the separation of the proteins of cell nuclei into several fractions which differ in solubilities and in glycine content. Twenty hours after intraperitoneal injection of glycine-l-C¹⁴ into adult rats bearing subcutaneously transplanted hepatomas the specific activities of the protein fractions of the nuclei of liver and tumor were in the descending order: residual lipoprotein > globulins > histone fraction III > histone fraction II. Each of the protein fractions of the nuclei of liver cells had higher specific activities than corresponding protein fractions of the hepatoma with the notable exception of the histones which were in the reverse relationship.

In experiments reported herein. 2 new methods have been utilized to compare the biologic effect of 3 dietary fats. (1) In Exp. 1 a study has been made in rabbits of the effect of single meals of butter, corn oil, coconut oil and water on fibrinolytic activity of blood (drawn 3 hours after feeding and measured by a technic utilizing streptokinase to activate profibrinolysin) in vitro under controlled conditions. Lysis time so measured was significantly prolonged in rabbits fed butter as compared with those fed either corn oil, coconut oil or water. Meals of corn oil and coconut oil had no more effect on lysis time than did water. (2) In Exp. 2 the effect of butter, corn oil, coconut oil and saline added in vitro on fibrinolytic activity of clotted human plasma was measured by a technic utilizing streptokinase to activate profibrinolysin under controlled conditions. Under these conditions butter markedly and significantly (p = 0.01) prolonged lysis time of human plasma, whereas corn oil and coconut oil had a lesser effect. (3) Other substances and approaches must be studied before definitive conclusions can be drawn regarding relationships of various fats to arteriosclerosis and thrombosis in man. Our experiments are significant in that an additional approach (which may be particularly useful for rapid screening) has been added to our armamentarium for study of biologic effects of various fats.

When phenethyldiguanide (PEDG) is administered to guinea pigs, it produces hypoglycemia accompanied by depletion of liver glycogen with no increase in muscle glycogen. PEDG causes decreased blood urea formation and decreased non-protein nitrogen excretion in the urine. It also produces an early and significant rise in blood lactic acid. From studies made on surviving slices of guinea pig liver, it is evident that PEDG increases lactic acid formation and increases glycogen depletion, but it does not affect glucose output. PEDG also exerts an effect on isolated rat diaphragm, increasing glucose uptake, increasing lactic acid production, decreasing glycogen formation, and decreasing oxygen uptake.

(1) Glycolytic enzymes and their relation to in vitro calcification of the preosseous cartilage of rachitic rat bone tissue have been studied. The findings indicate that although glycolytic enzymes are present in deep frozen tissue, ability to calcify in vitro is lost. However, if the tissue is treated first with calcium chloride and then stored in the deep freeze, the glycolytic enzymes are either absent or present only in trace amounts while the calcifying mechanism remains virtually intact. (2) Bone sections heated at 65°C and then treated with calcium chloride retain their ability to calcify in vitro despite the destruction of the glycolytic enzymes during the heating process. (3) Demineralization of bones results in a loss of glycolytic enzyme activity. However, when the demineralized bones are treated with chondroitin sulfate and then with calcium chloride, the calcifying mechanism is restored but not the glycolytic enzyme activity. (4) It is concluded that glycolytic enzymes are not part of the minimal system required for calcification.

It was demonstrated that intrinsic factor concentrate stimulates the uptake of radioactive vit. B₁₂ in slices of rat liver and kidney, in vitro. Evidence presented indicates the specificity of this stimulatory action. The stimulation is a function of concentration of intrinsic factor concentrate added, and may therefore be the basis of an in vitro assay for intrinsic factor. It was not necessary to use vit. B₁₂ deficient tissues to show the stimulation effect and it was observed that hyperthyroidism caused increased uptake of vit. B₁₂ in the absence of added intrinsic factor concentrate. A brief discussion of the possible function of intrinsic factor was presented.

The first recognized manifestation of a primary or transplanted pituitary tumor in the parakeet is a marked increase in a plasma protein component, tentatively identified as a globulin. Analysis by optical ultracentrifugation suggests that this protein is qualitatively different from any present in normal plasma. Several weeks after appearance of the abnormal protein there is often an associated hyperglycemia and obesity. It is suggested that all these changes express the response of target tissues to one or several hormones secreted by the tumor.

(1) Normal rats placed in a low oxygen atmosphere for time periods from 4 to 120 hours reveal definite elevation of plasma erythropoietin until 24 hours and after 48 hours erythropoietin drops back to normal levels as judged by Fe⁵⁹ uptake assay using hypophysectomized rats. (2) On the basis of this time-concentration relationship of plasma erythropoietin in hypoxic hypoxic rats, no significant alteration of erythropoietin titers is evident in hypoxic rats with or without kidneys and/or spleen. (3) Hence, neither the kidney nor spleen is the production site of erythropoietin under hypoxic hypoxia.

1. A fairly fixed amount of free cholesterol and cholesterol esters is found in the perchloric acid soluble (seromucoid) fraction of human serum. Phenomena of co-precipitation or of adsorption are probably responsible for this finding. 2. Seromucoid fraction, total serum cholesterol, cholesterol found in the seromucoid fraction were studied quantitatively in pathologic sera obtained from patients with lymphoma, disseminated malignancies, acute leukemia and from selected patients with liver disease. Seromucoid fraction was elevated in all diseases. Cholesterol found in the seromucoid fraction was relatively increased in sera of patients with lymphomas and metastatic carcinomas; relatively or absolutely decreased in acute leukemias and liver disease. 3. The significance of these findings consisted in the indication of possible effects of perchloric acid on cholesterol-containing components of serum. Such effects seemed to vary from normal to pathologic conditions, thus suggesting the possible future value of this study in the investigation of abnormalities of cholesterol metabolism in disease.

During the course of adaptation of the egg-adapted pseudo-rabies virus to tissue culture of monkey kidney cells, 2 types of cytopathogenic variations were encountered which appeared to be induced by 2 different strains of virus. One strain produced rounding degeneration, and the other cytolytic giant cell formation. Descriptions are given of the growth curve, plaque type in tissue culture, and infectivity for rats of these 2 strains.

The effects of estradiol and its benzoate ester on body weight and food consumption of immature male rats have been studied in experiments using controls which were either eating ad lib. or pair-fed to the treated animals. In animals eating ad lib., estrogen treatment for 3 weeks or more resulted in a depression of body weight accompanied by a voluntary restriction in food intake: body weight was more greatly depressed than food intake. Restriction of food intake in the controls to amounts eaten ad lib. by the treated animals duplicated the effects of the estrogen on depression of body weight, since the growth curves of pair-fed control and treated groups became parallel.

Tracings of the amino acid patterns obtained chromatographically on the tungstic acid acid filtrates of skeletal muscle, left ventricle and kidney from normal and potassium-deficient dogs have been presented. The amino acid patterns of the tissues from control and potassium-deficient dogs were found to be identical, even in the presence of marked changes in the electrolyte compositions of plasma and skeletal muscle. Differences between amino acid patterns of the skeletal muscle from potassium-deficient dogs and of the skeletal muscle from potassium-deficient rats previously reported were pointed out.

The sera from 7 animal species were found to contain globulin components which would function as “antigen'”for rheumatoid sera in the latex-fixation test. The effectiveness of these sera as “antigens'”depended on removal of albumin by either precipitation or dilution. Nine of 10 human sera behaved similarly. The addition of gelatin to gamma globulin resulted in loss of “antigenicity.”

(1) Pollen antigens (short ragweed) elute slowly from the surface of tanned sheep erythrocytes. (2) This elution does not apparently affect the titer obtained over a 2 hr incubation period. (3) Some antigenic fractions fix more permanently than others. (4) The elution is not a function of contact of the RBC-antigen with its specific antibody. (5) This method may possibly be used to separate various antigenic components in the search for antigens with fewer specificities.

Previous work has shown that a combined thyroxin-cortisone therapy in hypophysectomized rats will eliminate the anemia and induce a marked decrease in peripheral white cells; this was accompanied by severe hypoplasia of the bone marrow. It has also been shown that growth hormone increases peripheral white cell count in hypophysectomized rats but has no effect on peripheral anemia; the bone marrow was hyperplastic. Adding growth hormone to the thyroxin-cortisone therapy resulted in repair of post-hypophysectomy anemia, in normal histological appearance of bone marrow, in normal total number of cells/mm³ of marrow tissue, and in values for myeloid elements/mm³ of marrow and peripheral white cells slightly above normal. The values for erythroid elements/mm³ of marrow averaged 82% of normal. These hematological findings were accompanied by a normal oxygen consumption.

Complete regression of subcutaneously implanted Sarcoma 37 occurred in 62% of host mice treated with a polysaccharide derived from Candida Guilliermondi, in 67% of those treated with Fleischman's zymosan, and in 6% of the controls. No toxic symptoms developed following intraperitoneal injection of either of these polysaccharides, and no hemorrhagic reaction was noted in tumor tissues. Neither substance had any effect on the ascites form of Sarcoma 37 in vivo.

Heat stable antibodies to zymosan were produced in rabbits and measured by quantitative agglutination. The increased bactericidal activity for Escherichia coli B resulting from injections of zymosan could be absorbed by E. coli B. Some aspects of the antigenicity of zymosan are discussed.

1. Following partial hepatectomy in the rat, liver coenzyme A concentrations were found to be significantly reduced throughout the period of rapid liver repair. Neither fat nor water infiltration of liver, partial starvation, nor surgical stress with injury to the liver were thought to be important contributing factors. Evidence suggested that the reductions were related to alterations in carbohydrate metabolism which accompany rapid tissue growth and that coenzyme A may not play an important role in protein synthesis. 2. An unexplained rise in renal coenzyme A concentration was observed 24 hours after partial hepatectomy.

The presence in human erythrocyte stroma of a chromogenic substance giving absorption spectra (direct Ehrlich and Bial reactions) identical with those of crystalline sialic acid has been confirmed. A potent water-soluble inhibitor of viral hemagglutination, containing 7–11% by weight of this chromogen, has been isolated by partition dialysis from concentrated human erythrocyte stroma. 30–40% of the chromogen in the crude stroma concentrate and up to 56% of that in the purified inhibitor was rendered dialyzable following treatment with concentrated infective influenza virus, which in most instances also destroyed receptors for indicator viruses. Limited chromatographic analysis indicated that the dialyzable chromogen is most probably sialic acid. These observations represent the first direct demonstration of split products resulting from enzymic action of influenza virus on human erythrocyte components.

Four human transplantable tumors have been grown in hibernating hamsters. One human tumor strain failed to grow. During hibernation tumor growth was markedly inhibited, but resumed its normal rate when animals were returned to warm room temperatures.

These findings provide support for the hypothesis that the plasma iron level rises by night because of diminished utilization of iron for hemoglobin synthesis during sleep in the individual with normal erythropoietic activity. The conclusion is inferential in that no actual measurement of the newly formed hemoglobin was made but is clearly suggested by the findings of Wasserman and others(5) who interpret rate of removal of iron from the plasma as providing an index of erythropoietic activity. Bothwell and Mallett(9) found that plasma iron turnover varied throughout the day and was significantly affected by the plasma iron level. It is probable that an important consideration in assessing the findings of these authors concerns the state of the plasma iron level, i.e., whether it is rising or falling at the time of plasma iron turnover determination. An interesting finding by Bothwell and Mallett was that the fraction of iron removed per hour from the plasma was uniformly large in both morning and evening determinations in anemic subjects.

The plasma iron reaches its lowest level just after sleep begins and rises during sleep (10). This diurnal pattern is reversed in night-workers who sleep by day(10–13). The usual rhythm, with morning levels higher than evening levels, is found only if the subject has slept during the night(13). The diminished plasma iron turnover and increased T 1/2 by night suggest that diurnal variation of the plasma iron level is secondary to a diurnal variation in the rate of hemoglobin synthesis, which in turn is dependent upon the sleep rhythm. The high morning level of bilirubin in the plasma may be considered due to diurnal fluctuation in liver function rather than to increased hemolysis by night, as was held by Laurell(4).

1. A series of substituted ethylene glycols has been studied for anticonvulsant activity by both the maximal electroshock seizure and subcutaneous Metrazol seizure tests. 2. Fully substituted ethylene glycols are more potent anticonvulsant agents than mono-, di-, or trisubstituted analogues. 2-p-Chlorophenyl − 3 - methyl-2, 3-butanediol (phenaglycodol) has been selected for clinical trial since it has high potency and long duration of action. It is suggested that the long duration of action may be related to full substitution of the ethylene glycol nucleus which should reduce the probability of oxidative detoxification.

Release of heterogenetic infectious mononucleosis receptor from sheep and beef erythrocytes by receptor destroying enzyme, influenza viruses and plant proteases has been described. Numerous other enzymes were inactive. Material released by enzymatic action inhibited specifically infectious mononucleosis antibody and gave color reactions indicating that neuraminic acid or its derivatives are involved.

1) Defined media which allow heavy growth of 3 strains of group A streptococci have been developed. The medium consists of either a) 21 amino acids, glutamine, 6 vitamins, salts, purines, pyrimidines and glucose or b) 13 amino acids and 4 vitamins. 2) Cysteine is important both as an essential amino acid and as a reducing agent. As an amino acid only a small amount (10 μg ml) is needed and this can be substituted by an equivalent amount of cystine. As a reducing agent it can be replaced by ascorbic acid and less effectively by thiomalic or thioglycollic acids. Concentration of cysteine was critical for initiation of growth from small inocula. With less than 5 × 10⁶ ml cells at least 350 μg ml cysteine HCl are needed for obtaining visible growth. 3) Pyridoxal was necessary in a medium of 13 amino acids and 3 vitamins (nicotinic acid, pantothenate, riboflavin) whereas in a complete medium (22 amino acids) no need for pyridoxal was found. Pyridoxal could be replaced by L- or DL-alanine.

The life span of circulating platelets labeled in vitro with sodium chromate⁵¹ was normal in rabbits exposed to 750 r of X-ray subsequent to injection of the labeled platelets.

1) Newcastle disease virus was modified by serial passage of the California 11914 strain through tissue cultures using minced chicken embryos suspended in Simm-Sanders medium. 2) The modified virus was administered intramuscularly to over 20,000 chickens of all ages without producing any nervous or respiratory symptoms, but produced an immunity as demonstrated serologically and by intramuscular challenge with a virulent strain of ND virus. One dose of the vaccine given to susceptible chicks at 5 days of age or over induced an immunity lasting at least 12 weeks. Two doses from 7 to 9 weeks apart have resulted in a high degree of immunity which was still evident 33 weeks later. 3) When administered to 2 flocks of chickens, one in acute stage of infectious bronchitis and the other with chronic respiratory disease, the virus apparently did not increase the severity of the respiratory disease condition. 4) Repeated trials showed that vaccinated chickens did not transmit the infection to unvaccinated susceptible birds within the same pen during 33 weeks. 5) The modified virus can be propagated on HeLa cells to produce a vaccine for poultry, thus avoiding the introduction of egg-borne infections to poultry flocks through vaccines produced in chicken embryonated eggs.

1) Our data indicate that antibody against complement can be expressed as protein in specific combination with an immune precipitate to which complement has been affixed. 2) Anti-complement which has been labeled by injection of glycine-1-C¹⁴ into animals during active antibody production can be demonstrated as carbon-14 bound to precipitate-complement complexes. 3) Electrophoretic analysis of the sera of rabbits before and after immunization with guinea pig, human, and mouse complement shows that a consistent rise in γ globulin occurs during the immunization process. 4) Finally, electrochromatographic separation of antibody containing sera proved to be an effective means of concentration and partial purification of the antibody.

Plasma and thoracic lymph concentrations of thyroxine-1-C¹⁴ were followed in normal male rats after a single subcutaneous injection of 780 μg/kg body weight. Approximately 3% of administered dose appeared in the lymph during the 8 hour experimental period. The level of radioactivity in the lymph was lower than the plasma on a volume basis but significantly higher if calculated on the basis of protein content. Cellulose column chromatographic analysis of lymph samples taken between 2 and 4 hours, revealed the radioactivity present to be unaltered thyroxine.

Electroshock seizures have been studied in young rats and guinea pigs, and a correlation has been demonstrated between the developmental pattern and threshold of the induced seizures and the level of carbonic anhydrase activity in the brain. In young rats aged 20 to 35 days, the seizures were mainly tonic in pattern, whereas the clonic type of seizure predominated in rats aged 10 to 20 days. Failure to induce convulsions in the newborn rat was associated with a low level of carbonic anhydrase activity in the brain. The degree of maturation of the newborn guinea pig was equivalent to that of the 15-day-old rat, both with regard to the type of seizure response and the level of brain carbonic anhydrase activity. That brain carbonic anhydrase is of functional significance in the generalized spread of the seizure discharge and in the production of a maximal tonic convulsion is suggested by these data and by observations of the anticonvulsant activity of acetazolamide.

A method for determining the allantoic fluid volume of embryonated eggs by dilution of RISA is described. Experimental porphyria in chick embryos results in a marked increase in allantoic fluid volume.

Results of a comparative study of the cell responses induced by several strains of the Col SK group of viruses in various tissue culture substrates are reported. L cells, HeLa cells and KB cells supported cytopathogenic growth of EMC and of Mengo virus, F virus grew only on L cells, and Col SK and MM viruses failed to grow in any of the 3 substrates. HeLa tissue cultures inoculated with EMC virus, in remote serial passage, were infectious for HeLa cells and for mice at an ID₅₀ of about 10⁵ per 1 cc of culture fluid and had a hemagglutinating titer of between 1:2 and 1:4. Data permitting the establishment of a growth curve of EMC tissue culture virus on HeLa cells are submitted.

CBA mice protected against an otherwise lethal dose of irradiation with isologous CBA bone marrow showed normal rejection of skin grafts from the Cb strain. Irradiated CBA mice protected with Cb or (Cb x CBA) F₁ hybrid marrow were tolerant of skin grafts from the Cb strain. Irradiated CBA mice protected with a mixture of marrow from the Cb and CBA strain were not tolerant of skin grafts from the Cb strain. Late mortality (21 to 60 days post-irradiation), after good early protection (21 days) against doses of whole body X-irradiation above the LD₁₀₀, did not occur in mice protected with isologous bone marrow (where neither the host nor the donor can react immunologically against each other). It occurred in mice protected with homologous marrow from a foreign strain (where both the host strain and the donor strain can potentially react immunologically against each other). It did not occur in mice protected with marrow from an F, hybrid of the irradiated strain (where the host strain can potentially react against the donor strain but the donor strain has no potential to react against the host strain). It occurred in some but not all combinations of irradiated F₁ hybrids protected with marrow from a parent strain (where the host has no potential to react against the donor strain but the donor strain can potentially react against the recipient strain).

The terms “homologous disease'”and “heterologous disease'”are proposed to designate the complications specifically attributable to successful transplantation of homologous or heterologous, as opposed to isologous or autologous, bone marrow or lymphoid tissue into a tolerant host.

1. Tissue cultures of chick embryo liver, stomach, intestine and kidney, in which both epithelium and fibroblasts were growing for over 3 months produced hyaluronic acid, as shown by a positive mucin clot test and by the prevention of mucin clot formation by hyaluronidase. These findings are consistent with the demonstration of the spreading reaction in some of these organs and indicate that they belong to the group of hyaluronate-active spreading reactions. 2. In primary cultures production of hyaluronic acid may take place for very long periods of time, usually as long as the cultures do not deteriorate, while replica cultures, for instance strain L, sooner or later lose the ability to produce hyaluronic acid, despite their excellent appearance and good growth. 3. Although it has not yet been possible to separate factors promoting mucopolysaccharide production from factors promoting growth, it has been observed that cultures kept at room temperatures with considerably reduced growth, may sometimes continue to produce hyaluronic acid. 4. Hyaluronic acid of chick embryo extract is depolymerized in the first few days of incubation at 38°C (Table II), while hyaluronic acid produced in tissue culture is not depolymerized for months under the same conditions (Table IIa). A positive mucin clot test after a few days of incubation thus always indicates the presence of hyaluronic acid newly produced in vitro by fibroblasts. Chick embryo extract may therefore be used in any concentration in the culture medium in which production of hyaluronic acid in vitro will be tested by the mucin clot method.

Calcification of the rachitic metaphysis, with typical silver line test, was obtained in tibial slices that were transplanted to rats on a normal diet. Similar transplants to rats on a rachitogenic diet did not calcify. This difference was probably due to the higher Ca x P product in the body fluids of animals on a normal diet as compared to the product of animals on a rachitogenic diet. In vitro calcification was obtained in the sera of normal animals, in the sera of rachitic animals to which phosphate was added to raise the Ca x P product but not in the unmodified sera of rachitic animals. In vitro calcification was obtained in inorganic solutions with a Ca x P product of 50 but not with a Ca x P product of 20. Transplanted deep freeze stored bone slices treated with calcium chloride prior to deep freeze calcified more intensively than deep freeze stored bones not treated with calcium, as measured by the silver line test. Calcification in vitro studies indicated reversible inactivation of calcifiability of stored frozen bone which could be prevented or restored by calcium chloride treatment. While differences existed, calcification of the deep freeze stored bone indicated that there was a partial restoration of the calcifying mechanism or that some unknown ingredient exists in the body fluids which, if present, could have elicited restoration of the calcifying mechanism. Transplantation of rachitic bone, as proposed, may be an additional method for studying the differences of the actual body fluids on calcification of a given rachitic bone, or after treatment of such bone the changes in calcifiability with an actual standard body fluid such as that of an animal on a normal diet.

1. The plasma tide of AHF was measured after subcutaneous and intramuscular injections of bovine AHF fractions into hemophilic dogs. Better results were obtained with intramuscular than with subcutaneous administration; higher plasma AHF levels were maintained for longer times. Citrate in adequate concentration in the AHF solutions appeared to have an adjuvant action. 2. The rate of fall off of plasma AHF was followed after moderate levels of this clotting factor had been maintained for several hours. It is suggested that the biologic half-life of AHF in the dog may be at least 23 hours.

Nidation of embryos was delayed from 1 to 45 days in rats ovariectomized on the 3rd day after mating, by subcutaneous injection of 4 mg of progesterone per day. Implantation was delayed until such time as 1 μg of estrone per day was given in addition to the progesterone. Recovery of blastocysts from one horn of the uterus during progesterone treatment and subsequent implantation of the embryos in the other horn induced by the progesterone-estrogen indicates that the embryos are maintained in the uterus in a viable, but resting condition during progesterone treatment. If rats were ovariectomized on the 3rd day and untreated for 8 days, nidation of embryos was not observed when progesterone and estrogen was initiated on the 9th day and given for the next 5 to 12 days. Rats ovariectomized on the 4th day after mating were variable in their response to 4 mg of progesterone, some exhibited delay others implanted at the normal time. Delayed nidation up to at least 3 days was obtained in hypophysectomized rats when they were injected only with 4 mg progesterone per day.

A system in rat liver which oxidizes sulfide to thiosulfate is significantly elevated upon starvation, both in total activity and specific activity. This increase could not be induced in vivo by elevated substrate levels, and was not due to the loss of an inhibitor. Cysteine desulfhydrase levels in rat liver were not materially changed by starvation. These results have been discussed in the light of other findings.

It has been found that a single intracutaneous injection of vit. A in the rabbit causes an increase in thickness of the epidermis (excluding corneum) which is due to an increase in both number and size of the cells in the germinative and granular layers. The corneum becomes thinner and denser. The occurrence of large, young appearing nuclei in the absence of an increase in mitotic rate indicates that the maturation process is slowed.

In 44 tests, in which untreated human gingival tissue from cases of periodontitis simplex was incubated for 10 days in broth with its indigenous microbiota, no breakdown of collagen could be detected either chemically or histologically. All other tissue elements, however, were stripped away. In concurrent controls, bovine Achilles'tendon, stored frozen and sterilized by ethylene oxide as in a previous investigation, was used as a substrate for the same microorganisms and underwent collagenolysis in 37 of 44 tests. Freezing, thawing, and sterilization of human gingival tissue with ethylene oxide rendered its collagen susceptible to degradation by its indigenous flora in 17 of 23 tests. This action of ethylene oxide was paralleled by a striking alteration of the reaction of the tissues to the Mallory-azan stain for connective tissue. The participation of true bacterial collagenases in periodontal disease has not yet been substantiated. Conclusions to the contrary have been based on the digestion of altered collagen by other proteases.

1) Weanling, male, albino mice were more resistant to the skeletal effects of diets containing sweet peas or β-aminopropionitrile (BAPN) than were rats. They also showed a much greater individual variation in susceptibility to the skeletal effects of such diets. At sufficiently high levels of BAPN, however, both skeletal and aortic lesions occurred. The microscopic changes occurring in the aorta are described in detail. 2) Paraphymosis also occurred, but the paralysis and urinary retention often seen in rats receiving similar diets were not observed.

1. Percentage of diploid nuclei observed in livers of young adult hypophysectomized rats is greater than that seen in control animals of comparable age and weight. 2. In the hypophysectomized rat there is a small but significant increase in percentage of binucleate liver cells. 3. Total reduction in mitotic index in 125–135 g rats is no greater in 21 days hypophysectomized rats than in the unoperated animals; however, rate of change may be more rapid in operated animals.

Irradiation of the mouse fetus at 15.5 days gestation to only 10 r x-rays will give it a considerably better chance of survival if it is subjected to the LD/50/30 range of exposure when adult. Other fetal ages from 14.5 to 16.5 days show some benefit of 10 r exposure, but higher exposures (25 r to 300 r) to any age fetus will have a deleterious effect in later life if the mouse is exposed to x-rays.

A procedure is presented for determination of Tolbutamide in plasma. The method consists of a chloroform extraction of weakly acidified plasma, concentration of the extract to dryness, dissolution of the residue in a measured volume of ethanol, treatment of the resulting solution with Darco G-60, and measurement of the absorbance of the resulting charcoal filtrate at 228 mμ. Recoveries of Tolbutamide from human and dog plasmas have been satisfactory and plasma levels determined by this procedure have correlated well with the hypoglycemic response elicited by the drug.

Stress induced in mice by avoidance-learning or by restraint resulted in an increased susceptibility to infection with the virus of herpes simplex.

The effect of profound reduction of glomerular filtration rate on urinary osmolality of 2 subjects with diabetes insipidus was investigated. In both, reduction in filtration rate was associated with production of a significantly hypertonic urine. These observations support the animal experiments which suggest that ADH is not essential for the production of a hypertonic urine.

The serologic response of school children receiving the second inoculation of poliomyelitis vaccine 5 months after the first injection was studied. Thirteen percent had no demonstrable antibodies to any of the 3 types of virus prior to the second stimulus and many others were lacking in antibodies to one or two types. Only 33% had antibodies to all 3 types. Two weeks following the delayed second injection none was without antibodies to any type, and 85% had antibodies to all 3 types. The antibody levels attained by these children were at least as good if not better than those of other age groups following 2 injections at an interval of one month. The conclusion is made that a delay of 5 months between the first and second injections of poliomyelitis vaccine, although not normally recommended, actually proves advantageous in terms of serologic response.

1. Coagulation of rat semen seems to involve a diphasic reaction consisting of 1) flocculation and 2) gelation. 2. Velocity of the coagulation reaction can be timed with reproducible results, under the experimental conditions described. 3. The reaction is linear with dilution of the component secretions. 4. Optimum pH range for the reaction lies between 6.8 and 8.2. 5. Zn⁺⁺ and Cu⁺⁺ appear to inhibit the coagulation reaction. Blood clotting inhibitors have no effect. Na⁺ seems essential for clotting to occur. 6. Heating of the coagulating gland component destroys activity. 7. Slow freezing of the coagulating gland destroys activity, rapid freezing does not. 8. A crude preparation of the coagulating gland component has been obtained by (NH₄)₂SO₄ precipitation. Further work is in progress on purification of this substance.

The multiplication of cytopathogenic strain of Newcastle disease virus in monolayers of chick embryo cells was studied. The latent period in the growth cycle of this virus was found to last 8–9 1/2 hours, followed by a period of rise lasting 3–4 1/2 hours. The average yield of virus per cell in the tissue culture was found to be approximately 300.

A method for continuous intravenous infusion of dogs confined to metabolic cages has been described. The method was free of mechanical problems and was entirely satisfactory in our experience. It can be recommended for any experiment requiring continuous intravenous infusion of dogs for indefinite periods of time.

The conglutination of sensitized sheep erythrocytes or zymosan requires Ca⁺⁺. Mg⁺⁺ and Ba⁺⁺ are inactive but Sr⁺⁺ appears to substitute to a limited extent for Ca⁺⁺. Zymosan inhibits conglutination of sensitized sheep cells by bovine serum. Bovine properdin and zymosan react readily in the absence of Ca⁺⁺, but in the presence of Mg⁺⁺, without any interference due to conglutination.

Details of a technic are presented by means of which newly-hatched, bacteriologically sterile snails (Australorbis glabratus) can be secured for study. Snail eggs containing mature embryos are separated manually from the egg-mass, disinfected externally with dilute sodium hypochlorite, and isolated in sealed tubes containing a suitable salt solution and antibiotics. Using this procedure, 90–100% hatch occurs within 3–4 days of treatment, and tests have shown that only a negligible proportion are bacteriologically contaminated. Details of maintenance, behavior, and longevity of these snails are discussed.

Mononuclear phagocytes which have been altered by ingestion of various particles, take up tubercle bacilli less readily than the same type cell in an unaltered state. This decreased phagocytic activity is observed with both attenuated and virulent tubercle bacilli.

The adaptation of 2 strains of West Nile virus, of Tamil Nad virus and of Japanese B encephalitis virus to chick embryo cell and monkey kidney cell cultures is described. After a variable number of passages, cytopathogenicity was sufficiently enhanced that tube cultures could be used for virus titrations with all 3 viruses. Tissue-culture-adapted strains of West Nile virus could be studied by the plaque titration technic in bottle cultures of monkey kidney epithelial cells. This has not been possible so far with Japanese B virus. Neutralization tests with all 3 viruses were successfully performed in tube cultures and the plaque titration method was used for neutralization tests with 1 strain of West Nile. It is believed that the technics used in these studies have potentialities as tools in epidemiological research on West Nile, Tamil Nad, and Japanese B encephalitis viruses and presumably other members of the arbor virus group.

Incorporation of mevalonic acid into cholesterol by rat liver homogenates is favored by (a) use of tissues from very young rats, (b) use of a “loose'”homogenizer, and (c) saturation of the homogenate with straight oxygen and stoppering. Incorporation of mevalonic acid is less in the presence of compounds that may be intermediates or antimetabolites of cholesterol biosynthesis. With whole homogenates biosynthesis is not markedly influenced by the method of killing the rats or by additions of ATP.

Mumps virus, inoculated into embryonated chicken eggs of 48 hours incubation, produced a high percentage of cataracts in embryos surviving from 3 to 15 days after inoculation. Other defects observed included general growth inhibition, retardation in development of the amnion, mild axis twists, retardation of feathering, defects of the choroid layer of the optic cup and retarded development of the lids and skin around the eye. A decrease in the percentage of embryos showing defects was directly related to a decrease in the titer of the virus inoculated. Virus inoculated into the young embryos was shown to increase in titer over a period of 2 days after inoculation and was recoverable in high titer over a period of at least 5 days. Heat-inactivated mumps virus produced no cataracts or other defects.

The whole-blood B₁₂ values in 10 hyperthyroid subjects before and after a load dose of B₁₂ were lower than for a normal and a myxedematous group. The mean 8-hour urinary excretion after B₁₂ administration in hyperthyroid subjects was considerably lower than in the normal and myxedematous groups. These data suggest that B₁₂ turnover and demand is appreciably greater in hyperthyroidism and appreciably lower in myxedema.

Methods for determination of isocitric and 6-phosphogluconic dehydrogenases in human blood serum are described. The properties of these enzymes, and factors affecting their activity in the blood sera of normal human adults are discussed.

In the mesentery the number of mast cells increased in the perivascular region after exposing the animals for 2 and 4 weeks at 6°C; in the intervascular region the number decreased after 2 weeks but returned to the initial value after 4 weeks. The same changes were observed in animals exposed at 2°C. In the skin of the abdomen an increase was observed at both temperatures after 2 weeks. In the skin of the ear, which is much more exposed to the environmental temperature than the abdomen, a significant drop was observed after 2- and 4-week exposures to 2 and 6°C. However, the decrease was significantly larger at 2°C than at 6°C. The increases in number of mast cells in abdominal skin and the changes in the mesentery are considered to be systemic effects of cold; the decreases in the skin of the ear are considered to be local effects.

A significant increase in the succinoxidase activity of fetal swine ventricles was found to occur during the latter half of gestation, on both the wet weight and per unit of nitrogen basis. As gestation approached termination, an increase in activity occurred in the left ventricles of the maternal heart. These observations show that in large animals the fetal heart increases in succinoxidase activity with increased function, as previously demonstrated in the rat and chick; and that the later stages of gestation make increased requirements of the heart as indicated by greater maternal heart enzymatic activity. Two dietary levels of Mn and position of the fetuses in the uteri had no influence on the succinoxidase activity.

In tissue culture studies, the Edmonston strain of measles virus was neutralized by distemper antiserum prepared in ferrets with the egg-adapted and mouse-adapted strains of distemper virus. All normal ferret sera failed to show any neutralization of measles virus. In animal studies, ferrets immunized with measles virus and subsequently challenged with virulent distemper virus showed some evidence of protection as revealed by prolonged incubation periods, modified illnesses and survivals. The mouse-adapted distemper virus was completely neutralized by measles antiserum prepared in ferrets, whereas, normal serum failed to show any neutralization. Mouse-adapted distemper virus was also neutralized by human measles convalescent sera. These results suggest that common antigenic components are shared by the viruses of measles and distemper.

A group of male chickens exhibiting “aggressive'”and another group exhibiting “passive'”behavior were studied during and after a period of high cholesterol and fat feeding. Although the “aggressive'”groups exhibited more pronounced evidences of androgen activity, their plasma and aortic cholesterol contents were perhaps less than those of the “passive'”group. The extent and degree of coronary atherosclerosis was the same in the two groups.

1. Citric acid content (γ/g wet wt) of blood, diaphragm, duodenum, heart, kidney, liver, lung, and spleen was determined colorimetrically in the following groups of mice: (a) normal mice, (b) mice exposed to simulated altitude of 20,000 ft for 3 to 4 months, (c) mice given daily intraperitoneal injections of 1 mg cortisone acetate for 2 weeks, and (d) mice starved for 17 hours. 2. Mice of group (b) had 20–30% less tissue citrate than control mice. In group (c) there was no change in tissue citrate except in kidney, which increased, and in spleen, which decreased. Tissues from starved animals showed a significant increase in citrate in all tissues. Thus the change observed in group (b) cannot be correlated with cortisone injections nor with a reduced blood sugar level induced by inanition. 3. Altitude exposed mice are more susceptible to Sal. typhimurium infection than control mice.

The total volume of adrenocortical tissue regenerating in intramuscular autotransplants in rats during the first 2 weeks after operation is proportional to number of transplants growing. Thereafter, the total volume is about the same irrespective of the number of implants made or transplants regenerating, so the volumes per transplant are inversely proportional to the number of grafts. Average volume of regenerating tissue and cell depth increases with increasing age of transplant. At 90 days after operation the cell depth is like that of intact adrenal glands but the volume is less. Apparently transplants cannot deliver the amount of cortical secretion necessary to establish a pituitary-adrenal equilibrium until after 2 weeks of growth.

Emulsified fat infused into pancreatectomized dogs is cleared from the blood at a normal rate if the animal is receiving insulin whereas without insulin the injected fat is cleared slowly. It thus appears that the clearance of fat from the blood is dependent upon normal carbohydrate utilization. Serum cholesterol levels rise following infusion of fat in diabetic animals deprived of insulin. Serum cholesterol concentration falls following infusion of fat into pancreatectomized animals receiving insulin. Observations on fat metabolism in which the fat is administered intravenously give data comparable to those obtained following oral administration of fat and the mechanisms of clearance seem identical by the two methods.

An amperometric titration method for determining sulfhydryl groups was applied to mouse skin. The total sulfhydryl, the protein sulfhydryl and by subtraction, the soluble sulfhydryl contents were determined after painting with anthracene, DBA, DMBA, or a combination of the 2 carcinogens. The anthracene and the solvent, acetone, had no effect on the sulfhydryl levels. In general the shape of the curve of the total sulfhydryl content of the epidermis and dermis was the same, although the dermis contained approximately half that of the epidermis. For the most part, the effect on the protein sulfhydryl paralleled the corresponding total sulfhydryl curve. Most of the change in the soluble sulfhydryl level is presumed to occur in the epidermis. No cause and effect relationship to carcinogenesis or anticarcinogenesis is supported by these observations on the sulfhydryl groups of the skin.

The antifibromatogenic and antihysterotrophic activities of 19-nor-testo-sterone phenylpropionate and 19-nor-methyl-testosterone are 6 to 10 times greater than those of testosterone propionate and methyl-testosterone respectively. The activity of testosterone is decreased by Δ¹. No activity is obtained even with very considerable quantities of Δ¹-testosterone or Δ¹-androstenedione.

(1) A specific method for the determination of meprobamate in urine is described. (2) Metabolic studies indicate that about 12 to 20 percent of the unchanged drug is slowly excreted in the urine in 48 hours and that additional products are formed, including several conjugates at least one of which is a glucuronide. (3) Radioactive meprobamate has been prepared and metabolic studies have been performed. The liver is the principal site of metabolic alteration.

1. The effect of pyrazinamide and pyrazinoic acid on the renal clearance of urate, inulin and p-aminohippurate, in some instances also Tm_PAH, was studied in 10 human subjects. 2. A rapid and marked reduction in urate clearance was noted, without decrease in the filtered urate load. The implications as to the renal mechanisms regulating urate excretion are discussed. 3. Data indicating that the effect of pyrazinamide is ascribable largely to the action of pyrazinoic acid are presented.

Chondroitin sulfate extracted from human costal cartilage was prepared in 3 different ways for inoculation into rabbits, (1) a 1% solution, (2) a 1% solution incubated with heat-killed, group A, type 3, hemolytic streptococcus, and (3) a 1% solution incubated with heat-killed Staphylococcus aureus. These 3 preparations were injected into rabbits in an attempt to elicit antibody production. There was no evidence of the formation of precipitating antibodies against chondroitin sulfate in any of the rabbits'sera nor was there any clear indication of the acquisition of skin sensitivity by any of the rabbits.

Alpha-alanine inhibits antibacterial activity of cycloserine on staphylococci. The inhibition appears to be of a competitive nature. This amino acid does not inhibit activity of the antibiotic on other organisms studied. Alpha-alanine does not interfere with the action of other antibiotic agents.