Experimental Biology and Medicine

The effects of bile acid and cholesterol on the time-pattern of fecal steroid-C¹⁴ elimination was investigated in mice following a single injection of mevalonic acid-2-C¹⁴. In controls, excreted bile acid-C¹⁴ assayed 3 times sterol-C¹⁴ during the first 24 hours. This ratio reversed during the 2nd 24-hour period, and remained constant for the remainder of the 7-day experiment. Cholic acid depressed bile acid-C¹⁴ excretion, but failed to alter the time pattern of fecal sterol-C¹⁴ elimination. Cholesterol plus cholic acid depressed bile acid-C¹⁴ and increased α + β sterol-C¹⁴ excretion throughout the experiment. Hyodeoxycholic acid increased sterol-C¹⁴ excretion (shown to be of hepatic origin), had little effect on bile acid-C¹⁴ excretion, and increased total steroid-C¹⁴ eliminated. Lithocholic acid had a similar though smaller effect. After sacrifice, the livers of cholic acid-treated mice had twice the β-sterol-C¹⁴ concentration found in livers of controls, while those of mice treated with cholesterol plus cholic acid had β-sterol-C¹⁴ levels 4 times as high. Livers of lithocholic and hyodeoxycholic acid-treated animals showed reduced β-sterol-C¹⁴ levels. Carcass β-sterol-C¹⁴ levels were unaffected by any of the bile acids, but were lowered by the combination of cholesterol and cholic acid. The total amount of sterol-C¹⁴ synthesized from injected mevalonic acid was similar in all cases.

Substitution of acetyl for hydrogen in the NH₂ group of the N-terminal serine, NH₂ for OH” in the carboxyl of glutamine in position 5, and formyl for an amino hydrogen of lysine in positions 11, 15, 16, and 21 decreases or eliminates the ACTH-like activity of a synthetic tricosapeptide which duplicates the first 23 amino acids of naturally occurring ACTH.

Many of the sublethally irradiated BlF₁ and B6D2F₁ mice injected intraperitoneally with thymus cells from parental strain donors died within 90 days, owing presumably to an immunological reaction by the implanted cells against the recipient. Likewise, regenerated thymus tissue of lethally irradiated BIF₁ mice injected with C57BL bone marrow cells induced a fatal wasting disease on implantation in sublethally irradiated B6D2F₁ mice. It was concluded, therefore, that regenerated thymus tissue in lethally irradiated BlF₁ mice injected with C57BL bone marrow was repopulated by immunologically competent cells derived from the donor marrow.

Incubation of homograft sensitized lymphoid cells with ribonuclease inhibits their ability to produce the “transfer reaction” on intradermal injection into the antigen donor. Ribonuclease in the concentration used does not kill the cells as determined by trypan blue staining and culture in Millipore filler chambers. The data indicate that intact protein synthesis of transferred immunologically active cells is essential for the expression of the “transfer reaction.

1. A series of experiments with variously treated RBC stroma injected intra-peritoneally into mice clearly demonstrates that an eosinophile-stimulating substance previously noted is not a lipid. 2. The experiments further establish that the substance is either a protein or a protein-carbohydrate compound. 3. The possibility is raised that the physical state of material (gel or insoluble suspension) may determine the eosinophilic response.

Data have been presented on alterations in serum enzyme activity in patients with malignant and non-malignant diseases as compared to normal controls. Patterns of change have been established for the various groups. The patterns for the malignant groups differ statistically from those of the non-malignant ones. The malignant groups differ from each other.

Every individual in each group does not show all the changes described for the group. There is considerable overlap between pathological and control values. These discrepancies are discussed in terms of what is meant by “normal” range. Data are presented on the “normal” range of controls.

This study has demonstrated that compounds known to possess lathyrogenic properties may, or may not, block the periodic acid-Schiff reaction, and that for these compounds, no correlation exists between their aldehyde blocking capacity and their ability to produce lathyrism in tadpoles. The mechanism by which those compounds produce lathyrism therefore continues to be obscure.

Primary cultures of fibroblast-like cells from rheumatoid synovial tissue changed in vitro to a cell population exhibiting epithelial morphology, heteroploidy, altered growth characteristics, and decreased capacity to synthesize hyaluronic acid. Quantitative changes in the protein and nucleic acid composition of the cells were also demonstrated.

The effects of daily topical application of trypsin upon the dermal chemistry changes produced by croton oil induced local inflammation at the site of injury as well as distant from the site of injury, were compared with enzymatically untreated irritant injected control rats. Locally, trypsin treatment resulted in an inhibition of the increase in lesion hexosamine normally encountered in response to chemically induced inflammation. The collagen content of the lesion decreased independently of topical trypsin application. Although the eschar sloughed 10 days after injury with trypsin treatment, as opposed to the 13 days required by the controls, continued topical trypsin treatment inhibited the chemical healing of the lesion.

The serum nitrogen, hexosamine and hydroxyproline were significantly increased with trypsin treatment; evidence for the increased drainage of wound products into the circulation via proteolytically unblocked capillaries and lymphatics.

Local inflammation resulted in profound and specific decreases in concentration of collagen in uninjured skin distal to site of inflammation. Topical application of trypsin to the lesion appeared to partially inhibit this decrease in dermal collagen distant from the site of local injury. Topical trypsin application increased the amount of citrate soluble collagen found in uninjured skin distant from the site of local injury, but both groups of animals demonstrated a similar and profound loss in insoluble collagen. This dermal collagen loss was prolonged with continued trypsin treatment of the local lesion.

Modification of sodium and potassium levels of submaxillary and parotid, but not sublingual, glands was achieved by reduction of dietary potassium. Submaxillary gland exhibited a decrease in potassium of approximately 10 meq/kg w.w. when compared with normal animals of the same age, and parotid approximately 4 meq/kg w.w. Sodium increased in both glands (8 meq/kg w.w. for parotid; 13 for submaxillary). The major functional change observed in potassium deficiency was a marked reduction in fluid flow rate; electrolyte levels of the secretions were not influenced by changed gland levels.

1 Trypsin inhibitors injected intravenously at about the time of the provocative endotoxin injection, partially or completely inhibited the local Shwartzman phenomenon and prevented development of renal lesions (as seen in the generalized Shwartzman phenomenon). 2 Inhibitors administered intradermally mixed with the endotoxin skin preparatory dose, or intravenously at time of skin preparation, did not influence the reaction. 3 The data presented suggest that activated proteolytic enzymes are involved in the Shwartzman phenomenon. 4 The role of these enzymes in the pathogenesis of the phenomenon is discussed. Alterations of the clotting mechanism and vascular changes brought about by release of serotonin, histamine and activation of plasma kinins have been considered.

Trypsin inhibitor preparations isolated from human pregnancy urine inhibit hyaluronidase. The activity curve indicates formation of a dissociable compound. The trypsin inhibitor concentration in preparations of different purity parallels the content of hylauronidase inhibitor suggesting identity of the 2 inhibitors.

1. Intravenous administration of 9 α-fluorohydrocortisone and 2-methyl-9α-fluorohydrocortisone in doses sufficient to produce profound electrolyte effects in 6 salt-depleted and 6 salt-loaded subjects produced no significant change in glomerular filtration rate, with the exception of one salt-depleted subject, in whom there was an increase in both glomerular filtration rate and effective renal plasma flow following 2MeFF. 2. 2MeFF had a more profound sodium-retaining effect than did FF. Even in salt-depleted subjects, the sodium-retaining effects of both compounds were demonstrable. 3. FF had a more profound potassium-losing effect than did 2 MeFF. Kaliuresis was greater in the salt-loaded than in the salt-depleted subjects. 4. Plasma concentrations of sodium, potassium, and chloride did not change significantly during the 4 hour study after intravenous administration of the steroids.

The initially favorable results of the value of the mono-amine oxidase inhibitors in treatment of angina pectoris have latterly been tempered by further experiences of their use(l), and the response to therapy is probably, at best, unpredictable. However, it is of interest that Weiss et al.(2), using one of these drugs, iproniazid, in treatment of depressive patients, showed that those patients whose appetite, weight and sense of well being improved, demonstrated a definite hypoglycaemic response to the drug. It occurred to us that a similar correlation with hypoglycaemia may also exist in patients who showed improvement in the symptoms of angina pectoris as a result of such treatment. In considering the nature of this hypoglycaemia, the ganglion blocking action of iproniazid (3) may have resulted in a type of hypoglycaemic insensitivity due to inhibition of sympathetic discharge to the adrenal medulla and liver, such as follows hexamethonium therapy(4): or secondly, the slight galactose intolerance noted by Weiss et al. may have been a factor in production of hypoglycaemia, since it is known that an increased blood galactose may have some hypoglycaemic action in the normal individual(5). To try to elucidate these points it was decided to compare the response of total blood sugar and blood glucose levels to a mixed oral dose of glucose and galactose under the influence of ganglion blockade from mecamylamine, and mono-amine oxidase inhibition from iproniazid and nialamide respectively. The latter, unlike iproniazid, produces only minimal postural hypotension (6).

Methods. The following carbohydrate tolerance test was performed in all subjects. A fasting venous blood sample was collected. then a mixture of 25 g glucose and 25 g galactose in 250 ml water was given by mouth. Further venous samples were withdrawn 10, 20, 30, and 45 minutes later.

Synthetic isovalthine (S-(isopropyl-carboxymethyl)-cysteine) was added to urine from hypercholesterolemic patients that had received mevalonic acid-2-C¹⁴ intravenously. Isovalthine recovered from the urine was found to be devoid of radioactivity thus indicating that either (a) mevalonic acid is not a precursor of isovalthine, or (b) mevalonic acid may be a precursor of isovalthine but the metabolite is not encountered in all cases of hypercholesterolemia. The existence in the urine samples studied of an amphoteric metabolite of mevalonic acid was demonstrated.

When ovulation was induced by means of stainless steel electrodes in the preoptic area, delivering pulses of varied characteristics, a prominent zone of mild inflammation appeared at the end of the electrode track. When ovulation failed, the zone was characteristically small or absent. Electrolysis with direct current, 10 μA or more for 20 sec or longer, proved uniformly effective in causing ovulation. Substitution of platinum electrodes for stainless steel usually gave negative results. Microinjection of FeCl₃ solution into the preoptic tissue induced ovulation, while injection of similar amounts of acidified NaCl did not. Finally, electrolysis stimulated ovulation when electrodes of reagent grade iron wire were used, but not when similar electrodes of platinum were employed. It is concluded that iron and perhaps other metals electrolytically deposited from electrodes of stainless steel (or nichrome) produce irritative foci whose stimulative action on certain neurons continues long after the flow of electric current has ceased.

Hepatic vit. A levels are much higher in cold-acclimated rats than in rats maintained at 25°C. This increase in hepatic vit. A content does not stem from a sparing action induced by cold acclimation but is rather a simple corollary of increased food consumption that accompanies cold-exposure. Moreover, evidence is presented which suggests that the amount of vit. A needed for rats to live at 0-2 °C is not markedly greater than the amount needed to live at 25 °C.

Detailed studies were made of the chemical nature of carbon-14 activity found in acid-soluble nucleotide, ribonucleic acid, and deoxyribonucleic acid fractions isolated from kidney or liver tissue of rats rendered experimentally nephrotic with a series of 10 small daily injections of aminonucleoside-8-C¹⁴, and, in similar fractions obtained from rats sacrificed as early as 3 hours after administration of a single large dose of the labeled compound. No significant differences were noted. Only the adenosine and guanosine phosphates of the acid-soluble nucleotide fractions and the adenine and guanine bases of RNA and DNA hydrolysates were labeled with carbon-14. No evidence was obtained either for conversion of the intact aminonucleoside to the corresponding nucleotide or for incorporation of the 6-dimethylamino- or 6-methylamino-purine moieties into ribonucleic acid or deoxyribonucleic acid of kidney or liver tissue.

These results indicate that in rats under these conditions, presence or absence of adrenal tissue has no effect on the severity of hypertension produced by desoxy-corticosterone administration.

Hemadsorption of guinea pig erythrocytes by MKC cultures infected with a PPLO from the respiratory tract of laboratory rats is reported.

The 3-(5-Bromoindolyl)-β-D-glucopyranoside (I) was prepared in our laboratory for histochemical study of tissue β-glucosidase. The final reaction product following incubation of tissue sections was 5,5′-dibromoindigo which is highly chromogenic and precipitates in cells and tissue at the site of enzymic activity. The enzyme is readily destroyed in tissue by ferri-ferrocyanide and formalin, which is not the case with nonspecific esterase or glucuronidase. Saccharo-1-4-lactone, which has an inhibitory effect on β-glucuronidase, does not affect β-glucosidase. “Substrate inhibition” can also be demonstrated. Optimum pH is 5.4. The β-glucosidase can be demonstrated best in the preputial gland where activity is high. The older acini of the gland showed more intense reaction. Reticulum cells within the villous stalks of the small intestine showed a good reaction. Reticulum cells of the spleen, lymph node and Kupffer cells showed a varied reaction. Extremely small amounts are present in the liver and kidney.

Blood sera from patients suffering from castorbean-allergy will sensitize the skin of the Philippine crab-eating monkey, Macaca irus. The sensitized sites react to castorbean proteins, producing wheals and/or blanching characteristic of similar passive transfer tests (P.-K. tests) in nonallergic human volunteers. Skin reactions in the monkey are more easily interpreted when the colloidal dye Evans Blue is injected intravenously 5 or 10 minutes prior to challenge with the suspected allergenic material. The passive transfer of human serum antibodies into the skin of Macaca irus appears to be useful as a method which may be applied in lieu of the Prausnitz-Küstner test (on human volunteers) to avoid the grave risk of infection with serum hepatitis.

Thyroxine secretion rate (TSR) of 100 cross-bred cockerels was determined at 7 weeks of age without tapazole to block I¹³¹ uptake. At 9 weeks, 4 groups of equal TSR were separated. One group served as a control, whereas other groups were fed 0.025, 0.0375, and 0.05% tapazole in ration. TSR of groups were .74±.04, 1.11±.08, 1.18±.08, and 1.27±.07 μg/100 g body weight, respectively. Group not fed tapazole was significantly lower than where included, but differences between tapazole-fed birds were not significant. Tapazole appeared to increase TSR 63.5%. Analysis of these data showed that tapazole increased TSR to greatest extent in groups showing low TSR without tapazole. It is suggested that tapazole does not actually increase TSR but in its absence there is a tendency to underestimate the true TSR due to the slow release rate of thyroidal-I¹³¹. It is recommended that in determination of TSR sufficient goitrogen be fed to completely block I¹³¹ recycling.

Jensen cells grown in a continuous perfusion system produce extracellular material of two types. One is a muco- or glycoprotein or possibly a simple protein; the other, hyaluronic acid.

The present work described the cellularity, nitrogen content and kidney enzymes involved in gluconeogenesis, glucose production, glycogenesis, glycolysis and direct oxidative pathway in kidney cortex. The results were compared with values found in the liver. The kidney contained 50% more cells per unit weight; however, the average kidney cell contained only about half the nitrogen content found in the liver cell. These studies demonstrated that the enzyme systems involved in glycolysis and gluconeogenesis, phosphohexose isomerase, lactic dehydrogenase, glucose-6-phosphatase and fructose-1,6-diphosphatase, are present in the kidney cortex at relatively high levels. On the other hand, the low activities for kidney phosphoglucomutase, glucose-6-phosphate dehydrogenase and 6-phos-phogluconate dehydrogenase suggest that this tissue has poor glycogenic and direct oxidative pathways in comparison with the metabolic pathways of the liver. This investigation revealed a distinct carbohydrate enzyme pattern in kidney cortex and showed that the activities of these enzymes as determined in tissue homogenate and supernatant fluid can be well correlated with studies on the relative importance of glucose metabolic pathways as elucidated by isotope techniques on kidney slices.

The distribution of the cortical feedback fibers in the nuclei cuneatus and gracilis has been compared with the cyto-archi-tecture and the dendritic architecture of these nuclei. This comparison revealed that the cortical fibers distribute primarily to those parts of the nuclei containing multipolar, triangular, and fusiform cells maintaining long, sparsely ramifying dendrites. The cells with frequently ramifying dendrites, on the other hand, receive only few fibers. Furthermore, preliminary evidence suggests that some of the former cells maintain more collaterals than the latter.

1. Rats were fed diets which were either fat-free or contained 5% lipid and either without or with 2% cholesterol. Dietary lipids with 3 degrees of unsaturation were obtained by use of unhydrogenated oil, completely hydrogenated oil, and a 1:1 mixture of the two. 2. A dissociation of plasma and hepatic cholesterol was observed. Liver reflected changes in cholesterol metabolism more sensitively than did plasma. 3. Hepatic cholesterol concentrations were directly related to degree of unsaturation of dietary lipid when cholesterol was fed. The increase in hepatic cholesterol was confined to esterified cholesterol. 4. Dietary lipid was not obligatory for cholesterol absorption. However, absorption appeared to be dependent upon digestibility and fatty acid composition of dietary lipid.

At times of egg laying the serum of female pigeons showed a marked, significant increase in β-globulin and total calcium over the normal values found at all other times throughout the reproductive cycle, and at all times for the male. The relationship of calcium bound to β-globulin concentration was roughly in agreement with similar values calculable from previous work done by other methods. A fast moving component of mobility greater than that of albumin was present in many samples, male and female, particularly at times of milk formation.

An analysis of the cervical mucus from 182 women randomly selected, revealed that 23.7% contained isohemagglutinins when a single specimen was examined but when more than one specimen was obtained the percentage rose to 63.4%. Type 0 women had agglutinins in their cervical secretions more frequently than either type A or type B women, and, in general, the titers were higher. There was no significant correlation between cervical secretion titers and serum hemagglutinin levels. The occurrence of antibodies in the cervical mucus seemed to be unaffected by the secretor type. Observations on multiple samples obtained from 41 women failed to suggest any correlation between antibody titer and phase of the menstrual cycle. These findings extend the data which earlier suggested the hypothesis of ABO selection at the gametic level as a factor in explaining the deficiency of certain types of offspring in ABO incompatible matings.

Infusion of Syntocinon into pregnant ewes increased tonus but never induced rhythmic contraction of the gravid uterus. The infusion rates spanned a range of 0.76 to 1928 mμ per min. This is many times the effective infusion rate that elicits rhythmic uterine contractility in the human. Since sheep blood contains no pitocinase whereas that of the human does, it is suggested that pitocin is effective in the presence of pitocinase.

Polyoma virus (PY) and pneumonia virus of mice (PVM) are not related to each other serologically (1), but because of their similarity in size and their semi-ubiquitous presence in a latent form in mice, the possibility was considered that they belong to the same or related groups of viruses. Since sensitivity to ether serves to distinguish groups of viruses (2, 3), and since it is known that PY is ether resistant (4), the reaction of PVM towards ether was investigated. Ability of the 2 viruses to grow in tissue culture, and their in vivo effects on mouse lung were also compared.

Materials and Methods. Three strains of PY were used in these experiments. Strain LID-1 (5) was kindly provided by Dr. R. J. Huebner. Strain Lp or large plaque virus was derived by Dr. R. Dulbecco by single plaque isolation from a stock originally provided by Dr. W.P. Rowe(6,7). Sp or small plaque virus was isolated from transformed hamster embryo cells after exposure to Lp virus(7). Both Lp and Sp viruses were kindly provided by Dr. R. Dulbecco. The stocks of the various strains of polyoma assayed on secondary mouse embryo layers, according to the procedure of Dulbecco and Freeman (8), contained approximately the same number of plaque forming units, about 1×10⁸ PFU/ml.

A single stock of PVM was used. This had been prepared by Dr. F.L. Horsfall, Jr. in 1953 and had been stored at - 60 °C since that time.

Standard procedures were used for intranasal infection of mice(9) and for preparation and infection of tissue cultures. Ether treatment was done according to the procedure of Andrewes and Horstmann(2).

Lung specimens were fixed in 10% formalin and stained with hematoxylin-eosin.

Results. Results of experiments on ether sensitivity of PVM are recorded in Table 1.

Millipore diffusion chambers filled with gram-negative enteric bacilli and placed in the mouse peritoneum have been used to determine the fate of these bacilli in a cell-free environment. In contrast to findings in the guinea pig, all 7 strains of bacteria tested in the mouse grew in such diffusion chambers. These chambers also served to collect cell-free peritoneal fluid for bactericidal studies in vitro. Such studies showed mouse peritoneal fluid to have no bactericidal activity against gram-negative enteric bacilli.

In the normal albino rat the anti-proteolytic power of the plasma as measured by its ability to inhibit the tryptic digestion of Casein increases significantly with increasing body weight.

Some biological properties of 3 batches of a multichain polyamino acid, multi-(copoly-L-glutamyl-L-aspartyl) poly-L-lysine, were examined in vivo in dogs and in rabbits. The polymer was administered intravenously as 2 to 3% solutions of its sodium salt by one or by repeated injections. In some animals hypovolemia was produced by arterial bleeding prior to infusion. A low-molecular weight fraction of the polymer, amounting to about 10 to 20%, was excreted in the urine within a few hours. The rest of the injected polymer was not excreted in the urine, and disappeared gradually from the blood, with a constant half-concentration time of about 48 hours. As shown by its influence on blood pressure and on hematocrit, the polymer was able to combat the manifestations of hypovolemic shock. However, severe toxic manifestations appeared in many animals, consisting of diarrhea, vomiting, loss of appetite, a transient drop in blood pressure and a tendency to capillary bleeding. Progressive weakness of the animals was observed, leading to their death. The cause of toxicity of these polypeptides has not been found. Possibly their multiple negative charges may be responsible. It appears worthwhile to examine non-charged or isoelectric polyamino acids with regard to their plasma expanding properties and their toxicity.

Three live litters, totaling 13 rabbits, have been produced for the first time from frozen rabbit semen stored 24 hours at - 90°C. The diluter used was a modified CUE diluter containing 8% glycerine. Vasectomized males were used to induce ovulation and genetic proof was presented to show that the young could not have come from the vasectomized males.

These investigations do not bear out the assumed similarity between the biosynthesis of cholesterol in pregnant rats with the same process in tumor bearing animals. During pregnancy there is apparently no appreciable impairment or other change in rate of biosynthesis in the mother liver although total amounts of cholesterol produced increase with the increase of weights of the livers during pregnancy and more considerably so during lactation, when milk production requires the synthesis of large amounts of fat. It is doubtful whether the early embryonal cell produces any cholesterol from acetate at all or accumulates it. However, at the end of pregnancy the liver of the fetus synthesizes this substance at least at a similar rate as the adult animal, in contrast to the observation that cells of the Yoshida ascites tumor do not make cholesterol from acetate. The degradation of cholesterol in the fetal liver at the day before birth seems to be much less than in the normal controls or the pregnant mothers, thus causing the apparently tremendous incorporation of C¹⁴ from acetate into fetal liver cholesterol. The experiment shows that only a very minute transfer of this substance from the mother through the placenta takes place. At the day before birth gastrointestinal tracts and carcasses of the fetuses accumulate much higher radioactivities than in the mothers. Synthesis of fatty acids increases during pregnancy in the mothers'tissues and reaches high levels during lactation. At the day before birth the tissues of the fetus show very high levels of radioactivity in the fatty acids.

1. A massive inoculum non-growth carbohydrate testing procedure provides results not obtained previously with slow-growing mycobacteria. 2. The results obtained indicate usefulness of carbohydrate typing for aiding differentiation of species and strains of acid-fast organisms. 3. The trehalose and xylose reactions may correlate with pathogenicity for certain types of mycobacteria.

C¹⁴-labeled cholesterol was incubated with growing cells of A. aerogenes. Following hydrolysis of the incubation medium, a significant portion of the sterol was recovered in the saponifiable fraction. Apparently, the solubility of the sterol was modified by a substance produced during the growth of the Aerobacter.

1. X-Irradiation administered by the reported procedure significantly increased mortality over controls during a 40 week period. 2. Chlortetracycline administered in the feed at 20 parts per million significantly decreased irradiation mortality during the first 20 weeks of the experiment. 3. The marked chlortetracycline effect in Experiment 295 was due to modification of disease primarily due to Bartonella muris infection. 4. The lesser effect on mortality observed in Experiment 300 was probably due to antibacterial activity of the chlortetracycline on minor incident infections antagonized by the X-irradiation.

1. Nialamide increased the maximal electroshock seizure threshold in mice and rats. This effect was independent of the elevation of brain 5-HT and NE elicited by nialamide. 2. Diphenylhydantoin in doses from 6 to 55 mg/kg, which was almost 9 times the anticonvulsive dose, did not significantly alter the content of 5-HT or NE in the brain of mice. 3. Nialamide potentiated and reserpine decreased the anticonvulsant effect of diphenylhydantoin. No correlation could be found between these effects and brain amine levels.

In a test group of 8 rabbits, administration of digitoxin by intravenous injections of 0.1 mg/kg, given daily for 8 days, produced no significant changes in the external balance of magnesium. On the day following the last injection the exchangeable body content of magnesium, measured with Mg²⁸, was significantly increased. The results suggest that digitoxin produces subtle changes in the dynamics of magnesium metabolism which are not reflected in the external balance data.

To determine whether impairment of liver function interferes with release of I¹³¹ tag from radioiodinated antipyrine, groups of rats were functionally hepatectomized. poisoned with carbon tetrachloride or poisoned with dl-ethionine. RIAP was then injected intravenously and the diffusibility of the I¹³¹ tag through a cellophane membrane measured after one hour. Functional hepatectoray resulted in a marked decrease in diffusibility, while carbon tetrachloride and dl-ethionine poisoning produced a significant decrease in diffusibility. Return of liver function after carbon tetrachloride poisoning resulted in an increase of diffusibility. The data support the conclusion that the release of I¹³¹ tag from RIAP is a function of the liver and can be used to measure liver function.

Mice of A/Jax and C₃H strains were injected at birth with bone marrow cells from females of the opposite strain in an attempt to induce “tolerance.” Skin grafting experiments demonstrated that both males and females so treated did acquire a slight degree of tolerance. When the partially tolerant female mice were used as donors or hosts in homologous bone marrow transplantation experiments (with the opposite strain), the date of appearance of the secondary disease was delayed, an effect attributable to acquired tolerance. However, when partially tolerant males were used, survival did not increase, but actually decreased. The animals died in a very short time, as if hypersensitized. It has been proposed that the “tolerant” animals are chimeras; if so, the male animals may contain female cells already hypersensitized against “male,” and these “hypersensitized” cells may be responsible for the short survival.

By using 40% of brewers type dry yeast as the principal protein source, a low iodine diet has been designed that supports a good rate of body weight gain and also produces large goiters in rats. This diet appears to have a positive goitrogenic effect, over and above that attributable to its low iodine content.

A variety of cell lines, derived from both normal and tumor tissues, has been tested for possible stimulatory effects induced by sodium 5,5 diphenylhydantoin (Dilantin Sodium). Of these cells utilized, only fibroblasts responded by a stimulation of proliferation with the exception of the BW1081 mouse salivary gland tumor. The significance of these findings may be of considerable importance in our ultimate understanding of fibroblast function.

Profound hypo-and hypernatremia were produced in rats of varying age. Resulting changes in bone Na content were much more pronounced in the younger animals, and decreased progressively as the animals became older. Calculated Na “space” of bone averaged 91% of bone water content for the 3 age groups studied. Observed changes in bone Na content could be ascribed entirely to that portion within the “fluid phase” of bone. There was no evidence of a change in “crystal phase” Na. Observed changes in adult rats were so slight that rather large changes in serum Na would be necessary for their detection.

Experimental evidence is presented which demonstrates the occurrence of a definite sequence of events in the skin reactions of guinea pigs following repeated exposure to flea bites. The sequence was: I—induction of sensitivity; II—predominately delayed skin reactivity; III—delayed and immediate responses; IV—predominately immediate reactivity; V—non-reactivity.

Day old chicks were fed a diet borderline in methionine for 3 to 4 weeks. They excreted large amounts of formiminoglutamic acid (FGA). a metabolite of histidine, when either folic acid or Vit. B₁₂ was omitted from the diet. FGA was excreted in very small amounts by control chicks fed the complete diet. Incorporation of supplemental methionine at 1% of the diet caused a marked reduction in FGA excretion of Vit. B₁₂ deficient chicks and a moderate reduction in the folic acid deficient chicks. Further elevation of dietary histidine and continuation of the supplemental methionine resulted in increased FGA excretion which was relatively greater in the folic acid deficient than in the Vit. B₁₂ deficient group. Only on the last day of the experiment was the excretion elevated for the control group. Chicks fed diets severely deficient in methionine did not excrete large amounts of FGA until Vit. B₁₂ was omitted.

The aminonucleoside of puromycin (P-A), which produces the nephrotic syndrome in rats, was administered simultaneously with various nucleic acid intermediates. L-glutamine, D-glucosamine, and uridine intensified the nephrotoxicity, whereas guanine, adenylic acid, 4-amino-5-imidazole carboxamide, D-ribose, orotic acid, 2,6-diaminopurine, inosine, and thymine appeared to ameliorate the condition. Rats primed with L-triiodothyronine (T-3) developed proteinuria earlier than rats treated with P-A alone.

The R.E.S., which is the main defense against endotoxin, plays no part in the defense against staphylococcal toxin. The kidneys remove staphylococcal toxin from the circulating blood. The toxin or a modification of it is subsequently released, then exerts a lethal action elsewhere. For some hours after it is taken up by the kidney the toxin cannot act. Thus, the kidney is protective for some hours. Indeed nephrectomy prevents death from exotoxin. The renal necrosis produced by the toxin in itself is not the lethal lesion, because renal necrosis produced by arterial occlusion does not cause such rapid death as occurs in renal necrosis produced by the toxin.

Kidney renin and angiotensinase concentrations were determined for the following groups of rats: normals(7); rats with renal hypertension produced by unilateral clipping of the renal artery (12); and rats unilaterally clipped which failed to develop hypertension(11). Kidney extracts were assayed for renin by 2 methods: 1) direct intravenous injection into a rat sensitized by ganglionic blockade, and 2) in vitro production of angiotensin which was subsequently assayed in a similar manner. Angiotensinase concentration was determined by incubation of the kidney extract for 2 hours at 37° with a known amount of synthetic angiotensin and subsequent assay of the residual angiotensin. Renin concentration was reduced in kidneys of both groups of rats with renal artery clips. The reduction in the untouched kidney was greater than that in the clipped kidney. Concentration of angiotensinase in the kidneys of experimental animals which became hypertensive was equal to that in control animals.

The pattern of congenital transmission of a leukemia virus was studied in several inbred strains of mice varying in their susceptibilities to virus. Transmission of virus occurred through the maternal line in 3 C3H sublines, 2 low-leukemic and one high-leukemic. RFM mothers failed to transmit infection. There was no indication that parenterally-infected males, of any of the strains studied, transmitted virus to their offspring. Reciprocal foster-nursings of litters born to infected and non-infected mothers revealed that transfer through the mother's milk was the most effective route of transfer. Results of transmission during the prenatal period were equivocal. Leukemic lines were established in C3Hf/Lw and C3Hf/Fg strains through inoculation of virus into prospective parents in the neonatal period. The frequencies and ages at death from lymphocytic leukemia through 4 generations of C3Hf/Gs mice revealed no loss of virulence of virus. Apparently, low-leukemic and high-leukemic lines may be established simply through foster-nursing of litters. There was no evidence to support the concept for transmission of virus through germinal cells. The results reported here concerning the pattern for congenital transmission of Moloney virus stand in sharp contrast to the patterns for transfer of potentialities to develop leukemia in the high-leukemic strains of mice, AKR, C58, F and C3Hf/Fg.

A method has been described for plaque formation of parainfluenza type 2 virus in grivet kidney monolayer cell culture. The method has shown utility in the assay of virus infectivity. The plaquing technique should also be useful for cloning of this virus for genetic studies.

A procedure for concentration of albumin and globulins from normal urine is described. The albumin appears to be identical with serum albumin, and excretory rate is about 16 mg/day, under conditions of normal activity. Excretion of globulins amounts to less than 6 mg/day.

Fractions from Azotobacter agilis reduce TPN in the presence of 6-phosphoglu-conate. This reduction is due largely although perhaps not entirely, to oxidation of glycer-aldehyde-3-phosphate formed by the splitting system described by Mortenson, Hamilton, and Wilson. This organism possesses a TPN-linked triose-phosphate dehydrogenase, similar to that found in green leaves. The reduced TPN is reoxidized via the TPN-DPN trans-hydrogenase system.

Studies were made on the clinical acceptability and immunogenicity of live tularemia vaccine administered to the monkey and guinea pig by the dermal or respiratory route. Aerogenic as well as dermal vaccination proved innocuous. Animals vaccinated aerogenically showed higher levels of antibody and comparable if not greater protection against challenge with a highly virulent strain of P. tularensis than animals vaccinated dermally.

Mice injected with a rabbit antiserum to Ehrlich's ascites tumor developed hemorrhagic edematous lungs with a markedly increased serotonin content. Such lung findings were not seen in mouse anaphylaxis.

Intrinsic factor or a mixture of IF and Co⁶⁰B₁₂ was injected intravenously to rabbits and rats in which a biliary fistula had been prepared, and excretion of B₁₂ binding power or Co⁶⁰ in bile was measured. It was found that significant amounts of B₁₂ binding power or Co⁶⁰ appeared in the bile within one hour and that such excretion began almost immediately following intravenous administration. A possible mechanism of hepatic uptake and subsequent excretion into bile of intravenously administred IF or its complex has been discussed.

Spermine was active at a concentration of 3 μ moles per liter in allowing growth of a lysozyme-susceptible strain of B. anthracis in the presence of 1-4000 lysozyme. Spermidine. 1-3 propanediamine, putrescine, and cadaverine were respectively 1/2, 1/20, 1/40 and 1/200 as active as spermine. The activity of all of the substances was overcome by addition of 0.005M sodium bicarbonate to the medium.

Propagation of ECHO 9 virus in suspensions of human blood leucocytes has been described.

Gamma globulin (7S) isolated by chromatographic techniques from 2 normal individuals and from one patient with rheumatoid arthritis coats tannic acid-treated sheep erythrocytes and detects rheumatoid factors with a specificity not found using pooled human gamma globulin. The serological specificity of individual gamma globulin preparations is destroyed by heating the protein to 63°C for 5 minutes. The specificity is also observed in an inhibition system using whole rheumatoid serum or its pseudoglobulin fraction. The results may be explainable by the presence of multiple determinants in both the 7S gamma globulin isolated from individuals as well as in the rheumatoid factor complex found in individual rheumatoid sera.

A continuous 2-stage system for investigations of interactions between 2 or more species of bacteria is described. The system employs 2 or more chemostatically limited stages of pure cultures feeding to a non-limited mixed continuous growth cell. This permits comparison at any point on the growth curve of numbers and products of organisms in pure culture with numbers and metabolic products resulting from interactions between the same organisms in growing in mixed culture. The systems are mathematically defined and the data can be expressed in generation time. Results obtained with a mixture of S. salivarius and V. alcalescens indicate a distinct interaction betwen the strains employed. Generation time of each of these species was noticeably increased while growing in context with the other.

Variations in the pattern of liver circulation in the rat have been observed in different age groups. Hepatic blood flow decreased as age of the animal increased and was not dependent on vascular space and portal blood pressure. Vascular space values were higher in the middle age group but portal vein blood pressures were unaffected by variations in age.

Evidence presented shows that the influence of salt concentration of the environment on RNA infectivity for mammalian cells is due at least in part to alteration of the state of susceptibility of the host cell. The 3 different cell lines tested simultaneously responded differently when exposed to the same RNA preparation diluted in varying salt concentrations. Other factors which alter host cell susceptibility, sucrose concentration, age of host cell, nutritional environment of host cell and temperature during the adsorption interval, emphasize the importance of the physiological state of the cell at time of exposure to RNA to the degree of infectivity expressed.

A large dose of reserpine was given to dogs. The dogs were subsequently killed and gastrin was extracted from the antral mucosa. The activity of this gastrin was similar to that of gastrin prepared from normal dogs. This finding is thought to be evidence that histamine and serotonin are not constituents of gastrin, and that reserpine does not stimulate gastric secretion through release of gastrin.

Cells of tissues obtained from marine fish were shown to grow by the explant method at 20 °C in a medium designed for mammalian cells. Preparation of monolayers from cells dispersed by primary trypsinization was accomplished when additional sodium chloride was added to mammalian cell media. Cells in continuous culture were found to multiply more efficiently (as regards time required for sheet formation and number of cells required for satisfactory growth) than did cells in primary culture.

Following adsorption of influenza virus by Krebs 2 ascites cells there is a brief period of sensitivity to inhibition of viral replication by ammonia. After an hour at 35 °C addition of ammonium phosphate causes no inhibition. The period of ammonium sensitivity is prolonged by low temperature, cyanide, or 2-4 dinitrophenol, but the presence of glucose in the medium prevents this effect of CN and DNP. The results suggest that the ammonium-sensitive phase of infection is an energy-requiring process which occurs soon after adsorption and penetration, and comprises initial integration of the viral inoculum with cell components.

Previously described methods of transferring allergic encephalomyelitis in commercially available randomly bred, albino rats sensitized to nervous tissue plus adjuvant, entailed use of parabiosis or pretreatment of recipients with normal spleen cells of prospective sensitized lymph node cell donors. Further study indicates that the disease may be transferred using lymph node cells of splenectomized donors sensitized to nervous tissue and either intact or splenectomized recipients. Splenectomy of only the recipient does not appear to facilitate transfer of the disease.

The Mann-Williamson operation has been applied to the rat; gastric, marginal, and jejunal ulcers were seen in 85% of the animals. Early postoperative deaths may have been due to obstruction at the gastro-jejunal anastomosis; these animals developed a high incidence of ulcers in the prostomach. In rats that lived longer, the majority of ulcers were found at or nearby the gastro-jejunal anastomosis. It is felt that this latter group is representative of typical Mann-Williamson ulcer in the rat. A possible role of vascular factors in the production of ulcer is discussed.

Within 15 seconds after rapid administration of E. coli endotoxin in the rabbit a significant release of serotonin into platelet-free plasma occurs in vivo. The magnitude of immediate serotonin release was cor-related with amount of endotoxin administered, and there was no significant rise of plasma serotonin with a non-lethal dose.

1. Hypothermia rather than fever was the characteristic thermal response of mice to intranasal inoculation of the PR8 strain of type A influenza virus. 2. Mice showed marked hypothermia with fatal termination in response to inoculation of large amounts, e.g., 1,000 or more infectious doses (ID₅₀), of 2 sublines of the virus, one adapted to embryonated eggs and the other to mice. 3. Inoculation of mice with a small amount of virus, e.g., one to two ID₅₀, did not cause hypothermia. In mice inoculated with about 2 ID₅₀ of egg-line virus. the maximum level of virus in the lungs was lower than in mice inoculated with larger amounts of virus, and pulmonary lesions were present but failed to reach the level of 50% total lung-lesion score. 4. In mice inoculated with a moderate amount of egg-line virus. e.g., about 200 ID₅₀, the maximum virus level in the lungs was reached 3 to 4 days before the occurrence of hypothermia. Time of occurrence and degree of hypothermia appeared to be closely related to the extent of pulmonary lesions. 5. The mouse-passaged virus caused more rapid development of hypothermia than the egg-line virus. 6. It is concluded that hypothermia is related to the extent of pulmonary lesion rather than to the extent of virus increase per se.

A globulin concentrate from antiserum to bovine pituitary thyrotropin completly neutralized the effect on the thyroid of “long-acting thyroid stimulator” in the serum of patients with Graves' disease. Both early and late responses in the McKenzie assay were abolished. A globulin concentrate from normal rabbit serum had no neutralizing effect. About 10 times as much antibody was required to neutralize LATS as the approximately equivalent potency of human pituitary thyrotropin.

Rats in the advanced stages of vit. A depletion showed abnormal taste responses. A marked decrease in rejection of quinine and selection of NaCl solutions occurred. Administration of vit. A resulted in rapid recovery of NaCl selection but not of quinine rejection. These data suggest that vit. A may have a direct effect on the functioning of the taste cells.

Coronary and aortic atherosclerosis were studied in white leghorn cockerels fed an atherogenic diet and treated with parenteral estradiol benzoate or synthetic estrogen compounds of lower feminizing potency. The estradiol benzoate gave the expected feminization and protection against coronary atherosclerosis and enhanced thoracic aortic atherosclerosis. The 2 iodo-estrone acetate gave some suggested protection to the coronary vasculature, but was not clearly different from the other analogues in this respect. The 2 iodo-estrone enhanced aortic atherosclerosis and was significantly different in this effect from the other analogues. These observations suggest that the effects on atherosclerosis of the coronary arteries and aorta are mediated through molecular configurations different from those mediating feminization.

In a study of the relationship between blood thyroxine concentration and thyrotrophin (TSH) secretion, the time course of thyroid response to thyroxine injection has been examined. As measured by thyroid I¹³¹ release curves pituitary-thyroid inhibition occurred immediately following sudden increase in blood thyroxine concentration in 2 rabbits and within 3 hours in a third. Groups of rats were pre-treated with radioiodine and thyroid inhibition produced by injection of thyroxine. Protein bound iodine (PBI) levels in the blood were followed serially as the inhibitory effects of the injected material gradually decreased. Pituitary-thyroid activation (as measured by sharp increase in PBI¹³¹) was observed as PBI levels fell from 5.2 to 4.0 μg% (control PBI 4.0 μg%) over a 6-hour period.

Osteoarthritis of the knees was less frequent and severe in 16 month old female than male STR/1N mice. Orchiectomy, performed at 4-6 weeks of age, failed to reduce the amount of joint disease in males. Femur length was increased in the castrate animals.

Homogenates of rat liver, kidney, brain, spleen and muscle prepared in modified Tyrode's solution and injected into the rat are capable of stimulating erythropoiesis as measured by Fe⁵⁹ incorporation into red cells and reticulocyte counts. The activity of the homogenates does not appear to be a nonspecific effect. Certain points of similarity are noted between the homogenates and plasma erythropoietin.

Normal rat diet supplemented with potassium at concentrations of 5, 8, or 11% increased the excretion of a single intraperitioneal dose of Cs¹³⁷ from rats as compared with control rats ingesting normal rat diet without added potassium. Twenty days after injection, the Cs¹³⁷ burden of rats maintained on 5% potassium diet was half that of animals fed basal diet. Animals fed a semi-synthetic low potassium diet retained Cs¹³⁷ more tenaciously than animals on the normal diet.

Increasing dietary potassium intake simultaneously with exposure to Cs¹³⁷ does have the beneficial effect of enhancing Cs¹³⁷ excretion in the rat.

Suspensions of leucocytes from 2 normal donors and 18 patients with various diseases, including 3 patients with congenital agammaglobulinemia were exposed to Sendai or measles virus and maintained in vitro for several days. Production of interferon occurred in 21 of 22 preparations. It is suggested that interferon-like substances may be liberated by leucocytes in vivo, after infection with some viruses and that production of interferon by leucocytes may provide a criterion for presence of non-cytopathic viruses.

Fetal plasma potassium concentration is normally within the range reported for adults. Markedly elevated K concentrations are observed after brief periods of hypoxia. Concentrations up to 17 meq/1 may be anticipated if the fetus dies.

The first recognized active infections of western encephalitis (WE) in Massachusetts are described. The virus was isolated from 15 pools of unengorged and 11 pools of engorged Culiseta melanura and from 1 pool of engorged Aedes canadensis collected in southeastern Massachusetts during 1959. and from 3 pools of unengorged C. melanura collected in 1960. The virus also was isolated from the brain of an infected 3-year-old Impeyan pheasant and from the blood of a 3-week-old chicken in 1959. WE and EE (eastern encephalitis) antibodies were detected in domestic fowl hatched and reared in widely separated areas in eastern Massachusetts during 1959. Although no human or equine outbreaks of WE have occurred in the eastern United States, the potential for such an outbreak apparently exists.

In adult female CF no. 1 mice, kidney enlargement after unilateral nephrectomy is a consequence mainly of hypertrophy. New cell formation as evidenced by an increase in total DNA content of the kidney may be a contributing factor in young animals but this becomes negligible with increasing age. The compensatory response occurring after uninephrectomy in the adult mouse is quite radioresistant, whether irradiation occurs immediately or 5 days post-operatively. This contrasts with the well-known radiosensitivity of cell renewal systems.

Data are presented on the comparative tissue distribution of C¹⁴-free and esterified sterols following injection of cho-lesterol-4-C¹⁴ and C¹⁴-phytosterols to normal and bile-fistula rats. At the end of 24 hours a large proportion of the injected C¹⁴-phytosterols and cholesterol-4-C¹⁴ was recovered in the liver. All tissues examined (serum, liver, small intestine and adrenal) showed the presence of esterified C¹⁴-phytosterols, but less was esterified than with cholesterol-4-C¹⁴. A much larger proportion of the injected C¹⁴-phytosterols was recovered in the feces and intestinal contents than in the cholesterol-4-C¹⁴ group. Conversion of the injected C¹⁴-phytosterols to bile acid was observed; a larger proportion of the injected C¹⁴-activity was recovered in the bile as C¹⁴-sterol than with injected cholesterol-4-C¹⁴. No conversion of C¹⁴-phytosterols to cholesterol or related sterols could be demonstrated.

Destruction of chimpanzee kidney cells by 2 of 12 acute sera from patients with clinical infectious hepatitis was noted. The cellular effect has been seen in serial passage. The destructive agents, tentatively named SAM-1 and SAM-2, have been shown in fluorescent antibody and neutralization tests to be serologically cross-reactive with convalescent serum derived from clinical infectious hepatitis. The agents are resistant to heat treatment at 56°C for 30 minutes.

The effect of D-sorbitol on intestinal absorption of Co⁶⁰B₁₂ was studied in rats. The data demonstrated that sorbitol, when given by mouth in large quantities together with supraphysiological doses of B₁₂, enhanced absorption of B₁₂ in intact animals. However, absorption of small, physiological doses of B₁₂ was reduced by the same dose of sorbitol. The dependence of the sorbitol effect on the B₁₂ dose may be explained on the basis of the 2 different mechanisms for B₁₂ absorption. No intrinsic factor activity was demonstrated in sorbitol as studied with isolated intestinal loops. When the cecum was excluded from the absorptive route by ileostomy, the conditions that increased absorption in intact animals resulted in reduction of absorption. Possible involvement of the cecum and the altered physiological state of the intestinal contents in enhancement of B₁₂ absorption by sorbitol have been discussed.

The proportion of linoleic and arachidonic acid in liver and heart tissue of chickens fed fat-free diets decreased to near minimal values within the first 2 weeks after hatching. Addition of linoleic acid to a fat-free diet at 7 days of age resulted in a significant increase of both linoleic and arachidonic acid in the liver of chicks fed the supplement within 4 days. Chickens fed a diet free of unsaturated fatty acids from 1 to 28 days of age synthesized over 24 g of fatty acids. This consisted mainly of palmitate, palmitoleate and oleate. During the same period the linoleate and arachidonate content of the entire body decreased, resulting in a reduction in the percent of these acids to less than 1/16 of that originally present in the newly-hatched chick.

I. A stable EMC hemagglutinating antigen was prepared by alternate freezing and thawing of virus-infected HeLa cell cultures. 2. Optimal hemagglutination titers were obtained at 4° and 25 C with human and guinea pig erythrocytes when diluents containing potassium and boron ions were used. 3. A hemagglutination-inhibition test employing kaolin treated serum and tissue culture antigen is described and its practical usefulness in detecting antibody in human and swine serum is demonstrated.

The murine host range of Friend leukemia virus has been investigated quantitatively. Virus failed to multiply in C57BL and A strains. About 70% of C3H mice were susceptible, but overt disease developed in only 39%, the remainder being detectable by histologic examination only. Virus multiplied more slowly and to a lesser degree in C3H mice than in Swiss, DBA/2 and BALB/c, which were highly susceptible. Splenomegaly was more marked and more regular in BALB/c than in any of the other strains investigated; 100% of these mice being susceptible after initial adaptation.

A 30 day cotton rat assay was developed which gave results comparable to those obtained with rats assayed at 12 weeks. Age and growth rates were found to be factors which greatly affect the assay. Age and maturation of the tooth account for much of the biological variation encountered in experimental dental caries production and assay.