Experimental Biology and Medicine

Deoxyribonucleic acid was isolated from a mercaptopurine-resistant adeno-carcinoma 755 after treatment of the mice with 8-C¹⁴-labeled 6-MP. The DNA was degraded to the deoxyribonucleoside level with pancreatic deoxyribonuclease and the digest was subjected to 2-dimensional chroma tography. Deoxyadenosine and deoxyguanosine contained 20% and 10%, respectively, of the total radioactivity. The material of the major spot (60% of total radioactivity) migrated with authentic thioguanosme deoxyriboside, and after acid hydrolysis, with thioguanine. No 6-MP derivative could be detected.

The influence of hyper tonic glucose and mannitol on blood volume was tested in normovolemic-normotensive and hypovo-lemic-hypotensive dogs. Neither glucose nor mannitol had any effect on the blood volume of normovolemic dogs. In hypovolemic-hypo-tensive dogs, mannitol produced a significant increase in the plasma volume by mobilizing fluid from the extravascular spaces. The fluid entering the circulation was accompanied by a significant amount of protein. Also, plasma volume increased after hemorrhage with glucose, but the increase was small and was not accompanied by protein. Thus, both glucose and mannitol, but particularly the latter, increased the blood volume in hypovolemia.

Acetylsalicylic acid (ASA) stabilizes rat liver lysosomes in vitro from the labilizing influence of incubation at 37°. On a molar concentration basis, this stabilization is slightly greater than that achieved with hydrocortisone and chloroquine. Concentrations of ASA of 10²- M and below do not inhibit either free acid phosphatase or β-glu-curonidase enzyme activity.

The effects of TCAP on the incorporation of C¹⁴-adenine, C¹⁴-guanine, H³-uridine, H³-cytidine, and C¹⁴-valine into frog central and peripheral nervous tissue maintained in vitro was observed. TCAP inhibited C¹⁴-valine incorporation into the protein fraction of both tissues. However, H³-uridine incorporation into RNA was inhibited only in brain tissue. TCA.P caused changes in the nucleic acid metabolism of frog ganglia in general quite similar to those produced by DNP and azide. However, TCAP did inhibit adenine incorporation into RNA whereas DNP and azide did not.

Studies of homologous disease in newborn mice using axenic and conventional animals (a) provide evidence that for the same dosage of cells, populations of spleen cells have different effects according to their origin. They are more efficient if donors are conventional or immunized axenic, and less efficient if the donors are only axenic. The basis for these differences has not been determined. (b) Provide no evidence that super-infection plays a role in the G.V.H. reaction in this short time system; (c) definitely separate homologous disease of newborn from similar syndromes induced by other investigators with experimental infectious agents.

The cooperation and help of Dr. G. A. Voisin and Dr. R. Kinsky are gratefully acknowledged. We also appreciate the invaluable technical assistance given by Mrs. A. Collard and Miss J. Deloupe.

The effect of pinealectomy upon feed consumption and thyroid hormone secretion rate was studied in Sprague-Dawley-Rolfsmeyer female rats. Feed consumption was increased 5.77% in association with an increase of 11.8% in TSR. In a second group of 16 rats, daily injection of 40 μg/day of melatonin depressed feed consumption 12.0%, whereas injection of 20 μg/day of melatonin into young rats depressed TSR 16%.

Two series of unanesthetized rats were infused intraarterially with standardized volumes of 1.46 M (50% w/v) sucrose over the range 2.0 to 5.0 ml/100 g body weight. Animals in one of these series had been exposed 5 days earlier to the same concentration of sucrose at 2.0 ml/100 g body weight. These latter animals constituted the adapted groups. Comparison of the sucrose tolerance of adapted animals with nonadapted revealed that previous exposure to hypertonic sucrose significantly increased the resistance of twice-loaded animals to the damaging effects of the hypertonic solution. Evaluation of the excretion patterns of urinary solutes and the relationships between urine flow and solute concentrations suggest that a reduction in GFR may occur in nonadapted animals as load of hypertonic sucrose is increased from 2.0 to 4.5 ml/100 g b.w. Similar loads given to adapted animals appear less effective in their GFR depressing actions.

The authors gratefully acknowledge the excellent assistance of Mr. Don deSteiguer and Mr. Russell Moses.

Pyrrolidinomethyl tetracycline was administered to rats, rabbits, and monkeys for 10 days and changes in the utilization of glucose and distribution of lipids in the tissues were studied. Treated animals showed diminished glucose tolerance. They had decreased glycogen and increased cholesterol and total lipids in the liver. There was a rise in plasma levels of lipids such as cholesterol, phospholipids, triglycerides and free fatty acids. Changes indicated impaired metabolism of carbohydrate and lipids. Tetracycline moiety of the antibiotic seemed responsible for the changes observed. The drug should be used with caution as its therapeutic effect might disturb the normal metabolic patterns in the body.

The effects of fluoride intake on disuse atrophy of the femoral bone were investigated in rats after severing the right sciatic and femoral nerves. X-ray radiograms of the femora were made. Calcium content of the blood, specific gravity, ash percentage and calcium and fluoride content of both femora were determined in operated and non-operated rats drinking distilled or fluoridated (25 ppm) water for 2 months after starting the experiment. The results showed that fluoride counteracts the typical bone atrophy syndrome; it reduces the calcium content of the serum and prevents bone resorption. It is suggested that increased bone resorption is prevented by a reprecipitation of calcium as fluorapatite of low solubility on the surface of the bone crystals.

Acknowledgement is made gratefully to Ziporah Pinchevski for technical assistance in this study.

A known number of E. coli K-12 cells was injected in bruised and normal poultry tissues and examined daily thereafter. Normal tissues completely eliminated 48 × 10⁴ E. coli cells within 5 days but were unable to eliminate 12 × 10⁸ cells within 8 days. However, the number of cells recovered decreased with time and very few cells were recovered on the 8th day. Regardless of the number of cells injected, the majority of bruised tissue stimulated and supported E. coli growth for one week. At the end of 18 days post-injection, a small percentage of bruised tissues still harbored the test culture. Although extrastromal hemoglobin stimulated E. coli growth, increasing the severity of the bruise (and therefore the amount of blood in this tissue) did not affect E. coli growth significantly.

The effect of Triton on fat retention, blood lipids and growth in rats was studied by determining the amount of fed fat retained in the body, EFA levels of blood and increase in body weight, respectively, after its ingestion along with fat over a 12-week period. It was observed that rats retained less fat in the body when they were fed a diet containing fat and Triton as against the rats which were fed a diet with fat but no added Triton. It appears that Triton does decrease fat retention. An increase in Triton concentration showed little, if any, change in per cent fat retention. EFA levels were decreased at the end of 12 weeks indicating that Triton itself was not absorbed from the intestinal tract but the feeding of polyunsatu-rated fatty acids did decrease the level of the blood lipids. Average growth per week in all the rats on the different diets was almost the same, which suggests that Triton has no toxic effect as indicated by normal growth and well-being of the rats.

The IP or IV injection of 3 ml of plasma from hypoglycemic, hypophysecto-mized rats produced a lowering in hypophyse-al growth hormone (GH) on estimation 30 minutes later by the tibial epiphyseal cartilage test for GH. Plasma from untreated or hypoglycemic, intact rats and from untreated hypophysectomized rats was ineffective. No activity was found in plasma from hypogly-cemic, hypophysectomized rats with hypotha-lamic lesions in the ventral pre-mammillary area. The results are interpreted to mean that hypoglycemia results in the appearance of a circulating GH-releasing factor in the blood of hypophysectomized rats.

Evidence is presented that human erythrocyte glucose-6-phosphate dehy-drogenase is specific for the β anomer of glucose and glucose-6-phosphate. It is suggested that this specificity is responsible for the lack of stimulation of the hexose monophosphate pathway by methylene blue when galactose is used as a substrate.

Kidney cells from several different species of subhuman primates were cultured and infected with SV40. The cells were observed for 35 days, and the course of infection was studied. Cells from Cercopithecus mitis, Cercocebus atys, Papio cynocephalus and an unknown species of baboon were susceptible to the lytic effect of SV40 but were not quantitatively as sensitive as those from Cercopithecus aethiops.

The technical assistance of Beverly Cole is acknowledged.

Cultured kidney cells from a prosimian, mongoose lemur were infected with SV40, which induced a typical cytopath-ic effect after an extensive period of incubation. On the 119th day after infection, transformed cells developed from a focus of cells showing a certain degree of degeneration. The new growth of cells grew rapidly, lacked contact inhibition, formed multilayered cells, and synthesized SV40 and a soluble complement-fixing tumor antigen.

The authors thank Miss Beverly Cole for her technical assistance.

Adenovirus type 3 strain JF was propagated in its early passage history in rhesus monkey kidney cell cultures. Upon discovery of infectious SV40 in the adenovirus, further passages were carried out in African green monkey kidney cell cultures in the presence of SV40 antiserum. Following this procedure, two separate passage lines of the adenovirus were free of detectable infectious SV40, but vaccine seeds prepared from these lines (JF-1 and JF-2) induced in hamsters tumors which contained CF antigens identical to those found in tumors induced by SV40. These tumors, in turn, elicited serum antibodies that fixed complement with SV40 tumor and T antigens. In addition, both lots of adenovirus 3 strain JF induced the formation in human embryo kidney cells of SV40 antigen which was detected by the immuno-fluorescent technique. The induction of fluo-rescent-stainable SV40 T antigen in tissue culture was prevented by treatment of the virus with adenovirus type 3 neutralizing antiserum but not by treatment with SV40 neutralizing antiserum. This SV40 genetic material was presumably enclosed within adenovirus capsids. The findings are discussed as they related to the use of adenovirus vaccines in man.

The authors are indebted to Mr. Horace C. Turner for the performance of the complement fixation tests and to Mrs. Lena Wetherell for technical assistance.

The LD₅₀ of betazole for the pertussis-sensitized mouse is about 80 X that for histamine. Death following challenge is probably the result of a toxic effect other than a histamine-like shock. Betazole is considered to possess a negligible histamine-like action.

The ingestion of protein or other food does not appear to be a primary factor in the cessation of absorption of antibodies from the intestine of newborn puppies.

The authors thank Randy H. Mertens for technical assistance necessary to complete this work.

ACTH (100 U/mg) caused a 64% elevation of citrate and a 26% drop of serum calcium levels at 2 hours after administration of the hormone, intramuscularly, in fasting rabbits. One out of 12 rabbits died in convulsions. Chlordiazepoxide (2 mg/kilo) administered intramuscularly caused a 33% depression of serum citrate levels at 3 hours. This substance had no significant effect on serum calcium levels. ACTH administered one hour after chlordiazepoxide injection caused a greater elevation of citrate levels (110%) which persisted longer than with ACTH alone. In these experiments 7 out of 12 rabbits died in convulsions within 4 hours after ACTH administration. Calcium depression due to the ACTH in this experiment was the same as without the chlordiazepoxide but persisted for a longer period. Diphenylhy-dantoin (20 mg/kilo) did not cause marked changes in the serum citrate or calcium levels. ACTH administered intramuscularly after diphenylhydantoin injection intravenously depressed calcium levels and elevated citrate levels to the same degree as in the absence of the diphenylhydantoin. Diphenylhydantoin had no significant effect on the number of rabbits which survived the experiment.

Oral administration of the non-steroidal anti-inflammatory agents phenyl-butazone and indomethacin prevented or reversed a urate-induced synovitis in unanes-thetized dogs. Acetylsalicylic acid, codeine, promazine, chlorpromazine, topical counter-irritants and intravenous hydrocortisone were inactive. This model of gouty arthritis may be useful in detecting and evaluating orally active nonsteroidal anti-inflammatory agents.

Appreciation is expressed to Merck Sharp & Dohme for indomethacin; to Geigy Pharmaceuticals for phenylbutazone; and to Parke, Davis & Co. for CI-583.

Autofluorescent cells, having abundant lipoid granules, were previously found at the cortico-medullary junction in the thymi of 10-week-old rats. In this investigation, rats were treated variously to test possibilities concerning the undetermined nature of these cells. First, rats aged 5 weeks were adrenalectomized or orchiecto-mized and killed when aged 3 months. In their non-involuted thymi, the pattern of autofluorescent cells was as in the differentially involuted thymi of normal rats of the same age. This indicated that the development of these cells is not dependent on a process starting with thymic involution. Secondly, rats aged 3 weeks were placed on a fat-free diet until they reached 10 weeks. Their considerably involuted thymi contained as many autofluorescent cells as thymi of normal rats of the same age. This revealed the endogenous origin of the lipids of their granules as well as the fact that the formation of these cells is independent of thymic lympho-cytopoiesis and lympholysis, both phenomena having been considerably reduced by the inanition. Finally, rats aged 2 months were cortisone-treated or the thymus was irradiated. Thymic autofluorescent cells remained at the cortico-medullary junction while, in the cortex, macrophages were actively engulfing degenerating lymphocytes. This demonstrated that the thymic autofluorescent cells are not mobilizable macrophages.

The author thanks Mrs. M. Koekebakker for her devoted technical help.

The synthetic substrate, N-acetyl-L-phenylalanyl-L-diiodotyrosine was found to be hydrolyzed by pepsin but not at all by gastricsin. This difference has made possible the separate determination of pepsin and gastricsin in a mixture. The proteolytic activity of the mixture against APDT yielded the amount of pepsin present. The proteolytic activity of the mixture against bovine hemoglobin yielded the total amount of pepsin and gastricsin present. The amount of gastricsin was then simply calculated from the differences in these activities. The accuracy of the method was demonstrated using a reconstituted mixture of human pepsin and gastricsin. The content of pepsin and gastricsin in the native gastric juice was measured in 9 individuals both before and after histamine stimulation. Histamine stimulated the secretion of both pepsin and gastricsin.

The authors are grateful to Dr. John Mills, Mr. Gene May and Mr. Lee Poyer for helpful suggestions in preparing this manuscript.

A method has been described for obtaining isolated human glomerular basement membrane. Evidence for the purity of the isolated GBM includes electron microscopic and chemical data. Some of the membrane constituents, in particular hydroxypro-line, aminosugars, sialic acid, neutral sugars, and lipids have been identified. Hexuronic acid is absent as is DNA. Sixty percent of the nitrogen of the membrane can be accounted for by collagen; the remainder may be contributed by the protein moiety of a glycoprotein species.

The authors gratefully acknowledge the help of Dr. James B. Longley, Dept. of Anatomy, and Dr. G. R. Schrodt, Dept. of Pathology, in preparing the light microscopic and electron microscopic sections and photographs, and Dr. Charles Duncan, Dept. of Medicine, for his help in the thin layer chromatogra-phy of the lipids.

Monase indole-3(2-aminobu-tyl) acetate, a monoamine oxidase inhibitor, and 8 other structurally related compounds with different substitutions on the indole ring and amine side chain inhibited 5-HT uptake by platelets, degree of inhibition being directly related to concentration of inhibitor without particular relation to structure. C¹⁴-Mon-ase which differs from 5-HT by the absence of a hydroxyl group in position 5 of the indole ring and by the presence of a different side chain, was concentrated only slightly by platelets. It was concluded that the structural specificity for 5-HT uptake is largely determined by the hydroxyl group at position 5 of the indole ring.

BDB indirect hemagglutination was determined to be an effective measure of antibody to thermally denatured DNA. It was found equally as effective as bentonite agglutination in detecting anti-DNA in SLE sera. Antibodies to thermally denatured DNA were detected in the sera of experimentally-infected tuberculous rabbits. The antibodies appeared 4-6 weeks post-infection, reached a maximum titer at approximately 8 weeks, and declined thereafter. Similar anti-DNA antibodies were also detected in the sera from patients with tuberculosis or histoplasmosis.

Dr. Roy L. Donnerberg, Benjamin Franklin Tuberculosis Hospital, Columbus, Ohio, generously provided the human tuberculous sera employed in this investigation.

Human histoplasmosis sera were obtained through the courtesy of Dr. J. A. Schmitt, Department of Botany, Ohio State University; Dr. Tom D. Y. Chin, Kansas City Communicable Disease Center; Miss Jo Ann Sparks, Infections Disease Center, University Hospital, Columbus, Ohio.

Normal human sera were obtained from the University Hospital Blood Bank, Columbus, Ohio. Only those serum samples from acceptable blood donors who demonstrated no overt signs of disease were employed.

The observations presented above support the previously advanced concept of the embryogenesis of human and experimental cranioschisis(3-7). They also support the idea that any mesodermal alteration causing its derangement and collapse before the complete closure of the neural tube will necessarily be followed by failure of closure of the neuro-ectoderm, regardless of the nature of the teratogenic agent used. This concept supports the following facts: the closure of the cephalic neuro-ectoderm is a critical and significant period of embryonic development; the cephalic mesoderm plays an important role in its final closure; and cranioschisis (anencephaly), the most commonly induced experimental malformation, is the direct consequence of the failure of the cephalic mesoderm to fulfill its function of enclosing and lodging the cephalic neuro-ectoderm.

Experimental magnesium deficiency in rats is associated with an increased ability of the isolated everted intestine to transfer calcium against a concentration gradient. The augmented calcium accumulation of magnesium deficiency is not altered by replacement of magnesium in the bath. Although a direct effect of magnesium depletion on cellular transport mechanisms is not ruled out, the results are consistent with a humoral influence, like that of parathyroid hormone, on the gut in magnesium deficiency.

The authors gratefully acknowledge the technical assistance of Mrs. Lorraine Pozika.

Serum samples from patients of aseptic meningitis associated with Coxsackie-virus B5 were examined to detect both 2-mercaptoethanol (2-ME) sensitive and resistant neutralizing antibodies. In 75% of the sera obtained at an early stage after the onset of illness, the titer of 2-ME sensitive antibody was significantly higher than that of the 2-ME resistant antibody. During the late stage of infection the level of 2-ME resistant antibody exceeded the 2-ME sensitive titer; this was especially true of sera obtained 2 years after onset of illness. Fractionation of sera by a procedure of sucrose gradient centrifugation revealed that the rapidly sedi-menting peak of antibody corresponded to the 2-ME resistant antibody. The nature of the 2-ME sensitive antibody and the significance of its detection in diagnosis of the viral infection were discussed.

The authors wish to thank Drs. N. Ishida and D. S. Yohn for helpful discussion and advice in preparation of the manuscript.

1. Immuno-phoresis was performed in a series of 42 normal chickens from ages 1 to 210 days. Rabbit serum immunized against normal mature chicken serum was used for antibodies. Bar-bital buffer at pH 8.6, ionic strength 0.1 was used. 2. Identification of fractions was attempted by designating precipitation curves in the chick serum preparation with the names used for curves of known fractions in identical position and of identical shape in human serum preparations made under identical conditions. 3. Significant findings in chicken serum indicate that all fractions are more abundant in neonatal life than at later periods. 4. Prealbumin becomes less abundant after the 12th day of life, and is not detected in the preparations after the 20th day. 5. Precipitation curve in the position of the beta lipoprotein fraction is not observed in preparations after the 8th day of life. 6. Additional fractions of gamma globulin appear in some preparations after the 12th day of life. 7. The findings are significant for comparison with serological protein profiles of chickens on vit E-deficient diets.

1. Twenty-five newly hatched or very young chickens were subjected to a purified diet, deficient in vit E or any synthetic antioxidant and supplemented with 4.8% linoleic acid. If permitted to survive, these chicks developed encephalo-malacia between the 10th and 26th day. Forty-two chicks, fed on a conventional stock laboratory diet, developed normally and were well at the time blood samples were examined. 2. Immunophoresis patterns of blood sera from experimental and normal chickens were obtained. 3. In the experimental sera, an antigen-antibody precipitation curve with the mobility of beta lipoprotein persisted whereas this curve did not persist after the 10th day in normal chickens. This curve was interpreted as due to the presence of an abnormal protein, possibly a denatured beta lipoprotein. 4. The role of possible denaturation of beta lipoproteins in the blood serum in the presence of autoxidation products of linoleic acid in the serum as a possible mechanism for production of vit E deficiency syndromes is discussed.

1. Eleven one-day-old chickens were placed on diets deficient in vitamin E or any synthetic antioxi-dant and on sulfur amino acids. Eight chickens of the same hatch were kept on normal stock diets for comparison. All of the experimental chickens developed paralyses of breast muscles, and weakness of leg muscles in from 6 to 8 weeks. Normal chickens were free from symptoms. Both sets of chickens were sacrificed at the same time. Blood sera, obtained by cardiac puncture, were examined by immunophoresis, using anti-chicken serum antibodies in rabbit serum. 2. Significant im-munophoretic finding was the persistence of a precipitation curve in the beta lipoprotein mobility range in sera of experimental chickens, which is normally not found in chickens of the same age. The remainder of the im-munophoretic preparations pattern was alike in experimental and normal sera. 3. Interpretation of the altered serum protein in chronic vit E-deficient chickens is discussed in terms of altered lipid metabolism.

The effect of adeno-12 and adeno-5 viruses on the incorporation of thy-midine-H³ by cultures of human KB cells, newborn hamster kidney cells, and hamster embryo cells was investigated by radioautog-raphy. In KB cultures, infection with either adeno-5 or adeno-12 virus significantly increased the percentage of labelled cells, but the number of silver grains per labelled cell was somewhat lower than for control cultures. In hamster kidney cells, infection with either adeno-5 or adeno-12 decreased the mitotic index but significantly increased both the percentage of labelled cells and the intensity of label per labelled cell. Intranuclear viral inclusion bodies were formed in as high as 90% of the hamster kidney cells infected with adeno-5, which replicates in hamster cells. The incorporated label was closely associated with the intranuclear viral inclusion bodies. No viral inclusion bodies were found in the hamster kidney cells infected with adeno-12, which has an incomplete growth cycle in hamster cells. Hamster embryo cultures contained only a small percentage of epithelial cells and it was only in these that the intranuclear viral inclusion bodies were found following infection with adeno-5. Fibroblastic cells, and therefore hamster embryo cultures, were much less susceptible to adenovirus-5 (and probably to adeno-12) than the predominantly epithelial hamster kidney cultures. The percentage of labelled cells following infection of hamster embryo cultures with either adeno-5 or adeno-12 increased only slightly as compared to the hamster kidney cultures.

That viral DNA is formed even in the abortive infection of hamster cells with adeno-12 is suggested by a) decreased mitotic index but increased DNA synthesis induced by either adeno-5 or adeno-12, and b) association of the incorporated DNA-label with the viral intranuclear inclusions of hamster cells infected with adeno-5.

Reserpine in dosage sufficient to lower brain stem 5-hydroxytryptamine and norepinephrine suppresses endotoxin fever in rabbits. Pretreatment of rabbits with agents depleting only brain stem norepinephrine but not 5-hydroxytryptamine does not suppress febrile responses to endotoxin. An unexpected finding was the depletion of norepinephrine from the brain stem by guanethidine.

Previous studies in this laboratory have revealed that feeding excess quantities of phenylalanine to rats influences the activity of various liver enzymes(1,2,3), the quantity of brain serotonin(2,4), plasma amino acid levels, and affects these parameters in the developing fetus(5,6). Behavioral responses of rats fed such diets have also been evaluated (7,8). Other studies by Waisman and Har-low(9,10) have provided convincing data that the Rhesus monkey is a better animal than the rat for both biochemical and behavioral experiments designed to study the effect of hyperphenylalaninemia.

In attempts to develop still other animal models of hyperaminoacidemias, hepatic phenylalanine hydroxylase activities of animals of several species were determined and it was surprising to find that the Syrian hamster (Cricetus auratus) had but 18% of the hydroxylase activity of the rat(11). The purpose of this study was to determine the effect of different phenylalanine-supplemented diets on growth, plasma phenylalanine and tyrosine levels, phenylalanine hydroxylase, phenylala-nine-pyruvate transaminase, and tyrosine-a-ketoglutarate transaminase activity in the liver of the growing hamster.

Materials and methods. Weanling hamsters, 25 days of age, were obtained from a local vendor (Con Olson Co., Madison, Wis.) and placed in individual cages, in a room with 3-hour alternate dark-light cycles as previously described(12). The animals were randomized in groups of six, 3 ♂and 3 ♀ to a group, and fed different diets. The first 5 groups received commercial diet,‡ supplemented with varying amounts of L-phenylala-nine, or L-phenylalanine + DL-phenylala-nine; a group which received only commercial diet served as controls. Eight other groups received synthetic diets consisting of varying quantities of casein and dextrose plus corn oil, minerals,§ and vitamins. This basic diet was supplemented with 5% L-phenylalanine or a mixture of L-phenylalanine and DL-phenylalanine, as listed in Table I.

The action of morphine upon isolated mammalian cardiac preparations was reevaluated in order to test the hypothesis that morphine has the ability to antagonize the action of catecholamines on the heart. Over a wide range of concentrations (0.3-100 μg/ml) morphine slightly depressed contractile force, heart rate and coronary flow. Following exposure of the whole isolated heart preparation to morphine (100 μg/ml) for one hour, ventricular catecholamine levels were similar to their untreated controls. Furthermore, no consistent alterations in physiological response to epinephrine, isoproterenol or tyramine could be correlated with the presence or absence of morphine regardless of its concentration in the perfusate. These data fail to confirm the suggestion that morphine acts as a sympatholytic on the heart.

Laying hens were fed 312 to 5000 ppm iodine as KI in a practical laying diet and egg production and hatchability determined. Production ceased within the first week in hens fed the highest level and production was reduced at the lower levels. Fertility of the eggs produced was not affected but early embryonic death, reduced hatch-ability and delayed hatching resulted. Hens resumed egg production within seven days after removal from iodine feeding.

Utilizing enclosures covered with silicone rubber membrane, a new, safe, and simple method for producing polycythemia by hypoxia is presented. The polycythemic animals are excellent for use in erythropoietin assays.

Leucine-C¹⁴ was utilized to study the affects of 2 oral hypoglycemic agents (tol-butamide and phenethylbiguanide) on protein metabolism. Both compounds were seen to inhibit C¹⁴O₂ production and incorporation of the amino acid into protein by rat liver ho-mogenate. Differences in the mode of inhibition are noted and discussed. Neither compound appears to be dependent on the presence of glucose for its in vitro affects on protein metabolism.

Mice were injected with a 19 to 1 mixture of unlabeled and I¹²⁵ labeled beef insulins and sacrificed 2, 5, 10 or 25 minutes later. Insulin in especially prepared tissue extracts and fractions was estimated by: (a) immunoassay, and (b) I¹²⁵ count. Using either assay procedure, insulin (1) reached its highest concentration in liver sooner than it did in kidney, (2) was far more highly concentrated by kidney than by liver, (3) showed decrease in tissue concentration in the order plasma>liver>kidney, and (4) was rapidly and highly concentrated in liver particles brought down between 700 g and 105,000 g. Insulin added to liver ho-mogenate was not concentrated by these particles. These findings are in accord with the hypotheses that radioiodinated and unlabeled forms of insulin are metabolized in similar manner, and that both forms of insulin are capable of penetrating into liver and kidney cells.

We are indebted to Drs. James Ashmore and Peter Wright of our Department for guinea pig anti-insulin serum, to Dr. Mary Root of Eli Lilly Co. for beef insulin of low glucagon content, and to Dr. Howard Rickenberg, Microbiology Department, Indiana University, for use of a tissue ultrasonicator.

The inherited absence of an effective hemolytic complement system is linked with a deficiency in an antigen, MuBl, and constitutes an inborn error of metabolism. MuBl is completely lacking in animals of certain inbred strains of mice. The regulation of the concentration of MuBl as a function of age, of sex and of the number of gene copies per cell, was studied. The concentration of MuBl was identical in 3-week-old males and females. In older animals, it was approximately 1.5 times as high in the male as in the female. The concentration of the antigen was twice as great in homozygous as in heterozygous animals.

MuBl, a complement component of the mouse, occurs in higher concentration in male than in female animals. In the gonadectomized male, the level of MuBl is identical with that of the female, but can be restored to its original value and even increased above its normal concentration by administration of testosterone propionate. MuBl concentration of the female can also be increased by testosterone. Administration of medroxy progesterone acetate leads to a less pronounced increase in concentration. This is found in sham operated females, gonadectomized males and females, but not in sham operated males. Oestradiol monobenzoate slightly augments MuBl levels of gonadectomized female and male animals and of sham operated females. It stabilizes the MuBl concentration of sham operated males, thus causing a relatively lower concentration of MuBl in sham operated, oestrogen-treated males, than in sham operated uninjected males of identical age.

A tissue culture cell line of rabbit cornea (SIRC) has been discovered by Leerhoy(1) to show clear and readily detectable CPE when inoculated with rubella virus. This observation has been confirmed and extended. The SIRC cells have been shown to be as effective for primary isolation of rubella virus from throat swabs, rectal swabs, urine, cerebrospinal fluid, and lens tissue as the more widely used GMK cells. As previously noted for both GMK and primary human amnion cells(9), subcultivations are necessary with SIRC cells for maximum isolation of rubella virus. SIRC cells may be used for detecting rubella neutralizing antibody in human sera and titers obtained are comparable to those obtained in GMK. Serial passage of rubella virus in SIRC cells appears to yield higher virus titers than those obtained after serial passage in GMK.

SIRC cells offer several advantages. They are a continuous cell line easily maintained in the laboratory. They produce direct CPE, eliminating the need for challenge of cultures with a second virus, and they reduce the time necessary to obtain test results. They should prove useful in rubella virus diagnosis and investigation.

The reduction of tetrazolium by M. pneumoniae is inhibited by specific serum. Based on this, a method has been derived for measuring growth inhibiting antibody. This procedure was applied to post-vaccination and convalescent sera from animal and human subjects and compared with CF and IHA antibodies. The presence of TRI antibody correlated well with resistance to disease.

Two morphologically distinct types of MV plaques were produced on mono-layers of RK₁₃ cells under agar; the appearance of these plaques depended upon the presence or absence of sodium bicarbonate in the agar overlay medium. The numbers of plaques produced by a given suspension of myxoma virus were identical, regardless of the presence or absence of sodium bicarbonate. Moreover, the yield of virus from infected RK₁₃ cells was not altered by addition or omission of sodium bicarbonate. The specificity of the two plaque types was established by pathogenicity tests and neutralization of plaque forming units by anti-myxoma serum.

In polyacrylamide gel electro-phoresis of bovine I¹³¹-TSH the radioactivity and biological activity migrate similarly but with slight dissociation. Increasing quantities of antiserum shift increasing quantities of radioactivity to slower moving regions. Bovine TSH preparations interfere with I¹³¹-TSH binding to antibody in proportion to biological activity. Human TSH is much less active.

We thank Messrs. Frank Zodnik and Donald Eros for technical assistance; Drs. Robert W. Bates, Mary E. Carsten and John G. Pierce, and Peter G. Cond-liffe for supplying purified bovine TSH preparations.

Volunteers were given A2 influenza virus in a small-particle aerosol. Infection and typical influenza resulted from low doses of virus administered in this manner. Low levels of serum neutralizing antibody were not completely effective in preventing infection and illness. The human infectious dose of this influenza strain when administered by aerosol to subjects free of serum neutralizing antibody was approximately 3 TCID₅₀.

Gratitude is expressed to Dr. David W. Ailing for assistance with the statistics and in calculating dosages; Mr. Edward W. Harvey, Mr. Irven B. Stacy, Mr. Leonard P. Durocher, and Mr. James Turner for technical assistance; and Mr. Edward P. Derren-bacher aand Mr. Charles O. Masemore for assistance with volunteer inoculations.

The mechanism of the exaggerated natriuretic response to saline infusion exhibited by hypertensive subjects is not understood. An attempt was therefore made to reproduce this phenomenon in laboratory animals rendered hypertensive by pharmacologic means. Six mongrel dogs were studied. Their natriuretic response to saline infusion at a rate of 1 ml per minute was determined. The same dogs were restudied while their blood pressure was elevated with metaraminol. An enhanced natriuretic response to normal saline infusion was observed in 5 out of the 6 dogs made acutely hypertensive with metaraminol. The demonstration that exaggerated natriuresis may be induced in a species other than man makes available an experimental model for further study of this phenomenon. The present observations support previous evidence in man that exaggerated natriuresis depends on the presence of elevated blood pressure alone and is independent of the cause of the hypertension. From the acute nature of the present experiment the thesis that exaggerated natriuresis represents the shedding of sodium sequestered during the hypertensive state is rendered untenable.

Hemagglutinins (HA) of JBE virus prepared from infected HKC cultures were masked by serum, an essential ingredient of good nutrient medium. By removing the serum containing medium at the first sign of CPE and substituting a serum-free medium 199 at pH 8.0, until harvesting 16 hours later, a high titered HA was obtained. Reducing the amount of culture medium resulted in a further proportional increase in hemagglutinins and an HA titer of at least 1:1024 was easily obtained. Factors such as adaptation of the virus strain to tissue culture, multiplicity of infection as respects the inoculum, and delayed harvest did not seem to play any important role in HA titer. However, the presence of a stabilizer (H.A1.) in the serum-free medium may have a minor effect on HA titer at pH 7.0. A direct relationship was usually found between the infectious and HA titers in a freshly harvested serum-free medium. Stability of hemagglutinin was demonstrated for at least 8 days at 30° and 37°C in medium 199 containing 2% H.A1. at pH 7.8 though the infectivity titer decreased rapidly. At 5°C the HA titer was preserved without detectable decrease for at least 18 months and such stability was not dependent on the pH of the harvest or the presence of any added stabilizer in the medium. Such HA was non-infectious. The simple technique employed to achieve high-titered hemagglutinin from tissue culture and its stability together with loss of infectivity at refrigerator temperature offer a valuable substitute for the suckling mouse brain antigen for this, and at least some other group B arboviruses and probably others.

The Nakayama strain of JBE virus which, was grown in HKC and harvested in medium 199 with 2% H. Al., was inactivated by 1:4000 formalin and tested for potency. The inactivation curves at 30° and 37°C were biphasic with the inflection points occurring late in the reaction at a low survival ratio of 10-⁵ or less. The second phase of inactivation proceeded at a rate comparable to that of heat of the same degree alone, resulting in a great prolongation of the time required for inactivation. The antigenic potency appeared to decrease with prolongation of the inactivation time and when the triple inactivation period was achieved the potency of most lots was unacceptable. The OCT-attenuated strain of JBE virus prepared previously in the same manner and with comparable infectivity titers prior to inactivation was inactivated in less than half the time required by the Nakayama virus and consistently yielded a more potent vaccine.

(1) Chick and poult blood folates were measured microbiologically, using L. casei, S. jaecalis and P. cerevisiae, with and without incubation with chicken pancreas conjugase. Before incubation with conjugase, folate activity was low in blood of both chicks and poults. It increased greatly, particularly for L. casei, following incubation with chicken pancreas. (2) The use of pteroylmonoglutamic acid or pteroylheptagluta-mates (in brewer's dried yeast) as different sources of dietary folic acid did not alter the form of folate activities found by L. casei, S. jaecalis, or P. cerevisiae in chick or poult blood, either before or after incubation with chicken pancreas. (3) The major folate activity in both chick and poult blood was present as a form which was most active for L. casei following chicken pancreas conjugase treatment. Therefore, folate appears to be present in chick and poult blood mainly as a conjugate of N⁵-methyl-tetra-hydropteroyl-glutamic acid. (4) Although the extent of dilution of portal blood with systemic blood was not measured, the very low level of pteroylmonoglutamic acid in portal blood coupled with the approximately equivalent levels of conjugated folate active for L. casei in both portal and systemic blood indicate that both pteroylmonoglutamic acid and the pteroyl polyglutamates of brewer's yeast may be transformed to a conjugate of N⁵-methyl-tetra-hydropteroylglutamic acid in the intestinal mucosa.

The authors wish to acknowledge grants in support of this work by Agway, Inc., Syracuse, N. Y., and The Distillers Feed Research Council, Cincinnati, O.

Potent preparations of growth hormone-releasing factor (GRF) were prepared from bovine hypothalamic extracts, by the glacial acetic acid concentration procedure followed by gel filtration on Sephadex. Purified GRF is distinct from vasopressin, oxy-tocin, a-MSH and other hypothalamic-releasing factors.

We are grateful to Dr. E. Müller for advice on the use of his GRF assay and to Dr. William Locke for help in preparation of the manuscript.

Glucagon injected into the can-nulated sinus node artery of dogs produces a positive chronotropic effect. This effect appears due in part to local release of nodal norepinephrine and in part to a direct action by glucagon on the sinus node.

A technique whereby tumor cells can be introduced into the medullary cavity of the rat femur has been reported(l). This report describes the successful growth of mammary tumor in the same site.

Method and material. The experiments fell into two phases.

Phase I. Sprague-Dawley female rats were divided into 2 groups of 20. The first group received an homologous suspension of DMBA-induced breast cancer cells. Following the inoculation the tumor failed to grow, was at no time radiologically demonstrable and no viable tumor cells remained when the femora were histologically examined.

The second group received an autologous suspension of DMBA-induced breast cancer cells. All the animals in this group developed lytic lesions in the femur, well shown radio-logically and confirmed by histology. The difficulty, however, of establishing a uniform group of animals where inoculation of the femur could be performed in a fixed number, at a fixed time, with the same tumor, led us to seek another strain of animals. The inbred Wistar-Furth rat, in which a well-established breast tumor is easily transplanted and grows within a few weeks, was therefore chosen for the next phase. This allowed preparation of a uniform breast tumor suspension that could be used for inoculation of a group of animals. Phase II. Two major groups of transplant-able breast tumor are available with the Wistar-Furth animals: 1. A highly autonomous tumor. 2. A hormone-dependent tumor. These have been described by Kim(2) and it was decided ideal to see if the transplant-able tumors of type 2 could be utilized. Using the Wistar-Furth rats would thus eliminate the induction time of the tumor, a standard cell group could be used for each batch of rats and alterations of hormone environment, drugs and cytotoxic agents on the bone tumor, which is the main aim of the work, could be studied.

Using Boyden'sin vitro method, it was demonstrated that rabbit polymorpho-nuclear leukocytes reversed their direction of migration in response to a concentration gradient of chemotactic serum. The presence of a chemotactic factor or factors associated with the lysosomal fraction of leukocytes which does not require fresh serum for its activity was shown.

I am very grateful to Dr. Richard B. Crandall for the use of his laboratory and for his valuable advice, and also to Drs. E. Suter and J. J. Cebra for comments and suggestions.

Cell lines derived from adeno-virus-induced tumors or adenovirus-trans-formed cell lines clumped or retracted in 7.5 mM calcium or less, a characteristic generally not shown by cells derived from other virus-induced tumors or cells transformed by other viruses. Similarly, standard primary, diploid, and heteroploid cell cultures were not sensitive to 7.5 mM calcium. In support of these observations, the use of a low calcium medium facilitated the passaging of adenovirus-trans-formed cells in tissue culture; however, the use of a completely calcium-free medium resulted in a deficiency which caused detachment of the culture. The calcium effect may be useful as a marker to substantiate other evidence that a tumor was induced by adeno-virus or that a cell line was transformed by adenovirus.

The young chick actively converts sulfate-sulfur into taurine. The presence of optimum levels of inorganic sulfate in the diet appears to enhance the reaction more than with organic sulfur. Dietary cysteine represses the formation of total taurine in the liver and taurocholate-S³⁵in the bile fluid. These preliminary data suggest that one or more of the nonessential amino acids may combine with activated sulfate to form taurine without passing via cysteine. Components of one carbon metabolism and vitamin B¹². appear to be associated with the overall reaction.

A series of ω-hydroxyalkylfla-vins with 2 to 6 carbon chain lengths are shown to markedly enhance the utilization of normally suboptimal amounts of riboflavin given to growing rats. These analogs cannot function in place of the vitamin, but may decrease its metabolic destruction.

Nuclei isolated from rat liver cells can incorporate H³-cytidine triphosphate into RNA in vitro. Cortisol added to the incubation medium is taken up by the nuclei in small amounts, but does not stimulate nuclear RNA synthesis. Administration of the hormone in vivo, however, results in a marked increase in incorporation of H³-cytidine triphosphate incubated in vitro with nuclei isolated from 20 to 180 minutes after the injection. Synthesis of RNA in vitro is accompanied by a simultaneous partial degradation of RNA which affects the yields of RNA ex-tractable from nuclei even after very short periods of incubation.

Agglutinin titers for Macacus rhesus erythrocytes were higher in sera of infectious hepatitis patients than those of normal persons. The agglutinin was found to be a macroglobulin antibody (19 S), as shown by ultracentrifugation, zone electrophoresis, gel-filtration and sensitivity to heating and mercaptoethanol.

The authors wish to thank Dr. D. S. Yohn for reviewing this manuscript.

Ectopic calcification of the aortas of rats was induced by large doses of vit D₃. The calcification was accompanied by sudanophilia of the elastic fibers, where the calcification started, and by the presence of mucopolysaccharides between the fibers, as indicated by P.A.S. and alcian blue stains. Concurrent subcutaneous administration of condensed phosphates prevented the calcification as assessed by chemical determination of calcium and by silver nitrate staining. The development of the various matrix stains was inhibited as well. The most likely mechanism of this inhibition is thought to be the prevention of the formation of calcium phosphate crystals by the condensed phosphates. Possible mechanisms of the inhibition of the matrix changes are discussed.

We are grateful to Miss Isabelle Frossard and Miss Marianne Latscha for technical assistance.

The inhibitory effect of cysteine in the S. griseus phage-host system involved a sulfhydryl effect, which at a concentration of 50 μg/ml caused a 75% decrease in phage yield. A relatively short exposure time of 20 minutes during the early portion of the latent period was sufficient to produce near maximum inhibition. Presence of homoserine, methionine, threonine, or isoleucine did not alleviate the effect. The data obtained favor an interference mechanism whereby phage protein biosynthesis is inhibited through disulfide bond cleavage.

The effect of DMSO upon intestinal transport of glucose, galactose, and 3-0-methylglucose was examined. If glucose or galactose are present in the lumen in high initial concentrations, DMSO enhances the rate of absorption, and also changes the kinetics of transport from a saturation type (typical for carrier-mediated transport) to a diffusion type. Intestinal absorption of 3-0-methylglucose is not influenced by DMSO. If the sugar absorption is primarily an active transport, DMSO does not enhance it. It is concluded that DMSO causes glucose and galactose to become sufficiently fat-soluble to bypass carrier mediation.

Dimethyl sulfone has been detected and isolated from pasteurized cows'milk. The source of the sulfone is not yet known. Dimethyl sulfoxide was not detected.

Subcellular fractions and extracted lipopolysaccharide of P. aeruginosa were assayed for toxic activity by the mouse lethality technique. Toxic activity was demonstrated to be associated with the cell wall fraction. The isolated lipopolysaccharide was shown to be altered by treatment with lyso-zyme and with EDTA as evidenced by its loss of lethality for mice.

A technique for preparing neonatal rabbit blood for chromosome studies is described. It uses whole blood, Waymouth's tissue culture media, phytohemagglutinin M, and fetal calf serum. The advantages are the small quantity of blood required, the simplicity of equipment and facilities needed, and the efficiency of the combination of culture ingredients and procedures in the production of usable metaphases.

The distribution of lysosomal enzymes in conceptuses during resorption induced by ovariectomy was studied by histo-chemical methods. The histologic changes associated with ovariectomy-induced resorption were, in sequence, disruption of the maternal-fetal blood barrier (with mixing of fetal and maternal blood), simultaneous degeneration of the cells of the fetus, yolk sac, and placental labyrinth, and invasion of the fetal area with maternal blood. Histochemical study of acid phosphatase in the resorbing tissues showed a persistence of the lysosomes in the fetus, yolk sac, and placental labyrinth during tissue degeneration. There was a change in the distribution of the lysosomes in the fetal neural tube during the process of resorption.

Histochemical studies of the conceptus were made using the Barka-Ander-son and Gomori techniques for acid phospha-tase and periodic acid-Schiff reagent. Triton injection caused a marked vacuolation in the lysosomes of the visceral yolk sac cells, a pronounced increase in the amount of PAS-positive debris in the giant cell cytoplasm, and a loss of cell cohesion in general. A significant increase in resorption occurred with all dosage levels of Triton.

The heteroanthracenes acrifla-vine, eosin, Janus green, methylene blue, ribo-flavin, safranin and thionin inactivated ac-tinophage MSP2 when exposed to bright light. The extent of photoinactivation was determined by dye concentration, duration of exposure to light, ionic strength of the milieu, and availability of air. In general, combinations of photosensitizing dyes had an additive effect, indicating different mechanisms of action. The data suggest, however, that all of the dyes were damaging the phage deoxyribo-nucleic acid.

Dietary cholestyramine (MK 135) increased the rate of mobilization of blood and liver cholesterol in mice. The bile acid turnover rate in normal mice was 5 days, in mice treated with 1% cholestyramine, 1¼ days, or 4 times as fast. Bile acid pool size was 5.62 ± 1.09 mg in normals, 4.58 ± 0.65 in cholestyramine-treated mice. The pool was shown to consist almost exclusively of cholic acid and this spectrum was not altered by cholestyramine. The daily rate of synthesis of bile acids in normal mice was 0.56 mg, 1.84 mg in cholestyramine-treated mice. Digi-tonin precipitable fecal sterol excretion was 5.54 mg day in normal mice, 7.58 mg/day in mice treated with 1% MK 135. No conclusion could be reached as to whether the increased rate of mobilization of accumulated cholesterol affected by MK 135 was due to the increased excretion of either the fecal bile acid or sterol fraction exclusively, or was attributable to the excretion of both these fractions.

We wish to acknowledge the excellent technical assistance of Mrs. J. Bertasius. We also wish to thank Merck Sharp and Dohme Research Laboratories for a generous gift of cholestyramine.

Intravenous administration of sodium salicylate, 150 mg/kg twice within 2 hours to normal rabbits, resulted in a significant decrease in the tissue magnesium content of bone cortex, brain, appendix, heart, kidney, liver, and lung. The turnover of magnesium, as measured by Mg²⁸, was increased in the bone marrow, kidney, and liver.

In animals given sodium salicylate in daily doses of 40 mg/kg for 6 days, tissue magnesium content was significantly decreased in bone cortex, brain, appendix, adrenal, heart, kidney, liver, lung, muscle, skin, stomach, and testis. The relative specific activity was increased in the brain, appendix, heart, kidney, lung, and stomach. In rabbits, sodium salicylate depletes the tissues of magnesium and increases the turnover of this element in the body.

Cell-free extracts of Nocardia corallina were shown to contain glucose 6-phosphate and gluconate 6-phosphate de-hydrogenases which were NADP specific as well as enzymes for the conversion of ribose 5-phosphate into ketopentose, heptulose, and hexose. The results were interpreted as evidence for the major steps of the pentose pathway in N. corallina. Cells from various stages of the growth cycle differed in glucose 6-phosphate dehydrogenase specific activity which suggests that changes in pentose cycle activity may be correlated with differences in fat metabolism and the life cycle previously reported for this organism.

5-Hydroxytryptophol has been isolated, identified, and measured in urine of subjects with carcinoid tumors. In the urine of normal subjects and of patients with carcinoid tumors who ingested 5-HT-C¹⁴, 2.3% of the excreted C¹⁴ was attributable to 5-HTOH and its conjugates and 82.3% to 5-HIAA. These data show that 5-HTOH is a 5-HT metabolite in man as well as in animals.

1. The activity against M yco-bacterium leprae of 4,4-diaminodiphenyl sul-fone (DDS) was tested in mice by feeding a series of diets containing the drug in concentrations ranging from 0.03 to 0.00001%. Multiplication of M. leprae was completely suppressed at all levels. 2. The lowest DDS intake was a hundred times less that the least amount required to produce chemically detectable amounts in the blood (0.2 to 0.3 μg/ml).

The action of several peptide and amine hormones on their target tissues involves the generation of adenosine-3′-5′-monophosphate (3′5′-AMP). Inhibition of the enzymatic breakdown of 3′5′-AMP by high concentrations of theophylline results, in most of these hormone-tissue systems, in a simulation of the hormone effect or in a po-tentiation of its action. Adrenocorticotrophin (ACTH) action on steroidogenesis in adrenals, which involves 3′,5′-AMP formation; is not affected by theophylline to the same degree as the actions of other hormones on their target tissues. A possible explanation for this discrepancy may be found in the fact that high concentrations of theophylline inhibit protein synthesis in surviving rat adrenal tissue, a process which is known to be involved in the steroidogenic action of both ACTH and 3′,5′-AMP. It is suggested that theophylline action in rat adrenals has two antagonistic components, (1) potentiation of the steroidogenic action by protection of .3′,5′-AMP, and (2) reduction of the steroidogenic action by inhibition of protein synthesis.

Intracellular hydrogen ion changes in skeletal muscle from rats on a low-K regimen for 14 or 35 days were determined by DMO distribution. The objective was to determine whether changes in intracellular pH, plasma pH and plasma bicarbonate concentration would parallel the Na plus K deficit in K-deficient muscle. The results of this study were interpreted as indicating that the increased acidity in skeletal muscle from K-deficient rats is apparently related to the chronicity of K restriction but totally unrelated to the unequal replacement of muscle K loss by Na gain.

Serum gamma globulin levels of young NZW and NZB-NZC mice were found to be similar to those of 4 control inbred strains. Serum gamma globulin levels of young NZC and NZB-NZW mice were somewhat higher than those of the controls, whereas the levels were significantly higher in young NZB mice. In all strains studied the gamma and beta globulin levels were significantly higher at 16 to 19 months than at 3 or 9 months.

We wish to thank Mrs. Susan Vanek and Miss Mary Ellen Wilsterman for technical assistance.

On asynchronous and synchronized cultures of Chang's conjunctiva cells it was shown that cytotoxic antibodies (in the presence of complement) were most effective during the stage of cytoplasmic cell division, i.e., between mitosis and G₁-phase. All other stages of the cellular life cycle were relatively resistant in that longer incubation periods were required for cell destruction. A newly developed combined tetrazolium-Feulgen reaction was used in these studies with advantage.

Three γA myeloma proteins were encountered which were very similar but differed markedly in antigenic properties from the bulk of γA proteins. Several antisera from rabbits as well as primates served to distinguish these proteins as belonging to a well denned subgroup. The distinguishing characteristics were shown to be properties of the heavy chains. A general but not absolute relationship to electrophoretic mobility was demonstrated.

Addendum. Exchange of γA proteins with Drs. Vaerman and Her emails and also with Drs. Feinstein and Franklin indicate that these two groups of workers have also observed the same subgroups.

The authors are indebted to Dr. Thomas B. Tomasi for isolated γA proteins from salica, colostrum and ascitic fluid as well as for certain γA myeloma proteins and to Drs. R. S. Beckett and C. Rankin for a number of the γA myeloma sera.

Numerous reports have been concerned with the factors or properties that cause an inferior nutritive value of raw soybean meal as compared to properly cooked meal. These reports have been reviewed recently(1). Among such factors have been anti-enzymes, pancreatic hypertrophy, amino acid deficiency or unavailability, toxic proteins, and indigestible protein fractions, but the effects or even the existence of some are still matters of disagreement.

Proteins which are known to be highly indigestible, i.e., the keratins, have been shown to be improved in digestibility by steam cooking under pressure. A comprehensive report (2) has reviewed the literature in this field and presented specific data in respect to feather protein. It was shown that the principal changes on treatment with steam were loss of cystine, corresponding appearance of lanthionine and increased susceptibility to enzymatic hydrolysis. The conversion of cystine in part to lanthionine, which involves breakage of disulfide cross links between amino acid chains and loss of one-half the sulfur, was most rapid during the first 30 minutes of processing at 30 1b steam pressure. At the same time, amino nitrogen liberated by rupture of peptide bonds increased very slightly, if at all.

Some data (3) obtained during steam processing of hoof and horn meal are in agreement with the later studies on feather protein. Volatile sulfur, particularly H₂S, was detected in the exhaust gases. Neartly two-thirds of the original cystine was lost, while the apparent digestibility to pepsin and HCI improved from an original 13 to 73%. The loss of sulfur from proteins during cooking has been known for a long time. Recently, the cooking of cod muscle has been shown to result in a loss of cystine(4). We investigated the possibility that such changes could take place in other protein sources such as the soybean.

Lines of cells derived originally from various hamster and human tissues and transformed in vitro by SV₄₀ consistently exhibited increased resistance to infection with herpes simplex virus, as compared with control cells not exposed to SV₄₀. Increased resistance did not appear to depend upon differences in cellular capacity to adsorb HSV or to allow penetration into the cell; nor could it be related to enhanced production of interferon. It is suggested that the SV₄₀ genome in the nucleus of transformed cells interferes in a manner as yet undefined with the synthesis of HSV which occurs at the same site.

Normally well-tolerated doses of hypertonic NaCl produce extensive tissue necrosis in the rat if administered simultaneously with serotonin or such mast-cell dischargers as dextran, cpd. 48/80 or polymyxin. This predisposition for necrosis is demonstrable only if the conditioning agent is administered during a brief “critical period” simultaneously with or shortly before the hypertonic solution.

Angiotensinogen contained in mouse plasma reacts exclusively with mouse kidney extract containing renin, to release angiotensin. Renin or kidney extracts from other species (pig, rat, hamster, rabbit) as well as from man do not liberate angiotensin if incubated with mouse plasma substrate. Plasma from nephrectomized mice, when incubated with mouse kidney extract, gave about 6 to 7 times higher yields of angioten-sin-like substance. Administration of DCA together with salt loading for a period of 3 weeks reduced renin activity in mouse kidneys, but not as markedly as in rats. The resistance of mouse renin substrate to heter-ologous renin appears unique among non-primates.

Mucopolysaccharide levels have been measured in urine specimens of normal persons, patients with Hurler's syndrome and their relatives. In the samples from normal humans a marked decrease was observed in the amount of chondroitin sulfates and muco-polysaccharides per unit of creatinine with age until the late teens, after which the amount became constant. In normal humans a decrease in the chondroitin sulfate/muco-polysaccharide ratio with increased age was also observed. After making appropriate allowances for age and creatinine levels in the urine, mucopolysaccharide excretion in parents and siblings of Hurler's patients was found to be similar to normal individuals by all criteria used. Several patients with the heparitin sulfateuria type of mucopolysaccha-ridosis were found in this survey.

An established line of canine kidney cells (MDCK-USD) of male origin was shown to contain 3 metacentric chromosomes and possess a modal number of 81. It grew rapidly in Eagle's MEM, displayed contact inhibition and was susceptible to vaccinia, VSV and influenza (PR8) viruses. Two additional MDCK lines, the parent line and another derivative from other laboratories, were compared to the USD line and could be distinguished on the basis of modal number and morphology of the metacentric chromosomes observed in each line.

The acid-base composition of arterial blood and cerebrospinal fluid has been measured in 11 infants of low birth weight and in 13 children in order to establish normal values. The CSF data in children and the usually accepted normal data for adults were similar. The infants, however, showed a significant reduction in CSF pCO₂ when compared to either children or adults. This undoubtedly reflects the lower values for plasma pCO₂ which are typical of this age group.