Experimental Biology and Medicine

In a recent paper (1) in this Journal I presented data indicating that injection of a solution of the diazo dye azo blue into pregnant rats caused congenital malformations in the offspring. The experiment could not be repeated in the laboratory of Professor H. Tuchmann-Duplessis, University of Paris, who was gracious enough to inform me of this fact immediately. As a consequence, I returned the sample of my dye to the supplier for analysis. He finds that the substance purchased by me as azo blue was in reality the closely related dye trypan blue. This makes it necessary that I withdraw the claim that azo blue causes malformations as described in the published paper. The results attributed to the action of azo blue become valid if ascribed, instead, to trypan blue. All other observations in the same paper stand as presented.

An investigation into the manner in which the mistake was originally made revealed that the lot of dye in question was manufactured in 1941 as trypan blue. It was divided into two batches and placed in separate containers, each bearing the same lot number. For reasons not known, one of these containers was mislabelled azo blue while the other was correctly labelled trypan blue, although both retained the correct lot number. The company which manufactured the dye and originally mislabelled it is no longer in business. Needless to say, the company that has recently distributed the dye has made every effort to recall all samples of the erroneously labelled product, I am grateful to Professor Tuchmann-Duples-sis for informing me promptly of his negative results, and to the editor of the Proceedings for affording this opportunity to correct the error.

Virus-neutralization tests were carried out in embryonated hen's eggs employing the egg-adapted distemper virus and various samples of serum. Ferrets whose sera contained no demonstrable antibody at 1:40 dilution all succumbed to 100 MLD of ferret distemper virus, whereas ferrets showing antibody titer to 1:640 dilution of serum all survived the same challenge dose. The data presented demonstrate that the results of serum-neutralization tests in chick embryos have a definite correlation with ferret protection tests. Human gamma globulin and some samples of human serum have neutralizing substances in titers as high as those known to exist in immune ferret serum. On the other hand, samples of serum from normal premature infants did not show any neutralizing substances in 1:40 dilution of the serum.

1. The effect of dicumarol on the inhibition of the dermal Shwartzman reaction was studied. 2. It was found that unlike heparin, dicumarol did not inhibit the dermal Shwartzman reaction.

1. Toluidine blue and protamine, like inorganic cations, produce a reversible inactivation of the mechanism of calcification. This inactivation is a function of the inhibitor to calcium ratio. 2. Up to a concentration of about 15 meq/L, calcium ion in the dye solution increases the intensity of metachromatic staining. Above this concentration there is a gradual decrease of metachromasia. 3. Under certain conditions, the calcifiability of bone sections parallels metachromasia. But it is possible, by raising the calcium ion concentration, to destroy metachromasia while increasing calcifiability, or by treatment with certain protein denaturants, to destroy calcifiability while enhancing metachromasia. 4. Synthetic chondroitin sulfate-collagen complexes have been found to exhibit behavior similar to that of rachitic bone cartilage with regard to metachromasia and calcifiability.

1. Rickets was produced in rats thyroparathyroidectomized at 21 to 23 days of age as readily as in control animals. Both groups were on a rachitogenic diet with a calcium to phosphorus ratio of 10.3. X-ray routine histologic examination, and blood chemical analysis for calcium and phosphate were comparable in both groups of rachitic animals. 2. Rachitic bone cartilage from thyroparathyroidectomized rats calcified to the same degree as the rachitic bone cartilage from the controls. 3. Under the conditions of this experiment, neither the secretions from the parathyroid glands nor any enzyme system such as that involving bone citrogenase is essential for satisfactory in vitro calcification.

Intravenous insulin tests (0.1 U/kg of body weight) were carried out in 42 normal subjects and in 20 diabetics, blood sugar and serum inorganic phosphate being determined before and 15, 30, 45 and 60 minutes after administration of insulin. The blood sugar fall was found to be less in the diabetic patients at 15 minutes, similar in both groups at 30 minutes and more pronounced in the diabetic at 45 and 60 minutes. The phosphate fall was more pronounced in normals than in diabetics. At 45 minutes for instance, the average fall in diabetics was about 50% that found in normals. If diabetics are classified in 2 groups according to their normal or defective phosphate response to glucose no difference is seen between the 2 groups with regard to their response to insulin. It is suggested that the phosphate fall which takes place in a number of diabetics after the injection of glucose may be in part dependent upon an insulin secretion by the pancreas. It is pointed out that the experimental data thus far accumulated do not permit the assumption that the phosphate fall after glucose represents “peripheral utilization” of the carbohydrate.

The reduction of tetrazolium by serum is not a specific test for malignancy. It seems however to be correlated with the serum alkaline and acid phosphatase activity.

Hyla versicolor responds to light by melanophore expansion, and to darkness by contraction. The completenes of contraction depends upon duration of the period of dark-adaptation, generally more than 72 hours are required for all frogs to turn green. The change to brown after dark-adaptation is variable and no consistent relationship has been found between length of darkness and length of recovery period. Temperatures of 3-5° C darken frogs as does light. Light and cold appear to be stimuli which cause release of mel-anophorotropic hormone (ACTH?) from the pituitary gland. Light-induced changes, opposite to those shown by other frogs, seem explicable on the basis of natural selection during the diurnal period when these frogs are inactive.

Immunization with Proteus prior to LD₅ irradiation increased resistance of mice and rats to challenge with the living organism. Immunization with mixtures of the bacteria which occur most frequently in postmortem blood increased resistance to challenge in mice. Immunization with these mixtures did not increase survival in mice exposed to LD_65-95 radiation.

1. Our previous experiments showed that recuperation of regeneration in limbs of hypophysectomized newts may be obtained by replacement therapy with ACTH and with adreno-cortical hormones. We tested the hypothesis that an hyperadrenal state is necessary for regeneration. The study of effect of xenoplastically transplanted single or multiple adrenal frog glands upon regeneration of the fore limb in previously hypophysectomized newts was the method used. Control experiments showing that frog muscle tissue and also other tissues transplanted under similar conditions had no effect upon the non-regeneration typical for hypophysectomized newts indicated that xenoplastic transplants per se produced no observable effects upon regeneration. Frog adrenals, however, while not affecting regeneration in normal animals were found capable of restoring to hypophysectomized newts the ability to regenerate amputated limbs otherwise lost after extirpation of the pituitary. 2. Hypophysectomized animals are more susceptible to adrenal therapy 10 to 20 days after amputation than during earlier periods, since it is when transplanted at this time that frog adrenals most consistently produced regeneration. These results while not clearly understood, point to the existence of a critical stage within the post-amputational phases of regeneration when regeneration is particularly susceptible to adrenal hormones, endogenous or experimentally supplied. 3. Heretofore unsuspected involvement of adrenal secretions in limb regeneration of newts is thus demonstrated and the pituitary-adrenal synergism discovered for conditions of “stress” in mammals acquires a new importance for regenerative processes in amphibia.

1) A method for measuring mercurial diuresis in the rat is described. The effect of varying intravenous doses of mersalyl on hydrated rats has been studied. Maximum diuresis is obtained with a dose of 4 mg Hg/kg: and the height of diuresis is reached in 2 to 3 hours. The extent of tubular damage is proportional to the dose of the mercurial. No rats survived a dose of 12 mg Hg/kg and only 50% survived a dose of 8 mg. In the animals that survived, complete tubular regeneration took place within two weeks. 2) The results reported in this paper do not support the view that mersalyl, in therapeutic doses, has any effect on the oxidative enzymes of the Krebs cycle. Mercurial diuresis may be due to interaction of mercury with —SH groups or -SH enzymes in the kidneys. It is felt, however, that such -SH groups be sought elsewhere than among the Krebs cycle enzymes and coenzymes.

1. Three steroids with protein anabolic activity were tested in parallel with testosterone propionate to compare their relative properties of stimulating the levator ani muscles and seminal vesicles of weanling castrate male rats when injected subcutaneously in oil solution or suspension. The androgenic and myotrophic potencies of the test compounds, as well as their myotrophic/andro-genic ratios, were calculated by analysis of significant portions of the data. 2. The androgenic potencies of these steroids, expressed as percentages of the potency of testosterone propionate, were: methylandrostenediol 2.1, androstanolone 13.2, 19-nortestosterone cyclopentylpropionate 16.2. 3. The myotrophic potencies, expressed as percentages of the potency of testosterone propionate, were: methylandrostenediol 2.2, androstanolone 21.8, 19-nortestosterone cyclopentylpropionate 155. 4. The myotrophic/androgenic ratios of these compounds are, therefore: methylandrostenediol 1.0, androstanolone 1.7, 19-nortestosterone cyclopentylpropinate 9.5.

Data are presented to demonstrate that the serum levels of vit. B 12 activity in diabetics with or without retinopathy are widely different. Those with the retinal lesions have a much higher activity (292 ‡ 24) than those without retinopathy (162‡18). This difference is statistically significant.

The possible implication of vit. B12 in this disease is discussed.

Evidence has been presented to show that hog kidney dialkyl-fluorophosphatase (DFPase) liberates 1 mole of hydrofluoric acid and 1 mole of dialkyl phosphoric acid in the enzymatic hydrolysis of a mole of diisopropyl fluorophosphate, diethyl fluoro-phosphate or dibutyl fluorophosphate.

1. Antibodies in the sera from a group of tuberculin-negative cattle were destroyed by heating at 70°C for 10 minutes, while heat-stable antibodies were detected in some tuberculous sera. 2. Experimental tuberculosis in rabbits was accompanied by the appearance of heat-stable agglutinins early in the course of infection, and later by heat-stable hemolysins. 3. Similar findings were observed in rabbits injected with B.C.G. and M. phlei. 4. Several injections of P.P.D. were required to stimulate the production of heat-stable antibodies.

1. An eluate from BaSO₄-ad-sorption of normal serum is shown to have a marked effect on the clotting time in hemophilia without altering the defect in prothrombin utilization. 2. The eluate is shown to operate chiefly by reducing the latent period of prothrombin utilization. 3. The effect of this eluate on AHF assays is discussed. 4. The necessity of platelets for prothrombin conversion is demonstrated again under different experimental conditions. 5. AHF is shown to be required for the demonstration of maximal prothrombic activity in the prothrombin time test.

The method of Zlatkis, Zak, and Boyle for the direct determination of serum cholesterol has been adapted to the determination of total adrenal cholesterol. The values thus obtained do not differ significantly from those obtained by the method of Sperry and Webb in normal, hypophysectomized and cold stressed animals. The time required to analyze 10 samples and 5 standards by the Zlatkis, Zak? and Boyle procedure requires less than one hour compared to the 2 days usually necessary when methods based on the Liebermann-Burchard reaction are utilized.

1. S. pyogenes, strain S23, grows rapidly in glucose broth, forms large amounts of hyaluronic acid throughout the growth period and induces a rapid fall in the pH of the medium. In galactose broth growth is slower, polysaccharide formation is diminished, occurring only during the first 2 or 3 hours of growth and the reaction of the medium remains above pH 7.0 for a much longer period of time. 2. While no hyaluronidase could be detected in glucose culture supernates, a small amount of activity was detected in galactose supernates. It was not of sufficient magnitude to account for the low levels of hyaluronic acid in galactose cultures. 3. Maintenance of galactose broth cultures at pH 7.8-8.0 did not increase hyaluronic acid formation. 4. It has been shown that neither endogenous cell substance nor infusion reducing sugar serve as raw materials for capsule synthesis in galactose broth. 5. A direct correlation between hyaluronic acid formation and galactose concentration could not be established. 6. From the data obtained, it seems likely that galactose is not directly involved in hyaluronic acid formation, but that an unidentified component of the infusion is utilized for this purpose.

Parenteral administration of ethionine markedly depressed the AHF content of the blood in rats. This effect probably represents interference with synthesis of the protein rather than increased destruction. Although other coagulation factors were affected, the ethionine action did not involve all serum proteins, as shown by paper electrophoresis. Extensive tissue damage in ethionine-treated animals was confined to the pancreas, liver and bone marrow. Neither total pancreatectomy in dogs, nor carbon tetrachloride-induced liver damage in rats had any effect on plasma AHF levels. Studies are in progress to determine a possible role of the reticulo-endothelial system.

The surface area available for gas exchange in the lungs s an impotant factor in gas transfer between the alveoli and the blood 1 ., 2 Present estimates of this area are based on measurements of alveolar dimensions taken from fixed histologic specimens 3 , 4 , 5 . These estimates of total surgace area are made by measuring or calculating the area/volume ratio for a small portion of lung tissue and applying this ratio to an assumed total lung volume. The volume which must be assumed is uncertain because the degree of inflation represented in the particular specimen is difficult to determine, especially if the lungs are allowed to collapse before fixation is complete. Moreover, distortion of lung architecture inevitably occurs with standard histologic methods involving dessicatin and embedding, and such lobular subdivisions as the air-sacs and alveolar ducts, which contribute only slightly to the respiratory surface area, have in the inflated state a much larger volume than is generally realized 6 . for these reasons calculation by anatomic methods tend to result in overestimating the number of alveoli and the total lung surface.

The method of estimating lung surface described in this report is based on physical measurements of the lungs and avoids any assumption of size or configuration of respiratory structures. The calculation depends on consideration of the changes in free energy of the lungs as they are deflated. During inflation, energy is stored by deforming elasitc elements. and by the creation of a large air-liquid interface If the lungs are diflatd by allowing volume equilibrium at successive pressure decrements, the stored energy released during deflation can be measured.

1. The isotope concentration of the urinary hippuric acid of starved irradiated or non-irradiated rats given benzoic acid-carboxyl-C¹⁴ is much less than the dilution expected from the conjugation of the injected labeled benzoic acid with inert carbon atoms of the glycyl residue. 2. Evidence is presented that benzoic acid is a normally occurring metabolite in rats. 3. The effect of xradiation on the ability of starved rats to conjugate benzoic acid-carboxyl-C¹⁴ has been tested and no defect detected under the experimental conditions used.

1. The synthetic basic poly-α-amino acids, polylysine, polyornithine and polyarginine, accelerate conversion of fibrinogen to fibrin by thrombin; the acidic poly-α-amino acids, polyaspartic, polyglutamic and polycysteic acids, and also heparin, retard this reaction. 2. The acidic polyamino acids neutralize the clot accelerating activity of the basic polyamino acids. 3. The addition of polylysine makes possible the detection of concentrations of fibrinogen too low to be clotted by thrombin alone.

Insulin-I¹³¹ degradation in rats in vivo was not inhibited by the administration of zinc in the diet, or by treatment with the substance, polyphloretin phosphate. Marked inhibition was obtained by the intravenous or prolonged oral administration of 5-isopropylidene-2,4-dithiohydantoin and it was suggested that this may explain the protection afforded against diabetes by this substance in the partially depancreatized rat.

Addition of polyvinylpyrrolidone (PVP) to sucrose solutions used in extraction of subcellular components improved centrifugal resolution of particulates, helped to preserve morphologic integrity of subcellular elements, and aided in preservation of enzymic activities. PVP was particularly useful in counteracting irreversible agglutination of microsomes

Heat-killed Vi organisms, the saline extracts of their acetone precipitates and DNA fractions induced W-V variation in S. typhi, S. paratyphi C and Paracolobactrum Ballerup strains.

(1) Adrenocortical function of the dog is suppressed by administration of therapeutic doses of cortisone or hydrocortisone. (2) The suppression is prevented by simultaneous administration of ACTH.

The levels of amylase in the blood, urine, and tissues of the rat were not significantly altered by pancreatectomy. It is suggested that some organ or organs, other than the pancreas, is the principal source of blood, urinary, and tissue amylase in normal rats.

A method is described for determinations of cell and plasma volumes using Cr⁵¹ and I¹³¹ simultaneously in which hematocrit tubes are used for counting and separation of cells and plasma. In vitro determinations of volumes of blood were within 1% of actual volumes. Volumes and distributions found by this method in 4 goats are within the range of values found in the goat by other methods. Limitations of the method are also discussed.

A number of methionine analogs were studied for their ability to replace methionine and reverse methionine sulfoximine induced growth inhibition. Relative activity in replacing methionine and reversing methionine sulfoximine toxicity are not necessarily correlated. Some compounds which counteract methionine sulfoximine toxicity have no meth-ionine-like activity

1. The effect of soybean sterols A on liver deposition and fecal excretion of cholesterol-C₁₄ following a single feeding of I the labeled material has been studied using cholesterol-fed rats. Average recoveries of J labeled cholesterol in liver (measured as radioactivity recovered from liver non-saponifiable fraction) were 6.61% in a cholesterol-fed group, 1.80% in a cholesterol-fed group receiving one g of soy sterol immediately after cholesterol-C₁₄ administration and 0.47% in a group fed both cholesterol and soybean sterol for 120 days. 3. Average recoveries of radioactivity in the non-saponifiable fraction of feces were 20.14%, 25.13%, and 27.76%, respectively, for the 3 groups described above.

1. A decrease in the concentration of hexosamine in orbital connective tissue is observed with increasing age in normal rats but is abolished following thyroidectomy. It can be reversed, however, by the administration of thyroxin. The concentration of connective tissue hexosamine in hypophysectomized rats is similar to controls of the same age and is unaffected by either small or large doses of thyroxin. Growth hormone, on the other hand, produces an increase in the amounts of connective tissue and hexosamine with no change in the concentration of hexosamine. 2. It is suggested that the concentration and amount of hexosamine in orbital connective tissue in the rat is determined by a balance between the production of hormones of thyroid and anterior pituitary (growth hormone). 3. The response of rat orbital connective tissue to thyroidectomy or to the administration of pituitary extract appears to be less sensitive than that in the guinea pig.

Data have been presented which show that porcine gamma globulin, porcine and bovine serum solids and cowscolostrum do not cause a marked or consistent response in the serum gamma globulin level of one-day-old pigs when administered orally. However, parenteral administration of porcine gamma globulin gave a response which approached that obtained with nursing pigs. The inclusion of trypsin inhibitor with orally administered porcine gamma globulin and bovine serum solids did not affect the serum gamma globulin level.

L type colonies have been isolated from group A streptococci by the combined effect of penicillin and concentrations of various salts considerably higher than usually incorporated in bacteriological media. The appearance and morphology of these cultures correspond to those isolated from the alpha streptococci. There is reason to believe that the technic described will be helpful in the isolation of L forms of other bacterial species.

1. Oxidation of tyrosine by the tyrosine oxidase system from livers of thiamine-deficient animals is markedly higher than that by preparations from normal animals. 2. After dialysis the activity of the normal preparation cannot be restored by the addition of ascorbic acid, glutathione, or 2,6-dichloro-phenolindophenol (DCPP). The thiamine-deficient preparations are still very active under these conditions, however. 3. Injection of thiamine into normal and deficient animals one day before assay of the livers for tyrosine oxidase has little effect on the high activity of the thiamine-dencient preparations. Feeding a complete ration for 2 weeks to thiamine-dencient animals, however, decreases the activity to the level present in the normal preparations. 4. Inanition produces an increase in tyrosine oxidase activity by liver enzymes. This may be a partial explanation for the fact that thiamine-dencient rats (which are anorexic) possess a higher activity of this enzyme. The reason for the increase under either of these conditions, however, cannot be explained at present.

Six individuals taken from sea level to 14,900 feet showed a normal red cell life span during the period of acclimatization; therefore, the polycythemia secondary to altitude is due entirely to an increase in the rate of red cell formation.

Five-ml samples of normal, human plasma have been separated by a low temperature-low salt-ethanol procedure into 4 primary fractions, I + III, II, IV + V, and VI. The concentrations of protein-bound carbohydrate (hexose) and of protein in whole plasma and the 4 primary fractions and the protein-bound carbohydrate content of sero-mucoid were determined. Fraction IV + V, of the primary fractions, contained the greatest amount of protein-bound carbohydrate and of protein. The percentage of protein-bound carbohydrate with respect to protein was highest in Fraction VI.

1. Rabbits on severe caloric restriction were fed cholesterol and showed significantly increased blood cholesterol, fatty acids, lipoproteins and phospholipids. The degree of these biochemical changes and of the atherosclerosis was greater than that of a control group fed similar amounts of cholesterol in addition to a normal diet. 2. Therefore, severe caloric restriction does not protect, but enhances cholesterol induced atherosclerosis in rabbits. This effect of weight loss should be taken into consideration in experiments on atherosclerosis.

Daily administration of large doses of glucagon to rats and rabbits for 6 months did not produce any toxic reactions. The rate of weight gain of treated animals was the same as that of control animals. Diabetes was not produced and there was no evidence of chronic hyperglycemia. The liver glycogen concentration of the treated animals was significantly greater than that of the untreated animals.

Type specific neutralizing antibodies were produced in rabbits inoculated with the 3 types of poliomyelitis virus grown in tissue culture. Rabbits were successfully immunized by repeated intravenous or intramuscular inoculations of active or ultraviolet irradiated virus suspensions. The possibilities of a rabbit test for the assay of poliomyelitis vaccine are discussed.

1. Single, 90-minute irradiation with fast neutrons within the lethal range (175-250 rep) resulted in early deaths among CF No. 1 female mice with a significant peak in the mortality curve 4-8 days after exposure. Among 129 neutron-irradiated mice (216-265 rep: LD_60-98—30 days), sacrificed for bacteriological examination, positive cultures of heart's blood and/or spleen were first obtained on the 4th day, and coincided with the onset of mortality. Between the 4th and 8th days after exposure, approximately 40% of the sacrificed mice showed positive cultures. 2. After comparable irradiation with gamma rays (Co⁶⁰), from 750-1300 r, deaths occurred later, with a peak approximately 12-14 days post-exposure. Gamma-irradiated mice (900-940 r) were found to have positive blood and/or spleen cultures during the latter part of the second week. There seems to be a close correlation between bacteremia and time of death following these 2 ionizing radiations. 3. All the microorganisms recovered in cultures were found to be members of the normal enteric flora of the mouse. 4. The bacteremia, as measured by colony counts on heart's blood was less severe in the neutron-irradiated than in the γ-irradiated mice.

Inulin, exogenous and endogenous creatinine, and ferrocyanide ion are all cleared at the same rate by the dog kidney, and probably exclusively by glomerular filtration

1. Growth of birds fed a pantothenic acid-low diet was decreased by addition of vit. B₁₂. The degree of severity of pantothenic acid deficiency symptoms observed was increased by addition of vit. B₁₂. The lowest level of supplemental pantothenic acid that supported growth and prevented deficiency symptoms was 6.64 mg/kg of diet or 17.14 mg/kg when pantothenic acid contributed by the protein is added. When the supplemental pantothenic acid level was increased to 9.96 mg/kg; no additional effect was observed. 2. The B₁₂ content of liver was increased by addition of that vitamin to the chick diet and this was proportional to the level of B₁₂ added. Additional increase in B₁₂ content of the liver was observed when the pantothenic acid level in the diet was increased. This indicates that pantothenic acid may have had a sparing effect on B₁₂ when the pantothenic acid content of the diet was adequate. 3. The pantothenic acid content of the liver was influenced only when the level of supplemental pantothenic acid was raised to 6.64 mg/kg or a total of 17.14 mg/kg or higher.

Dogs, well hydrated with saline became anuric or oliguric when their respiration was suspended with overdoses of thiopental sodium. This anuria could either be reversed by administration of an isotonic mannitol solution, or did not occur when mannitol was administered throughout the experiment. The ability to reverse an experimental anuria with an osmotic diuretic presents a potentially valuable tool for the study of this type of renal dysfunction.

Papain and ficin digest collagen in the pH 2.0-4.5 range at low salt concentration. Evidence is presented that collagen undergoes a reversible alteration at low pH which renders it susceptible to digestion by a number of proteolytic enzymes active at acid pH's. A mechanism for the enzymatic breakdown of collagen in areas of suppuration is suggested. No evidence has been obtained to support a preliminary alteration in collagen as part of the mechanism by which Clostridium histolyticum collagenase preparations degrade collagen.

(1) The DPN content and choline dehydrogenase activity were low in livers of young rats and increased with the age of the animal. (2) The DPN content approached the adult level when the animals were 9 weeks of age. (3) Choline dehydrogenase activity reached 85% of the adult level by the time the animals were 13 weeks old. (4) The betaine aldehyde dehydrogenase activity in livers of newborn rats was 90% of that found in adult animals. (5) It appears that the low rate of oxidation of choline encountered in young rats may be due to a lack of choline apodehydrogenase. (6) The low level of DPN found in the livers of young rats may also be a limiting factor in the in vivo oxidation of choline to betaine.

Epinephrine in dilute solution is destroyed by x-irradiation to yield what appear to be pharmacologically inert decomposition products. The presence of 0.9% sodium chloride offered a moderate protective action against this effect. The extent of loss of pharmacologic activity of epinephrine is exponentially related to the dose of x-irradiation.

Immature mice fed a purified ration containing 4% glucoascorbic acid exhibited loss in weight, diarrhea, an unthrifty appearance, irritation and swelling of the anal region and alopecia. These effects were completely counteracted by the addition of dried alfalfa to the purified ration at a 10% level in the diet. The protective effects of alfalfa were not due to its mineral content or to its content of any of the known vitamins. Supplements of casein or cottonseed oil were without beneficial effect. Purified cellulose completely counteracted all symptoms of glucoascorbic acid toxicity; the weight increment of mice fed this supplement, however, was significantly less than that of animals fed dried alfalfa.

1. Long-time castrated male rats had a mean circulating red blood cell count of 8.70 ‡ .43 million cells/cu mm before treatment. Treatment with testosterone propionate (TP) or nortestosterone cyclopentylpropionate (NTCP) at daily doses of .25 mg or 1 mg for 27 days increased the number of cells to group averages of 9.52 to 10.14 mil-lion/cu mm. Blank-injected controls had RBC counts averaging 8.42 million at the end of the experimental period. 2. Hemoglobin concentration rose from a pretreatment mean of 17.88 g/100 ml to group averages of 19.1 to 20.0 g/100 ml during steroid treatment. Blank-injected controls had 17.8 g/100 ml at the end of the treatment period. 3. It is concluded that nortestosterone cyclopentylpropionate is similar to testosterone propionate in causing increases in red cell count and Hb in the castrate rat.

The administration of estrogen and progesterone to castrate female rats for 15 days or more stimulated an increase of DNA comparable to that found at a corresponding period of pregnancy. Continued injection of these hormones for periods up to 32 days increased only slightly the DNA content. This is interpreted as indicating a cessation of further gland proliferation even in the presence of these hormones. In the hormone stimulated glands the PNA content increased comparable to pregnancy but since profuse lactation was not stimulated it did not increase compared to the latter stages of pregnancy or lactation. However, when the hormones were administered for 26 days and then stopped, lactation set in sufficient to nourish foster litters of rats.

A highly potent sodium-retaining steroid has been isolated from adrenal venous blood and demonstrated to be identical with aldosterone with regard to: 1) infra-red spectrum, 2) chromatographic behavior of the parent compound and 2 derivatives, 3) biological activity in inducing sodium retention in adrenalectomized rats, 4) ultraviolet absorption and 5) sulfuric acid chromogen.

Crystalline aldosterone suppressed the release of pituitary ACTH in animals exposed to the stress of cold as judged by adrenal ascorbic acid determinations. Its activity in this respect approximated one-third that of cortisone and eight times that of desoxycorticosterone

(1) Dogs have been passively sensitized by administration of antiserum to bovine serum albumin developed in the rabbit. 21 mg of antibody nitrogen sensitized each of 2 5 dogs. All manifested symptoms of anaphylaxis and 13 of these died; 6 (of 8) following a dose of 17.3 mg of antigen nitrogen, 1 (of 4) after 10.88 mg, 6 (of 12) after 5.4 mg, and 1 following a dose of 4.33 mg of bovine serum albumin nitrogen. (2) Latent periods varied from 3% to 36 hours. No correlation could be shown between latent period and shocking dose. (3) Sensitizing doses of 10.5-16.8 mg antibody nitrogen followed by antigen in amounts of 4.33-13.0 mg of nitrogen produced symptoms of anaphylaxis in 7 dogs but all recovered, (4) An immediate and rapid fall in blood pressure was the one symptom exhibited by all 32 animals.

Following the adaptation of Type I, Mahoney strain, poliomyelitis virus to mice, further passages in tissue cultures, mice, and alternate monkey skin to tissue cultures resulted in the establishment of 4 passage lines of virus, 3 of which appear to be distinct variants. These strains have been designated as LS, LS-a, LS-b and LS-c and are differentiated on the basis of their range of host reactions. LS and LS-a react similarly and are so-called mouse and monkey spinal variants. LS-b is a mouse cerebral strain and LS-c is a non-neurotropic strain which is avirulent for both mice and monkeys by all routes. These variants have unaltered antigenic and tissue culture characteristics. Preliminary experiments with the LS-c strain have shown it to be capable of inducing good antibody titers and a high degree of resistance to virulent virus challenge in monkeys. There is suggestive evidence that continued skin passages of the LS-c strain may yield a dermal poliomyelitis virus variant.

These studies have demonstrated that all 3 immunologic types of poliomyelitis virus may be propagated successfully in monkey kidney and skin tissue grafted onto embryonated egg membrane. No preliminary “adaptation” is necessary for virus multiplication in this system. Presence of virus is not limited to the grafted tissue but is found also in moderate concentration in the allantoic fluid, the CAM, and the embryo. However, this is probably due to spillage and dissemination from the grafts where virus growth apparently occurs. More precise knowledge is needed with reference to the exact sites of multiplication and the specific cells participating in this activity.

Further studies are desirable to determine the practical and theoretical applications of this technic and whether simplification and improvements are possible.

1. The effect of free iodide (Nal or KI) and phenothiazine N.F. (green) on the suppression of I131 uptake by thyroid in rats is studied and compared. Various related compounds and derivatives of phenothiazine are also surveyed. 2. Column chromatographic fractionation of phenothiazine N.F. (green) reveals that the activity described is strongly associated with its iodine content; however, phenothiazine N.F. (purified) with negligible amounts of iodine has some suppressing quality. 3. In continuous feeding experiments phenothiazine N.F. (green) when given in doses sufficient to partially suppress I131 uptake did not affect the weight of the thyroid gland, total body weight or any of the other organs studied. No component of phenothiazine N.F. (green) is goitrogenic. 4. A chromatographic method is described for studying the renal excretory levels of free iodide after rats are treated with Nal, phenothiazine N.F. (green) or a combination of both. Those animals receiving Nal or Nal and phenothiazine N.F. (green) showed an equal but elevated level of iodide excretion. Those rats given phenothiazine N.F. (green) alone had little or no iodide in their urine over a 24-hour period. 5. It is concluded that the effect of phenothiazine N.F. (green) on reducing the uptake of I¹³¹ by the rat thyroid is not due solely to the availability of free iodide to this gland as a result of dissociation from an organic combination.

1. With reference to “A” group, all tocopherol deficient animals died during the course of the 3 decompression exposures. None of the normal diet group, and only one of the tocopherol supplemented animals died. 2. Group “B”. No deficient diet animal survived 3 consecutive decompression experiences under the conditions of this experiment while 23.9% of the normal diet group survived these 3 experiences and 35.5% of the alpha-tocopherol supplemented synthetic diet group survived. 3. It can therefore be concluded that rabbits deficient in tocopherol to a dystrophic level did not elicit as much resistance to the hypoxic factor as did those animals on the normal or tocopherol supplemented diet. Those animals on the tocopherol supplemented diet had a greater survival rate (11z.6%) than did those on the normal diet.

A standardized laparotomy wound was produced in mice following a whole body x-ray dose of 500 r. The tensile strength of the healing wounds was compared on several post-operative days to wounds in unirradiated mice. In addition, 30 day mortality was determined for mice subjected to wounding, to irradiation, and to wounding plus irradiation. Irradiated animals showed a significant retardation of wound healing from the 6th through the 11th day after wounding, but not thereafter. Wounding did not alter post-irradiation mortality.

Administration of pituitary growth hormone to 4 normal and to 1 depan-creatized dog produced a decrease in the lipid content of the serum and of the albumin-alphai lipoprotein fraction within the first 6 hours. After repeated daily injections of GH for 5 to 10 days, a progressive rise in the lipid content of this fraction took place and resulted, in some animals, in a marked hypercholesterolemia. No evidence of impairment of carbohydrate utilization was found in normal dogs, while fasting blood sugar and glycosuria increased in the diabetic animal. The possible significance of these findings is discussed.

At concentrations of lO-⁴ and 10-⁵ M, methylbis (^β-chloroethyl) amine stimulates the aerobic glycolysis of thymus lymphocytes in vitro. This phenomenon is discussed in relation to the lymphocytolytic activity of this compound.

1. Following oral administration of vit. B₁₂-Co⁶⁰ to normal individuals and subsequent “flushing” by subcutaneous injection of cobalamin, the radioactive material could be extracted from urine by n-butanol; the Co⁶⁰ containing substance had the same distribution coefficient between ammonium sulfate saturated urine and n-butanol as authentic vit. B₁₂-Co^60. 2. In an aqueous 2, 4-lutidine system the radioactive material in the urine has the same Rf value as pure vit. B₁₂ and vit. B₁₂-Co^60. 3. Our studies support the view that the Schilling test measures absorption of vit. B₁₂-Co⁶⁰ from the gut rather than absorption of decomposition products. Its use therefore as an assay of intrinsic factor appears justified.

1. A B-virus strain isolated from “normal” monkey kidney was propagated in cultures of monkey kidney and chick embryo tissues. 2. The B-virus strain studied was relatively stable in tissue culture medium when stored at 4°C. Freezing seemed to destroy some activity, but there was no further loss demonstrable on storage at −20°C and −72°C. 3. Approximately 27% of 513 rhesus monkey sera showed neutralizing activity for the isolated B-virus strain.

1. The distribution and breakdown of insulin-I¹³¹ in rats under various changes in carbohydrate metabolism has been studied. 2. An intravenous glucose load exerted no detectable effect other than to reduce renal radioactivity. 3. Intravenous fructose markedly reduced renal precipitable radioactivity, and increased hepatic and muscle radioactivity. 4. Intraperitoneal glucose or fructose has the same effects as intravenous fructose but to a lesser degree. 5. Alloxan diabetic animals concentrated more precipitable radioactivity in the liver and blood. 6. Phloridzin exerted no effect on insulin distribution and degradation. 7. Fasting effected little change; there was some decrease in renal and some increase in hepatic precipitable radioactivity. 8. The limitations and implications of the study are discussed.

Radiation-induced lymphoid tumor grafts were able to take and grow progressively in young hypophysectomized C57BL mice. The rate of tumor growth was significantly diminished in the hypophysectomized animals. Factors influencing tumor growth have been discussed.

104 Puerto Rican individuals known to be infected with Schistosoma man-soni were skin tested with antigen prepared from lyophilized eggs and cercariae. 35.6% reacted to egg antigen while 95.2% reacted to the cercarial antigen. The discrepancy was found to lie in those who were passing living eggs and had no skin sensitivity to egg antigen while those who had been treated more than a year before approached in percentage the skin reactivity with cercarial antigen.

Studies were made on the levels of Ca+ +, Na+, Mg++ and K+ in the plasma of birds with erythromyeloblastic leukosis. No changes from the normal were observed with the extracellular elements Ca++ and Na+. In contrast, the ordinarily intracellular elements Mg+ + and K+ were elevated in close relationship to the capacity of the plasmas to dephos-phorylate adenosine triphosphate and, consequently, to the virus content of the plasmas. The source of the abnormal amounts of Mg+ + and K+ have not been determined. The possible origin of the elements was discussed.

A complement fixation method based on the reaction between CRP in human test sera and rabbit CRP antisera has been described. This technic provides a precise and reproducible estimate of the relative amounts of CRP in human serum. The procedure has greater sensitivity than the precipitin method and may be of greater value in following the efficacy of therapy.

An attempt has been made to show some changes under hypothermia with control studies on two factors, repeated plasma volume determination and hyperventilation. A moderate amount of blood seemed to be trapped away from circulation at lowered body temperature (4-6). Since blood clotting involves the action of enzymes it might be expected that coagulation would be retarded by lowered temperature. Increases in bleeding and clotting times at 23‡-25°C were highly singnificant.

The onset of sexual maturity in female rats, as measured by vaginal patency, was found to be delayed in vit. B₁₂-dencient animals as compared to vit. B ^-supplemented littermates when their mothers were fed a B₁₂-dencient ration during lactation. No such differences were observed when the mothers were fed B₁₂ during lactation. The nature of the carbohydrate and protein in the ration was found to have an effect on the differences observed.

Pantothenic acid is more active for growth promotion in Tetrahymena and Colpidium than are any of the conjugated forms of the vitamin. For Glaucoma pantethine is the most active form.

There is a descending conduction pathway from the shivering suppression “center” in the diencephalon extending caud-ally through the medulla. Electrical stimulation along this pathway suppresses shivering. One interpretation of these results might be that central control of shivering is accomplished over this route. Instead of acting on the site of origin of the shivering impulses (presumably the posterior hypothalamus), the inhibitory influence may be exerted at lower motor centers, perhaps at the ventral horn outflow, although the conduction pathway in the cord has not been investigated. Direct suppression of the ventral horn cells innervating-particular muscle groups would be a means; of obtaining the graded response that is characteristic of the shivering process.

Administration of gonadotropic substances simultaneously with Enheptin (2-amino, 5-nitrothiazole) sustained sexual activity in most cases. Failure to restore sexual normality in all cases is probably due to the quality and quantity of administered gonadotropic hormone. The drug Enheptin was not able to suppress thyroidal hypertrophy during thiouracil treatment. Pituitary gonadotropic hormone potency is reduced in intact as well as capons treated with Enheptin.

Tumor extracts obtained by centrifugation, if incubated with hyaluronidase and recentrifuged, will pass readily through Pyrex glass ultrafme fritted discs and Selas #03 porcelain filters. They pass more slowly through a Berkefeld N filter, and least rapidly through a Seitz S-l asbestos filter, with over 50% loss of nitrogen-containing material. Filtrates from the first two filters, when injected into the prospective host prior to implanting a tumor homograft, are fully effective in abrogating the animal's normal resistance to the graft. Filtrates of the latter 2 filters show a slight effect, in terms of greater growth of the homografts prior to regression, as compared with uninjected controls.

The customary method of transmission of toxoplasmsis to man and to animals remains unknown. This report shows that infections are transmitted between swine and rodents when either animal is fed infected tissue from the other, likewise when pigs ingest infected swine offal. The feeding cycles described are reminiscent of those known in trichinosis. Similarities and differences are discussed, and implications for human disease pointed out. Whether there is a risk to the public, and if so, its extent, will require future studies on swine and on pork, under natural as well as experimental conditions.

Pancreatic cholesterol esterase catalyzes the esterincation and hydrolysis of a number of different sterols and sterol esters. The order of activity observed in the esterifying system with oleic acid was as follows: dihydrocholesterol † cholesterol † β-sitosterol † sitosterol † stigmasterol † ergosterol. Of these sterols, only dihydrocholesterol and cholesterol were esterified with butyric acid. In the hydrolytic system all of the sterol butyrates tested were split very rapidly, with the exception of ergosterol butyrate. the order of splitting of the oleates in the first 4 hours was: cholesterol oleate † sitosterol oleate † stigmasterol oleate † ergosterol oleate. The relation of enzyme activity to sterol structure is discussed.

The toxic action of phenylethylamine and tyramine, but not of ephedrine and D-a-methylphenylethylamine is increased by pretreating guinea pigs with iproniazid. Isoniazid is less effective. The results are explained by the blocking of MO by iproniazid.

1. When whole blood from polonium-injected rats or dogs was centrifugally fractionated, it was found that approximately 90% of the polonium was associated with the red blood cells. 2. Isolation of hemoglobin from similar blood indicated that the polonium was associated with this portion of the red blood cell. Fractionation of hemoglobin into globin and heme indicated that almost all of the polonium was associated with the globin. Measurement of the affinity of major red blood cell components for polonium demonstrated that globin had as much affinity as lysed whole red cells, while heme residue had practically none. 3. Chemical methods which rely on a change in hydrogen ion concentration for the isolation of polonium containing red blood cell components were not satisfactory.

Human Sarcoma #1 (H. S. #1), Human Epidermoid Carcinoma #3 (H. Ep. #3) and Human Embryonal Rhabdomyosarcoma #1 (H. Emb. Rh. #1) have been grown by serial passage on the chorioallantoic membrane of the chick embryo, and the H.S. #1 and H. Ep. #3 by serial passage in the yolk sac. Metastases of the H. Ep. #3 to the liver and kidneys of the host chick have been observed. The H. S. #1 and H. Ep. #3, when treated with a nitrogen mustard, HN2, showed the same pattern of histological injury previously described for the mouse sarcoma 180 growing in chick embryos and mice.

The supernatant fraction of chick liver is the principal source of the PGA to CF converting enzyme system. The latter is precipitated by 40% alcohol. Homocysteine consistently enhances PGA to CF conversion by the chick liver fractions. The enzymatic synthesis of CF from PGA by acetone powders of chick liver is optimal in presence of homocysteine, serine, and Mg⁺⁺. Under these conditions the glycine content of the acetone powder is increased 4-fold.

1. The “acid-active” DNAase of rat spleen has been found to increase markedly after 500 r of total body x-irradiation. 2. Such a change is not apparent in the “neutral” enzyme of liver or spleen. 3. The maximum in activity of th elevated DNAase occurs 24-48 hours post-irradiation. 4. Maximal response is elicited at a dose of 200 r.

The effects of 5 members of the morphinan series have been studied on the electroencephalogram, electrocardiogram and blood pressure of the dog. Levorphan produced maximum central effects with mild cardiovascular depression. Dextrorphan produced severe cardiovascular depression with little or no direct central effect. Levomethorphan and dextromethorphan had mild central and peripheral actions. Levallorphan, which had little action by itself, antagonized both the central and peripheral effects of levorphan.

1. Male mice of the C₃H, C, A and D₈ strains are susceptible to the diabetogenic action of alloxan. A difference in susceptibility depended upon the strain, the D₈ was the most susceptible and the C₃H, the most resistant, to a given amount of alloxan injected. 2. Glucose tolerance tests in diabetic as well as in non-diabetic mice are presented. The blood sugar changes after intravenous injection of alloxan showed the triphasic type curve described in most experimental animals.

A simple and rapid method is described for estimating pyruvate-activated glutaminase II activity of liver tissue. Glutaminase II activity was found in livers of rats, mice and rabbits but was absent in livers of monkeys and was low in livers of guinea pigs. Using human liver tissue taken at autopsy, glutaminase II activity could not be detected although arginase activity was found in these livers.

The C-reactive protein response in human blood following intravenous injections of typhoid vaccine is described. No significant alteration in the promptness, intensity or duration of this response was noted during the administration of cortisone, ACTH or salicylates. 2. In rabbits the response of the analogous Cx-reactive protein to experimental pneumococcal inf ection is similarly not modified by the adrenal cortical hormones. 3. The experimental results support the clinical impression that the disappearance of C-reactive protein from human blood during antirheumatic therapy with adrenalcortical hormones or with salicylates is secondary to suppression of the inflammatory process rather than a primary effect of these agents upon the metabolism of the C-reactive protein.

1. Two isoniazid derivatives, N-isonicotinoyl - N'- (salicylidene) hydrazine (INSH) and N-isonicotinoyl-N'-(2, 2-dimeth-yl-3-hydroxypropylidene) hydrazine (IHPH), were found to have an in vitro tuberculostatic effect similar to that of INH but both exhibited cross resistance with the parent compound. H37Rv tubercle bacilli were easily made resistant to INSH by serial passage in drug-containing liquid medium, after which the strain was found to have lost its former virulence for guinea pigs. Resistance was much slower to appear, however, in medium containing a combination of INH plus INSH. 2. P-amino-salicylic acid hydrazide (PASH) showed about the same in vitro effect as, and cross resistance with, PAS, but there was no cross resistance with INH. Tubercle bacilli could easily be made resistant to PASH by serial passage in drug-containing medium. This compound demonstrated very little beneficial effect in mice infected intravenously with the H37Rv strain. 3. INSH produced therapeutic results similar to those seen with INH in experimental tuberculosis of rabbits infected intravenously with a virulent bovine strain. 4. Guinea pigs were infected subcutaneously with 4 INH-resistant strains of tubercle bacilli; 3 were human strains isolated from patients treated with the drug, and the 4th, a bovine strain isolated from a rabbit treated with INH. Treatment with INH, INSH, or IHPH produced good results in the animals infected with the 2 strains showing least in vitro resistance, but very little beneficial effect in those infected with the 2 strains containing at least 1.0% of individual organisms resistant to 5 μg/ml. There was no significant difference in the results obtained in any of the 4 groups with INH or either of its 2 derivatives.

Details of a technic for repeated partial hepatectomy in the rat are presented. Up to 80% of the liver can be excised and aspirated at the first stage. Any one of the 3 principal lobes (right or left lateral or the median) will supported life after removal of all other remaining hepatic tissue. The caudate lobe will not support life if all other parts of liver are removed at the first stage. Following the restoration of liver additional partial removal of liver can be done by excision at the second and third stages. At all later stages the method of removing liver is that of aspiration. Careful attention to prevention of hypoglycemia, hemorrhage and infection insures uniform success of the procedure.

1. Antisera specific for either NDV or influenza virus-treated chicken erythrocytes were prepared. 2. Virus modified red cells were detected in chickiens infected with NDV by various routes of inoculation by use of the specific antiserum. 3. Hemagglutinins for NDV-treated chicken red cells were also detected in the serum of infected chickens.

A method has been developed by which individual variations in urinary excertion of purines may be investigated on a qualitative andsemi-quantitative basis. The method employs an ion exchange step to remove uric acid, and separates by paper chromatography the purines precipitated by silver nitrate. Two normal subjects on purine-free diet excreted a total of more than 30 mg/day of non-uric acid purine derivatives. Those identified, in order of diminishing abundance, are hypoxanthine, xanthine, 7-methylguanine, adenine, 1-methylguanine and guanine.

Administration of SKF Compound 525A to mice (50 mg/kg, 75 mg/kg) prolongs significantly Pentothal-induced sleeping time, the duration of which is dependent on the dose of SKF-525A. The excretion of tracer amounts of Pentothal-S³⁵ has been shown to be delayed by pretreatment with SKF-525A. Over a 7 hour period control animals excreted the maximum percentage of the injected dose (13.455%) within one hour, while treated animals retained S³⁵ until the fourth hour after injection (11.228%). At the end of the 7 hour observation period, 31.1 and 30.08% of the injected doses were recovered from treated and control series, respectively. These observations are being extended to other barbiturates and the possible mechanism of action of SKF-525A.