Experimental Biology and Medicine

An autoradiographic study of mouse skeletal cell proliferation rates using increasing doses of H³TDR were reported. The data showed that in both the osteogenic cells of the femoral periosteum and epiphyseal plate the labeling indices increased rapidly with increasing doses of H³TDR to 1.0 μc/g body weight and less rapidly with larger doses. It was concluded from this study that previously reported estimates of labeling indices based on lower doses of administered H³TDR are lower than the actual values. A dose of 1.0 μc H³TDR/g body weight is recommended for the estimates of labeling indices using the “flash labeling” technique. However, doses of 1.0 μc and above are not to be used for cell migration or cell kinetic studies.

Ouabain, norepinephrine, and angiotensin were added to papillary muscles removed from normal cats and from cats in shock. Solutions used in the muscle bath were Krebs-Henseleit solution (KH), normal plasma and shock plasma which contained a myocardial depressant factor (MDF). The MDF depressed developed tension in shock muscles by 50% of the value in KH while it only depressed control muscles by 33%. Ouabain and norepinephrine, but not angiotensin, completely reversed the effect of MDF in shock muscles, restoring contractile force to that present in control plasma. Shock muscles differed from control muscles in that they were more depressed by MDF, and they were more responsive to ouabain. Thus, shock muscles, although functionally altered and depressed by MDF, respond well to ouabain and to norepinephrine. Therefore, cardiac glycosides and β adrenergic drugs may be useful in the therapy of shock.

The effect of in vivo CPZ administration on the in vitro metabolism of the rat epididymal fat pad has been studied. Animals maintained at 22°C for 48 hours sustain a hypothermia following CPZ and their fat pads show increased weight, increased glucose uptake, and decreased glycerol release. When animals are similarly treated and maintained at 31°C, there is no hypothermia and no difference in fat pad weight or metabolism from those of placebo treated animals maintained at 22°C or 31°C. It is concluded that the metabolic changes observed are a consequence of the hypothermia induced by CPZ administration.

The secretion of growth hormone in response to hypoglycemia and the administration of arginine in man has been shown to be greatly influenced by certain non-pituitary hormones. Corticosteroids decrease growth hormone response to these stimuli in man (1,2) while estrogens and the synthetic estrogenic compound diethylstilbestrol facilitate the release of growth hormone(3,4). The mechanism of action of these modifying effects has not been established. Corticosteroids have little effect on the concentration of growth hormone in the pituitary of the normal rat(5). If corticosteroids are administered during the induction of hypoglycemia, the expected drop in growth hormone content is inhibited(5). Estrogens on the other hand decrease the concentration of growth hormone even when compared to pair fed control rats(6).

The modifying effects of estrogens and corticosteroids on growth hormone secretion could be exerted either at the level of the hypothalamus by affecting the secretion of the somatotropin releasing factor or these hormones could affect the secretion of growth hormone by acting directly on the somatotropic cells themselves. The observations of Pecile and Müeller (5) have suggested that cortisol may deplete the hypothalamus of somatotropin releasing factor. In their experiments hypothalamic extracts from cortisol treated animals had a much reduced capacity to cause a depletion of bioassayable pituitary growth hormone in recipient animals when compared to hypothalamic extracts from normal rats. Cortisol-treated recipient rats retained their ability to respond with a depletion of bioassayable growth hormone when injected with hypothalamic extracts from normal rats. Although one can not have complete confidence in the specificity of bioassay of crude pituitary extracts, these observations suggest that cortisol affects growth hormone release at the level of the hypothalamus.

Despite these observations we felt that it was important to study possible influences of corticosteroids and estrogens directly on the pituitary in the absence of hypothalamic tissue.

The chronic, disseminated inflammatory reaction observed in rats following a single intradermal injection of Freund's-type adjuvant was associated with a mild, non-progressive anemia which morphologically, biochemically and kinetically resembled the anemia associated with chronic disorders in man. It is suggested that “adjuvant disease” in the rat is an appropriate experimental model in which to study the complex pathogenesis of this distinctive but not uncommon form of anemia in man.

The results of measurements of potassium-39/potassium-41 isotope ratios on biological samples presented in this paper do not support earlier claims (4,7,8) that the isotopes of potassium are fractionated by biological systems. Ratios obtained for potassium nitrate isolated from normal and malignant human tissues, pig and human foetal tissue and plant tissue did not differ significantly from the mean value found for A.R. potassium nitrate 14.09 ± .01 (s.e.). The ratios obtained for impure (ashed) tissue samples similarly were not significantly different from the mean found for the A.R. potassium salt. This differs from the results presented by Reutersward(12) and Kendall(15) who found a higher ratio for samples from which the potassium was not chemically isolated.

DMSO causes ultrastructural changes in rat wound fibroblasts of dilation of the endoplasmic reticulum and diminution of ribosomes. Such features are similar to those described in the wound fibroblast of scorbutic guinea pigs. Ascorbic acid partly prevents DMSO damage. DMSO probably acts by interfering with cell membrane electron transfer systems and this may be the basis of its beneficial effect in clinical situations.

In vitro exposure of erythrocytes to ozone resulted in an increased osmotic fragility associated with the formation of TBA reactants. This suggests that lipid peroxidation may be involved in the mechanism of ozone toxicity.

Human plasma was subjected to starch electrophoresis with veronal buffer at pH 8.6, μ = 0.05. The veronal buffer eluates of each of the 40 sections into which the block was cut were used for the study of caseinolytic and fibrinolytic activity after activation with streptokinase or urokinase. A high fibrinolytic activity was induced by streptokinase in the γ-globulin fraction. A high caseinolytic activity was induced by urokinase in the β-globulin fraction and by streptokinase in the α₁-globulin fraction. Caseinolytic activity in the α₁-globulin fraction was not inhibited by a concentration of epsilon-aminocaproic acid (2 × 10^-3 M) which gave a marked inhibition of the fibrinolytic activity elicitable in the β- and γ-globulin fractions. Inhibition of the caseinolytic activity in the α₁-globulin fraction was effected by dicarbobenzoxy-lysine (2 × 10^-3 M) which had only a slight inhibitory effect on the fibrinolytic activity of the β- and γ-globulin fractions.

Hydrocortisone administered to tourniquet traumatized rats, in infusion fluids, was effective in lowering the levels of beta-glucuronidase in the serum. However, survival in hydrocortisone-infused animals was less than in saline-infused animals. Moreover, an effective infusate containing Cohn Fraction II of lyophilized, reconstituted, human plasma did not significantly alter the level of beta-glucuronidase in the blood. Hydrocortisone, as given in these experiments, proved an ineffective agent for resuscitation from tourniquet shock. Serum levels of beta-glucuronidase, presumed to reflect lysosomal enzyme release, did not correlate with survival.

The rate of initial entry of both radioactive calcium and strontium from the gastrointestinal tract into the vascular system has been determined in man by adapting an integral equation approach for use with a digital computer. A single tracer dose of Ca⁴⁷ and of Sr⁸⁵ was given both by the oral and intravenous route in separate studies and serial samples of plasma were obtained for radioassay. The maximal rate of initial entry of Ca⁴⁷ per 15-minute interval ranged from 2.2% to 3.8% of the dose and was reached within 1 1/2 hours. This rate was sustained for about one hour and decreased thereafter with time. The rate of initial entry for Sr⁸⁵ was less than that for Ca⁴⁷, not exceeding 1.5% of the dose in any 15-minute interval, but was relatively more sustained from the first to the fourth hour After the fourth hour, the rates of initial entry of Sr⁸⁵ and Ca⁴⁷ were almost proportional.

The superimposition of infection by the dwarf tapeworm, Hymenolepis nana, on mice exposed to either 450 or 650 R of whole body x-irradiation considerably lowered host survival. Larger worm loads were observed in mice surviving radiation exposure and infection. Infections persisted for longer periods of time and at higher levels (in terms of worm number) in irradiated hosts as compared to those not irradiated. However, increased susceptibility and lowered immunity, as exhibited by irradiated (450 R) hosts, did not appear to permit autoinfection to take place. Streptomycin therapy produced a striking increase in the survival of irradiated, infected mice.

Groups of rats were fed tryptophan-deficient diets and niacin-tryptophan deficient pellagragenic diets. The former group showed significantly decreased concentrations of serotonin in the small intestine, brain, and colon, while pellagrous rats showed a significant increase in the small intestinal serotonin levels and no decrease in the brain or colonic concentration was observed. The elevated small intestinal concentration of serotonin in the N-T deficient rat is considered to be compatible with a diarrhea based on increased intestinal motility.

An investigation of the trace element zinc in serum as an indicator of exposure to ionizing radiation was predicated upon the role of certain trace elements in important biochemical processes in the living organism. Specifically zinc has been identified in several enzyme systems, i.e., dehydrogenases and pancreatic carboxypeptidase(1,2), and alcohol dehydrogenase and glutamic dehydrogenase (3) in mammalian liver with evidence that zinc is the active enzymatic site and its presence is indispensable to their activity (4,5).

Alteration of enzyme levels in the serum of various mammalian species including man have been reported following exposure to significant amounts of ionizing radiation. In certain of these reports the enzymes were of the zinc containing metalloenzyme groups (6-8). By an investigation of the common denominator of these systems, i.e., zinc, following the radiation insult, it was postulated that a useful pattern of response might evolve allowing application of this parameter as a biologic indicator of exposure to ionizing radiation. Additional support to this concept was given by Wolff(9) who noted increased serum zinc levels in patients undergoing radiation therapy.

Materials and methods. Pure bred Beagle dogs (equal numbers of male and female) in experimental groups of 30 each were utilized. The experimental subjects were individually irradiated using a Cobalt 60 teletherapy unit containing 989 curies of activity while the control group received sham irradiation. The clinical status of the experimental subjects was monitored throughout the post-exposure period. Radiation was delivered at 5.0 R/min, Entrance Air Dose. The target skin distance was 162.5 cm. One half the total dose was delivered to each side of the animal. Constant geometry was maintained by a restraining box constructed of one half inch thickness of lucite (24″×12″×20″). A Siemens-Reinigewerke R-meter was used to calibrate the irradiatior unit and to monitor each exposure.

Rates of C¹⁴O₂ formation from ethanol-1-C¹⁴ were measured in rats that had been allowed only water, 20%, 10%, or 5% solutions of ethanol to drink. Rats which had drunk 10% ethanol for 6 to 11 months metabolized it 18% faster than controls or animals drinking 5% ethanol. Rats drinking 20% ethanol were not different from controls. None of the groups forced to drink ethanol differed significantly from controls in rate of C¹⁴O₂ formation from acetate-1-C¹⁴ and glucose-U-C¹⁴.

Evidence is presented for a common antigenic specificity on the Fd region of human IgG, IgA, and IgM. The relationship of this finding to the evolution of immunoglobulin classes is briefly discussed.

Phenol extracted RNA, unlike other preparations, is less soluble than DNA in aqueous-alcohol mixtures. Further, the RNA so prepared is associated with a high molecular weight polyglucose (CHO). These components were separated by acid precipitation and their solubility determined separately and in reconstituted mixtures. RNA, free of CHO, remains largely precipitable with 20% ethanol. Isolated CHO precipitates from 20% ethanol only upon addition of RNA which can effect the precipitation of six times its weight of CHO. Denatured fractions of RNA, soluble under these conditions, also precipitate in the presence of CHO. Despite the interaction of these macromolecules in solution, the unusual solubility properties of phenol extracted RNA seem to be a consequence of its native state rather than the presence of CHO.

Exposure of male rats to hyperthermia (55°-57°C) for a 15 minute period caused an immediate thrombocytosis lasting for 1 hour after removal from heat stress, a delayed thrombocytosis starting 3 hours after removal from heat stress and lasting through day 2. and a thrombocytopenia appearing 7 days after heat stress. Speculation as to the different physiological mechanisms which might have caused the thrombocytosis at these 2 time periods points toward an immediate release of platelets from their followed by a hemoconcentration due to the pituitary-adrenal stress response. The delayed thrombocytopenia has been attributed to decreased production and/or release of new platelets.

The experiments indicate that calcium movement across the avian shell gland in vitro is in part dependent on metabolic energy derived from oxidative metabolism, requires the generation of phosphate-bond energy, and is predetermined by the physiological state of the shell gland at time of sacrifice.

Radiochemical procedures with 31 castrated male sheep employed to investigate relationships between thyroxine degradation rate and total red cell volume (RCV) permitted procurement of accurate and dependable results without use of substitution therapy. The significant relationship (P< 0.01) between degradation and RCV in these intact animals, under physiological conditions and independent of body weight, would suggest environmental factors to affect red cells through the thyroid axis. The nature and mechanisms of these relationships, though considered speculative, were discussed.

These results demonstrate the impermeability of Artemia cysts to DMSO. There was no evidence of DMSO penetration even after 9 days, and hydrated cysts shrunk rapidly when placed into hyperosmotic DMSO solutions. DMSO, at 2.1%, appears to have no particular toxicity to the eggs or to the free-swimming nauplii.

An attachment was designed which made it possible to use the Allo-Kramer shear press to determine the breaking strength of the tibia of 4-week old chicks. Breaking strength determined in this manner was found to be highly correlated with tibia ash. It is suggested that the breaking strength is as good a measure of dietary calcium and phosphorus levels as is tibia ash. Also the breaking strength of bones as determined in this manner may be useful in evaluating factors influencing fragility of bones.

A comparison of the histocom-patibility characteristics of S-180, S-180/MP, S-180/B₆ and ECA was carried out by transplantation techniques in 7 strains of mice. S-180/MP and S-180/B₆ appeared to be less immunogenic than S-180; ECA was less immunogenic than the two S-180 sublines. S-180/MP, S-180/B₆ and ECA appeared to have antigenic properties in common with S-180. The therapeutic effects of 6MP and KTS against S-180/B₆ were greater in C57B1/6 than in Swiss HalCR mice. The effects of 6MP and vitamin B₆ deficiency against ECA were evident only in Swiss HaICR sensitized to S-180. Thus, in this case, the effectiveness of therapeutic treatments was influenced by the degree of the host response to the tumor.

An iodination procedure for human growth hormone using ¹²⁵I and the double-antibody immunoassay utilizing this labelled preparation as tracer are described. The mean specific activity was estimated to be 190 mC/mg. The growth hormone determinations were made on 0.1 ml of 1:3 diluted plasma. The results showed satisfactory recovery, accuracy and a sensitivity of 0.375 m/μg/ml.

RNA polymerase activity, measured in vitro in the nuclear fraction of livers from mice infected with MHV-3 virus, was decreased 23% and 50% at 24 and 36 hours respectively after inoculation of the virus. The in vivo incorporation of orotate radioactivity in the nuclear RNA of liver cells showed no change from the normal during the first 24 hours after infection when viral multiplication had already reached its peak level, but a significant increase was evident at 36 hours. The cytoplasmic RNA of liver cells showed no change in in vivo incorporation of orotate radioactivity even at 36 hours after infection when the hepatocellular lesions were already marked.

A toxic factor was demonstrated in living and disrupted Leptospira pomona and in the supernatant fluids of cultures by alterations in the permeability of guinea pig peritoneal macrophages; the supernatant fluids also inhibited the uptake of oxygen by porcine kidney cells. Virulent and avirulent L. pomona were equally toxic. The soluble toxic factor was nondialyzable and thermolabile; it was not specifically neutralized by antiserum.

Extensive allelic substitutions at the a and b loci in mice reveal that multiple forms of tyrosinase separable by acryl-amide-gel electrophoresis are subject to complex genetic control. Depending upon genic constitution at the a and b loci, a maximum of three electrophoretically separable forms of tyrosinase are demonstrable. Specific alleles at these loci have also been shown to influence tyrosinase activity.

1. Tension-length diagrams of excised human coronary orifices resemble those obtained from human femoral artery. 2. Wide differences can exist in the two coronary orifices of one individual. These are not directly related to the occurrence of atherosclerosis and may affect coronary blood flow.

In the present paper the anti-hemostatic effect of heparin on rat tail preparations was studied. It was confirmed that the antihemostatic effect of heparin is not removed after several washes with saline solutions and it was shown that its activity is closely related to the sulfate groups of the heparin molecule. The coagulation time of samples collected from oozing blood in the heparin-treated tail remained in the normal range.

Several salts, nucleotides and proteins were tested for ability to remove this antihemostatic effect. Of the substances tested, phosphate and sulfate ions, ATP, ADP and myosin were able to remove the antihemostatic activity while salts and proteins involved in blood coagulation were without effect. The effect of ATP could be shown to be accompanied by the release of heparin to the bath fluid by a microelectrophoresis technique.

The inhibition by heparin of myosin ATP-ase activity was demonstrated in vitro.

The role of muscular contraction, coagulation, and platelet adhesiveness on hemostasis is discussed.

A cell line has been established from a nodule of human giant and spindle cell carcinoma of the thyroid metastatic to a kidney. The culture has been maintained through more than 24 passages over a period of 1 1/2 years. Its identity as a cancer cell line was established by its growth on hetero-transplantation into immunologically tolerant rats with reproduction of the histologic appearance of the original tumor. Chromosomal studies showed a high degree of aneuploidy and polyploidy with numerous abnormalities of chromosomal form.

Mature male fowls were divided into 4 groups of equal TSR. One group served as controls whereas the other 3 groups were injected daily with 0.5 mg tapazole/100 g bw/day for 30, 60 and 90 days. The mean wet thyroid gland weight of the control group was 134.8 mg. After 30 days, the thyroid weight increased 66.8%, after 60 days, the increase was 187.9%, and after 90 days the increased weight was 136.5% above the controls. The determination of the total DNA of each group of thyroid glands indicated the extent of cell multiplication involved and from these data it was possible to estimate the proportion of the weight increase which was due to cell multiplication and the increase due to cell size.

After 30 days the cell multiplication accounted for 68% and cell size for 32% of the increase, after 60 days 57% to cell multiplication and 43% to cell size and after 90 days 42% due to cell multiplication and 58% due to cell size.

1. Pinealectomy when performed in male rats results in a significant increase in weight of the testes, the prostates and the seminal vesicles. This suggests that the pineal gland usually inhibits the secretion of LH and of FSH. 2. Melatonin administered to male rats diminishes the weights of prostates and of seminal vesicles, but does not change testicular weight, indicating that exogenous melatonin suppresses the secretion of LH, but does not interfere with FSH release. 3. In prepuberal female rats melatonin retards puberty and decreases pituitary weight as well as the weights of the ovaries and the uteri, confirming that melatonin reduces LH secretion. 4. It is suggested that the pineal gland exerts an anti-FSH effect through compounds different from melatonin.

The intraperitoneal administration of the “P” form of PHA resulted in a marked depression of RES phagocytic activity. The alteration of phagocytosis occurred in temporal relationship to an enlargement of the spleen. In the majority of the animals hepatomegaly was also observed. Doses of PHA-P of 70-100 mg/100 g of body weight were found to be toxic to mice.

The kinetics of proline and hydroxyproline and their respective incorporation into dentine collagen during rapid dentinogenesis has been reported. The incorporation of proline into collagen and the subsequent hydroxylation of this imino acid takes place as an intracellular event, demonstrated by the rapid rise in the specific activity of hydroxyproline in the neutral salt collagen fraction and the intracellular localization of the grain density. Four hours after administration of proline C¹⁴, the biosynthesis of collagen and its export from the odontoblast into the extracellular organic matrix was completed. Both proline and hydroxyproline exhibit similar specific activities in the insoluble collagen fractions throughout the experimental period. The developing rodent molar was found to be an excellent experimental model for the study of rapid collagen synthesis and its role in the formation of the dentine organic matrix.

Autorepopulation studies using a radiobiological method provide evidence that endogenous hemopoietic stem cell proliferation in the shielded mouse occurs during the first 5 days after irradiation. Exposure to hypoxia, sufficient to produce elevated levels of endogenous erythropoietin, failed to induce erythroid differentiation during the early proliferative phase of marrow repopulation. The data support the concept that the replication of stem cells, per se, renders them refractory to the induction of erythroid differentiation by erythropoietin.

The generalized Shwartzman reaction has been produced in the non-pregnant rat by the injection of bacterial endotoxin and infusion of dextrose/saline over a 4 hour period. Intravenous infusion of dextrose/saline acts to “prepare” the rat for the generalized Shwartzman reaction. Alpha-adrenergic blockade by dibenzyline prevents the generalized Shwartzman reaction produced in this way and suggests that the combined effect of endotoxin plus dextrose/saline infusion acts to localize thrombi in glomerular capillaries by stimulation of alpha-adrenergic receptor sites in the kidney. Exogenous norepinephrine infusion has little or no effect on the incidence of glomerular capillary thrombosis under the condition of these experiments.

Sublethal X-irradiation of guinea pigs 12 days before to 3 days after sc vaccination with viable P. tularensis LVS resulted in a maximum mortality of 7.5% at 2 intervals. Surviving animals, however, displayed an agglutinin response and resistance to respiratory challenge similar to that of nonirradiated vaccinated animals.

Congenital malformations of the type produced by maternal ingestion of the alkaloid veratramine occurred from maternal ingestion of the plant Veratrum californicum. A preliminary identification of veratramine in benzene extracts from the plant by thin layer chromatography was achieved.

Repeated attacks of experimental allergic encephalomyelitis (EAE) were induced in rats by 2, 3 or 4 consecutive biweekly passive transfers of living, encephalitogenic, lymphoid cells. The incidence and severity of clinical signs of EAE decreased progressively during successive attacks. A decrease in spinal cord lesions was found histologically only after the fourth transfer. Unexpectedly, the cerebellum revealed a marked increase of EAE lesions after the second, third or fourth transfers. This redistribution of lesions may be due to local blockade at the sites of original attack in the spinal cord.

The effect of a single dose of TSH on the incorporation in vivo of ¹⁴C labeled amino acids into thyroid proteins of the rat have been studied. Soluble thyroid proteins were analyzed by sucrose density-gradient ultracentrifugation. TSH, injected 2 to 4 hours before pulse-labeling, increased the total amount of ¹⁴C in protein and accelerated the appearance of label in 19 S thyroglobulin. When TSH was given within one hour of labeling or two hours afterwards, total ¹⁴C incorporation was unaffected, but the label appeared in mature (18-19 S) thyroglobulin at the expense of label in an immature (15-16 S) precursor protein. The results suggest that TSH produces an early stimulation of several phases of thyroglobulin biosynthesis.

The lack of a uniform and reproducible plaque assay for rhinoviruses (now totaling about one hundred types) has handicapped characterization of these important human pathogenic agents. Different procedures involving variations in the optimal cell culture, composition of the overlay medium and incorporation of anti-inhibitor and/or enhancing substances (e.g., DEAE dextran and Mg⁺⁺ ions) have been described by various workers. In an attempt to develop a simple and reproducible procedure for plaquing a substantial number of rhinoviruses, we found the following conditions to be optimal for plaque formation by all 16 rhinoviruses that have been tested: (1) fully grown WI-38 cell monolayers in prescription bottles; (2) an overlay medium consisting of HCAEM containing 10% newborn calf serum and 0.7% Ionagar No. 2; (3) double agar overlay technique with the second overlay (containing neutral red) added 6 days after the first; and (4) incorporation of 25 μg/ml of DEAE dextran to the first overlay medium as an additional means to improve plaque formation.

Hypophysectomy or castration of male rats results in a diminution of biosynthetic enzymic activities and tissue concentrations of ascorbic acid to levels characteristic of females. The data support the hypothesis that the sexual difference in tissue concentration of ascorbic acid in intact rats is dependent upon a difference in the rate of hepatic biosynthesis. The sexual difference in hepatic enzymic activities is interpreted to be dependent upon testicular secretion of androgens.

1. The Jerne hemolytic plaque assay method readily detects antigenic cross-reactivity between sheep, goat, and calf erythrocytes. 2. Naturally occurring plaque-forming cells against sheep erythrocytes are more frequent in spleens of conventional colony mice than of specific-pathogen-free mice of the same genotype. 3. Vaccines prepared from each of 5 isolates of E. coli and A. aerogenes from the gastrointestinal tracts of conventional colony mice significantly increased the number of sheep erythrocyte hemolytic plaque-forming cells in the spleens of SPF mice. 4. It is postulated that the naturally occurring hemolytic antibody-forming cells found in the lymphoid tissues are probably caused by cross-reactivity between erythrocyte and bacterial antigens.

The intraperitoneal injection into mice with Ehrlich ascites tumor of trace amounts of H³-methyl N-carbamyl maleamate, a substance which in larger amounts produces destruction of spindle fibers, resulted in the appearance on autoradiography of grains chiefly in the cytoplasm and only rarely in the nucleus. In a number of instances the grains appeared to be distributed in a perinuclear fashion. Somewhat similar results were obtained when H³-methanol was administered. The administration of cold methyl N-carbamyl maleamate (1 mg) together with the labeled substance resulted in a marked enhancement of the uptake of the labeled material into the cells, while a similar experiment with non-radioactive methyl N-carbamyl maleamate and H³-methanol showed no increase in uptake of isotope by the tumor cells. It was concluded that H³-methyl carbamyl N-maleamate enters the tumor cells as an intact molecule and associates with cytoplasmic components. The relationship of some of these components to spindle fiber structures or precursors is suggested.

Isolated islets obtained 1 to 2.5 hours after injection of guinea pig anti-insulin serum into normal rats secrete insulin in vitro at an abnormally high rate in response to glucose. Twenty-four hours after induction of the insulin-deficiency, the absolute rate of insulin secretion is subnormal, but is still increased in relation to the low insulin content of these islets. This sequence of changes is seen in pancreatic tissue removed from spontaneously diabetic animals and suggests that this system could be used as a model for the study of islet function during the development of insulin-deficiency.

Combined effects of OVAR-X and HYPO-X (double operations) on feed intake were studied. In addition, the effect of triple operations on mammary gland DNA was reported. While OVAR-X in addition to HYPO-X had little or no effect, OVAR-X and ADRE-X in addition to HYPO-X caused a marked initial decrease in feed intake. However, the feed intake gradually returned to the preoperative control level at longer intervals while body weight remained at a reduced level. It was suggested that the presence of 1% NaCl in the drinking water was unable to compensate for the lack of aldosterone which might have caused the initial decrease in feed intake.

With DNA as a measure of mammary gland tissue, a significant involution of the duct system from the ovariectomized level was observed following HYPO-X and triple operations. Although triple operations caused a lower DNA value than that of HYPO-X alone, there was no significant difference between the two groups. This suggests that the ovaries and adrenals are of secondary importance on maintenance of mammary glands after hypophysectomy.

The isoenzymes of hexokinase of several human tissues have been examined by disc electrophoresis. An infant with hemolytic anemia was found to have a band that migrated faster than hemoglobin. His father displayed the same abnormality whereas the mother's zymogram was normal.

Ethionine resistance in E. coli ATCC 9637 occurs at a high frequency and requires the constant presence of the analogue to maintain resistance. Resistant cells exhibited a lag phase 6 to 8 hours longer than sensitive cell controls when cultured in a glucose salts medium. This extended lag was reduced to that of sensitive cell controls by ethionine, methionine, homocysteine or cystathionine. A similar prolonged lag occurred when sensitive cells, previously grown in the presence of methionine, homocysteine or cystathionine, were inoculated in basal glucose salts media. The extended lag in all cases tested was due to the rapid death of a significant portion of the initial inoculum during the first hour of incubation. Resistant cells were repressed for cystathionine synthetase to the same degree as sensitive cells grown in media containing methionine. The data indicate that ethionine resistance in this strain occurs by an induced ability to convert ethionine to methionine via homocysteine, which results in repression of cystathionine synthetase. The viability loss apparently occurred in inocula repressed for de novo methionine synthesis due to metabolic imbalances brought about by the rapid growth conditions employed in this study.

1. The results of mouse serum level studies are presented in terms of total and unbound antibiotic concentrations. DOOTC administered at one-half the oral dosage of MOTC, TC, and DMCT produced higher and longer lasting total and unbound drug concentrations than the latter antibiotics. 2. It is suggested that the previously reported chemotherapeutic superiority of DOOTC over these drugs results from the achievement and maintenance of superior unbound drug levels. 3. The more efficient oral absorption of DOOTC over the other tetracyclines (3.5-4.5 times) in the mouse was demonstrated by comparing cummulative urinary excretion ratios obtained after oral and intravenous drug administration.

Studies conducted to follow biosynthesis of deoxyribonucleic acid (DNA) in cultures of African green monkey kidney (GMK) cells inoculated with PARA (SV40)-adenoviruses 2, 7, and 12 showed an initial depression in incorporation of H³-thymidine into the DNA. Levels of DNA started to increase after 15 hours, reaching a maximum in the virus inoculated cultures 24 hours post-infection. Extraction of the DNA from infected cells, followed by their identification by isopycnic analysis in CsCl gradients, revealed the presence of virus DNA with a density corresponding to the adenovirus serotype used. DNA samples from GMK cultures inoculated with PARA-adenovirus 7 or its transcapsidants, PARA-adenovirus types 2 and 12, yielded bands with peak densities of 1.710 g/ml [adenovirus type 7 DNA with 51 moles % guanine + cytosine (GC)], 1.715 g/ml (adenovirus type 2 DNA with 56 moles % GC), or 1.706 g/ml (adenovirus type 12 DNA with 47 moles % GC), respectively as well as a band at a density of 1.699 g/ml (GMK DNA with 40 moles % GC). A separate band that might have been distinctive for the DNA of the PARA particle was not detected.

1. Statolon, a broad spectrum antiviral agent, effectively inhibits the development of adjuvant-arthritis in Fischer strain rats. 2. It is suggested that a virus is involved in the pathogenesis of the disease, and it probably has some relationship to lymphoid cells. 3. Evidence against an anti-inflammatory or anti-immunological effect of statolon in adjuvant-arthritis is presented. 4. The possible participation of other factors in the pathogenesis of this experimental model of arthritis requires further study.

Male weanling rats were fed casein or purified amino acid (AA) diets equivalent to 10% protein (N X 6.25) to determine the effect of the addition of 50% water, nonessential amino acids (NEAA) or other N sources on growth. The daily weight gain of rats fed the wet casein diet (6.0 g) or the wet AA diet of Ranhotra and Johnson (5) (5.8 g) was significantly greater than that of rats fed comparable dry diets (5.3 and 4.5 g, respectively). However, the addition of water to a variety of other diets containing different AA mixtures but the same non-nitrogenous ingredients did not significantly improve growth. The carcass fat was significantly higher with the wet AA diets than with the dry AA diets. The data indicate that the response to added water is related to the balance of the AA in the diet. Rats fed the dry casein or AA diet at the 15% protein equivalent level had a significantly faster rate of gain and improved food efficiency over those fed the diets reduced to 10% protein equivalent, even though the essential AA were supplied in adequate amounts. The addition of a combination of glycine and glutamic acid, urea, or a mixture of NEAA significantly improved growth, but diammonium citrate or other NEAA added singly did not give a significant response.

In man, the polypeptides brady-kinin and kallidin, but not eledoisin, produce pain on intraperitoneal injection. None of these substances produces pain when injected subcutaneously or intramuscularly in higher concentrations. Therefore, none of these polypeptides can be considered as a universal mediator of pain.

A new technique was used to measure the heart rate of developing chick embryos. Ballistocardiography should afford ideal conditions for observing embryonic heartbeats with a minimum of disturbance to the embryo. The same embryos can be measured on each day of incubation. The heart rate increased from day 4 through day 10 and then decreased until hatching time. The average frequency at 10 days was 280 bpm and 263 bpm at 19 days of incubation. Male embryos consistently had slower average heart rates than did females. No significant difference was observed in the average daily heart rate of embryos from 4 different genotypes, even though differences are apparent between adults of these genotypes. Mechanical disturbances or alterations in temperature had a marked effect on the heart rate of the embryo. Brief exposure to room temperature or slight injuries, such as those following insertion of electrodes, usually result in a decreased heart rate.

Protein-bound iodine levels were determined on CFW mice inoculated with Moloney Leukemia Virus. When frank microscopic evidence of leukemia appeared in several animals, all inoculated animals and their controls were sacrificed. Hematoxylin and eosin sections were made from the spleen adn thymus of each animal, and the degree of microscopic involvement with leukemia noted. The difference in the mean PBI between virgin controls and virgin inoculated mice was not significant at the 5% level. Even though the mean PBI between the control animals and the inoculated animals as a group was not significant, a significant inverse correlation at the 5% level was demonstrated between the amount of leukemic involvement of the spleen or thymus and the PBI. The maximum increase in PBI occurred prior to microscopic involvement with leukemia and trended lower as microscopic evidence of splenic and thymic involvement appeared.

The size and numbers of vaccinia plaques in chick embryo cell cultures are enhanced by trypsin.

Mammalian prolactin inhibits thyroxin-induced resorption of the tail in Rana catesbeiana tadpoles. Mammalian growth hormone does not show this effect. The possible importance of prolactin as a larval growth hormone in amphibians is emphasized.

A simple method for cannulating the mouse thoracic duct is described that enables the collection of lymph for periods of up to five days. A pure lymphocyte population is obtained that is useful for immunologic and antibody synthesis studies.

After 4 days of treatment with U-19425, rats no longer respond to the drug with a decrease in plasma FFA and blood sugar 2 hours after treatment. The initial plasma FFA response to the drug is similar, but pretreated rats escape from antilipolytic effects of U-19425 faster than nonpretreated controls. Tachyphylaxis to 5-methylpyrazole-3-carboxylic acid may be due to a physiological compensatory mechanism which is mediated by the adrenal and/or pituitary glands.

Vegetative cells of the common yeast Saccharomyces cerevisiae grown in a synthetic medium were incubated in the presence of 2 × 10^-6 g/ml of Benzo(a)pyrene. Ultraviolet microscopic studies of cells in the logarithmic growth phase show isolated fluorescence of intracytoplasmic organelles which appear to be mitochondria on the basis of comparison with intravitally stained cells.

Fresh isolates of trachoma-inclusion conjunctivitis (TRIC) agents infect the human eye, but undergo only very limited, if any, replication in cell culture. In the course of laboratory passage, variants arise which proliferate freely in cell culture. The ability of such cell culture-adapted variants to infect the human eye was investigated. Of 20 volunteers infected with 2 such variants, only one developed clinical eye disease. In 16 others, limited replication of the agent was proven by immunofluorescent staining of conjunctival scrapings, with the largest amount of TRIC antigen present during the second week after infection. The cell culture-adapted strains LB-1 and BOUR-J appeared to be 100-1000 times less infective for the human conjunctiva than fresh TRIC isolates. The limitations of such variant strains as potential live vaccines are considered.

Streptovitacin A, a highly efficient inhibitor of protein synthesis, at 10 μg/ml of medium completely prevented synthesis of infectious poliovirus (type 1) and of CF antigen, as well as of CPE, in BS-C-1 cells when it was added up to about an hour before the end of the eclipse phase and was then left in the medium for the duration of the experiment. When the drug was added at the very end of the eclipse period, before new infectious virus had appeared, and was then left in the medium, a small amount of new virus appeared in the cells. When the drug was added during the exponential phase of viral multiplication, there was always some further increase in infectious virus but never at the level achieved in the untreated cultures. When the drug was added one or two hours after infection and washed away one or two hours later, the subsequent delay in viral synthesis was always considerably longer than the time the drug was present in the culture medium. These results fit the assumption that protein synthesis in BS-C-1 cells is not completely inhibited for 15-30 minutes after the addition of the drug and is not restored for a number of hours after its removal, as was found by Dales for L cells. The data also indicate that the drug-induced interference with protein synthesis during any phase of the poliovirus replication cycle did not result in an irreversible progression in the synthesis of infectious virus.

The REF has been demonstrated in the kidneys of rats, rabbits, dogs, sheep, pigs and humans. In these 6 species, approximately equal quantities of the REF were found in the renal cortical and medullary tissues. Exposure of rats to hypoxia induced an increase in both cortical and medullary REF activity.

No significant difference in serum interferon titers could be demonstrated in neonatally thymectomized and litter mate control mice inoculated with NDV intravenously. Although the blood lymphocyte counts of the thymectomized mice were not consistently depressed, there was other evidence that the lymphoid tissue of these animals was markedly altered.

Adenovirus type 5 viral antigens were separated on DEAE cellulose. These antigens were reacted with various homotypic and heterotypic adenovirus antisera in order to determine which of the viral antigens were reactive in the passive hemagglutination test. It was found that the fiber antigen was the principal antigen responsible for this activity. Some activity was also associated with the penton antigen. The hexon antigen could not be assayed by this method. Heterotypic responses were due primarily to the penton and fiber antigens.

The diurnal variation in TSH content of the plasma and pituitary of the female adult rat under conditions of 14 hours of light and 10 hours of darkness was determined by an immunochemical technique. The plasma TSH level rose gradually from 3 A.M. to 3 P.M., then declined; whereas the pituitary TSH was highest at 3 A.M. and then declined until 3 P.M. These data suggest that the thyroid hormones would be lowest some time prior to 3 A.M. and reach a peak some time after 3 P.M., thus stimulating maximal activity during the night.

Methyl S (or 4) - (3,3 - dimethyl −1 - triazeno) imidazole-4 (or 5)-carboxylate(1), designated NSC 87982 by the Cancer Chemotherapy National Service Center, National Cancer Institute, National Institutes of Health, has been reported to be a potent inhibitor of Gram-positive and Gram-negative bacteria, yeasts, filamentous fungi and algae in vitro and to protect mice against an experimental infection of Staphylococcus aureus (2). Microbiological assay of various mouse tissues indicates that not only are significantly high blood levels obtained following oral, intraperitoneal, or intravenous administration of the drug, but also that detectable concentrations of NSC 87982 are found in the brain and other organs of the mice(3). The broad-spectrum antimicrobial activity of NSC 87982 and the knowledge that it is being considered for clinical trial prompted a study of the mechanism of action of this compound.

Materials and methods. The inhibitory activity of NSC 87982 against numerous strains of Escherichia coli and Streptococcus faecalis which are resistant to various antimicrobial agents or radiation(4) was determined using previously described procedures (5); the spread-plate procedure(6) was used to determine the effect of metabolites (amino acids, B vitamins, purines, pyrimidines) on inhibition of E. coli by NSC 87982-details of this technique have been reported (7,8). Protein determinations were made by the method of Lowry et al (9). Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were determined by procedures previously described (10,11). By serial transfer in a simple glucose-salts medium containing increasing concentrations of NSC 87982, a culture of E. coli ATCC 9637 was obtained, (designated E. coli/NSC 87982) which is 30-foldresistant to the inhibitor.

Results and discussion. Strains of E. coli or S. faecalis resistant to various antibiotics, antimetabolites, or irradiation were not cross-resistant to NSC 87982.

Intradermal, intraperitoneal and intravenous injections of horseradish peroxidase solutions induce increased vascular permeability in rats. The vascular leakage is confined to venules, is of short duration, can be inhibited by a combination of histamine and serotonin antagonists and is associated with mast cell degranulation. This phenomenon occurs also in the guinea pig but is minimal or non-existent in the mouse.

The isolated perfused cirrhotic rat liver which is the site of mesenchymal cell proliferation incorporates 3 times as much C¹⁴ lysine into serum gamma globulin as the normal liver from litter mate controls. This data is interpreted as further evidence that hypergammaglobulinemia in active cirrhosis results partly from persistent increase of gamma globulin producing mesenchymal cells in the liver.

The isolated perfused cirrhotic rat liver which is the site of mesenchymal cell proliferation incorporates 3 times as much C¹⁴ lysine into serum gamma globulin as the normal liver from litter mate controls. This data is interpreted as further evidence that hypergammaglobulinemia in active cirrhosis results partly from persistent increase of gamma globulin producing mesenchymal cells in the liver.

1. A method for the separation of a cytotoxic factor active against Ehrlich ascites tumor cells and of plasminogen from Cohn Fraction I-III is described. The method involves extraction by fluorocarbon, gel-filtration on Sephadex G-25, and ion-exchange chromatography on DEAE-Sephadex. 2. The fluorocarbon extract contains both CyF and plasminogen. Heating the extract to 58°C for 1 hour inactivates plasminogen but leaves the CyF unaltered. 3. Separation of CyF from plasminogen in the fluorocarbon extract could be done on Sephadex G-25, using a column equilibrated with a buffer of such low pH and ionic strength as to precipitate the plasminogen selectively, and then recovering this with an eluting buffer in which plasminogen is soluble.

A plaque assay method for the HF strain of herpes simplex virus using L-cells under methyl cellulose has been described. Its ease in preparation, the reproducibility of results, the distinctness of the plaques, and the short period of time required for plaques to appear make it a suitable technic for use in classroom experiments and in research.

Cell cultures prepared from dog lungs without trypsinization contained viable lung macrophages. When such cultures were inoculated with CDV, giant cells were seen 2 to 6 days after inoculation. When virulent CDV was titrated in DL cell cultures and in dogs, comparable endpoints were obtained. Giant cell formation in lung macrophage cultures, therefore, permitted demonstration and assay of virulent CDV.

Endotoxin was detected serially in the circulating blood of dogs with a bioassay by injecting canine plasma intravenously into New Zealand albino rabbits and simultaneously 50 μg epinephrine intradermally. The presence of rabbit dermal necrosis 24 hours later indicated the presence of endotoxin in the canine plasma. Prior to the injection of endotoxin all canine plasmas gave a negative reaction. Within 3 minutes post-endotoxin all plasmas showed a positive test. At 3 hours when the blood pressures had stabilized in 7 surviving animals no endotoxin was in the plasmas. However at 6 hours endotoxin was detected in the plasma of 6 out of 7 surviving animals. The persistence of endotoxin in the circulating blood could not be correlated with survival or death.

A method was described for the separation of vit B₁₂ binders in normal serum on a 15 × 4 cm DEAE-cellulose column by stepwise elution with 0.005 M pH 8.0, 0.0175 M pH 6.3, 0.04 M pH 5.9, 0.1 M pH 5.8 and 0.4 M pH 5.2 sodium phosphate buffers. Two radioactive peaks appeared when 300 μμg Co⁵⁷ B₁₂ per ml of serum had been added in vitro. With the addition of 2,000 μμ/ml of Co⁵⁷ B₁₂ to the serum, 5 radioactive peaks appeared.

Survival time was increased over 90% in mice with hereditary muscular dystrophy treated orally with a serotonin antagonist, methysergide bimaleate. There was a concurrent increase in body weight in treated animals. Short term treatment for 3 weeks with the drug caused a significant enlargement of the ovaries of dystrophic mice, but had no effect on body weight or other endocrine organ weights of normal mice.

The cytological effects of 16 quinone compounds were tested on Ehrlich ascites tumor cells in vivo. Of these only juglone (5-hydroxy-1.4-naphthoquinone) produced mitotic abnormalities. An accumulation of abnormal metaphase figures occurred and the cells in metaphase were prevented from entering ana-telophase. Chromosomes in prophase cells appeared diffuse and sticky. Juglone administration, either intraperitoneal or oral, caused decreases in the amount of ascitic fluid at a time that marked increases were taking place in control animals.

When 10-day-old chick embryo femora were cultured for 2 days in medium containing methyl or propyl paraben, there resulted an increase in dry weight as compared with femora cultured in the control medium. The methyl paraben concentrations found to stimulate growth were 10^-5 and 10^-6 M. Propyl paraben stimulated at 10^-5 to 10^-7 M. The effects of the two were not additive. It is suggested that these compounds may stabilize lysosomes.

The formation of pyrogen by rabbit leukocytes can be inhibited significantly by treating animals with high doses of chloramphenicol. The mechanism of this effect probably involves inhibition of protein synthesis at some point during the development of granulocytes. On the basis of experiments with inhibitors of protein synthesis it appears that leukocytes from inflammatory exudates do not synthesize much additional pyrogen during incubation in vitro.

Intracellular pH was determined by distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO) in skeletal muscle of dogs 1) infused with .3 N HCl, 2) infused with .3 N gluconic acid, 3) infused with HCl and hyperventilated to maintain extracellular fluid pH constant, 4) infused with .6 N HCO₃ and given CO₂ and O₂ to breathe to maintain extracellular pH constant, and 5) maintained in hemorrhagic shock. After the one to two hours required to reach equilibrium distribution of DMO there was not a striking difference in the intracellular hydrogen ion-extracellular hydrogen ion gradient between animals infused with HCl and those in hemorrhagic shock. Those infused with gluconic acid did show a significant difference from the shock animals. The differences were in the direction that would be expected from slower distribution across cell membranes of H⁺ and/or HCO₃- than CO_2. The experiments in which extracellular pH was maintained constant in the face of infusion of HCl or NaHCO₃ also demonstrate the primary dependence of intracellular pH on Pco₂ although in these experiments there is additional clear evidence of transfer of H⁺ or HCO₃- between intracellular and extracellular fluid over these short time intervals. With these time intervals of 1 to 3 hours, the intracellular acidosis of hemorrhagic shock is well reflected in the extracellular fluid, and the extracellular acidosis produced by infusion of acid into the blood is reflected in the intracellular fluid.

The screening of maternal sera for lymphocytotoxic antibodies against a panel of 10 cells showed a 46.7% overall incidence of antibody formation. Positive reactions were obtained with 25% of the anti-sera obtained from women after a single pregnancy and 68% with antisera from women having had 8 or more pregnancies. As judged by the number of panel cells with which an antiserum reacts, there was no discernible tendency toward decreasing specificity with increasing numbers of pregnancies. It was observed, however, that antisera from first and second pregnancies differed in specificity from those antisera obtained after 3 or more pregnancies. A basis for this, and related observations, is discussed.

Two experiments were conducted to test the effect of diet composition on heat tolerance. The addition of 12% animal fat to the diet significantly reduced the survival time of chicks when they were exposed to 40.8 ± 0.3°C and 75 ± 5% relative humidity. The level of protein in the diets had no effect. The birds used had been previously selected for either a high or low tolerance to heat. In Experiment 2, the differences between runs and the run X diet interaction were both highly significant. The interaction obscured the main effect of diet which was not significant. This population which was unselected with regard to heat tolerance had such great variation that large numbers of birds would be required to test effects of diet on heat tolerance.

Isolated myocardial strips of rabbit atrium were exposed to an oxygenated, modified Ringer-Locke's solution having various levels of Pco_2, pH, and bicarbonate. Peak contraction force and maximum rate of tension development were used to evaluate myocardial contractility. Exposure to a low Pco₂ and control pH environment did not cause a significant change in contractility. Myocardial performance was significantly increased by exposure to a bathing solution containing a low Pco₂ and high pH. The results are interpreted to indicate that the change in pH is the significant factor in the positive inotropic response to low Pco_2.