Experimental Biology and Medicine

Studies of the toxicity disclose that sulfathiazole administered per os to rabbits is less toxic than sulfapyridine. On the other hand, administered the same way to mice sulfapyridine appeared to be less toxic than sulfathiazole. It is obvious that the absorption and elimination, and the resulting toxicity of the drug depend upon the species of animal used. In Types II and III pneumococcus infection, the therapeutic effect of sulfapyridine is superior to that of sulfathiazole, based on the oral dose only and with no blood level determinations.

1. A colorimetric method for the determination of p-caproylaminobenzenesulfonhydroxamide has been described. 2. This drug is approximately equal, weight for weight, to sulfanilamide in antistreptococcal activity when fed at intervals of 4 hours, but it is somewhat more effective than sulfanilamide when fed only once daily. 3. When the sulfonhydroxamide and sulfanilamide were given in equal-weight oral dosage, the former gives the lower and more constant blood levels.

When dogs were placed on a diet deficient in nicotinic acid, the addition of nicotinic acid corrected the deficiency, while the addition of nicotinic acid and sulfapyridine failed to do so. When raw liver and sulfapyridine were added to the diet, the deficiency disappeared. It seems possible, but not proven, that sulfapyridine inhibits the action of nicotinic acid but not of preformed coenzymes.

Early studies on influenza suggested that the virus agents of the disease, although not antigenically identical were closely enough related for the infection to evoke a demonstrable rise in antibodies reactive against the recognized strains of the influenza virus. The first convincing indication that influenza might be due to antigenically unrelated viruses was presented by Stuart-Harris, Smith and Andrewes, 1 who found that a rise in antibodies against the usual strains of influenza virus occurred in only 33% of the cases they studied in England in 1939; although they failed to isolate the actual virus, they concluded that a number of their cases must have been caused by an agent other than the usual influenza virus. More direct evidence has recently been obtained independently by Francis and by ourselves. In February, 1940, we 2 isolated a virus (termed TM) from 2 cases of influenzå and showed that in each instance the infection evoked an increase in antibodies against the homologous (TM) virus but caused no detectable increase in antibodies reactive against the PR8 3 strain of influenza virus. At about the same time Francis 4 isolated a strain (termed “Lee”) which was also distinct from the PR8 and from other previously recognized strains. He showed that the convalescent serums from cases of influenza which had occurred in various places during the early part of 1940 and also serums from 2 cases that had occurred in 1936 had increased titers of antibodies against his Lee strain but not against the PR8 strain. Francis concluded that his Lee strain was a new type of virus and proposed that it be termed influenza B to distinguish it from the older strains (PR8, WS, etc.) for which the term influenza A had already been suggested.

The observations reported here indicate that the urinary excretion of nicotinic acid in pellagra is not significantly different from that of the normal.

Well nourished hypophysectomized rats did not show mammary development when stimulated by large doses of estradiol benzoate for a period of 28 days. When this result is correlated with those of other workers it appears that both estrogens and some pituitary factor must act directly on the mammary gland to produce normal development.

Surviving frog skin is the seat of an electrical potential which has been related to factors affecting its metabolic activity. 1 2 To determine the efficiency of electrical energy production through an external circuit by surviving frog skin it is necessary to measure the joules of electrical energy and the oxygen consumption of the skin during the same interval of time. An iodine coulometer 3 of low resistance (18–22 ohms) with sufficient electrode area to minimize polarization within the range of skin potentials, was employed to integrate the quantity of current passing through the external circuit. The coulometer was calibrated against known, constant current densities for measured intervals with a microammeter in series as a check. Average voltage was determined with a standard potentiometer by readings at 5 to 10 minute intervals.

The oxygen consumption was found by difference between initial and final oxygen concentrations in 25 ml samples of the Ringer's solution surrounding the skin, using the Winkler method for analysis.

Experiments were carried out in duplicate on symmetrical pieces of frog skin (Rana catesbiana) of 24 cm² area. Each piece of skin was clamped between a pair of iso-electric cup electrodes with 60 ml of Ringer's solution on each side. The electrodes were of lead amalgam-lead chloride covered by a surface layer of saline agar. The external circuit consisted of Ringer's solution, the electrodes, a coulometer and a microammeter in series. A potentiometer was connected across the electrode terminals.

Results of a typical one-hour determination at 25°C: The coulometer measured 1.497 coulombs. The average potential for 6 readings was 0.039 volts. The oxygen consumption was 0.239 ml at standard temperature and pressure. The respiratory quotient of the isolated skin was not determined. The highest and lowest respiratory quotients for the intact frog range from 0.94 to 0.72 4 .

The impression is prevalent that administration of adrenalin is harmful in clinical shock. 1 Furthermore, various investigators including Freeman 2 3 have reported that a condition which they claim is analogous to shock can be produced in experimental animals by continuous injection of large doses of adrenalin. However, other workers have been unable to produce shock by prolonged administration of this drug. 4 5 In a recent study of traumatic shock, 6 physiologic doses of adrenalin were shown to have a more pronounced pressor action in the course of shock than before trauma.

In view of the controversy concerning the rôle of epinephrine in shock, the effect of administration of slowly absorbed epinephrine in experimental shock was investigated. Shock was produced in anesthetized cats by exposure and manipulation of the small intestine for 30 minutes. The manipulation consisted of stripping the gut forcefully throughout its length. In the control experiments this procedure produced a marked momentary spasm of the segment of gut stripped. There was considerable oozing of sanguinous fluid and the bowel became purple in color. The visible fluid loss at the time of manipulation never exceeded 5 cc. In the animals receiving adrenalin, the identical procedure resulted in less spasm, no visible fluid loss, and less discoloration of the gut.

In 14 cats, anesthetized with chloralose, 80 mg/kilo, the bowel was manipulated without aseptic precautions, using bare hands, and blood pressure was recorded with a mercury manometer from a cannula in the carotid artery. In 6 control experiments, a fall in blood pressure to shock level (60 mm Hg) was produced in an average of one hour and 33 minutes after beginning intestinal trauma, with a range from 35 to 195 minutes. These animals survived an average of 3 hours with a range from 47 minutes to 4 hours and 23 minutes.

Our data show that injections of thyroxin do not increase the rate of intestinal absorption of thiamin chloride in rats as they do in the case of dextrose and other substances susceptible to phosphorylation. This observation would indicate that phosphorylation plays no dominant part in determining the rate of intestinal absorption of thiamin or that thyroxin has no stimulating effect on the rate of enzymic formation of the pyrophosphate ester linkage. The stimulating effect of thyroxin may be limited to the phosphorylating processes involving the mono-phosphoric acid ester, as in the transport of dextrose across the intestinal mucosa. The conclusion that phosphorylation plays no part in the intestinal absorption of thiamin is supported also by the work of Ochoa, 10 who found that in vitro intestinal tissue, in contrast to liver tissue, possesses little ability to phosphorylate thiamin.

Two observations made during the course of our experiments indicate that absorption of thiamin in the intestine probably takes place by means of simple diffusion. First, the rapid initial absorption of thiamin is succeeded by a much slower rate of absorption, presumably after the diffusion equilibrium between the lumen of the gut and the intestinal mucosa has become established. Secondly, absorption of thiamin is roughly proportional to its concentration in the intestine, as was seen when the dose of thiamin was increased from 100 μg to 1 mg. On the other hand, if the absorption of a substance is aided by phosphorylation, as, for instance, is the case with dextrose, the rate of absorption remains constant and is within wide limits independent of the concentration of the substance which is being absorbed.

Glycogen deposition in rat diaphragm in vitro varies directly with the concentration of glucose in the medium. Insulin catalyzes this process greatly at low sugar concentrations but very little at high concentrations. The bearing of these results on the mechanism of insulin action is briefly discussed.

In previous communications 1 2 the theory was advanced that, after monaster formation, the cortex of the fertilized egg of Arbacia punctulata consists in part of a bound calcium compound. Data were presented showing that this labile combination can be broken down in the living cell by various chemical agents, with the subsequent release of free calcium ions. For detecting calcium release, use was made of the observation that the characteristic red pigment “granules”of the egg disintegrate in the presence of calcium or other divalent cations. 3 The purpose of this paper is to describe briefly two observations which indicate that, in addition to the chemical procedures discussed in the previous papers, simple physical means are capable of breaking down the cell cortex and causing calcium release.

When immature eggs, or mature unfertilized eggs, or fertilized eggs that have not reached the monaster stage of development, are slowly crushed by withdrawing the sea water from under the cover slip with filter paper, considerable flattening of the egg can occur without rupture of the cell membrane. The compression can attain such a degree that the cell in many places is only a few micra thick. The red pigment granules remain intact as long as the cell membrane is unbroken. The moment that the cell membrane ruptures, the sea water and protoplasm intermingle and the red chromatophores disintegrate. The results are quite different when fertilized eggs that have reached the monaster stage (10–12 minutes after fertilization) are compressed in a similar manner. The slightest pressure on this egg causes the red pigment granules to “explode”in the region of the cell where the pressure is applied. The cell membrane remains intact and many of the red chromatophores are unbroken.

Earlier studies in this laboratory demonstrated that warming or cooling portions of the external surface of the ventricles caused characteristic alterations in the T wave of the electrocardiogram. 1 The basis of these T wave changes was found to be alteration in the duration of one or the other of the two components of the electrocardiogram, i. e., of the dextro- or the levocardiogram. Cold prolonged and heat shortened the duration of these components. The following experiments were undertaken to determine the cause of these alterations in the duration of the dextro- and levocardiograms.

Electrograms and monophasic action potentials were studied in 5 dogs by the method previously described, 2 one lead being taken from the anterior surface of the right ventricle and the other from the anterior surface of the left ventricle. Negativity at the lead on the right ventricle gave an upward deflection of the beam. The influence of temperature was determined first on the electrogram, and then on the monophasic action potential. Variations in temperature were produced by applying a small tin chamber to the surface of the heart under one or the other electrode, and circulating through the chamber water at 5°C or 55°C.

The results of heating or cooling the surface of the heart under one or the other electrode are shown in Fig. 1. When the region under the right ventricle electrode was cooled, an upright end deflection was obtained, while when this region was warmed, the end-deflection was sharply inverted. The duration of the whole electrogram was prolonged when the heart was cooled, and was not materially altered by warming. Exactly opposite effects followed cooling and heating under the electrode on the left ventricle. Cooling produced a prolonged downward end-deflection, while heating produced an upward end-deflection of normal duration.

It has not been possible to interfere with the process of reproduction in female rats on an adequate diet by administering various oxidation products of fats, by mouth, subcutaneously, or intraperitoneally. These products included rancid animal fats, their volatile oxidation products with extremely high peroxide content, the unsaponifiable portion of irradiated fats and some aldehydes. Mortality of the young was very great. Unlike mice, rats were not susceptible to the damaging effect of heptaldehyde on reproduction; unless the mothers succumbed to systemic intoxication they bore litters even with serious lesions at the site of injection. An adequate stock diet (Purina dog chow, finely ground) treated with ethereal ferric chloride supported reproduction in the second generation, indicating that the coupled oxidation of tocopherol in the presence of rancid fats requires adequate contact for an adequate time.

The observations that tumors are high in arginase 1 and mono- and polynucleotidase 2 activities, and recent studies with radiophosphorus, 3 4 indicate rapid metabolism of the nuclear substance of neoplastic tissues. However, in these experiments, except those comparing lymphoma with normal lymph node, 4 it has been necessary to contrast cancer cells with various kinds of normal cells of different types. The liver carcinoma which can be produced by feeding azo dyes 5 is admirably suited to such experiments since the analogous normal tissue is readily available.

In Experiment I three groups of stock albino rats were used, one receiving a semi-synthetic diet (dextrin 79, casein 10, butter 5, salts 4, yeast 1, cod liver oil 1), another receiving in addition 0.06% dimethylaminoazobenzene (DAAB), and the third receiving 10% whole dried liver in addition to the basal ration and the azo dye. In Experiment II two groups of stock albino mice were used, one receiving a stock diet, 6 and the other in addition 0.05% p-aminoazotoluene (AAT). The rats were kept on the diet for 5 months and the mice for 7 months before the experiments, at which time all the animals receiving the azo dyes but no liver had developed hepatomata. Of 10 rats receiving both the azo dye and whole liver, only one developed liver tumors, and was discarded.

Each animal received subcutaneously 0.5 cc of a neutral radiophosphate solution. After 24 hours the animals were killed and the livers extracted 2–3 times with cold 0.01–2 N NH₄OH. The entire protein fraction was precipitated with cold trichloracetic acid, and the precipitate washed several times each with cold dilute trichloracetic acid, acetone, alcohol at 70°, and ether, in order to remove all non-nucleic phosphorus. The residue was then wet-ashed, the total phosphorus determined colorimetrically, and the radioactivity measured with a Geiger-Müller counter.

Human blood from which the riboflavin has been removed by irradiation contains some factor which enhances the growth-promoting (acid production) activity of riboflavin on Lactobacillus casei є. In the future, investigators who make determinations of the riboflavin content of blood or other biological materials must take into consideration the presence or absence of this factor.

1. Pregnant mare serum hormone, when injected into normal and hypophysectomized males, causes an elevation in red cell count. Injections of testosterone into castrated females or hypophysectomized males and females likewise increase red cell count. 2. The red cell counts of normal females treated with pregnant mare serum hormone are lowered, those of castrate or hypophysectomized females, similarly treated, remain unchanged. Estradiol lowers the counts of normal females. 3. It seems possible that the gonadal secretions are responsible, to some extent, for the sex difference in normal red cell count encountered in many species of animals.

1. The growth in body weight of female rats thymectomized at the age of 8 days and followed to the age of 6 months is the same as that of sham operated littermate controls. 2. The response of hypophysectomized-thymectomized female rats to a preparation of the pituitary growth hormone is the same as that of similarly treated hypophysectomized littermate controls which had been subjected to a sham thymectomy. 3. The response of plateaued thymectomized female rats with intact pituitaries to a potent growth hormone preparation of the anterior pituitary is the same as that of sham operated littermate controls. 4. Under the conditions of this experiment, therefore, the thymus gland was not necessary either for the growth in weight of otherwise normal animals or for the marked increase in body weight produced by the administration of anterior pituitary growth hormone.

It is now well established that deoxycholic acid is necessary for the absorption of vitamin K. The fact that certain water soluble compounds possess vitamin K activity has raised the question as to whether or not these substances are absorbed from the intestinal tract in the absence of bile. Smith and Owen 1 have shown that oral administration of 4-amino-2-methyl-1-naphthol to patients suffering from chronic obstructive jaundice led to an increase in the level of prothrombin. Similar results were reported by Warner and Flynn 2 on administration of the potassium salt of the disulfuric acid ester of 2-methyl-1,4-naphtho-hydroquinone to vitamin K depleted bile-obstructed rats. The present experiments are in harmony with these two observations, although the compounds used and the experimental technic employed were somewhat different.

Choledochocolonostomized rats were maintained on a low vitamin K diet and were kept in cages with wide mesh screen bottoms to prevent their having access to their feces. Three to 6 weeks following the operation there was a decrease in the prothrombin content of the blood. For the prothrombin estimations, blood was obtained by heart puncture. In this connection it should be pointed out that it is not feasible to obtain blood in this manner when the prothrombin levels are much lower than about 50%, due to the possibility of hemorrhage. The prothrombin levels were estimated according to the method of Almquist and Klose, 3 modified so that only 0.5 cc of blood was required. When the prothrombin level had fallen to about 50%, the compound under test was administered orally. The data are given in Table I.

The data indicate that both phthiocol and 2-methyl-1,4-naphthoquinone are absorbed from the intestinal tract of bile fistula rats, and that deoxycholic acid is not necessary to insure the absorption of these compounds.

Females of Ascaris lumbricoides excrete in their eggs during a 5 days'starvation period at 37°C under aerobic conditions, a noticeable amount of ether soluble material, indicating the presence of a fat metabolism of some intensity. The sum of the ether extracts derived from the body and the eggs of the starved worms corresponds closely to that found in unstarved'worms, showing that during the starvation period no fat had been used for the production of energy.

In 1937 a specific heavy protein was isolated 1 by differential ultracentrifugation from infectious extracts of cottontail-rabbit papillomas. 2 Purified by several cycles of alternate low- and high-speed ultracentrifugation the material sediments in the analytical ultracentrifuge with the sharp boundary characteristic of a single molecular species and with a sedimentation constant of S₂₀° = 265 × 10^-13 cm sec^-1 dynes^-1. Studies on infectivity, 3 complement-fixing capacity, 4 and neutralization 5 with specific immune sera have demonstrated a high degree of uniformity in the biological behavior of the protein and its intimate relation to the papilloma virus. In the present work, further evidence of the homogeneity of the macromolecular protein is furnished by examination of its electrophoretic behavior.

The studies were made on about 150 mg of protein freshly prepared from 350 g of warts from many cottontail rabbits, extracted and purified by 3 ultracentrifugal cycles by the routine procedure previously described.¹ The purified product was dissolved in buffer solution consisting of 0.05 M sodium chloride and 0.05 M sodium veronal-sodium acetate to give a solution of 0.1 ionic strength and adjusted to the required pH with NaOH or HCl. Studies were made on dialyzed solutions at protein concentrations of 4.5 to 2.7 mg per cc. The instrument† employed was of the Tiselius 6 type. Movement of the protein boundary in the 11 cc cell under the influence of constant electric potential at 0°C and changes in boundary shape were recorded photographically by the refractive index method of Svensson. 7

The pH regions available for study of the intact protein are determined 8 by the limits of molecular stability in both acid and alkaline ranges and by insolubility near the isoelectric point. An example of the results obtained in the acid range of solubility is shown in Fig. 1 in the series of curves photographed after successive time intervals over a period of 5.5 hours at pH 3.78.

In recent papers Almquist 1 2 emphasizes the precautions necessary in the care and housing of test animals in order to prevent bacterial K-vitamin synthesis in wet feeds and in droppings, and states that heated diets 3 are not growth-promoting. These reports prompt us to present additional data on this type of diets.

Experiments conducted during the past months have shown that ration K-11 (Table I) containing p-aminobenzoic acid 4 is more suitable than the ration K-7, previously described 3 as being deficient in the rat anti-gray-hair factor, because it permits better growth of the test animals and an earlier incidence of the hemorrhagic diathesis. It was observed also that vitamin K is apparently a growth factor, since chicks lose weight when the deficient symptoms occur and grow at a greater rate on ration K-11 supplemented with vitamin K than on ration K-11 alone.

The type of casein in ration K-11 is an important factor. The SMACO† Vitamin-free Casein proved to be an excellent source of protein in a vitamin K-deficient diet, but Labco‡ Vitamin-free Casein is apparently not entirely devoid of vitamin K, as the hemorrhagic diathesis does not develop in all the test animals reared on this diet in which this brand of casein is used as the basal protein. A similar observation has already been made in a comparative study of rations containing the Labco product and fish meal, respectively. 5

The ration K-11 can be used even when wet. One hundred day-old chicks were placed on a diet consisting of ration K-11 thoroughly mixed with an equal part of fresh ripe bananas. All the birds on this diet showed the typical K-deficiency symptoms within the same period of time as the control birds on ration K-11.

There was no correlation between the blood prothrombin level and a single estimation of the urinary excretion of hippuric acid after ingestion of known amounts of sodium benzoate in 12 patients with liver disorders after treatment with synthetic vitamin K analogues.

A granulomatous fungus infection simulating scrofuloderma was described by Gilchrist in 1894. Since the fungus was seen in the diseased tissues as a budding, yeast-like organism, associated with a skin infection, it was called Blastomyces dermatitidis 1 and the disease blastomycosis. A number of different species of Blastomyces 2 and a number of variously named fungi 3 since reported from cases of North American blastomycosis have been shown to be either slight variations of Gilchrist's organism or to be Coccidioides immitis, 4 5 the causative agent of San Joaquin Valley Fever or coccidioidal granuloma.

South American investigators have described a granulomatous fungus infection as pseudococcidioidal granuloma 6 caused by a fungus first confused with Coccidioides immitis, 7 8 later named Paracoccidioides brasiliensis. 9 Moore, 10 however, showed that Paracoccidioides reproduced in the tissues by multiple-budding, thus indicating its relationship to Blastomyces, but retained the name Paracoccidioides and placed it in his family Coccidioideaceae in equal rank with Coccidioides and Rhinosporidium.

Although the stages in the life cycle of Blastomyces dermatitidis and Paracoccidioides brasiliensis have been studied by numerous investigators, 1 – 11 and Paracoccidioides has been compared with Coccidioides, 8 – 10 12 no detailed comparative study of the various forms of B. dermatitidis and P. brasiliensis has been made. In order to establish the identity of the South American fungus the present study was undertaken.

Seven strains‡ of Blastomyces, isolated from lesions of North American blastomycosis, and 6 strains‡ of Paracoccidioides, isolated from lesions of the South American disease, were compared as to their appearance in tissue, and their growth and microscopic appearance on various artificial culture media both at room and at incubator temperature. Blastomyces dermatitidis was seen in the tissues as single-budding, thick-walled, yeast-like organisms. When diseased tissue or pus from blastomycotic lesions was placed on Sabouraud's glucose agar, after varying periods of time at room temperature, the fungus developed into a cottony-mold-like growth.

In Table I are summarized the number and type of “immunizing”injections administered and the number of survivors of 3 subsequent tumor implants given 1 month apart. The “immunizing”treatment in (1) and (2) killed over half of the animals, most dying of lymphoma, some of toxemia. The injections in (3) and (4) resulted in a mortality rate little more than would be expected in an untreated group in 4 months (9.5%). None developed tumors following injections of nuclei.

Of the 100 mice receiving a single injection of 500 viable lymphoma cells only 2 survived. Of these, one survived 2 implantations before succumbing to pneumonia and the other survived 3 implantations and at the time of this writing is still alive without tumor. Since the number of survivors is so small no deductions can be drawn regarding the efficacy of this treatment as means for “immunizing”the animals to the tumor. In group (2) receiving fragmented cells, 4 of the 43 survivors (9.3%) showed “immunity”to 3 successive implantations. Group (3) receiving tumor nuclei showed 2.4% “immunity”while in group (4) receiving liver nuclei there was 4.8% immunity.

Since no failures to “take”were observed in 2900 control animals inoculated with the tumor, the incidence of immune animals in this strain must be less than .035%. As a result of the treatments described, the incidence of immunity has been raised to 2.4–9.3% (which seems to be a significant increase). The injections of nuclei produce about as much “immunity”in this population of mice as the other two methods used (injection of viable and of ground frozen cells), without the risk of killing a large proportion of the animals entailed in these latter procedures. Furthermore the “immunization”obtained in treatments (3) and (4) must be attributed to a response to nuclear material only.

Attempts to transmit the virus of poliomyelitis, both old and newly isolated strains, to the cotton rats, Sigmodon hispidus cremicus and S. h. texianus and the mice, Microtus californicus and M. montanus, were unsuccessful by the methods employed. The virus, however, could survive in the brain of the Microtus for 3 to 4 days and be transferred to monkeys but not mice. It was found that the convulsive spasms that developed among both inoculated and the normal rats were probably due to a deficient diet and not to a virus disease. Microtus californicus is highly resistant to intracerebral inoculation of the virus of lymphocytic choriomeningitis, M. montanus less so, while the Sigmodon species are more susceptible.

A thermostable enzyme, first noted by Jones 1 to be present in the pancreas, has received recent attention at the hands of Dubos and Thompson, 2 Schmidt and Levene, 3 Kunitz, 4 and Allen and Eiler. 5 Dubos and Thompson found the enzyme to effect the decomposition of ribonucleic acid from yeast. The enzyme was noted to be without action on the following substances: desoxyribonucleic acid from thymus, egg albumin, hemoglobin, Witte's peptone, a number of plant, animal, and bacterial polysaccharides, ethyl acetate, tributyrin, and an ether-soluble fraction extracted from pneumococci.

The enzyme was named “ribonuclease”and claimed not to be a phosphatase. Schmidt and Levene describe the action of the enzyme to be that of a depolymerase, and consider the name “ribonucleodepolymerase”to offer a more appropriate description of the mode of action. Kunitz has isolated the enzyme in the crystalline state, and claims that its mode of action appears to correspond to the nuclease activity described by Dubos and Thompson. Allen and Eiler have shown that the enzyme effects the liberation of an acidic group of ribonucleic acid. Titration data place the liberated acidic group in the range of a secondary phosphoric acid dissociation. An examination of the structures of the known components of the ribonucleic acid molecule shows that the secondary hydroxyl of the phosphate group conceivably may be linked with any of the following reactive groups: (a) the hydroxyl group of guanine or uracil; (b) the amino group of guanine, adenine, or cytosine: (c) the hydroxyl group of position 5 or of position 2 of the ribose; (d) the hydroxyl of another phosphate group. This last possibility, seemingly, is ruled out by the data of Allen and Eiler. However, the indications are that certain characteristics of the action of the enzyme are those of a phosphatase.

The application of spirits of turpentine for 7 days to the nipples and adjoining skin of lactating mice weaned on the 4th day after parturition was shown to retard the rate of involution of the mammary lobule-alveolar systems. Similar applications of turpentine to castrate and normal females failed to stimulate the growth of alveoli. Pseudo-pregnancy was not stimulated in the normal female. It is suggested that the retardation in the involution process is due to the great subcutaneous hyperemia produced by the turpentine applications.

Fresh pregnant cattle pituitary in amounts which will stimulate growth of the lobule-alveolar system in castrate female mice was found to contain insufficient progesterone to give a positive response by the sensitive McGinty technic. Lipid extracts of the AP which stimulate duct growth in the male mouse were also found to be negative for progesterone. These observations are taken to indicate that neither the mammogenic duct nor lobule-alveolar effects of the AP are due to the presence of progesterone.

The synthesis of carbohydrate from glucose by resting yeast is completely inhibited by M/1000 2:4-dinitrophenol. Fermentation, however, is markedly stimulated, while respiration appears to be unaffected. Both synthesis and respiration are inhibited by M/10,000 sodium azide, but a portion of the alcohol formed by fermentation of the glucose is later oxidized. Higher concentrations of azide also inhibit the oxidation of alcohol, thus permitting the accumulation of alcohol as an end product of the utilization of glucose.

In the course of a study of experimental gas gangrene in guinea pigs, the relative resistance of nerves to invasion by Cl. welchii was frequently noted. The literature on the histopathology of gas gangrene of the peripheral nervous system gives few details of conditions within nerves. In one case of human gas gangrene from which we obtained postmortem material, however, examination of microscopic sections of the groin musculature showed a single Cl. welchii lying between axis cylinder and myelin sheath. This nerve was contained within an edematous nerve bundle and the latter was in the midst of a necrotic area. This one observed instance of an organism in this position so emphasized the usual absence of these organisms from very close proximity to nerve fibers in both experimental infection in guinea pigs and natural infection in man, that an attempt was made to induce growth and proliferation of the organisms by depositing them directly within the nerve bundles.

The growth∗ from 5 cc of the supernatant of a 24-hour chopped meat culture of Cl. welchii (strain WX) was suspended in 1 cc of 10% CaCl_2. Of this suspension, 0.1 cc was injected directly into the sciatic nerve of the guinea pig on one side in 6 animals. The needle, 27 gauge, was directed proximally. It penetrated the nerve about 2 cm distal to the sciatic notch and was pushed forward into the nerve for a distance of about 0.5 cm. The object was to deposit the organisms either within the nerve bundle or very close to a nerve bundle. This succeeded in most cases and was checked by following the rupture of epineurium and perineurium caused by the needle, and also by occasionally finding the organisms between nerve fiber and myelin sheath at the place of injection.

The tested living Gram-positive organisms were more susceptible to the antagonistic action of sporogenic aerobic bacilli than the living Gram-negative. In contrast, all tested dead Gram-negative bacteria were susceptible to the action of all sporogenic strains, while dead Gram-positive remained unaffected.

A wide variety of bacterial species and several molds grown in synthetic biotin-free media have been shown to synthesize biotin to a greater or lesser degree as measured by the yeast-growth biotin assay method. This evidence suggests that biotin may be of widespread importance in microbial nutrition.

Anestrus occurred as long as a pellet of testosterone propionate was present in the subcutaneous tissues of normal female rats. No significant changes occurred in the estrous cycles of normal adult female rats when pellets of testosterone propionate were implanted in the spleen in which the normal portal circulation was intact. When the blood from the spleen containing a pellet of testosterone propionate entered the systemic circulation without passing through the liver, estrus was inhibited.

The technical assistance of Ruth Helmuth is gratefully acknowledged.

Measurement of the pyrogenic activity of horse antipneumococcic sera in rabbits will aid in the production of concentrated serum free of chill-producing substances.

1. Extra-cellular respiration was exhibited by seminal plasma of the boar. Oxygen consumption of seminal plasma ranged from 5 to 22% of that of the whole semen. The R.Q. was unity. After the plasma had been held for 5 minutes at 100°C respiration at reduced intensity was observed. Respiration was not exhibited by that fraction of the seminal plasma which passed through a porcelain filter. 2. The metabolic rate of the ejaculate of a cryptorchid boar was of the same order as that of seminal plasma. 3. No errors in measurement of oxygen consumption were detected which could be attributed to the presence of bacteria.

Sulfanilamide is promptly absorbed from the peritoneal cavity in dogs. Following the intraperitoneal injection of sulfanilamide the levels of concentration of the drug in the peripheral blood are not as high as those reported by other investigators following the oral or subcutaneous administration of smaller doses.

Forssmann 1 first used catheterization of the right heart on himself, after exposure of a vein of the arm by a surgeon. Numerous other investigators since have used right heart catheterization for visualization of the right chamber of the heart and pulmonary vascular trees by means of contrast substance. 2 3 4 5 6 7 The introduction of the Robb and Steinberg method, 8 however, renders this method unnecessary for the latter purpose. Collection of right heart blood by catheterization of the right auricle for determining cardiac output in man 9 is mentioned by Grollman, 10 who discredits it because of the possible dangers and numerous misleading factors associated with it. In animal experimentation it is widely used and its innocuity established.

Because it is apparently the soundest method for obtaining mixed venous blood for respiratory gas determinations, and because of the numerous problems of hemodynamics it might help solve, a method of right heart catheterization was developed which attempts to overcome objections to former methods. The principal objections included the possibility of venous thrombi and thrombophlebitis that might be associated with introduction of a foreign body in the blood stream, the formation of thrombi within the catheter, and the psychic effects accompanying the procedure with possible alterations in the cardiac output.

The following equipment was used in our method: a specially made 10 gauge Lindeman type of needle; a 3-way stopcock with a Luer lock, tightly fitting adapter; a No. 8 French flexible radiopaque ureteral catheter with 2 holes, one at the rounded tip and another about 1 cm from the tip. The catheter is silk with a smooth varnish finish. A saline reservoir with rubber tubing and clamp for controlling the rate of flow was also used.

Under the strictest asepsis a nick is made in the skin over the median basilic vein of either the right or left arm after a preliminary infiltration with 2% novocain.

Rats have been fed diets containing 1% cholesterol from weaning throughout their life span. Their growth, health and time of survival have not differed significantly from those of control animals on the same basic diet without the cholesterol.

Of a series of substituted ethanols and ethanol derivatives, only ethylene chlorohydrin, ethylene glycol, and β-hydroxypropionitrile are oxidized by liver preparations containing an active alcohol oxidase.

Analysis of the livers of white rats for arginase activity after the administration of thyroxine have been made. The data show that, although the hormone in the dosage used caused male rats to lose body weight, the arginase content of the livers remained unchanged. Livers from pregnant and nonpregnant female rats given thyroxine contained quantities of the enzyme comparable to those found after feeding diets high in protein or after long fasting. Possible relations of these changes in enzyme concentration to protein catabolism are discussed.

In investigations undertaken by one of us (S.S.K.) it was found that sodium citrate in dilute solution exerts a powerful solvent effect on tertiary lead phosphate, 1 that citrate removes lead ion from solution by the formation of a soluble complex of extremely low dissociation, 2 and that the administration of sodium citrate to lead poisoned rats produced a significant increase in the excretion of lead. 1 The junior author (T.V.L.) independently observed that in a case of plumbism receiving potassium citrate incidental to a chloride excretion test there was a sharp drop in the blood lead level and a rise in the urinary output of lead. (Chart I, No. 7.) These observations led to the present study of the effects of the administration of sodium citrate in plumbism.

Six cases of lead poisoning seen at the Philadelphia General Hospital since August, 1940, provided the basis of this study.∗ All presented clinical evidence of mild to severe lead intoxication and all had abnormally high lead concentrations in whole blood on admission. Each adult received from 2 to 4 g of sodium citrate and the children from 1 to 2 g by mouth 3 times daily. Blood lead concentrations and, in 3 cases, urinary lead excretions were determined before and at intervals during the course of treatment by the method of Letonoff and Reinhold 3 slightly modified. 4

The results are presented in Chart I. There was a rapid disapearance of toxic symptoms without untoward reactions in every instance and the blood lead concentration fell to normal or nearly normal levels, (normal range as determined by this method is from 0 to 0.05 mg lead per 100 g of whole blood). Urinary excretion studies gave variable results with a trend toward increased urinary excretion of lead during treatment.

It is shown herein that infection with B. violaceous may readily occur through various types of surface injury of the feet of animals. It is suggested, therefore, that ingress through external injury forms the usual portal of entry of B. violaceous in the natural infection. These infections are fulminating and usually fatal in a short period of time. In this connection, it appears probable that both the lymph and blood streams may form the routes of host invasion although the lymphatic pathway is emphasized. The lesions produced are similar to those found in the natural infection of carabao as well as those observed in the human infection. Although B. violaceous has in general been regarded as innocuous, it is further stressed herein that certain strains are definitely pathogenic and present evidence of distinct virulence. Furthermore, a likely portal of entry of the natural infection is indicated.

All attempts to grow Tetrahymena geleii in a medium of known composition have, so far, failed. Failure to obtain growth in anything but peptones led Lwoff 1 to formulate the hypothesis that the ciliate requires polypeptides and cannot utilize free amino acids. Earlier 2 he had hinted that the failure of growth in a mixture of silk peptone, fibrin peptone and gelatin, which contains all the known amino acids, is the result of the lack of some supplementary substance. Gelatin 3 will support growth if supplemented with a small amount of yeast extract; indeed, it is stated 4 that transplantable growth is obtained in gelatin plus thiamin and riboflavin. Silk peptone gives slight, but transplantable, growth if thiamin is added. 5

It is clear, then, that the question of the amino acid requirement of T. geleii cannot be answered until more is known of the supplementary needs.

In order to discover the vitamin (or supplement) requirements, a basal medium is necessary. Since it had been found that the ciliate will grow well in a 1% solution of crude casein (Eimer and Amend), a similar solution of Casein-Harris (free from fat and water soluble vitamins) was tested. It gave no growth beyond the first or second transplant.

Slight improvement in growth was noted upon the addition of thiamin to this medium. All possible combinations of thiamin, riboflavin, nicotinic acid, pyridoxin, pantothenic acid, biotin (as a concentrate) and inositol† were added. Growth was better in the media containing riboflavin and pyridoxin in addition to thiamin, but it was slow and never reached the concentration obtained in crude casein. It was evident that some other factor is required for optimum growth. The possibility of toxicity of the medium was eliminated, since good growth occurred upon the addition of 0.1% yeast concentrate (Yeast Vitamin Harris) to the vitamin-free casein, whether or not other supplements were present.

Two grams of sulfanilamide, sulfapyridine and sulfathiazol were ingested in successive experiments by 4 patients from whom bile was draining through tubes inserted in the bile duct after operations upon the biliary tract. Intervals of 3 or 4 days separated the experiments upon each subject. A satisfactory test with sulfapyridine was not obtained in one instance. Specimens of bile were collected for 4 successive 4-hour periods in each experiment. The first period preceded, and the others followed the ingestion of the drug. Samples of urine were collected simultaneously with the bile specimens after the drug had been given, and a sample of blood was drawn at the midpoint of each of these 3 4-hour periods. The free compounds were determined in the specimens of bile by the diazo technic of Bratton and Marshall 1 in the presence of acetone. Since the usual methods for clarifying solutions, including the one used upon bile by Hubbard and Anderson 2 precipitated a rather high proportion of added sulfathiazol, the bile was clarified by the use of 2 successive precipitations with barium—first in alkaline solution as the phosphotungstate and then in acid solution as the sulfate. The sulfonamide drugs could be determined with an accuracy of about 5%, or, if present in very low concentrations, to the nearest 0.1 mg per 100 cc by this procedure. Control specimens, analyzed as a part of each experiment, gave no red color by the technic. The concentrations of the drugs in the urine were determined after dilution, one part to 100, by the same diazo technic. Blood analyses were carried out as described by Bratton and Marshall. 1

The results upon the blood and bile of different subjects were qualitatively similar, except for such variations in actual blood concentrations as would be expected from the varying ease of absorption of the different compounds studied.

Partial adrenalectomy increases the susceptibility of guinea pigs and rats to anaphylactic shock. 1 2 3 The lethal dose of antigen has been found to be inversely proportional to the degree of adrenal insufficiency. Wolfram and Zwemer 4 report that “cortin”protects normal sensitized guinea pigs against fatal anaphylaxis. In the dog the severity of anaphylactic shock is said to be lessened by the previous injection of cortical hormone. 5 Perla and his coworkers 6 report an increased resistance in rats to histamine shock by the use of “cortin”. These observations parallel others in which cortical extract is said to improve the lowered resistance of adrenalectomized animals to bacterial infection. 7 8 This has been explained as due to increased antibody production, especially in the earlier stages of the immunizing process. 9

Since anaphylactic shock, whether actively or passively transmitted, is accompanied by a fall in serum complement, 10 it is of interest to see whether the reported protective effect of cortical hormone can be attributed to an alteration in the titer of the serum complement.

Method. Healthy adult guinea pigs were kept on a high vitamin C diet for several weeks prior to use, in order to exclude any effect of vitamin C deficiency on the level of serum complement. 11 Blood for titration of complement was obtained by cardiac puncture, with or without ether anesthesia. (In a series of preliminary experiments the use of ether anesthesia did not change the complementing activities of the sera. This confirms the observations of Ecker. 12 ) The method of titration of complement was exactly as previously reported. 13 Sera were diluted 1:30 with Brooks”solution and titrated immediately after dilution for the point of initial hemolysis.

Two different preparations of cortical hormone were used: (1) adrenal cortical extract in 10% alcohol (50 dog units per cc) “Cortin”† and (2) desoxycorticosterone acetate in sesame oil (5 mg per cc) “Percorten.”

Nelson 1 first reported the cultivation of Trichomonas foetus in chick embryos after inoculations of 1 cc of a culture into the allantoic sac. The eggs were opened 3 to 4 days after inoculation. Allantoic fluid from the most heavily infected embryos was used to carry the protozoan through 14 subcultures. Later, Levine, Brandly, and Graham, 2 following the same procedure, carried T. foetus through 23 passages and found that 12-day embryos were better than others in an 8- to 16-day series tested. Hogue 3 inoculated T. foetus into the allantoic sac of developing chick embryos and allowed the embryos to hatch or die. In one of 4 chicks that hatched, the protozoa were found in the yolk sac, esophagus, intestines, gall bladder, and cloaca 2 days after hatching. The other 3 gave negative results on fecal examinations. In embryos that died after inoculation, the trichomonads were also found in the digestive tract, yolk, and gall bladder.

The present report concerns a comparison of the growth of bacteria-free cultures of Trichomonas foetus 4 and Trichomonas vaginalis 5 in chick embryos. Also, successful subculturing of T. vaginalis in chick embryos is recorded for the first time.

After a few preliminary trials, T. foetus was established in chick embryos and was carried through 14 passages in 78 days. The inoculations were made into the allantoic sac as described below for T. vaginalis. The organisms seemed to grow better after the first few subcultures. Seven- to 14-day-old embryos were employed and were inoculated with 0.1 cc to 0.4 cc of trichomonas-containing material. The small inoculum may explain the slight difficulty encountered in establishing the infection. The eggs were opened from 4 to 7 days following inoculation. Observations and experiences were essentially the same as those already reported by others. Examination of the different egg parts showed that the protozoa developed in the allantoic fluid or the amnionic fluid, but were not found in the egg white, the yolk sac, the actual embryo or its blood stream.

Liver injury, mainly in the form of acute diffuse necrosis combined with fat infiltration, has occurred irregularly in young rats fed a diet devoid of vitamin B (casein 18%, sucrose 68, melted butter fat 8, cod liver oil 2, salt mixture 4) and supplemented with thiamine, riboflavin and pyridoxine. 1 In some of these livers there was diffuse periportal fibrosis. Rats fed the same basal diet plus yeast, or concentrated yeast extract, were free from any pathological changes in the liver. Later, the occurrence of cirrhosis of the liver on a nutritional basis and its prevention similarly by the addition of yeast to the diet were reported in rabbits 2 and in guinea pigs. 3

The unpredictable and irregular incidence of liver injury in rats has been indirectly regarded as a basis for the assumption that the proper experimental conditions for the regular production of liver injury have not been provided by the experimental technic used.

In view of the well known lipotropic activity of casein, 4 the high proportion (18%) of casein in the basal diet was considered to be possibly one important factor which might counteract other conditions that would be conducive to liver injury. Consequently, rats weighing between 100 and 250 g were put on a diet that had the following composition: casein 10%, sucrose 64, lard 20, cod liver oil 2 and salt mixture 4. This modified basal diet was supplemented with thiamine, riboflavin, pyridoxine and pantothenic acid. In this group of rats the incidence of liver injury rose from an irregular occurrence, as it was in the rats on the original diet (with casein 18% and butter instead of lard), to a regular complication. Necrosis with or without cirrhosis and cirrhosis without necrosis were observed in rats that died between the 100th and 150th experimental days or were killed on the 150th day.

Vitamins B₁ and B₆ have an inhibiting influence on the development of Eimeria nieschulzi infections in rats on a special ration when administered by a parenteral route, one at least as striking as when the vitamins are fed.

The intravenous injection of Koagamin and oxalic acid in the doses used in this study had no effect on the coagulation time in 5 cases of hemophilia.

Grateful appreciation is expressed to Dr. John Lawrence for criticisms and suggestions.

The results obtained by Boyden 1 with the Libby photronreflectometer 2 indicate clearly the value of this instrument in studies of precipitin. The instrument affords an accurate measurement of the precipitate formed by antigen and antibody. Light rays passing through the turbid system are reflected by the suspended particles, and falling upon a photronic cell generate a current of electricity which is recorded by a galvanometer. The galvanometric readings, obtained with doubling dilutions of antigen and a constant amount of antiserum, furnish the points for a curve, the area under which is used to indicate the total reaction between the antigen and antiserum. A comparison of the areas of the homologous and heterologous reaction-curves is used herein to indicate the chemical similarity. In the interfacial tests, relationship-values were determined in the manner described by Boyden, 3 the end-point being the all-important factor.

The graphs show the results obtained when several antigens were tested with a diluted (1+2) anti-leghorn serum. Each antigen represents the pooled sera of from 4 to 95 birds, with the exception of turkey-buzzard which is the serum of one individual. In a similar manner the same antigens were tested with the precipitating antisera to the serum-proteins of turkey, guinea hen, duck, and pelican. The table shows the values obtained by the interfacial (ring) and nephelometric methods.

The injection of a mixture of antigens, such as serum, may result in the production of: (a) antibodies to one fraction alone; (b) antibodies to several or all fractions, with more to one fraction than to another. The fractions are rarely present in equal amounts, so the interfacial endpoints may, in some cases, be the result of one fraction and its antibodies, and in other cases the result of another fraction with its antibodies.

Adult male albino rats administered a vitamin B-complex deficient diet over a period of from 6 to 13 weeks failed to develop histologic evidence of hepatic cirrhosis, in spite of the fact that they did develop the usual signs of B-complex deficiency. Data as to food and water intake as well as weight changes are presented.

The effect of pantothenic acid in cure of nutritional achromotrichia is difficult to assess at present because other factors are clearly involved. Certain liver extracts providing as low as 40 μg of pantothenic acid per day caused complete remission, while the effect of synthetic d-calcium pantothenate was negligible at best. The addition of d-calcium pantothenate to extracts low in this factor, however, increased their anti-graying potency. Age or weight increase of animals did not seem to influence the curative effect of supplements for graying.

Although rats were raised to maturity on highly synthetic diets supplying adequate d-calcium pantothenate, successful matings were not observed.

During the course of observations on mice which had received injections of various estrogens for prolonged periods an enlargement of the bile ducts was observed in many animals. In untreated mice the bile ducts, cystic duct, common duct and its branches, were small thread-like structures. Occasionally a slight cystic distension of the common bile duct was apparent at autopsy. The walls were delicate and when slightly distended almost transparent. Histologically the ducts had a thin and irregular muscularis, and a well-developed mucosa. The tall columnar epithelium was moderately folded on the loose underlying tissue of the lamina propria, and a few small glands extended to the serosa, especially in the common bile duct (Fig. 3).

The bile ducts of many mice which had received estrogens were grossly thickened to several times the size of the ducts of the untreated (Fig. 1). They are also rather rigid, white and somewhat nodular. Enlargement was usually greatest at the points of junction with the branches leading to the several lobes of the liver. The main duct tended to become smaller as it approached the gut. The cystic duct was usually thickened up to the neck of the gall bladder. The gall bladder was involved in only a few mice and in these it was reduced in size and histologically resembled the upper end of the cystic duct.

Microscopic examination of the enlarged ducts of the estrogen-treated mice uniformly showed an extensive increase of the epithelial folds and gland-like processes which completely penetrated the thickened mucosa, the muscularis and frequently projected to the serosa (Fig. 2). The epithelium consisted of tall columnar cells which showed slight mitotic activity. In many ducts 2 types of cells were found, tall cells with an eosinophilic cytoplasm and cells with a clear cytoplasm.

Pulmonary circulation time is prolonged in humans whose internal temperature is reduced toward 85°F when exposed to low environmental temperatures. It is suggested that diminished output of the right heart is a large factor. Correlations between fall in rectal temperature and prolongation in circulation time exist only when the femoral vein is the site of injection. Shivering shortens circulation time or minimizes prolongation due to hypothermia.

The celiac ganglia are anatomically and functionally related to the splanchnic nerves. The peripheral links in splanchnic efferent conduction pathways are made up of celiac ganglion cells. Splanchnic nerve components, however, are not the only nerve fibers which terminate in the celiac ganglia. In a recent experimental anatomical study, terminal branches of axons have been demonstrated in preparations of the celiac ganglia of the cat in which all the splanchnic nerve fibers which enter these ganglia had undergone degeneration, following bilateral section of the splanchnic nerves. Intact nerve fibers also have been demonstrated in sections of the distal segments of divided mesenteric nerves arising from the celiac plexus, after the fibers separated from their cells of origin had undergone degeneration. 1 These findings support the assumption that axons of enteric origin enter the celiac ganglia. If such fibers effect synaptic contacts with celiac ganglion cells, they might constitute the afferent limbs of reflex arcs with central connections in the celiac ganglia. The present investigation has been undertaken to test the hypothesis that reflex reactions can be mediated through the celiac plexus in the absence of intact nerve fiber connections with the central nervous system.

Methods. The experiments have been carried out on cats under nembutal anesthesia. The splanchnic and vagus nerves were divided bilaterally and the intestine transected in the lower portion of the jejunum. In one series of experiments, the common bile duct was cannulated and the drops of bile recorded. After the rate of flow was established, mesenteric nerves were stimulated either by means of a faradic current or distention of the ileum and colon. In another series, a balloon was placed in the proximal segment of the intestine and a kymographic record taken of the intestinal motility.

The effect of sulfanilamide and sulfapyridine on some of the bacterial infections has been so striking that these drugs have been tried on many others, including some of the malarias, both of man and of animals. The results obtained have been of considerable interest, and not the less so because they have varied a great deal with the species. Coggeshall, 1 for example, found that sulfanilamide was effective in eradicating Plasmodium knowlesi infection in monkeys, but was apparently without any action on Plasmodium inui in the same host. He suggested that the difference might be due to the much more rapid metabolism of the former species. This seems probable in view of the generally accepted idea that sulfanilamide is effective because oxidation products are formed from it which destroy catalase, and hence interfere with the metabolic activities of the cell. Since the malaria parasites are present in the blood stream they are presumably more exposed to this effect than if they were segregated somewhere in the tissues.

Against the malaria parasites of man sulfanilamide and its derivatives appear not to be of great value, although the reports are conflicting, and there seems to be but one report of its use in avian malaria. Coggeshall 2 states that when administered to chicks infected with Plasmodium lophurœ and canaries infected with Plasmodium cathemerium it was without noticeable influence. It has also been used on Hemoproteus columbœ, but failed to exert any effect (Durand and Villain 3 ).

Since it is well-recognized that the various species of malaria plasmodia react quite differently to even the same drug, trial of sulfapyridine and ssulfanilamide on some of the other species of avian malaria has been uundertaken. So far all the tests have been made on blood-induced infections because such infections are more easily produced in the numbers required for extensive experimenting, and also because mosquitocs have not been available.