Experimental Biology and Medicine

1. Ulcers in the forestomach were induced in rats: (a) Recovering from vitamin A-deficiency with 2, 30, or 300 units vit. A daily, (b) Recovering from a vit. B6 deficiency with suboptimal amounts (1, 2, or 4 γ daily) of pyridoxine. (c) Recovering from an essential fatty acid deficiency with 20 mg of linoleate daily.

2. Deficiencies of these 3 essentials did not, in themselves, result in lesions. The ulcers occurred only during the curative period in which the specific nutrient was fed.

3. α-Tocopherol fed daily to the rats during the cure of the specific deficiency gave complete protection. γ-Tocopherol orally was also effective, but a-tocopheryl phosphate injected was not.

4. The amount of fat in the diet was related to the incidence of stomach ulcers in the vitamin A experiments. Ten per cent gave good protection. Five per cent allowed moderately severe lesions, which were preventable by tocopherol. Fat-low diets gave very severe lesions not preventable by the level of tocopherol fed, 0.5 mg daily.

Morphological and experimental studies suggest a vascular disorder to be the primary injury observed in the kidney of the acutely choline-deficient

In the preparation of antiorgan sera the administration of small amounts of antigen (spleen and bone marrow) was found more effective than the rapid immunization of rabbits with massive antigen dosage. A single injection of the antigen 30-40 days after the last dose gives rise to an anamnestic response of the host and to a serum of higher potency. This can be evaluated by complement fixation titres and by the outgrowth of tissue explants exposed to the action of the “anamnestic” serum.

1. Benadryl offers effective protection against histamine shock in rabbits. 2. Benadryl did not protect against anaphylactic shock in rabbits sensitized to egg white. 3. Benadryl did not protect guinea pigs actively sensitized to egg white against anaphylaxis induced by intraperitoneal administration of the antigen under the dosage conditions employed.

Choline has been shown in our previous studies to have a decholesterolizing effect in old hens. 1 Methionine has been found to take part in a reversible reaction with choline in the animal body 2 and to substitute for choline 3 and be effective in lipid metabolism disturbances in the liver of mammals. 4 , 5 It therefore seemed logical to study methionine, just as we have choline and inositol, as a possible decholesterolizing agent in old hens with hypercholesterosis.

Forty-four 2-year-old white leghorn hens were individually caged and fed the standard Texas A & M laying mash. The initial blood lipid levels were determined by the methods of Bloor 6 for total cholesterol, Bloor and Knudson 7 for cholesterol esters, and King 8 for total and inorganic phosphorus.

Methionine, 0.5 g, was placed daily in the gullet of each of 24 of the old hens. One hen died of liver disease. After feeding the methionine for 18 to 29 days, the hens were bled a second time and the blood lipids were again determined, but, as shown in Table I, very little decholesterolization had occurred. The administration of methionine was continued, third bleedings were made after 36 to 51 days, and the bloods chemically analyzed as before.

When random samples of blood showed that the cholesterol levels were singnificantly lowered, the hens were sacrificed and blood collected fro lipid analysis. The aorta, about a quarter gram of heart muscle and a similar mass of liver were individually and accurately weighed, then maceratd with sand and 1 to 3 ether alcohol solution. The extract of each organ was analyzed and the amounts of total cholesterol and cholesterol esters in 100 g of each tissue calculted. Twenty old hens fed as simultaneous controls were also sacrificed and the blood and tissues likewise treatd. The results are set down as controls in Table I.

It is evident that the total blood cholesterol and cholesterol ester levels definitely decreased and the phospholipids rose after the old hens had been given 0.5 g methionine daily for about 3 weeks. After 5 to 7 weeks treatment, significantly lower levels of the blood cholesterol and cholesterol esters were established, and the aorta, heart, and liver showed signifificantly lower lipid levels than those of the control series. Methionine there-fore, seems to act as a decholesterolizing agent in old hens.

The present study is based upon the use of a fibrinogenolytic (rather than fibrinolytic) technic for demonstration of a natural plasma protease (tryptase) and its precursor (tryptogen) in the fibrinogen-rich plasma fractions. The value of streptokinase (miscalled “streptococcal fibrinolysin”) for activation of the enzyme precursor is confirmed and tests are presented to show that failure to secure proteolytic effects e.g. in bovine plasma Fraction I, is due to lack of tryptogen (tryptase precursor) rather than to any significant presence of tryptase-in-hibitor.

Heparin, unaided, shows no enzyme-inhibitory effects in the cited tests. Antitryptase effects of (a) crystalline inhibitors, from pancreas and soybean, and (b) crude and fractionated egg-albumen, favor the characterization of the plasma protease as a “tryptase.”

Confirmation of the existence of tryptogen, together with a trace of active tryptase, in H.F. (“antihemophilic globulin”) is further, but still inconclusive, evidence that the source of the coagulation defect in hemophilia is to be sought for in the complexities of the plasma protease system.

Study of the complement-fixation antigens obtained from chick embryos infected with mumps virus revealed 2 anti-genically distinct components separable by high speed centrifugation. One antigen is closely linked with the virus and found predominantly in the allantoic and amniotic fluids; the other component is smaller and present mainly in the allantoic and amniotic membranes. The 2 antigens can be differentiated by serum absorption technic. Some human sera contain antibodies to the 2 antigens (early convalescent sera) whereas others may show antibodies only to the virus-bound antigen but not to the soluble material (long past infection).

The exact mode in which ions are distributed in protoplasm is at present unknown. Using radioactive isotopes, several authors have attempted, by placing photographic film in close contact with tissues and thus obtaining a radio-autograph, to record where added ions are localized in the cell. Unfortunately this procedure has not yielded any great amount of information on cellular localization, although excellent differentiation of different tissue masses has been obtained. 1 The difficulty in obtaining clear radio-autographs of cells is that the cell diameters are of the same order of magnitude as the distance that β-rays, not normal to the film. travel before striking the film. The net result is a general fog which obscures the desired detail. To obtain pictures with more detail it is necessary therefore either to col-limate or to focus the electrons coming from the tissue to the film, or else to use cells of much larger dimensions. In this study the second alternative has been employed in that cells of Nitella or Chara sp., which have diameters of the order of 1-2 mm and lengths of about 40 mm, were used.

Experimental. Cells of Chara or Nitella were prepared for use as previously described. 2 The cells were removed from pond water, blotted to remove excess water and placed on end in a tube containing 0.001 M radioactive sodium phosphate solution at 15°C and pH 7. In some experiments only enough phosphate solution was used to cover 1-2 mm of the cell as measured along its axis. The tube was then filled with washed paraffin oil. By this arrangement it was possible to expose only a minimal part of the surface of the cell to phosphate solution and hence obtain some idea of the rapidity of diffusion and possible circulation of the phosphate due to cyclosis. To be sure there must be a thin film of water extending up from the P04 solution along the cell surface but this method probably lets ion diffusion proceed as asymmetrically as possible. After various times of immersion the cells were removed from the tubes, washed thoroughly in running distilled water, placed on a glass slide and rapidly frozen and dried in vacuo. A piece of X-ray film was then placed over the dried cell and exposed for a suitable time.

The studies referred to, and our own observations in experimental brucellosis are in our opinion, good evidence of the ability of Brucella to use the cytoplasm of a variety of cells as a source of material for its growth. The fact that even in the early stages of the infection, when the hosl has not developed immunity against brucellae, important groups of this organism are found only within the cytoplasm of certain cells, seems to indicate that the intracellular growth is the most convenient for the preservation of the infecting agent. When the intracellular colony has reached its maximum, the cells are destroyed and the brucellae exposed to unfavorable conditions, namely to phagocytosis by polymorphonuclear leucocytes. From the scarcity of extracellular organisms one may think that the intracellular fluids are not suitable for an active multiplication of Brucella, exception is made of zones where the tissues have been subject to considerable damage. In these zones one may find extracellular Brucella in large numbers. One would consider that the organism could be placed together with true intracellular parasites, but this is not the case since Brucella have its own enzyme systems with which it is able to use rather ordinary material for its metabolism.

Considering as likely that in man Brucella grow in a manner similar to that which has been observed in the experimental infection, the pathogenesis of the disease seems to us better understood.

The natural protease (tryptase) in several plasma protein fractions resembles a weak pancreatic trypsin in its fibrino-genolytic, fibrinolytic, and “thromboplastic” effects. These actions are inhibited by crystalline trypsin-inhibitors from pancreas and soybean. The clear demonstration of thromboplastic action in these quantitatively-controlled tests is direct proof of participation of plasma tryptase in the blood-clotting system.

When ANTU was fed chronically to rats for 2 years in concentrations of 50 to 800 ppm in the diet, the concentrations from 100 to 800 ppm were toxic. Externally, the rats were stunted and showed spectacle eyes, thinning and coarsening of the hair and deformities of the legs and feet. Histopathological changes consisted of hyperplasia of the thyroid, hyperplasia of the splenic pulp, hyaline changes in the cytoplasm of the centrolobular hepatic cells, a slight to moderate decrease in the thickness of the adrenal cortex, calcified tubular casts in the renal medulla, slight myeloid hyperplasia of the bone marrow, and slight modifications in bone structure. The degree of injury in each case was related to the concentration of the AXTU. The concentrations of 600 and 800 ppm ANTU produced a marked tolerance.

The tritocerebral commissure of the shrimp, Crago, has been chemically fractionated to separate 2 mutually antagonistic chromatophorotropins influencing body-coloration, a general darkening one (CDH) and a body-lightening one (CBLH).

The tissue section containing radioactive material is mounted directly on the photographic emulsion in the dark room. After suitable exposure, the plate is developed, and the tissue stained etc. The preparation may be studied microscopically as the autographic image is in place just below the tissue.

High oral intakes of folic acid are stored inefficiently in chick livers and have little if any effect on the content of this vitamin in chick muscle tissue. Chicks given large oral doses of folic acid and then placed on a folic acid-deficient diet grew as well as chicks receiving an equivalent amount of folic acid by injection. The distribution of folic acid in some representative chick tissues has been studied.

Thirty-two successful testicular homo transplants were recovered from 40 castrated male and female mice of the A strain bearing the intrasplenic transplants for a period ranging from 23 to 275 days. The testicular transplants showed the general histological features of cryptorchid testes, although spermatids and sperm heads were noted in one case after 243 days of transplantation. The present material gave no indication of tumor development in the intrasplenic testicular transplants. The experimental data suggested that the endogenous androgen produced by the intrasplenic testicular transplant was inactivated when passing through the hepatic portal system.

Rutin, a flavonol glycoside with vitamin P activity, was studied for signs of acute and chronic toxicity. In rats and guinea pigs, intravenous and intraperitoneal injections of 30 to 50 mg/kg, and intravenous injections in rabbits of 100 to 200 mg/kg, were inocuous.

The rate of growth of albino rats was not affected when the diet contained as much as 1%percnt; of rutin, and after 400 days on such a diet, histological examination of the tissues showed no evidence of injury which could be definitely related to rutin administration. Organ weights of the experimental animals were normal. The length of the estrous cycle was the same in rats eating a 1%percnt; rutin diet as in control animals, reproduction was as good, and the young appeared to be as healthy. On the basis of the criteria employed, rutin is nontoxic both acutely and chronically.

The toxic doses (LD₁₀₀) of actinorubin and lavendulin have been determined for white mice by intraperitoneal injection. In the case of lavendulin it was 0.5 mg, while in the case of actinorubin it was 1.37 mg. The ability of the antibiotics to protect mice from death following the intraperitoneal injection of 100 to 1000 M.L.D. of K. pneumoniae was determined. In the case of lavendulin this therapeutic dose was 25 μg and in the case of actinorubin it was found to be 13.7 μg in one experiment. In a second experiment 0.68 mg of actinorubin killed 50%percnt; of the mice while 30 μg protected 50%percnt; of the mice against 100 lethal doses of K. pneumoniae. Further testing of the 2 antibiotics has not been possible due to the limited supplies of the substances.

Exposure of the peritoneal cavity of the rat to irradiation in excessive amount will produce an increase in capillary fragility.

Under the conditions of the experiment, rutin appeared to hasten the recovery time after irradiation.

Chick embryo heart fibroblasts cultured in vitro, when immersed in colchicine solutions 10^-4 or 10^-5 M, show a tendency to assume a spherical shape. Their surfaces display a considerable activity as manifested by rapid protrusion and withdrawal of pseudopodia. Subsequently the zone of out-growth of the culture splits up into detached elements, comprising one or more cells. This whole process is not lethal to the cells and is, to a great extent, reversible.

Polycythemia can be rapidly induced in rabbits by exposure to altitudes of 20,000 feet 16 hours a day, 5 days a week. It would appear that there is no additive effect when cobalt injections are combined with the altitude exposure.

There may be 2 stages in the development of polycythemia. The first phase is characterized by a normal whole blood volume with a subnormal plasma volume. The second phase shows a normal rate of increase in plasma volume with a further increase in whole blood volume.

Subcutaneous cobalt injections slowly induce polycythemia in rabbits but they are accompanied by anorexia, sterile abscesses and other symptoms of toxic effects.

Streptomycin in amounts of from 0.5 to 2.0 mg injected into infected embryonated eggs has a definite slight inhibitory action on the growth of R. prowazeki. In the case of R. rnooserl, 2.0 mg of streptomycin had a. small but significant effect. With R. orientalis, a dose of 2.0 mg per egg appeared to have no significant effect on the multiplication of the rickettsiae. By contrast, appropriate doses of PABA (5.5 mg per egg for R. prowazeki, and 11.0 mg per egg for R. mooseri and R. orientalis) produced a striking inhibition of growth, reducing the numbers of viable rickettsiae by 10,000 to over 10,000,000 times as compared with untreated controls.

Curare, which affects the electrical activity of the brain has also a distinct effect on the temperature in the brain in combination with pentobarbital narcosis. These temperature changes in the brain indicating changes of oxidation processes are increased by curare under light pentobarbital narcosis, and decreased under deep pentobarbital narcosis. This latter central action may be involved in the synergistic narcotic effect.

Chicks fed a completely synthetic (chemically-defined) diet in which amino acids are the sole source of nitrogen show a definite subnormal growth response.

Casein is superior to its component amino acids as a source of nitrogen for the chick.

Diets containing some of the nonessential amino acids are superior to diets containing the 11 amino acids now recognized as essential for the chick.

Under the experimental conditions chosen, methionine at a level of 50 mg per day (0.8%percnt; of the diet) did not prevent the development of fatty livers in rats fed a diet in which a mixture of essential amino acids replaced protein.

1. A method is described for the preparation of riboflavin pellets that can be implanted in animals and in humans.

2. The effect of these riboflavin pellets (containing cholesterol) in cases of retarded riboflavin resorption lasts from 1-1 ½ months.

3. The riboflavin pellets implanted in a patient suffering from riboflavin deficiency brought about complete relief of relatively long duration from deficiency symptoms.

4. The method may be of practical value for patients suffering from riboflavin deficiency due to inadequate resorption, and when repeated injections are difficult to carry out.

Sodium salicylate has been found to inhibit the spreading effect of hy-1 aluronidase in vivo. Hyaluronidase derived from CI. perfringens and bull testis was also inhibited by sodium salicylate in vitro. The concentrations necessary for in vitro inhibition are considerably higher than those obtained in vivo.

1. An infectious agent was recovered from the central nervous system of a rabbit which had been inoculated 9 months previously with Herpes simplex virus and which had suffered 3 acute relapses of the disease. The virus recovered exhibited characteristics identical with those of the original virus. 2. A second instance of virus recovery 4 months after inoculation is reported.

Electron micrographs of gold shadow-cast specimens of tobacco mosaic virus show that artifacts, consisting of segmented or even globular particles, are formed when the specimen is exposed to the beam of electrons at high intensity. It is suggested that these are caused by the thin film of gold becoming fluid and forming globules on the surface of the virus rods and supporting membrane. Tobacco mosaic virus not treated with gold did not show any change after having been subjected to the same treatment. The results indicate that gold covered specimens should not be subjected to prolonged exposure to an electron beam of high intensity.

In the rat a single intravenous or intraperitoneal injection of egg white elicits a reaction characterized by hyperemia, followed by edema of the face, tongue and paws. Although only these regions respond, they are not necessarily affected to an equal extent. Continued injections of the same dose produce variable reactions in the same animals but there is no acquired tolerance. Age does not play a great influence, but females seem less sensitive than males.

Several investigators have recently recently reported changes in plasma, or whole blood cholinesterase content in anemias and leukemia. Values reported were expressed in terms of cholinesterase activity per volume of sample. Sabine 1 has calculated the cholinesterase content per cc of red cells but was unable to secure accurate values by direct measurement.

The red cells contain only specific cholinesterase and none of the nonspecific type found in plasma or serum. It is unlikely, therefore, that the cholinesterase in the red cells is derived from the circulating plasma, but, more probably, their esterase content is produced by the tissues from which they are derived or with which they are closely associated. Any change of esterase content per cell may be important, therefore, in reflecting the activity of the reticuloendothelial elements of bone-marrow. The production of the esterase by the red cell itself cannot at this time be excluded.

Preliminary to some observations of the red cell content in various pathological states, we have determined the cholinesterase content per million cells in 18 normal human subjects. We have been able to secure accurate values by using volumes of washed, resuspended red cells calculated to contain the same number of cells as determined by cell count. The cells were lysed with 10%percnt; NaCl at 2°C. The measurements were then conducted at 37.5° by a modification of the method of Ammon 2 in which the bicarbonate-Ringers solution and the acetylcholine chloride solution were both placed in the large space and the blood sample in the side cup of the Warburg vessels. Values of cholinesterase activity thus secured are expressed as cmm of CO2 produced per hour per million red cells.

(1) A simple kennel and dietary routine for the maintenance of the health and vigor of dogs with pancreatic fistulas is outlined. (2) Pancreatic fistula dogs supported by this regimen tolerated major abdominal surgery (gastrectomy).

The erythrocytes of 2 patients with acquired hemolytic anemia and spherocytosis have been shown to be readily agglutinated by an immune rabbit serum containing an anti-human globulin antibody. Since both patients have exhibited abnormal hemolytic activity for transfused cells this finding is compatible with the existence of an immune body type of hemolytic agent as the cause of the accelerated hemolysis.

Evidence has been presented to show that the immune substance can be separated from patients' cells by heating the cells to 56° C for 5 minutes and can then be combined with normal cells. The similarity of this immune body to the Rh blocking antibody is discussed. The demonstration of the presence or absence of an immune body attached to the erythrocytes should become a routine procedure in the study of hemolytic anemias.

Conditioned reactions which had been inhibited by lack of reinforcement in rats may be restored by insulin coma in normal as well as in adrenodemedulMted rats. Apparently a secretion of adrenalin which is observed under conditions of insulin coma and electroshock does not play an integral part in the mechanism of the restoration of inhibited conditioned reactions. This interpretation is supported by the fact that the injection of adrenalin remains ineffective in the normal animals as far as restoration of inhibited conditioned reactions is concerned, whereas insulin coma and electroshock restore these reactions. It is inferred from these experiments that the restitution of inhibited conditioned reactions is linked up with a hypothalamic-cortical discharge previously established by Murphy and Gellhorn and is not due to the liberation of adrenalin which invariably accompanies the excitation of hypothalamic centers under conditions of insulin coma and electroshock.

A method for the determination of biological values of proteins with Tetrahymena geleii H has been described. The values found for casein, lactalbumin, a commercial protein hydrolyzate and gelatin have been compared with each other and with those reported in the literature.

Four nonhypertensive patients with idiopathic paroxysmal tachycardia of supraventricular origin were given neo-synephrine intravenously in amounts sufficient to increase the systolic blood pressure 30 to 40 mm of Hg. In 2 of these the attacks had persisted for several hours in spite of all mechanical methods of treatment and administration of mecholyl. In all 4 cases the rhythm reverted to normal within one minute after the injection of neosynephrine. This is considered to be produced by reflexes from the carotid sinuses and aortic arch. Occasional ventricular beats occurred during the 2-minute period after the return to the normal sinus rhythm. Blood pressure returned to normal within 4 to 8 minutes after the injection.

Intravenous injection of neosynephrine or some other brief-acting vasopressor compound may prove to be the treatment of choice in selected cases of auricular or nodal paroxysmal tachycardia.

Incubation of lymphocytes in suspension at 37° C produced primary and secondary vacuoles visible on dark-field examination. Preliminary irradiation of the suspensions greatly accelerated this process. The addition of distilled water to lymphocytes produced enlargement and vacuolization of the nuclei. The hypothesis is proposed that irradiation caused the absorption of water into one or more foci in the nucleus.