Experimental Biology and Medicine

1. The permeability of the synovial membrane as measured by speed of absorption and excretion into the urine of phenolsulphonphthalein (PSP) instilled into the joint was found to be surprisingly constant in a group of 16 normal rabbits.

2. Hyaluronidase markedly increased permeability of the synovial membrane. The effect was maximal and was not augmented by desoxycorticosterone acetate (DOCA).

3. Adrenal cortical extract decreased permeability of the synovial membrane and an tagonized the effect of hyaluronidase. This effect of adrenal steroids was more pronounced when they were released endogenously by the alarm reaction. Estrone also decreased the permeability of synovial membrane.

4. Desoxycorticosterone increased maximally the permeability of synovial membrane to the same extent as hyaluronidase and could not be augmented by hyaluronidase.

5. It is suggested that the normal permeability of the synovial membrane is in part controlled by the balance between adrenal steroids of the DOCA type and of the Compound E type.

6. These findings are consistent with current views of the etiology of rheumatoid arthritis either by hyaluronidases or from exposure to stressing stimuli. They are also consistent with the therapeutic effects of Compound E or of adrenocorticotrophic hormone in the treatment of arthritis.

Since the development of adrenal cortical adenomas in gonadectomized mice has been inhibited by the implantation of pellets of estrogenic hormone, 1 it might be inferred that withdrawal of gonadal secretion is of primary importance in precipitating the development of the castration-induced adenoma of the mouse adrenal cortex. The objective of the experiments being reported was to determine the effect of the administration of regulated, “physiologic” amounts of estrogenic or androgenic hormone on the induction of adrenal cortical tumors in susceptible ovariectomized mice.

Mice of the NH strain were used in these experiments. NH females develop adrenal cortical tumors spontaneously following ovarian regression, and precociously if gonadectomized. 2 Adrenal cortical adenomas are present in most NH mice of both sexes 5 months after removal of the gonads.

Four NH males and one female whihc had been gonadectomized at 23 days of age received (by subcutaneous injection beginning 2 weeks after operation) 0.05 microgram of estradiol dipropionate‡ in sesame oil at intervals of from 5 to 9 days. This treatment with estrogenic hormone provided for constant estrus, as determined by vaginal smear. These 5 animals were autopsied 6 months after the institution of hormone treatment. Administration of hormone was suspended 53 days before autopsy in 2 NH females which received similar treatment.

Two ovariectomized NH females received the same treatment with the estrogen and in addition were injected weekly with 150 μg of progesterone in oil. Two additional ovariectomized females received the estrogen plus progesterone, but treatment was suspended 47 days before autopsy.

Two NH females ovariectomized at 21 days of age were given 17.5 μg of testosterone propionate in oil weekly by subcutaneous injection beginning 2 weeks after operation. This treatment was continued until the animals were autopsied 5 1/2 months after ovariectomy. Three mice were similarly treated except that they received no injections of hormone for the 46 days preceding autopsy.

In a search for antibiotics active against Mycobacterium tuberculosis var. hominis several hundred antagonists have been encountered and one called Streptomyces sp. EI₅ in our records was chosen for intensive study because of its marked activity against the mycobacteria and the low animal toxicity of its crude filtrate. 1 The present report is the account of a study of this organism and the antibiotic it produces.

Methods and Results. A. Morphological and Cultural Characteristics of Streptomyces sp. EI_5. Since preliminary tests of solubility and antibacterial activity indicated that the antibiotic produced by S.spEI₅ is similar to streptomycin and streptothricin, a comparison of the organisms that produce these antibiotics was made.

Streptomyces sp. EI₅ was morphologically similar to Streptomyces griseus, and both organisms produced a soluble yellow to yellow-brown pigment when grown at room temperature on nutrient agar, glucose agar, starch agar, and gelatin. Only S. griseus produced pigment on potato agar. The color of the aerial mycelium varied from white to tea-green with S.griseus and from white to gray with S. sp. EI₅. The vegetative mycelium of S. griseus was white to cream colored and that of S.sp. EI₅ yellow to tannish orange. Streptomyces sp. EI₅ was not susceptible to the S. griseus actinophage of Reilly, Harris and Waksman. 2 These observations indicate that S. griseus and S. sp. EI₅, are similar but significantly different organisms.

Streptomyces lavendulae produced a darker brown pigment on glucose agar, nutrient agar and glucose broth than S. sp. EI₅, and in addition produced pigment on the potato plug. Further, slide cultures on Czapek's medium at 30°C showed the aerial mycelium of S. lavendulae to be pink to lavender, and the hyphae often coiled terminally, in contrast to the white to gray color and the straight hyphae of S. sp. EI₅. The conidia of S. sp. EI₅ were observed to be oval to globose and considerably smaller than those of S. lavendulae. These differences indicate that S. lavendulae and S. sp. EI₅ are not identical.

Application of the technic of Oudin of ragweed pollen extracts and their antisera demonstrated the presence of at least 5 different antigens in such extracts.

The dialysate of benzene-ether extracted brain tissue is capable of causing signs and lesions of allergic encephalitis in guinea pigs. The material recovered in the dialysate is free of protein and presumably contains only substances of low molecular weight.

The administration of Rose Bengal and ultraviolet irradiation in rabbits results in rapid and marked increase in the excretion of urinary coproporphyrin Type III to values 17 to 40 times those in the controls, returning to normal by the 5th day. Neither the dye alone nor the irradiation alone had any effect.

Simple hemolysis by distilled water had no effect upon the urinary coproporphyrin excretion. Phenylhydrazine produced a slight delayed increase of coproporphyrin excretion believed to be due largely to an increase of coproporphyrin Type I, as a result of increased erythropoiesis.

Additional studies are being undertaken in an attempt to discover the origin of the coproporphyrin excreted and the effects of other photodynamic compounds.

These results constitute the first demonstration that photodynamic activity causes a marked increase in the excretion of coproporphyrin III.

Studies were made of serum polysaccharide levels in dogs following the production of sterile turpentine abscesses, of bacterial abscesses, and of talc granulomas, the injection of turpentine intrapleurally, and experimental surgical operations. In all cases the polysaccharide level rose and the maximal level depended somewhat upon the type of inflammation. Elevation occurred both in the presence and absence of fever. The maximal elevation appeared within 3 to 6 days after the initial injury. The suggestion is made that this phenomenon is correlated in some way with tissue proliferation or repair.

Bacterial glucuronidase which hydrolyzes the glucuronides of estriol, pregnanediol, menthol and phenolphthalein liberates a large proportion of estrogen, ketosteroid and corticosteroid of human urine.

Prostigmin was given to young rats in daily amounts of 0.04 to 0.1 mg. The ovaries were slightly increased in weight. The uteri if mature as the result of age or estrogens showed increase in weight following injections of prostigmin and these changes were present after desiccation. Estrous cycles showed no significant change from normal. Cellular elements of inflammatory cells were more abundant in the submucosa after prostigmin. On the whole, pathological changes were not marked; therefore, from the pathological viewpoint effects of prostigmin are minor as far as changes in the ovaries and uterus are concerned.

In hypertensive patients Veriloid is an active hypotensive agent when administered acutely, or chronically for periods as long as 5 months. Meticulous regulation of dosage is important in obtaining optimum results. After several weeks of continuous therapy it is frequently necessary to reduce the dosage in order to avoid nausea and vomiting. Nevertheless, relative to its hypotensive effects Veriloid seems to produce fewer toxic reactions than any other oral preparation of Veratrum viride presently available.

1. Rats and mice were intubated with single doses of a new miticidal compound, di-(p-chlorophenyl) methylcarbinol (DMC). The effect of graded doses on ultimate survival is discussed and the ability of large doses to produce signs suggestive of nervous impairment is described.

2. A powdered diet containing 0.1% DMC was well tolerated by growing rats during a ten week period.

3. The results compare favorably with those obtained in a similar limited study of DDT in another laboratory.

Daily inunction for 24 days of 270 rat units of alpha-estradiol (Progynon D-H) to normal and castrated immature male rats produced noticeable changes in the thyroid glands and in the testes of the noncastrated males. The thyroid glands appeared, histologically, in a hypoactive state. They exhibited an increased colloid content and a low follicular epithelium and destruction to the follicles. The hypoactivity appeared greater in castrated rats than in the non-castrated. Descent of the testes in immature males was prevented by the alphaestradiol.

India ink, suspensions of blood charcoal, carmine, and trypan blue were injected subcutaneously and intraperitoneally. Carmine was injected also intramuscularly. After subcutaneous and intramuscular injection a rise in body-temperature was observed. After intraperitoneal injection a fall in body-temperature was observed. As judged by relative engorgement of capillary beds, and changes in blood cell volume (hematocrit) and plasma protein concentration, these animals were not in shock during hypothermia, Intraperitoneal injections of ox bile, and solutions of peptone produced a less intense hypothermia although ox bile caused severe shock.

A procedure has been described 1 for analyzing blood coagulation in three experimental steps: 1. activation of prothrombokinase; 2. activation of prothrombin; 3. coagulation of fibrinogen. Crude thrombokinase, besides activating prothrombin, also accelerated conversion of prothrombokinase. Both activities where lost upon adsorption with barium sulfate.

It is now reported that a material, prepared by elution from barium sulfate, promotes the activation of prothrombin, and also the conversion of crude prothrombokinase. Beginning with a solution of bovine plasma globulins, fraction “A” was adsorbed on Hyflo (Johns-Manville) and eluted with phosphate-potassium chloride solution. The unadsorbed globulins were exposed to adsorption by barium sulfate. From the latter, two successive fractions were eluted, with O.2-0.3M phosphate, pH 6.6, and with 0.6M phosphate, pH 7.9. The pH 6.6 eluate had more prothrombin. From the pH 7.9 eluate, proteins soluble in 0.35, but precipitable by 0.45 saturated ammonium sulfate were selected as the “converter fraction.” This involved 3 extractions and 20 precipitations. Proteins soluble in 0.45, but precipitable by 0.60 saturated ammonium sulfate, constituted the “thrombin fraction” (3 extractions, 7 precipitations). Fifty-five liters of plasma yielded 10 ml of each fraction. Both were dialyzed simultaneously in the same beaker.

Activations were studied with calcium present, giving results summarized in Table I. At high dilution, the converter hastened both the activation of prothrombin and the conversion of crude prothrombokinase. In comparing different preparations, converter activities estimated by the 2-stage and 3-stage procedures were roughly parallel. This is compatible with the view that the tests measured two different effects of the same substance.

In contrast, converter activity did not parallel thrombin activity. The ratio of converter to thrombin activity was 100 times as great in the converter fraction as in the thrombin fraction. Conceivably, varieties of thrombin can exist with different converter activities, or some factor modifies the behavior of thrombin in one of the fractions. If, as must be contemplated, the converter is distinct from thrombin, the results suggest how difficult would be its elimination from thrombin by salting-out. Moreover, the converter is manifest in sufficiently minute amount to cause confusion, even as a small contamination in a highly purified thrombin.

1. The hemaggutination test for rheumatoid arthritis is modified by the selective absorption of non-specific factors. The details of this modification are described.

2. Of 39 cases with active peripheral rheumatoid arthritis with x-ray evidence of joint changes, 35 were positive by this test.

3. All cases of inactive peripheral rheumatoid arthritis, rheumatoid spondylitis without peripheral joint involvement and psoriatic arthritis tested were negative.

4. In presumptive cases of early rheumatoid arthritis without x-ray evidence of joint changes, the test was negative.

5. In 67 control cases, including a variety of non-rheumatoid arthropathies, the test was negative except in two cases of infectious hepatitis.

6. The modified test was positive in 90% of the proven cases of active peripheral rheumatoid arthritis tested; the Rose test in 61%; the streptococcus agglutination test in 58%. In all cases in which the modified test was negative, the Rose test and the streptococcus agglutination test were also negative.

The authors express their thanks to Dr. R. Schuman, Miss E. C. Arrowsmith, and Mr. O. Vasquez of the Clinical Laboratory Service for technical assistance.

The methanol precipitative technic of Cox, et al., when applied to the purification and concentration of papilloma virus from naturally occurring cottontail rabbit papillomas, domestic rabbit papillomas and neutral virus-antibody mixtures readily yielded active papilloma virus from the cottontail papillomas but it did not yield virus from either domestic rabbit papillomas or from papilloma virus and its antibody in neutral mixture.

The addition of 0.5% of dlglyceric aldehyde to a caries-producing ration or of 0.2% dl-glyceric acid to the drinking water did not reduce the initiation nor the rate of development of carious lesions in caries-susceptible white rats and cotton rats.

Nine of 14 strains of PPLO of human origin and one strain of rat origin were observed to grow on beef heart infusion agar without ascitic fluid or serum only in symbiosis with Staphylococcus albus and all 15 strains grew in symbiosis with Proteus vulgaris.

A technically simple, rapid method for the quantitative titration of trypsin or similar proteolytic enzymes, and of protease inhibitors, suitable for critical tests on a large scale is described. The procedure utilizes technics familiar to serologists, rather than to skilled biochemists only, and gives definitive, reproducible results that can be expressed in simple figures. A feature is the provision for the making of a permanent record of each titration. Since the reagents are stable, and readily standardized, comparable findings can be obtained in separate experiments conducted in the same or in different laboratories at any time.

The assistance of Mrs. David A. Cook with records and statistics is gratefully acknowledged.

Two methods are described which permit the estimation of colored substances on one-dimensional and two-dimensional paper chromatograms with a minimum expenditure of time and material.

1. Progressive acute disseminated encephalomyelitis was produced in 90% of a group of guinea pigs by the subcutaneous injection of water in oil emulsions of homologous brain tissue plus adjuvants.

2. While prophylaxis and treatment with moderate dosages of either para-aminobenzoic acid or sodium salicylate alone provided no protection of guinea pigs against experimental allergic encephalomyelitis, the use of larger dosages of salicylate as well as combined salicylate and para-aminobenzoic acid therapy were effective in preventing this disease.

3. Effective prophylaxis was demonstrated only when the medication was started before or shortly after the sensitizing injection of brain tissue was given.

4. No therapeutic effect of sodium salicylate alone or combination of para-aminobenzoic acid with sodium salicylate could be shown.

Based on the determination of radioactivity for periods of 10, 20, 30, and 40 minutes following a subcutaneous injection of 10 mg/kg of C^-14 labelled methadone HBr into rats, it was found that:

(a) Methadone∗ makes its appearance in the blood within 10 minutes, and is confined to the plasma.

(b) There was no activity in the cerebrum before 10 minutes, and even thereafter the concentration is very low. The rate of accumulation roughly parallels that of the plasma indicating no special affinity of the brain for the drug.

(c) The adrenals and liver show a marked ability to accumulate methadone∗ at a rate in excess of that shown by either the cerebrum or plasma.

(d) There was no activity in the sciatic nerve within the period studied.

Animals fed a niacin-tryptophan deficient ration supplemented with 0.208% DL-phenylalanine and 0.2% DL-methionine for a period of 12 weeks developed a nervous syndrome. Generous quantities of tryptophan or niacin in the ration protected the animals from this syndrome, but neither compound cured the symptoms once they had progressed beyond a given stage.

A modification of Harrison's diphenylamine method for fructose or inulin determination is described, heating being carried out at 75°C for 60 minutes. With this procedure the ratio of fructose to glucose color production is 64, as compared with a ratio of 8 at 100°C for 30 minutes. Inulin hydrolysis is complete, the standard error of the method is less than 1 percent, the color is light stable and the low color production by glucose permits one to omit fermentation except in the presence of unusual glucose fluctuations.

Patients with certain neoplastic diseases involving the reticuloendothelial system (Hodgkin's disease, lymphosarcoma and acute leukemia) exhibited a high percentage of false positive trichina precipitin reactions. Such reactions were much less frequently encountered in sera from patients with metastatic carcinomas and no positive reactions were observed in the normal controls. The false-positive reaction does not appear to be related to medication.

1. DDD and DDT when administered orally are stored in the tissues of the dog, the greatest storage appearing in the fat.

2. Similarly DDD and DDT are both stored in the fat of the rat.

3. Both DDD and DDT are transported across the placental barrier in the dog.

4. In the dog, a small proportion of orally administered DDD or DDT is excreted in the feces. Of the 2, DDD appears in greater amount.

5. When administered orally to the dog, neither DDD nor DDT are excreted as such in the urine. DDA is a urinary end-product common to both DDD and DDT, appearing in greater amount following DDD administration.

We wish to extend our thanks to Dr. E. L. Stanley of the Rohm and Haas Company for performing the analytical procedures connected with this study.

1. The urinary and fecal excretion of coproporphyrin was studied at varying intervals in 21 young rats maintained on a diet containing 18% yeast as a source of protein.

2. Fourteen of the rats died suddenly between the 30th and the 125th day of acute massive hepatic necrosis. The gross and microscopic findings were identical with those described by Himsworth as characteristic of this lesion.

3. No significant increase in the excretion of coproporphyrin was observed during the course of these experiments. This is believed to minimize the likelihood of chemical or metal toxicity as the cause of the necrosis.

Both aureomycin and Chloromycetin prolong the life of chick embryos infected with the 6BC strain of psittacosis virus. There is a direct relation between the dose of antibiotic and the prolongation of life of the embryos, the infecting dose being kept constant. With equal doses of the antibiotics, aureomycin prolonged the life of the embryos for an average of from 2 to more than 3 days longer than Chloromycetin, depending on the dose used.

On a weight basis, aureomycin was more than 3 times as effective in protecting chick embryos against the dose of virus that was used in this study. On a molecular basis the aureomycin was more than 5 times as effective as Chloromycetin in this infection.

Following the administration of desoxycorticosterone acetate to persons with nutritive failure and hypotension, there was a prompt increase in blood pressure readings and in body weight (the increase in body weight is associated with retention of sodium in the tissues and this problem is being studied further). When the administration of this substance was discontinued, the blood pressure readings promptly returned to normal. During the first week after the administration of desoxycorticosterone acetate was begun, one patient stated that she had great symptomatic improvement; one complained of severe headaches, and she developed a “moon-faced” appearance which gradually disappeared when the injections were discontinued; the third patient noticed only swelling of the ankles. While our findings are of general interest, we do not recommend the use of desoxycorticosterone acetate as a therapeutic agent for hypertension.

1. The blood picture of 14, 15 and 16 day White Leghorn chick embryos injected into the allantois with aqueous adrenal cortex extract, shows no significant change in the number of lymphocytes but a market absolute polymorph leukocytosis, 24 hours after injection. The erythrocytes are not affected by the extract.

2. Large individual variation in the lymphocyte count and greater difficulty involved in their identification may have obscured the effect of the hormonal preparation on the lymphocyte count.

3. ACE treatment induced a tendency (not statistically significant) toward weight loss in the 14 day embryos, a significant decrease in the hatchability rate, and an apparent defective feather development. However the blood picture and weight in comparison with the controls was normal after hatching.

It has been demonstrated for the first time, histochemically, that the normal kidney of the frog, Rana pipiens, possesses a strong alkaline glycerophosphatase reaction in its proximal tubules, especially at their brush borders, whereas the distal convolutions are negative. The glomeruli show a moderate alkaline reaction, categorizing the frog in a class with the cat (the only other animal thus far investigated that gives this reaction). With regards to the neoplastic kidney, the tumor cells themselves are alkaline phosphatase negative with an accompanying depletion of the enzyme in adjacent non-tumorous portions of the renal tissue.

Both normal and tumorous kidneys are essentially negative for acid glycerophosphatase. The significance of these results is discussed.

Inositol is an effective agent in lowering serum cholesterol and lipid phosphorus in hypercholesteremic diabetics. Further study of its action and applications is warranted.

The absorption, excretion, and tissue-distribution of 4 compounds of the 9-aminoacridine series have been studied in rats at oral dose levels ranging from 50 to 500 mg/kg. At any dose level Acranil concentrates in the tissues to the greatest extent, while the other compounds follow in this order: Win 2390, 2389, 2333. For any single compound, the tissue concentrations ordinarily increase with the dose, the largest amount being found in the liver. In order of increasing rate of absorption the compounds are Win 2390, Win 2389, Acranil, Win 2333 but none of these is as completely absorbed: within 24 hours as has previously been reported for Atabrine.

In previous experiments on grafted adult salamander eyes, it has been shown by the author, 1 , 2 , 3 that degeneration of the neural elements of the retina is followed by regeneration. The retina can be regenerated with subsequent return of vision at least 4 times in the same eye repeatedly transplanted. 2 The functional patterns associated with each of the retinal quadrants are reestablished and the visuomotor responses of the host are determined by the orientation of the graft. 4 , 5 , 6 , 7

Regeneration of the retina in amphibians especially in larval urodeles has been reported previously by other investigators. As to its source of origin iris tissue has been claimed by some authors. 8 Retinal tissue at the ciliary margin 8 , 11 and ganglion cells in the fully differentiated retina 12 have been proposed as the source. One of the above mentioned investigators 8 believed that the regenerating retina in larval urodeles could also be derived from the pigmented epithelial layer.

An extensive study has been made to settle the question of the source of origin of the regenerating neural portion of the retina in the eye of the adult urodele, Triturus v. viridescens. In various groups of experiments over 500 grafts of iris tissue have been placed in the aqueous and vitreous chambers of the eye. None of them has given rise to retinal tissue. A detailed study was made of the degeneration and regeneration of the retina in 200 grafted eyes preserved at close intervals for at least 3 months after operation. Degeneration spreads throughout the neural retina during the first 3 weeks. In some cases destruction proceeds at different rates in various parts of the same eye and when degeneration overlaps early stages of regeneration it is more difficult to obtain a clear picture of the origin of the new retina.

In the course of an investigation designed to test the ability of various substances to prevent the deterioration of complement, it was found that certain of the polyethylene glycols greatly enhance the hemolytic activity of complement. The polyethylene glycols, a series of polymers with the general formula HOCH₂(CH₂OCH₂)xCH₂OH, are soluble in water and in many of the aromatic hydrocarbons. The compounds with average molecular weight up to 700 are liquids; those with average molecular weight above 1000 are wax-like solids, sold under the trade name “Carbowax”.† This paper constitutes a preliminary report on the effect of the polyethylene glycol carbowax-4000 on the hemolytic activity of complement, as determined by complement titrations in the presence and absence off this compound.

Procedure. Complement titrations were carried out according to the 50% hemolysis end-point method developed by Mayer et al., 1 and Kent et al. 2 The reaction mixtures, 20 ml in volume, had the following composition: 1) 1 part complement, the dilution so adjusted as to yield final dilution values ranging from 1:3300 to 1:950; 2) 1 part standardized suspension of sensitized erythrocytes; 3) either 2 parts of buffered saline, 3 or 2 parts of an 8% solution of carbowax-4000 in buffered saline. The sensitized erythrocytes were well mixed with either buffered saline or carbowax-4000 solution, and then the complement added dropwise with constant agitation. All components were kept in ice-water until time of incubation. The reaction mixtures thus prepared were incubated at 37°C for 60 minutes, with frequent agitation. After centrifugation of the mixtures, readings of per cent hemolysis in the supernates were made at 5500 Å in a Coleman spectrophotometer, an optical density of .500 representing complete hemolysis. Titrations were carried out in duplicate. Controls were set up of 1) sensitized erythrocytes and carbowax-4000.

It has been observed that a methionine deficiency in the rat reduces liver succinic dehydrogenase activity slightly, completely reduces liver xanthine oxidase activity, and has practically no effect upon endogenous respiration of liver tissue in vitro.

A concentrate of vitamin B₁₂ exerted a marked lipotropic effect when injected into rats receiving a high-fat diet. This lipotropic effect was not due to the small amount of choline present in the concentrate.

A micro modification of the standard Warburg vessel for the study of gaseous exchange of tissues has been described. As would be anticipated from the decrease in size from 17 ml to about 3 ml, the amounts of tissue required for satisfactory results can be reduced to about one-fifth those usually used.

An advantage over other more sensitive apparatus is that the manometer supports are interchangeable with the standard Barcroft-Warburg supports, so the same water-bath and shaking device can be used for both.

A study of long-term folic acid administration in 70 non-macrocytic anemia patients was made. Signs related to sub-acute combined sclerosis or peripheral neuritis did not develop in any of the group.

A method for preparing blood group specific substance A from Difco neo-peptone by the Folin-Wu technic is described and comparisons with other preparations are made. The method is simple and the product obtained compares favorably in purity and potency with products obtained by other methods.

Eleven steroid compounds have been studied as to their influence on sodium excretion in adrenalectomized rats. Desoxycorticosterone caused significant retention at one microgram, and the acetate was effective at 25 micrograms, the lowest concentration studied. Δ⁴-pregnenol-21-trione-3,12,20-21-acetate gave a significant retention at 1340 micrograms. 17-Hydroxycorticosterone caused a significant excretion of sodium.

The following steroids were found to be negative: 11-dehydrocorticosterone acetate (100 μg), pregnanetrione-3,11,20 (200 μg), corticosterone (400 μg), allopregnanepentol-3 (β), 11(β), 17(α), 20, 21-3(β), 20, 21-tri-acetate (800 μg), allopregiianetriol-3(β), 17 (α), 21-one-20 (800 μg), testosterone (2000 μg), estradiol (2000 μg), and pregnanetrione-3,12,20 (2000 μg).

The effectiveness of the autolyzed noirmal mouse brain method for isolation and adaptation of poliomyelitis virus was investigated in 19 experiments with mice and 4 experiments with cotton rats. Three monkey adapted strains (Tennessee, Mahoney and Buffalo) and 12 stool specimens from poliomyelitis patients were used. The Lansing strain was also included in the experiments.

The administration of large doses of 2,6-diaminopurine in mice and rats can result in death within a few hours. In dogs, large doses caused protracted vomiting, hemorrhagic diarrhea, dehydration and death within 48 hours. Doses permitting survival of rats and dogs for 24 hours or longer produced depletion of bone marrow and damage to epithelium of colon and ileum. The course of intoxication and changes in bone marrow permit a differentiation between the actions of 2,6-diammopurine and a folic acid-antagonist like 4-aminopteroylglutamic acid.

The intravenous administration of 78 mg of organically bound mercury (2 cc mercuzanthin) causes a significant decrease in the Tm_PAH but no significant change in the Tm6 in man. It is concluded that under the conditions of this study mercury had no effect on the renal tubular glucose transfer system in man.

Several attempts were made to sensitize 4 dogs to their own red blood cells. Three dogs showed no definite evidence of autosensitization. Although 2 of these 3 dogs showed occasional marked fluctuation in mechanical fragility unaccompanied by other changes during these studies this was considered to be due to technical difficulties. However, for a short period in one dog, 25 d'ays after the injection of a modified Freund antigen, there was agglutination of washed red blood cells when they were suspended in anti-dog serum rabbit serum. During this period of apparent autosensitization there was no anemia, reticuloeytosis or jaundice. The red blood cells showed no alteration in osmotic or mechanical fragility. There was no fall in complement titer. Cold and warm agglutinins did not appear. There was no evidence of a serum complement-fixing antibody for the dog's own red blood cells. Such apparent autosensitization supports the explanation that in certain instances of acquired hemolytic anemia in man serum autoagglutinins may be formed in response to an antigen in red blood cells.

In a study of 288 samples of blood from 152 cases, a gradual appearance of Ach toward the 2nd trimester of pregnancy, an increase of Ach with the onset of labor, and a disappearance 48 hours or more after labor were demonstrated. Blood of 20 normal subjects of both sexes showed no evidence of Ach with the same test.

1. Casein, amandin, arachin, myosin, and the crystalline globulins of pumpkin seed and watermelon seed give marked to moderately strong diacetyl monoxime reactions for citrulline when tested directly but their acid hydrolysates give completely negative results.

2. Citrulline itself is relatively slowly destroyed by treatment with 6N hydrochloric add at 110°C.

3. Only very small amounts of ureide compounds are found in enzymatic hydrolysates of the 6 proteins; however; when citrulline is added to such hydrolysates, its “recovery” in terms of colorimetric units corresponds to several times the theoretical value. The reason for this anomalous result has yet to be determined.

4. It is probable that citrulline is either absent or present only in minimal quantities in these proteins.

1. Whole Arctic sticklebacks and ordinary guppies were analyzed for fatty acids, cholesterol and lipid phosphorus.

2. It was found that diet does not have an appreciable effect on the lipids in the guppies.

3. Cholesterol and fatty acid are higher in the guppies than in the Arctic fish. On the other hand phospholipid is essentially the same in all the fish examined.

4. There is more phospholipid in relation to cholesterol in the Arctic sticklebacks than in the guppies, a fact which may be interpreted as indicating a higher level of fat turnover in the former than in the latter.

5. The ratio of cholesterol/lipid phosphorus is lower in the stickleback than in the guppy, indicating a lower water content in the tissues of the former. This may be correlated with the reported ability of the stickleback to resist freezing injuries.

This work was undertaken to ascertain whcther the perception of pain is associated with a specific change in the electroencephalogram (E.E.G.), which might be used as an objective criterion of pain.

Methods. A Grass Electoencephalograph was used with 6 or 8 electrodes in 17 experiments on as many subjects. In addition to the usual 8 electrodes (2 frontals, 2 parietals, 2 temporals and 2 occipitals) the Grinker needte eIectlrodel in the sphenoid btone was employed tin 2 experiments, and a saline saturated cotton elelctrode prlessed against the posteritor pharyngeal wall in one. For the purpose of exploring any changes from the region of the base of the skull or hypothalamws near the foramm ovale in sbill another experiment an, insiullated needle electrode was inserted (under infiltration anesthesia) between thle mandible and zygoma to a depth of 5.5 cm. Monopolar registration was employed with the lobe of the ear as indifferent electrode. Pain was pnolduced in several ways in each experiment. (a) A Von Frey hair stimulator was applied to1 a previoudy determind pain point on the right forearm with a pressure of 8 g. (Ib) The webl of the skin between the thumb and index finger of the right hand was pressed between, a forceps. (c) As a control in two experiments heat, cold and touch were applied to the rigbt foreairm without causing pain.

Results. In all experiments a definite chang? in the E.E.G. occurred. It consisted of a decrease in amplitude, which was in the nature of a loss of electrical entropy and must be separatld flrom the attention wave, which frequently appeared as an addlitioaal factor. The change was not specific for a pain stimulus or sensation since a similar response was obtained with heat, cold and touch.

Koenig and Koenig 1 have described an acute pulmonary edema following the administration of ammonium salts to different species. It appears to be specifically a result of the ammonium ion. The occurrence and gross appearance of this edema resembles epinephrine induced pulmonary edema and in an effort to obtain information about the mechanism of the ammonium pathology a study has been made of its relation to edema due to epinephrine and adrenergic stimuli.

Experimental. Epinephrine induced pulmonary edema may be prevented by adrenergic blocking agents. 2 , 3 Their influence was observed upon ammonium pulmonary edema. Unfasted guinea pigs were divided into groups with the same sex distribution. All groups were lightly etherized to pass a stomach tube. Group 1, the controls, were given a sham dose of 10 cc water per kg body weight. All other groups received 1200 mg/kg of a 12% solution of NH4CI by oral gavage. Ten minutes before receiving the ammonium salt groups 3, 4 and 5 respectively received by intravenous injection in 1 cc saline/kg; 2 mg/kg of a B-haloethylamine, N-(9-fluorenyl) -N-ethyl-B-chloroethylamine-HCl (“SKF-501”); 2.5 mg/kg N-(2-chloroethyl)-N-ethylbenzhydrylamine-HCl (“SY-2”), which like “SKF-501” is an effective adrenergic blocking agent; and 1.5 mg/kg of the potent anttihistaminic N-B-dimethylaminopropylthiodiphenylamine - HCI (“Phenergao”, 3277 R.P.). The presence of pulmonary edema was determined at an autopsy by gross inspection and lung weight was used as a measure of its extent.

The summarized data comprise Table I. Group 1 was killed with ether while all of the animals in the other groups succumbed with convulsions 5-30 minutes after receiving the ammonium salt. The two adrenergic blocking agents and the antihistaminic all were without influence upon mortality. Since both of the adrenergic blocking agents completely prevented the ammonium edema but not the mortality, we may conclude that pulmonary edema need not be the cause of death fromtoxic doses of ammonium salts. The anthistaminic was without effect on the ammonium edema, which in this respect resembles the fulminating pulmonary edema due to epinephrine. 2

Normal mice, or mice bearing a transplantable lymphosarcoma, were given single injection s of adrenal cortical extract or exposed to a single radiation dose of 200 r (total body radiation). Examination of the serum protein patterns of these mice at 3 and at 6 hours after hormone injection, or at 6 hours after exposure to X-rays, revealed a striking increase in total serum protein levels. However, electrophoretic examination did not reveal significant alterations in the relative distribution of the serum proteins.

A choleretic response to cinchophen was observed in guinea pigs.

The chinchophen output in the bile was found to be greatest in guinea pigs and to decrease in the following order: rats, cats and rabbits.

The following is the order found for susceptibility to cinchophen ulceration: cat, dog, guinea pig and rat. Rabbits were fund completely resistant.

There appears to be no abvious relationship between the species susceptibility to cinchophen ulceration and the choleretic response of that species to cinchophen or to the concentration of cinchophen in the bile after intravenous administration.

It would appear that the ulcer susceptible animals investigated have a low basal bile flow whilst the unsusceptible have a high flow.

A technic is described of producing focal lesions in the cerebral cortex of rabbits by microwave irradiation without incising the scalp or skull. The results on two animals are reported and future possibilities of this work are discusse.

The original strain of Coxsackie virus, serologic type 1, has been measured using elford's ultrafiltration technic. Ultracentrifugation furnished a rough check. The virus is very small, the estimated size bening 10 mμ or slightly less.

1. In an effort to find whether the “virtual castration” of vit. A deficient male rats is due to prevention of the synthesis of androgens or to the lack of response of target organs, the effects of administration of testosterone in deficient non-castrates and castrates were compared with the effects on their controls.

The response of the deficient animals was found to be essentially normal. The accessory sex organs in particular were extraordinarily hypertrophied under the influence of testosterone administration even in the face of severe losses of weight.

The evidence obtained suggests that vitamin A deficiency interferes with the synthesis or release of androgens.

It appears that in rabbits histamine is linked via the histamine containing platelets with blood coagulation, and this is connected via heparin with the heparin producing basophiles. All these elements are involved in serum disease. The first injection of serum is followed by a rise in histamine and platelets, the second by a drop in these elements. The destruction of the white cell layer following reinjection calls forth an outpouring of heparin which in turn is followed by basophilia in the blood. Only the changes following reinjection can be regarded as allergic. Neohetramine was found to effect only the primary reactions following the first administration of serum. It suppressed the rise in blood histamine and the proteinuria observed during the first week following the first injection, and also impeded the primary mesenchymal reactions in lungs and heart which are believed to be instrumental in antibody formation. There are reasons to believe that this was not a specific antihistaminic effect, but that it was due to a non-specific action of the drug upon the permeability of tissue membranes possibly via the hyaluronidasehyaluronic acid mechanism.

Acute toxicity studies were made on 3 different samples of cottonseed pigment glands administered orally to 228 rats in the form of slurries made with either distilled water or 2% ferrous sulfate solution. The LD50 values for the samples when administered in water were, respectively, 1140, 1490 and 2290 mg/kg. When given in 2% ferrous sulfate solution the samples were detoxified so that even such high doses as 7000 mg/kg for each were no longer toxic. There was little effect on the body weight of all 99 rats which survived doses of ferrous sulfate-treated cottonseed pigment glands 3 to 6 times the LD50 value for water-suspended glands. Administration of pigment glands in 0.5% FeSO₄ did not detoxify them, and led to the death of all 24 rats given this preparation.

Tolserol has a definite effect upon the electrical discharges of the human cerebrum; the most pronounced changes were found in teh electrical activity of the hypothalamus and of the midbrain.

The pattern of distribution of alkaline phosphatase activity in tissues is, when 5-nucleotide is used as a substrate, different from patterns obtained with any other substrate. The specificity of'5-nucleotidase appears to be an established fact.

Streptomycin PAS may be given intramuscularly without undue irritation. Chemical assay of PAS, and bioassay of streptomycin, each give virtually quantitative recovery of streptomycin PAS in vitro. Following intramuscular administration of streptomycin PAS in human subjects, streptomycin was demonstrated in the plasma and urine within the expected range. PAS also appeared in significant concentrations, but the quantitative relation to streptomycin was altered.

In a quantitative chemical study of developing muscle there is a greater activity of adenosinetriphosphatase than is warranted by the increase in amount of myosin proteins. In order to ascertain the reason for this discrepancy determinations of phosphatase activity were made on fractions of developing muscle obtained from rat embryos of 14 days, fetuses of 19 days, rats of 1-2 days and 9-10 days of age. The activity was calculated in micrograms of phosphorus liberated in 15 min. at 37.5° per mg N.

1) Pyridoxine deficiency in mice produces granulocytosis and lymphopenia. 2). The leukemia of the Ak mice now being bred in this laboratory is participated in by both granulocytes and lymphocytes. In pyridoxine deficiency the leukemic granulocytosis is more severe and the survival time of the animals is decreased. 3) The hypothesis is advanced that the granulocytosis in pyridoxine-deficient mice is due to the failure on the part of the animals to perform metabolic functions in which pyridoxine derivatives play a catalytic role; thus, supplies of compounds used in the formation of granulocytes accumulate, and, as a result, the animals utilize this plethora in producing excess numbers of granulocytes. The implications of this hypothesis are discussed.

A device for measuring blood pressure in the aorta of the unanesthetized rat is described. It is simple and permits an unlimited number of readings over a long observation period.

Crystalline egg lysozyme precipitates insulin. The complex produces a prompt lowering of the blood-sugar level in rabbits. Precipitates formed by mixing insulin with lysozyme which has been heated, or treated photodynamically so that 90 to 100% of its lytic activity on M. lysodeikticus is destroyed, but about 60% of its precipitating power for insulin is left, produce a hypoglycemic response which, up to 7 hours after injection, is intermediate between that resulting from the injection of insulin in solution and that following administration of protamine-zinc-insulin.

The quantitative detection of orally administered digitoxin in the urine of subjects receiving the drug has been accomplished. Approximately 14% of the given dose is excreted in the urine of normal subjects during the first 72 hours after its oral ingestion.

Using a specific and sensitive assay procedure, we have tested the urine of normal persons and of patients having a variety of disease states for its adrenotrophic activity. Tests using both fresh urine and urine concentrates prepared in three different ways were carried out. Contrary to previous reports, no adrenotrophic activity could be consistently demonstrated.

Curves of plasma radioactivity were determined for 25 patients after the ingestion of 100 microcuries of radioiodine. The patients were divided into 11 cases of myxedema, 5 cases of hyperthyroidism and 9 euthyroid individuals. The curves were constructed from plasma samples drawn at 30 minutes 1, 3, 6, 12, 24, and 48 hours. It was not possible to statistically separate, at these time intervals, the curves of the hyperthyroid and euthyroid individuals. The curves of the myxedematous patients, however, were significantly different from those of the non-myxedematous patients; thus the myxedematous curves show a later peak level, a lower peak level and a fall more delayed than do the curves of the non-myxedematous patients.

Rapid adaptation occurs in adult rats exposed to anoxia at low temperatures. This enables the animals to tolerate extreme anoxia (32 mm Hg pressure of O₂) at various temperatures (14-31°C) for a period more than 40 times longer than unadapted controls.

In an attempt to decide whether or not the yolk or the egg white is the carrier of a growth factor transmitted through the egg from the hen's feed to the chick, the experimental technic described here was devised. In the past it has been assumed that egg white has a purely nutritional function in the growth and development of the chick embryo, but no experimental evidence exists to support this supposition.

The yolk also furnishes food, but in addition the yolk-sac serves as an organ for the digestion and assimilation of food by the chick embryo. This function of the yolk-sac indicates a structural relationship between the embryo and the yolk.

In this experiment fertile hen's eggs from crosses of Rhode Island Reds and White Leghorns were used. A circular window approximately 1/4 inch in diameter was bored in the small end of each egg and the egg-white was removed into a glass ground-joint wash bottle by suction. To combat bacterial infection 0.1 cc of an aqueous solution containing 3000 I.U. of penicillin and 3.3 mg of streptomycin was injected into each of the eggs so treated. The egg white from another fertile egg was then transferred by air pressure from the wash bottle into the egg from which the egg white had previously been removed. When one egg white was not sufficient to completely refill the shell, that from another fertile egg was added. Thus, at times egg white from more than 1 egg was used for replacement in a single egg.

After the egg was completely filled with egg white, the window in the shell was closed with a strip of scotch tape and the egg placed into an incubator for 3 weeks. At intervals of 7 days, the condition and development of the chick embryo was observed by candling the eggs.

In the first successful series, 12 such eggs were prepared; of these 3 chicks hatched which were normal in every respect. Of the rest of the embryos, two lived 2 days, one 4, two 6, one 7, one 13, one 15 and one 21 days.

These are probably the first live chicks produced after such drastic treatment. This experiment demonstrates that the chief function of the white of the hen egg is mainly to furnish food for the developing embryo.

It has been previously held that the two chalazae serve to orient the embryo in a proper position for hatching by keeping the yolk more or less fixed. In these experiments, however, the two chalazae were removed with the egg white and thus the position of the developing embryo depended upon gravity. Further work is in progress on the nutrition of the chick embryo employing this technic.

The vitamin B₁₂ content of a number of natural materials as determined by the rat assay is presented. Fish solubles, streptomycin “slops”, sheep rumen contents and glandular meats are excellent sources of vitamin B₁₂. Muscle tissue, eggs and milk products contain lesser amounts, whereas plant materials show no measurable activity.

1. No reactivation of the left diaphragm was seen by us in 15 dogs up to 20 months after complete section of the left phrenic nerve.

2. Microscopically all diaphragms showed complete muscular atrophy with degeneration or disappearance of nerve fibers, 14 to 20 months after complete section of the left phrenic nerve. It is difficult to conceive how such atrophic muscles could ever regenerate.

3. It is highly possible that phrenic exeresis in the neck of the human does not always result in complete phrenicotomy with subsequent atrophy of the diaphragm, but in a number of instances results in diaphragmatic palsy.

4. We found no evidence that the twelfth thoracic intercostal nerve or sympathetic innervation could influence the reactivation of the diaphragm in the dog.

5. Our experience with one diaphragm leads us to believe that a hemifunction of one half of a diaphragm may lead the fluoroscopist to assume that complete reactivation of that diaphragm has occurred.

6. The phrenic nerve above the point of section was able to convey reflexes 20 months after section; this was demonstrated by rise in blood pressure and increased respiration upon stimulation of the central end of the nerve, which were abolished when the nerve was cut central to the stimulating electrode.

7. No evidence of the gastro-cardiac symptom-complex of Roemheld was seen.

Acute coronary artery ligation in the dog results in loss of coenzyme A in the infarcted myocardial area. Pretreatment with pantothenic acid does not influence this loss.

1. Protein-depletion in young adult male rats, as produced by a weight loss of 25% on a protein-poor diet, materially increases susceptibility to total body irradiation. 2. Age or weight in normally nourished adult rats, in the 150 to 280 g weight range, have little effect on susceptibility to radiation.

The author wishes to express his appreciation for advice and assistance to the following men: P. R. Cannon, R. W. Wissler, and E. P. Benditt, Department of Pathology, University of Chicago; A. M. Brues, Biology Division, Argonne National Laboratory, Chicago; L. J. Savage, Department of Mathematics, University of Chicago; and H. D. Landahl, Committee on Mathematical Biology, University of Chicago.

The animals used in this experiment were irradiated through the collaboration of the Biology Division, Argonne National Laboratory Chicago.

(1) A saline extract of the liver of fetal guinea pig possesses greater hemolytic activity than a similarly prepared extract of maternal liver, as determined after 4 hours incubation at 38°C.

(2) The activity, for the most part, is due to a heat-sensitive agent and, to a lesser extent, to a heat-resistant substance.

(3) The lytic action of such extracts is not species-specific. It will lyse red cells from the same fetus, as well as those from the mother or from another species.

1. The perfused livers from adult rats fed either a basal diet containing 12% of casein or the same diet plus 4-dimethylaminoazobenzene for 4 weeks, and the liver tumors induced by this dye were fractionated into nuclear, large granule (mitochondria), small granule (microsome), and supernatant fluid fractions. The amounts of 17 amino acids were determined in the total proteins from the original homogenate and in each of the fractions. The importance of using perfused livers for these analyses was demonstrated experimentally.

2. When the basal diet was fed, the proteins from the nuclear fraction contained lower concentrations of histidine, tryptophan, phenylalanine, and cystine and a higher concentration of glycine than the whole liver proteins. The large granule proteins contained less histidine, the small granule proteins less methionine and glycine and more tryptophan, and the supernatant fluid proteins less tryptophan and more cystine than the proteins of the original homogenate.

3. The livers of rats fed the dye contained higher concentrations of glutamic acid in the nuclear proteins, arginine in the large granule proteins, and cystine and tryptophan in the supernatant fluid proteins than normal livers.

4. The proteins from each fraction of liver tumor contained less methionine and more cystine than the proteins in the corresponding

The effectiveness of the triazole analogue of guanine, azaguanine, in inhibiting the growth of subcutaneous transplants of mammary adenocarcinoma Eo771 1 in mice of the C57 black strain has been reported by Kidder, Dewey, Parks and Woodside 2 and confirmed for this tumor. 3 It was suggested by Kidder and co-workers 2 that their results indicated that the tumor tissue utilizes guanine for nucleic acid synthesis while normal tissue does not.

We have investigated the incorporation of isotopically labeled guanine (containing N¹⁵ in the 1 and 3 nitrogens and in the 2-amino group 4 ) into the nucleic acids of this strain of mouse bearing this tumor. The guanine was administered in five intraperitoneal injections, of 2.4 mg each, over a 6 day period, beginning on the 7th day after implantation of the tumor. Inasmuch as isotopic nitrogen, particularly from the 2-amino group, can be contributed to body pool ammonia 4 it is not unlikely that the isotope found in the protein residue, pyrimidines and even the adenine (Table I) arose from this source. There was, however, a small but definite incorporation into the guanine of the nucleic acids, but there is no evidence of a specific uptake by the tumor since the incorporation into the guanine of the tumor tissue and into that of the non-tumorous viscera of the same mice was significant in each case.

When guanine was adminisered to an unspecified strain of the white rat 5 or to Sherman strain rats (Rockland Farms), either orally 4 or intraperitoneally, 6 there was no significant incorporation of the guanine into the tissue nucleic acids. The species difference in the purine metabolism of the Sherman rat and the C57 black mouse (bearing adenocarcinoma Eo771) makes it necessary to be conscious of other such possibilities in mammalian purine metabolism.

Rhenium is preferentially and actively filtered from the blood by the thyroid. After injecting a single dose, the Re∗ concentration in that organ is 25 to 100 or more times greater than that found in any other tissue. These findings therefore add support to our hypothesis that the thyroid will selectively filter from the blood all the elements of the seventh periodic group. The greatest concentration of Re∗ in tissues occurs within one hour. For several hours it decreases slowly and then more rapidly so that more than 90% is recovered from the urine in 24 hours.

Antisera prepared in hamsters by the intranasal instillation of allantoic fluids containing influenza virus were type-specific when used in the complement-fixation test with the soluble antigens; group- or subtype-specific (e.g. A prime) when used in the complement fixation test with the allantoic fluid antigen, and strain-specific in hemagglutination-inhibition tests.

It appears that the hamster is an animal best suited for quick identification of influenza viruses. It is suggested that their sera be used for systematic classification of strains.

Epinephrine protects adrenal insufficient rats from the edema and mortality caused by the intraperitoneal administration of egg white. Mortality is prevented by doses (0.04 mg/kg) which are not able to prevent edema in rats with intact adrenals.

The antihistaminic Phenergan, N-dimethyl-amino-2-propyl-l-thiodiphenylamine (“3277 RP”), in doses as low as 5 mg/kg prevent death from egg white edema in adrenalectomized rats.

“SY-28”, N-(2-bromoethyl)-1-naphthalene-methylamine hydrobromide, a Dibenamine congener possessing both antihistaminic and adrenergic blocking activity, also prevents the edema and death, but is in itself toxic to adrenalectomized rats.

“SKF-501”, N-(9-fluorenyl)-N-ethyl-β-chloroethylamine, a Dibenamine cogener with potent adrenergic blocking powers but with a low toxicity and without antihistamine action, also inhibits the edema and decreases the mortality.

The possible modes of action are discussed.

A microbiological assay for vitamin B₁₂ using Lactobacillus leichmannii (ATCC 4797) has been developed. The assay medium contains crystalline amino acids as the nitrogen source and adsorbed tomato juice filtrate as a source of unidentified growth factors. A high level of cysteine and ferrous sulfate are present in the medium to keep it in the necessary reduced state. Data are given to show the degree of precision and reproducibility to be expected from the assay.