Experimental Biology and Medicine

Sulfanilamide may not be used as a measure of total water in the dog.

Weanling rats receiving a diet severely deficient in choline required approximately 5 mg of choline chloride per rat daily to prevent the fatal hemorrhagic kidney disease. Choline deficiency was produced within 7 to 14 days at any time during the rapid growing period when choline was omitted from the diet. Weanling rats fed a stock diet for periods of 3 or 5 weeks were somewhat less susceptible to choline deficiency than rats fed the diet low in choline and supplemented with 20 mg of choline chloride daily for similar periods.

The use of diffractograms in the study of molecular structure has been discussed extensively by Clark. 1 Numerous studies on bone by this technic have confirmed that there is a characteristic pattern most nearly agreeing with the type pattern of dahlite. Not many reports have given consideration to the possibility that this characteristic pattern may undergo physiological alteration. Roseberry, Hastings and Morse 2 described briefly some age changes in cultures of osseous tissue. Clark and Mrgudich 3 described characteristic changes in rickets, quantitatively related to the degree of severity.

There was no fundamental change in chemical composition, since the lattice spacings were not altered. Both inorganic and organic elements were disorganized as evidenced by the much darker central area with its sharply defined boundaries and by the extension of the orientation arcs, sometimes forming a complete circle.

Several other reports have been made of qualitative alterations in diffraction patterns of bones under various experimental conditions. 4

In an effort to study the influence of healed rickets on the subsequent behavior of bone, our laboratory first made a study of mechanical efficiency, already reported. 5 A summary of findings in the first 5 series of those experiments is presented in Table I. From this it will be seen that in all groups on a rachitic diet for 2-3 weeks, there were no significant differences in mechanical efficiency as manifested by breaking stress, regardless of the duration of the recovery period. Significant differences were seen only when the period of rachitic feeding was extended to 6 weeks.

These same bones were used in the present study. Sections of cortex were ground from the tibial mid-shaft adjacent to the point of fracture.

The absence of renin in the aglomerular kidney of the midshipman, a marine fish, cannot now be construed as suggestive evidence of the probable formation of renin by the glomerular or arterial component of a kidney because the glomerular kidneys of other marine fish studied, also lack renin. The presence of renin in the glomerular kidneys of the catfish and carp, however, suggests that the kidney of most fresh water fish contains renin. Just why the kidney of marine fish, whether glomerular or aglomerular, should be devoid of detectable renin and the kidney of fresh water fish richly supplied with this same substance is a question which cannot be answered at this time. As has been noted, already, there is evidence that the tubular function in the kidneys of these two varieties of fish is probably not identical.

It is well known that various narcotics and anesthetics have a depressing effect upon respiration but this has not been systematically investigated as a function of temperature. Thus, while something is known of the influence of this class of drugs on the absolute respiratory rate, little information is available for changes in the relative respiratory rate.

The newt, Triturus viridescens. was used in the present experiments both because it is a poikilothermous animal and therefore useful for temperature analyses and because its respiration has already been investigated by means of a Warburg apparatus suitably modified for this purpose.

The minimum dose required to maintain newts immobile for 24 hours under the conditions of temperature variations was determined experimentally for two suitable drugs. After preliminary anesthesia for 15 minutes in a 1-10 dilution of a saturated aqueous solution of chloretone, animals were transferred to modified Warburg vessels containing 1 cc of a 1-20 dilution of a saturated solution of chloretone. Comparable fluid was introduced into one unit serving as a thermobarometer. Nembutal (sodium pentobarbital) produced the desired effect with an intraperitoneal injection of 0.2 cc of a 0.6% solution.

Thermal adaptation and the procedure for measuring oxygen consumption followed that described by Pomerat and Zarrow. 1

All data reported were based on animals which remained immobile during the entire period of measurement and which recovered from the anesthetic at its conclusion.

Arrhenius plots of the data for oxygen consumption during chloretone anesthesia yielded a temperature characteristic (μ) of 17,200± (Fig. 1). This is the same value as that found for unan-esthetized animals by Pomerat and Zarrow 1 and for newts studied in relation to their endocrine system by Pomerat. 2 A similar treatment of the values obtained for 10 different animals under nembutal anesthesia is represented in Fig. 2. A distinct shift in the μ. is seen to have occurred.

Information relative to the physical character of the papilloma virus protein 1 has been obtained by means of ultracentrifugation, 2 electrophoresis, 3 , 4 diffusion and viscosity. 2 Recently, more direct studies of this animal virus have been undertaken with the electron microscope. In the present paper are described the preliminary observations dealing chiefly with data gathered with this instrument relative to the appearance and size of the virus.

Papilloma virus protein isolated in 0.05 M phosphate buffer pH 6.5 by previously described ultracentrifugal procedures was diluted with water to concentrations of the order of 0.05 to 0.5 mg per cc. The salt concentration in the final solutions was approximately 0.005 M. Preparations for study were made by pipetting the solutions onto collodion membranes supported on 200-mesh wire gauze. Free fluid was removed with the pipette, and the resulting thin film allowed to dry in the air. For comparison, examinations have been made also of mixtures of papilloma and tobacco mosaic viruses† the latter in concentration of about 0.4 mg per cc of the mixture.

The essential findings are illustrated in Figs. 1 and 2. In Fig. 1 are shown the results obtained with papilloma virus alone. Repeated micrographs of 3 different batches of the purified protein have resulted consistently in observation of the circular images shown in Fig. 1. When the protein concentration was kept in the region of approximately 0.1 mg per cc, the images were for the most part single, though grouping was frequent as seen in Fig. 1. In preparations of higher concentration, large groups of particles occurred and fewer single images were seen. A regular arrangement of images in the groups was not observed.

In our experiments, administration of thyroid extract to normal guinea pigs greatly enhanced their sensitivity to histamine shock. We believe that the enhanced sensitivity to histamine which follows administration of thyroid extract explains the enhanced anaphylactic response observed after administration of this hormone.

Administration of thyroid extract to normal guinea pigs led to marked decrease of the ascorbic acid content of the adrenals. Depletion of the ascorbic acid stores of the adrenals of guinea pigs leads to depletion of their cholesterol and cortin contents. We suggest that the enhanced sensitivity to histamine observed in our guinea pigs is due to depletion of the cortin content of their adrenals.

One percent of an aqueous-alcoholic yeast extract protects rat skin against the toxic effects of phenylmercuric nitrate in a dilution of 1 :100.000 as determined by respiration measurements. A similar amount of yeast extract does not impair the germicidal efficiency of phenylmercuric nitrate against S. aureus in dilutions as low as 1 :500,000 under the test conditions. Protection of host tissue against toxic action of germicides with maintenance of germicidal properties is thus suggested.

Yellow O.B. (1-0-tolylazo-2-naphthylamine) is not a carcinogenic substance.

Confirming previous observations 1 on nutritionally dystrophic rabbits and rats, Houchin 2 recently reported the oxygen consumption of dystrophic hamster muscle to be much above that of normal muscle. It was also demonstrated that within 27 hours after administration of α-tocopherol acetate to the dystrophic hamster, the Qo₂ of the muscle was reduced toward normal value without significant regeneration of normal muscle status as indicated by a persistent high chloride content.

These observations naturally suggested that α-tocopherol may play a role in the normal processes of muscle metabolism. The data in this paper establish the fact that α-tocopherol phosphate† acts in vitro on muscle cells from vitamin E-deficient animals to lower the excessive oxygen consumption toward the. normal.

Vitamin E-deficient rabbits, hamsters, and adolescent rats were studied. The oxygen consumption of muscle slices was determined in the usual manner in the Warburg apparatus. The control medium was a modified Locke-Ringer's solution; to prepare the experimental medium 5 mg α-tocopherol phosphate were dissolved in a few drops of water and made up to 100 cc with the Locke-Ringer's solution. The solution was well shaken immediately before the required amount was measured out. The results are expressed as Qo₂ (cubic millimeters of oxygen per milligram dry weight per hour).

As seen in Table I the oxygen consumption of muscle from E-deficient rabbits and hamsters in the medium containing α-tocopherol phosphate was 41.3% and 35.7% lower than that of slices from the same muscle in the unsupplemented medium. The oxygen consumption was thus considerably reduced toward that of normal animals.

The oxygen consumption of the muscle of control animals was not altered beyond experimental error by the medium containing α-tocopherol phosphate. When the Qo₂ had already been lowered by previous administration of α-tocopherol to dystrophic rabbits, the addition of a-tocopherol phosphate to the medium caused no significant change.

The use of pectin as a blood substitute in the treatment of patients in shock has been proposed. 1 Since the rationale for the administration of pectin is to increase the plasma volume, information regarding the effect of this material upon the plasma volume is desirable. The present investigation was undertaken to determine the influence of the intravenous injection of a 0.75% solution of pectin† upon the plasma volume, and to compare the results with similar observations following the intravenous injection of physiologic saline.

Nine male patients, who were normal with respect to the cardiovascular system were studied. The plasma volume was determined by the method of Gibson and Evans 2 as modified by Gibson and Evelyn 3 using “Evans Blue” dye (T-1824). In 5 patients after the initial determinations were made, pectin solution was injected intravenously at a rate of 10 cc per minute. In 2 patients dye was reinjected and the plasma volume was redetermined immediately upon completion of the pectin infusion. In 3 patients the second plasma volume determination was made 4 hours after the end of the pectin infusion. In all cases a final determination was made at the end of 24 hours. Similar determinations at corresponding time intervals were made in 4 patients following the intravenous injection of 1000 cc of physiologic saline.

Results. Table I shows the results obtained following the injection of an average of 690 cc of pectin in 5 patients as compared with the results in 4 patients each of whom received an intravenous injection of 1000 cc of physiologic saline. In the first 2 cases the plasma volume immediately following the injection of pectin was increased by an amount corresponding to the volume of pectin solution injected.

1. Pyridoxine-deficient dogs excrete small amounts of a compound which can be converted to a green pigment by ferric ammonium sulphate. 2. Pyridoxine-deficient rats do not develop as profound anemia as the deficient dogs.

Seven species of avian malaria plasmodia have been successfully preserved for periods up to 90 days by low-temperature freezing. Very rapid freezing” and thawing seem of more importance than the occurrence of fairly considerable temperature variations. For maintaining stocks of malaria for research or class use this method of preservation has much potential importance. It is probable that all species of plasmodia can be preserved in this way equally well. And it does not appear that different stages of the parasite are much differently affected.

Developing chick embryos are now used extensively for the study of viruses and rickettsiae. At present they are used in large numbers for the preparation of vaccines against virus and rickettsial diseases. However, it has always been extremely difficult to sterilize thoroughly the eggshell with ordinary chemical disinfecting agents. In 1939 Penna 1 described a technic in which he employed an oxyacetylene torch for burning the eggshell open while the egg was rotated slowly in an adjustable clamp turned by an electric motor. Penna's technic is satisfactory except that it is too slow. The purpose of this report is to describe a modification of Penna's apparatus which permits a considerably faster removal of embryos under sterile condition.

The apparatus is illustrated in Figs. 1, 2, and 3. The eggs are handled in groups of four carried in shallow metal trays (A). The flame of an oxyacetylene torch (B) is applied at the assumed position of the air sac's margin as each egg is made to rotate through one complete revolution; the optimum rate of turning is dependent on the resistance to burning shown by the shells of a particular lot of eggs, but is usually of the order of 45 rpm. The metal trays serve to retain the embryonic, fluids which occasionally issue forth when a shell cap breaks loose while being flamed. They also perform the same function while the caps are being displaced with a sterile instrument and while the embryos are being removed. The trays and holders can be quickly rinsed in running tap water and then put back into service.

The details of the holders and trays can be seen in Fig. 1. Each holder (C) consists of a 1 5/8- in. length of heavy rubber tubing into one end of which is inserted a 1-in. length of brass tubing (D).

(1) The median lethal doses of Dicumarol have been determined intravenously or by mouth in mice, rats, and guinea pigs. (2) Death uniformly occurs in rabbits with intravenous injection of daily doses of 1-2 mg per kg; in dogs with oral administration of daily doses of 5-50 mg per kg; and in mice and rats with the feeding of 0.01-1% Dicumarol in food. The majority of rabbits can tolerate daily doses of 0.1-0.5 mg per kg by vein for 6 weeks, and a few mice and rats can survive 30 days on a diet containing 0.005% Dicumarol. (3) Most animals dying from Dicumarol develop hemorrhage into various tissues and organs, and pulmonary edema. Central necrosis of the liver has been observed in about one-half of the rats examined, and occasionally in rabbits, mice, and dogs.

The anesthetic activity in mice of seven new 5,5-dialkyl-barbituric acids has been studied. Intravenous anesthetic doses, intraperitoneal anesthetic doses, intraperitoneal lethal doses, and duration of action were measured.

Rapid recovery from severe carbon monoxide poisoning under X-ray treatment 1 , 2 and spectrographic evidence of the reduction of blood CO after X-ray treatment 3 have previously been reported. A recent publication has described progressive discontinuous stages of decreased resistance to CO among young rats and rabbits during the first few days after birth. 4

Some attempts have lately been made to determine the factors responsible for these phenomena. Each of several kinds of effects has been considered and there may be others not yet investigated.

The release of reserve red cells from the spleen in response to X-ray injury to circulating cells has often been described, 5 but a latent period, or lag, of 6 hours or more is associated with this process. In CO recovery the effect must be maximal within so short a time after the onset of asphyxia that the release of this reserve seems to be ruled out.

Possible recombination of CO and O₂ in the blood and tissues to form CO₂ ereduce the amount of CO present was checked in 12 experiments carried on in CO and O₂ saturated water at body temperature. In none of these tests was the amount of CO₂ (determined as carbonate) significantly increased by X-ray exposures of 500-2,000 γ . Actual differences in cc of standardized HC1 used were 0.4-0.55; 0.45-0.5; 0.07-0.27; 0.35-0.3; 0.45-0.42; 0.4-0.25; 0.18-0.1; 0.1-0.15; 0.2-0.25; 0.1-0.05; 0.2-0.15; 0.3-0.41, an average of 0.09 when a difference of 0.2 might be significant. Dr. Daniel Mazia and Dr. Lloyd B. Thomas kindly advised me in this work. Dr. L. J. Stadler, of the U. S. D. A. and the Department of Field Crops, University of Missouri, generously provided the X-ray services for the work discussed here.

3,3′-methyIene-bis-(4-hydroxycoumarin), Dicumarol, was shown in our experiments to be able to greatly reduce the incidence and degree of thrombus formation following the intravenous injection of Monolate in dogs. In dogs and monkeys some signs of necrosis of the liver were found in a number of animals, but since most of them suffered from a severe anemia, the significance of this finding is questionable. The livers of guinea pigs treated with the sweet clover factor showed changes as they occur in vitamin C-depleted animals. Intravenous glucose tolerance and bromsulfalein retention tests failed to show liver damage in animals not suffering from bleeding due to the administration of the drug. While this is in agreement with clinical findings, further studies of the physiology of this substance with particular reference to liver function appear indicated.

Our present experience shows that there is a considerable difference in tolerance between various species of animals and also among the individuals of the same species. The observation that single blood transfusions in dogs in a state of extreme depression of the pro-thrombin level were of only temporary benefit, is paralleled by similar clinical findings 11 , 8 and calls for particular care in avoiding excessively high dosage.

1. No significant difference was observed in the length of time required by diabetic and non-diabetic rats on a thiamine-free diet to develop signs of marked deficiency (39 as compared with 41 to 45 days respectively). Following treatment with thiamine, the diabetic rats recovered as quickly as did the non-diabetic animals. 2. During the period of deprivation, the glycosuria of the diabetic rats diminished, presumably because of the lowered food intake. However, urinary sugar tended to reappear at the final stage of deficiency at the time of “polyneuritis”. Carbohydrate tolerance in both the diabetic and non-diabetic rats, as judged by glucose tolerance tests, showed little change until signs of marked deficiency appeared, when there was impairment of tolerance. 3. Following-deprivation, a thiamine intake 4 to 8 times the maintenance dosage appeared to improve carbohydrate tolerance.

Alkali-treated bovine serum albumin when administered by vein or by other parenteral routes is not toxic to human beings, dogs, rabbits, guinea pigs, or mice. It possesses little or no antigenicity. It is capable of raising and maintaining the blood pressure of dogs subjected to severe hemorrhage. These facts suggest that alkali-treated bovine serum albumin may prove useful as a substitute for blood in man.

Sodium nitrite, when administered orally 4 mg per 100 g or intraperitoneally 2.5 mg per 100 g, reduces the arterial pressure of unanesthetized hypertensive rats.

In patients with pernicious anemia, the excretion of riboflavin, both before and after oral administration of 5 mg of the vitamin, was of the same order as that eliminated by healthy-individuals. There is, thus, no indication of impaired absorption of this compound by such subjects as a result of their lack of hydrochloric acid.

Arthritis of mice, caused by pleuropneumonia-like microorganisms has been treated with gold sodium thiomalate, sodium thiomalate, the disulphicle form of sodium thiomalate and sodium succinimido-aurate. The compounds containing gold, gold sodium thiomalate and sodium succinimido-aurate, were effective in curing this disease, the others had no therapeutic effect. Sulfur alone or in sulfhydryl linkage is not necessary in order that gold-containing compounds have a therapeutic effect.

In 9 children the concentration of amino acid nitrogen in the plasma decreased on the average of 0.38 mg per 100 ml following anesthesia with a further decline averaging 0.46 mg per 100 ml during a surgical procedure. In 5 of the 9, the plasma amino acid concentration was lower 2 hours after operation than at the conclusion of the operative procedure.

The pattern of change of plasma amino acid concentration in one patient with chronic hypoaminoacidemia was similar to those with normal concentrations preoperatively.

Additional studies are being prosecuted to elucidate an explanation of the factors causing the observed changes.

Mall 1 , 2 appears to have been the first to record the portal venous pressure in animals under experimental conditions. The later determinations by Burton-Opitz 3 , 4 , 5 were shortly followed by Tiger-stedt's 6 records. These observers used the technically difficult T-cannulation of the portal vein, although this itself probably interfered with the normal pressure under investigation. In the experimental animal, we 7 have employed cannulation of the divided inferior mesenteric vein, cannulation of a divided splenic vein (as suggested by Bayliss and Starling), and T-cannulation of the portal vein itself. The height of a column of physiologic saline solution above the portal vein sustained in a vertical tube attached to the cannula was taken as the portal pressure. In our animal, the portal pressure was always about 10 cm saline higher than the venous pressure simultaneously determined in the lower extremity. This communication is intended to record a series of portal venous pressures determined at laparotomy in man.

Rousselot 8 measured the portal venous pressure in 4 patients with Bantis' syndrome of congestive splenomegaly without cirrhosis and observed the presence of portal hypertension—the pressures varying between 330 and 465 mm.

Method. A modification of the method of Griffith, Chamberlain, and Kitchell 9 was used. The manometer consists essentially of a vertical glass tube with a T-cock at the lower end mounted on a firm base. One horizontal limb of the cock permits filling the manometer with saline to a level above the expected venous pressure. The other limb is attached by sterile rubber tubing to a glass adapter and hypodermic needle which are filled with saline solution. With the abdomen open, the needle is inserted into an omental vein by the surgeon, whereupon the T-cock is turned by the observer to allow the vertical manometer to communicate with the vein.

In a series of studies it was shown that convulsions induced by metrazol and other convulsant agents or by electroshock as well as insulin hypoglycemia are accompanied by an increased excitability of autonomic centers at the hypothalamic and medullary level. 1 It was thought that this action is of greatest importance for the shock therapy of psychoses and may explain the fact that these procedures have similar therapeutic effects although their action on the cortex as measured by the electroencephalogram is quite dissimilar. 2

It seemed probable that hypoglycemic coma and convulsions may have in addition to their temporary influence on the cortex as revealed by the electroencephalogram far reaching effects on cortical processes which would be detected best by chronic experiments. Therefore the effects of these procedures on the conditioning process were studied.

The experiments were performed on 17 rats which received as an unconditioned stimulus a shock inducing them to jump from a compartment A across a small partition into the compartment B. This reaction was established by a few shocks. Thereafter a bell was sounded 2 seconds prior to shock. The sound continued during the shock. After the conditioned response (C.R.) was established at nearly 100% for 1 to 3 days the C.R. was inhibited by lack of reinforcement. When the C.R. response had either completely disappeared or was present only in 10-20% of the tests the rats were subjected either to metrazol convulsions, electroshock or insulin coma. Whereas control animals not subjected to these procedures showed no spontaneous recovery of the C.R. it was observed that the experimental group treated with coma or convulsions regained the inhibited C.R. in spite of continued lack of reinforcement. The duration of this effect was variable and depended on the number of “treatments” and in the case of insulin probably on the depth and the duration of the coma.

Whether the urea effect is an enhancement of the sodium sulfadiazine effect alone, or a neutralization of the methionine effect, or both, cannot be established from this preliminary data, although we are investigating this point further. However, under the conditions of our experiment, the following conclusions seem to be justified:

1. With the concentration of sodium sulfadiazine used, approximately 80% inhibition of growth of E. coli in synthetic medium occurred. 2. In the concentrations used, methionine and urea alone, or in combination, displayed no effect on the growth of the test organism. 3. Methionine almost completely inhibited the bacterio-static effect of sodium sulfadiazine, thus confirming previous reports. 4. Urea and sodium sulfadiazine together showed a greater growth inhibitory effect than the latter alone. 5. Urea neutralized this inhibitory effect of methionine on sodium sulfadiazine and in conjunction with the sulfonamide, even though in the presence of methionine, displayed a greater growth inhibitory effect than the sulfadiazine alone.

Work is in progress to elucidate the action of urea on the effect of other inhibitors and on other sulfonamides. We are also investigating the action of urea on sulfonamide-fast pathogens in synthetic medium, and on the bacteriostatic action of sulfonamides in experimental and clinical treatment of infected wounds.

Clinical experience 9 has confirmed the work of Holder and Mac-Kay, and we feel that the beneficial action of urea in combination with the sulfonamides in the treatment of infections may be due partly to the effects described in this paper.

In CCl₄ poisoning vitamin A is found in damaged but not in uninvolved liver areas. The pathologic areas take it up faster and release it slower than normal ones. High vitamin A therapy repletes the uninvolved areas.

The reports of Marshall, et al., 1 , 2 on the use of sulfaguanidine in the treatment of enteric infections suggested a trial with this drug on infectious enteritis in swine. This disease, frequently referred to under such terms as necrotic enteritis or “necro”, diphtheritic enteritis, infectious diarrhea, bloody scours, and Salmonellosis, is a widespread and important disease of swine. Salmonella types of organisms are usually considered as the principal causative agent.

Table I, Group 1, summarizes controlled experiments in 12 lots of pigs containing from 2 to 14 animals manifesting various forms of the disease (acute to chronic). The drug was administered in capsules and the daily dose divided between morning and evening. Group 2 represents several lots of animals treated under field conditions in which it was not possible to leave some as controls. Daily observations were made of them, however. The drug was administered in capsules or mixed with small amounts of feed.

In addition to the above experiments a total of 333 swine were treated under conditions of a general veterinary practice and with the cooperation of local practicing veterinarians. The results obtained were similar to those shown in Table I.

In general, the diarrhea was checked and the feces returned to normal consistency by the fourth or fifth day and the treatment was continued for an additional 3 or 4 days before the animals were released. A corresponding improvement in physical condition occurred.

These preliminary trials suggest that an effective dose is within the range of 0.165 to 0.33 g per kilo body weight (or 0.75 to 1.5 g/10 1b). In preliminary toxicity trials no evidence of ill effects was noted with dosages below 0.66 g./kg.

Lowering blood sugar below 120 mg % may influence the response of the electroencephalogram to hyperventilation. Consequently it is advisable to keep blood sugar at a level of 120 mg % when the electroencephalogram is recorded during overventilation.

At least three local anesthetics, procaine, pontocaine and nupercaine can inhibit experimental auricular fibrillation in dogs.

The resistance of the chick embryo to toxic substances is a subject which has been little explored in spite of the fact that the embryo is well adapted to a variety of bacteriological and physiological experiments. The developing egg consists of rapidly growing embryonic tissue in a perfect nutritional environment. It comes sealed in an ampule, the shell, which is readily opened for inoculation or other manipulation and easily sealed again for the period of rein-cubation.

The original purpose of the present study was to determine whether the fertile egg could be used as a medium for testing the inactivating effect of antiseptics on viruses. Such studies could be made only if the normal physiology of the embryo is not greatly disturbed by the injection of such agents.

Three tests were performed on different days with each substance. Embryonated eggs which had been incubated for 6 or 7 days were candled and the location of the embryo marked on the shell. This mark was kept uppermost until the inoculation was completed. A hole was drilled through the shell over the air sac by means of a 2 mm dental bur or a carborundum disc. The injection was made by means of a 2-inch, 20 gauge needle fitted to a tuberculin syringe. The needle was directed slightly upward and inserted 3 cm into the egg. The amount injected was 0.05 cc in each instance. This represents 1 part in 1000 of total egg weight, or the equivalent of 70 cc for a man weighing 70 kilos. The eggs were candled daily and the day of death noted. The results are shown in Table I.

The minimal lethal dose for man has not been determined for many of these substances.

The observations of Florey, et al. 3 on the antibacterial activity of penicillin against Gram positive organisms are confirmed and extended. Preparations have been obtained of such activity that 0.03 γ inhibits the growth of 2 to 4 million hemolytic streptococci. This represents an equivalent of 240-250 Oxford units per mg.

Penicillin acts either as a bacteriostatic or bactericidal agent depending on the experimental conditions. The number of organisms decreases at a constant rate until 99% of the organisms have been destroyed. The rate of killing varies with different organisms. The action of penicillin on hemolytic streptococci is not accompanied by lysis of the organisms. No detectable amount of penicillin is destroyed or absorbed from solution by the organisms. It appears to be effective only when active multiplication takes place.

Penicillin is highly effective against hemolytic streptococcus and pneumococcus infections in mice when given sub-cutaneously, intravenously or intraperitoneally. When given in the form of oil suspensions or dry pellets, a single subcutaneous injection confers a high degree of protection. Penicillin is apparently non-toxic within the range of therapeutic dosage.

Recent studies of the mechanisms involved in the production of experimental renal hypertension show that several humoral agents may be involved. Following the hypothesis that phenolic amines may be involved in renal hypertension, Schroeder and Adams 1 found tyrosinase preparations from mushrooms to be effective in lowering the blood pressures of hypertensive animals. Their studies were extended to the effects of tyrosinase preparations on arterial hypertension in man and reported results in 17 patients, with significant falls in blood pressures in 13 of these following daily administration of unspecified amounts of their tyrosinase preparations. Some phenolic substance was considered to be altered by the injected enzyme to account for the lowered blood pressures observed.

The present experiments were carried out with mushroom tyro-sinase preparations made by modifications of the purification procedures of Keilin and Mann. 3 The valuation of the enzymic activity was made upon a catechol-hydroquinone substrate in terms of “catecholase” units as defined by Adams and Nelson. 4 The final preparations were made up to contain 500 “catecholase” units per ml and contained about 2 mg per ml of non-dialyzable total solids. By heating such preparations for 40 minutes to 60°C, about 95% of the “catecholase” activity was destroyed, and such preparations are referred to as heat-inactivated.

Four patients with malignant or premalignant hypertension were hospitalized and observed before treatment for 7 to 10 days, with blood pressures taken twice or more daily. In the first patient studied, the marked local reactions from injections of the unheated enzyme preparations suggested that the hypotensive response might be due to non-enzymic substances in the preparation. Treatment was then continued with heat-inactivated preparations and it was observed that, while the enzymic activity was almost completely absent and the local reactions were somewhat less, these preparations were still very effective in lowering blood pressure.

Complement-fixation tests with the virus of meningo-pneumonitis in Michigan revealed that 61 of 100 pigeons reacted with the antigen; 41 of them in dilutions of 1 :32 or greater. Pigeon serum fails to give the cross-reaction with lymphogranuloma vene-reum antigen which is commonly seen with human serum.

Reactions of variable intensity were obtained with 22 of 109 sera from domestic turkeys, with 5 of 24 sera from domestic ducks and, in the lowest dilution only, with 9 of 45 chicken sera.

No reactions were observed with the sera of 90 wild fowl.

In view of the increasing volume of literature pertaining to endocrine activities of the crustacean sinus gland, it is unfortunate that the only simple and sure method of removal of these glands from the body has involved the removal of the total eyestalk. The eyestalk constituting the most important light receptor of the organism, contains, in addition to the sinus gland, several important nerve ganglia, the x-organ, and other possible endocrine sources. For this reason the usual removal of so much additional tissue with the glands leaves the interpretation of the results of eyestalk removal somewhat uncertain, to say the least. Furthermore, such a function as control of retinal pigment migration, which is believed by a number of endo-crinologists to be normally under the control of the sinus gland, has not yet been confirmed by such a crucial experiment as gland extirpation since this latter has also always involved removal of the effector mechanism as well. Nearly every recent investigator in crustacean endocrinology has at one time or another attempted to remove the sinus gland while leaving the rest of the stalk intact. The only published attempt to date 1 dealt with the highly transparent shrimp, Palaemonetes. The technic developed in this case was not sufficiently satisfactory to be used as a routine laboratory method, and furthermore could not be used successfully upon the great majority of crustaceans whose exoskeletons were relatively opaque.

The simple technic described below has been devised to extirpate the sinus glands from crayfishes without eyestalk removal, and, in fact, with little apparent disturbance to the remaining functions of the stalk including vision. The operation can be performed rapidly, and after a little practice, bilateral extirpation of the glands in an animal can be accomplished in less than 5 minutes.

Although Ott and Scott 1 demonstrated that an extract of the posterior hypophysis would cause an ejection of milk from the alveoli of the mammary gland this phenomenon was not associated with the natural let clown of milk until the work of Ely and Petersen, 2 who demonstrated that both the oxytocic and vasopressor substances of the posterior pituitary would cause an ejection of milk in the cow. Turner and Cooper 3 reported the same for the rabbit. Ely and Petersen observed fright or the injection of epinephrin inhibited the natural response to the milking” stimulus. Petersen and Ludwiek 4 showed that the substance responsible for the ejection of milk is humoral in nature and that the blood from excited cows has great vasoconstricting properties in perfused bovine mammary glands.

As a result of these observations, it was deemed advisable to test the effects of a number of substances upon the blood flow and the milk ejection mechanism in the bovine mammary gland. This report deals with 9 substances: oxytocin, pitressin, epinephrin, his-tamine, atropine, ergonovine, acetylcholine, Mecholyl (acetyl-β-methylcholine) and Lentin (carbamylcholine).

The experiments here reported were on bovine mammary glands perfused according to the technic of Petersen, Shaw and Visscher. 5 The glands were obtained from a packing plant. The teats were cannulated to permit drainage of the milk immediately after the perfusion started and the cannula left in situ during the experiment. The blood pressure was maintained at 110 mg Hg pressure at all times. The substances tested were injected into the arterial blood as it entered the gland.

The number of experiments, dosage of the substance, effect on the blood flow and the effect on the ejection of milk are given in Table I for each substance tested.

1. Intravenous administration of the protamine, salmine sulfate, to untreated guinea pigs and rats produced death at dose levels of 6-12 mg/100 g. Intraperitoneal injection delayed these effects. 2. The addition of salmine sulfate to whole blood or plasma resulted in the formation of a flocculent protein precipitate. Addition to whole blood caused hemagglutination in the human, dog, cat, and rat. 3. The addition of salmine sulfate to whole blood perfusing isolated organs caused cessation of flow. Addition of comparable amounts to Ringer's fluid and serum perfusing organs was without effect upon the rate of Mow. 4. These experiments suggest the possibility that protamines exert toxic effects through embolic vascular phenomena.

1. Aluminum hydroxide, aluminum phosphate, magnesium trisilicate, and mucin inhibited peptic activity by altering the pH, the percent of inhibition being directly related to change in pH. 2. Of these 4, magnesium trisilicate was the most effective inhibiting agent, exerting the greatest effect on the pH. 3. Of the substances used, sodium lauryl sulfate alone completely inhibited peptic activity without any alteration in pH.

Ovulation of the normally growing follicle of the hen's ovary can be induced by as much as 17 hours prior to time of expected normal ovulation by intravenous injection of appropriate hormones. A luteinizing preparation from horse anterior pituitaries effected ovulation of follicles 10 to 11 hours prior to time of normally expected ovulation in 100% of injected hens at a level of 4 rat units (Fevold rat seminal vesicle test). The commercial preparations Prephysin, Gonadin serum and Anteron were effective when administered at sufficiently high levels. At appropriate submaximal injection levels the percentage of ovulating hens decreased with increasing prematurity of the ovarian follicles; this effect is more pronounced in follicles of relatively great prematurity at the time of effect of the injected hormone.

The time at which ovulation occurs following intravenous injection of appropriate hormone preparations into pretreated and normally laying (non-pretreated) hens has been determined. The interval in pretreated hens averaging more than 2 ovulations per hen at time of autopsy was 6.8 hours, range 6.1-7.2 hours. Ninety percent of all normally laying hens (69) ovulated from 6.5 to 8.5 hours following ovulatory injection, the remaining 10% of injected hens requiring up to 9.6 hours for ovulation. The non-pretreated hens were injected with 4 different hormone preparations administered at differing dosage levels and at times calculated to effect ovulation of follicles at varying degrees of prematurity. The minimal ovulatory interval recorded for pretreated hens was 6.1 hours, for non-pretreated hens, 6.5 hours.

Tocantins 1 recently reported that plasma exposed to air currents showed a rapid diminution of prothrombin activity and that addition of CO₂ restored this loss. In view of the importance that these results have in the quantitative determination of prothrombin, and in the possible solution of the problem of preventing the spontaneous loss of this clotting factor in stored blood, it seemed important that these experiments be repeated.

Four cc of oxalated plasma were put in a pyrex test tube (25×100 mm) fitted with a 2-hole rubber stopper through which were inserted 2 glass tubes. One of the tubes nearly reached the surface of the plasma. The test tube was tilted to expose a maximum area of plasma surface, and a stream of air (first passed through a dilute solution of sodium hydroxide to remove CO₂) was blown over the plasma, kept at 38°C. The “prothrombin time” was determined by the author's method 2 and the CO₂ content by Van Slyke's original volumetric method. The loss of volume by evaporation was restored by adding distilled water.

The results indicate that the prothrombin of dog and rabbit plasma is not demonstrably altered by one hour aeration even though considerable CO₂ is lost. Human plasma aerated at room temperature likewise does not lose any significant amount of prothrombin activity in one hour. At 38°C, however, a definite loss of prothrombin occurs, which the addition of CO₂ does not restore. It appears, therefore, that the decrease of prothrombin is due to direct oxidative destruction or to the formation of an oxidation product in plasma which inactivates prothrombin, rather than to the loss of CO₂ from the plasma.

The excretion of hippuric acid in normal adults following intravenous injection is exceedingly high: about 44% is eliminated in 15 minutes when 2.26 g of sodium hippurate are administered. In various renal disorders the speed of excretion is diminished.

Injection of a renin-activator preparation of ox-plasma restored the blood pressure of dogs after hemorrhage. This restoration was observed with quantities of the preparation containing only one-tenth or less of the total plasma protein removed.

Corresponding quantities of 10% gelatin or of control plasma concentrations failed to restore the blood pressure.

From these observations it is concluded that the secretion of renin in severe hemorrhage (and perhaps in other forms of shock) is sufficient to produce exhaustion of renin activator. The resulting failure of the renopressor system is followed by a fatal collapse of blood pressure. This collapse may be staved off and the blood pressure restored by replacing the exhausted activator by a suitable preparation of ox plasma.

These investigations suggest the possibility of improving the transfusion therapy of hemorrhage and shock by fortifying the plasma with suitable preparations of renin-activator, or possibly by substituting relatively small quantities of activator preparations for plasma in emergency treatment. Work in this direction is in progress.

Experimental and clinical observations have shown that heparin inhibits the clotting of blood and the formation of thrombi. Recently we have studied the effect of heparin on the coagulation of citrated blood and plasma by staphylococci. 1 Some strains of staphylococci will coagulate human plasma. 2 , 3 One, 0.5 and 0.1 cc of heparin† (Liquamine 1.0 cc = 10 mg) was added to a series of tubes containing 3.0 cc of both rabbit and human plasma and sterile saline (3 parts of plasma and one of saline). One-tenth of a cubic centimeter of a broth culture of staphylococci was added to each of these tubes. The medium in each of the tubes was completely coagulated during 24 hours of incubation at 37.5°C. Seven strains of staphylococci (4 aureus, 2 albus and 1 citrus) were then used to study the effect of heparin on the coagulation of plasma. Each of the series of 3 tubes containing the different dilutions of heparin was coagulated by 4 of these 7 strains of staphylococci. Physiological saline in quantities equal to that of heparin was added to a series of tubes of the citrated plasma for the controls. They were inoculated simultaneously with the different strains of staphylococci. Each of the strains of staphylococci that coagulated the heparinized citrated plasma also coagulated the control medium. This citrated plasma medium, containing both heparin and saline, did not coagulate during the time of incubation when staphylococci were not added. When citrated blood was substituted for the plasma, coagulation occurred in a similar manner with the four of the seven strains of staphylococci.

It is evident, therefore, from these observations that heparin, even in a 25.0% concentration, does not inhibit the coagulation of citrated human and rabbit plasma and also blood by the strains of staphylococci containing the so-called coagulase factor.

The possible use of bovine albumin in man 1 , 2 , 3 makes the behavior of bovine albumin as an antigen of some interest. Previous reports 4 , 5 , 6 indicate that bovine albumin behaves as a potent antigen in rabbits and guinea pigs. This paper is to report some studies on the antigenic behavior of bovine albumin in rabbits, guinea pigs, and in man.

Bovine albumin was prepared as described elsewhere 3 by fraction-ation of plasma with ammonium sulfate. Electrophoretic analysis was carried out by the methods of Tiselius 7 and Longsworth. 8

Three rabbits were given a course of 6 injections of bovine albumin over a period of 4 weeks. Serum from blood drawn at the end of this period gave a titer as high as 24,000. Sixteen guinea pigs were given a sensitizing dose of 50 mg of albumin per kg and were tested with a shock dose of the same size administered intracardially. Eleven guinea pigs were similarly treated with globulin. All animals which died after the shock dose were posted. The results are given in Table I.

The bovine albumin had a longer sensitization period than globulin. A similar difference has been reported between horse albumin and globulin. 9

The following case history is definite evidence that bovine albumin may act as an antigen in man. Patient X. R. was given 10 cc of 6% albumin intravenously on November 11, 1939. The skin test to albumin, which had been previously negative, was positive on December 18, 1939, at which time 3 cc of 8.8% albumin solution were administered intravenously over a 5-minute period. At the end of this time the pulse was weak, the respiration was shallow, and cynanosis developed.

1. Methyl alcohol is preferable to ammonium sulfate for the preparation of bovine albumin for intravenous use. 2. Bovine albumin can be prepared which gives a low incidence of reactions when administered intravenously to man.

Baker, Harrison and Miller 1 have studied the effect of synthetic surface active agents on the metabolism of bacteria as measured in the Warburg manometric apparatus. Cationic surface active agents were found to inhibit equally the metabolism of both Gram positive and Gram negative bacteria. Anionic agents, in general, inhibit the metabolism of Gram positive bacteria only. In a subsequent paper 2 studies on bactericidal effects were reported. Dubos 3 discussed the differential susceptibility of Gram positive and Gram negative bacteria to different injurious agents, and suggested the use of anionic and cationic surface active agents as tools for the study of bacterial structure.

In this paper we report the effects of an anionic surface active agent, the dioctyl ester of sodium sulfosuccinate, and of a cationic agent, cetyl pyridinium chloride, on a Gram positive organism and a Gram negative organism. As an index of the action of these agents we used the oxidation of lactate by molecular oxygen as compared with anaerobic oxidation by methylene blue.

Methods. Two organisms were used, the Insecticide Board strain of Staphylococcus aureus, a Gram positive organism, and Escherichia coli, University of Washington No. 406, a Gram negative organism. These two cultures were selected because they possessed relatively active lactic dehydrogenases as determined by the Thunberg technic.

Staphylococcus aureus was grown on the broth prescribed for the Food and Drug Administration procedure for the phenol coefficient. 4 After 24 hours' incubation, the organisms were collected by means of a continuous flow' Sharpies centrifuge and washed 3 times with M/100 phosphate buffer at pH 7.0. Escherichia coli was grown on Blake bottles containing F. D. A. broth to which 2% agar had been added.

Strains of pneumococci. gonococci, E. coli and S. aureus have been rendered resistant in vitro to the bacteriostatic action of sulfonamide drugs. 1 , 2 , 3 , 4 , 5 , 6 Rammelkamp 7 has also observed that S. aureus may become “fast” to tyrothricin, both in vitro and in vivo. The present report is concerned with the in vitro and in vivo production of sulfonamide resistant strains of staphylococci.

The in vitro observations were made with 2 strains of S. aureus isolated from patients with severe staphylococcal infections. A chemically-defined medium as described by Gladstone was used throughout. 8 These strains were made resistant by exposure to increasing concentrations of sodium sulfathiazole in this medium. Initial growth was established with a minute inoculum from an agar slant. Subsequent transfers of the cultures in the synthetic medium were made with a 5 mm wire loop. The two strains showed an initial difference in susceptibility to the action of sulfathiazole. Strain 14 grew readily for several generations in the presence of 0.25 mg per 100 ml of sulfathiazole, but strain 7 was unable to institute growth in this concentration after the second transfer. It was necessary to start strain 7 in 0.1 mg per 100 ml. Control observations were carried out with the parent strains transferred an equal number of times in a drug-free medium.

The degree of growth was estimated regularly by measuring the density of the culture in the Evelyn photoelectric colorimeter, as previously described. 9 The development of drug resistance was determined by seeding approximately 1,000 organisms per ml to tubes of medium containing varying concentrations of sulfanilamide, sulfapyridine, sulfadiazine, and sulfathiazole.

The extent to which sulfanilamide is acetylated by the liver was found not to be of any definite value as a test of liver function.

Earlier workers have reported studies on the hematology of vitamin B deficient monkeys. 1 , 2 The current observations were undertaken in an attempt to determine which, if any, of the presently recognized components of the vitamin B complex will maintain or restore specific blood cell and nutritional equilibria.

Methods. Healthy Macaca mulatta, receiving a normal diet, were isolated in individual cages and observed clinically and hematologi-cally for a control period of 2 to 3 weeks. The animals showing satisfactory equilibria were then divided into 3 groups and placed on experimental diets: (1) No. 600 1 (modified Goldberger), (2) No. 1 and (3) No. 2 (Table I). The controls have included observations on each experimental animal before it was placed on diet and on a number of monkeys of the same species and approximate age maintained on stock diet. These animals showed no hematologic changes comparable to those to be described.

Results. All of the monkeys on the experimental diets developed leucopenia. In the 4 animals on diet 600, this appeared between the 42nd and 77th days. In the 6 animals receiving diet 1 and in the 5 receiving diet 2, the time of appearance of the initial leucopenia varied between the 30th and 103rd days.

One interesting feature of the response to these diets was a “plateau” period of remarkably stabilized total white cell counts preceding the first appearance of leucopenia. The relative constancy of the total white cell counts between the 14th and 45th diet days seen in monkey No. 4 (Fig. 1) is representative. Similar well-defined periods of stability varying in duration from 24 to 100 days were seen in 3 of the 4 monkeys on diet 600, 4 of the 6 on diet 1, and 2 of the 5 on diet 2.

The evidence increases that circulating antibodies play a minor part in the resistance to poliomyelitis both in man and in monkeys. Antisera have commonly failed to prevent the disease 1 even when used in large quantities or to modify its course when used thera-peutically; 2 the disease has developed both in monkeys and in man even though they possessed neutralizing blood of high titer; 3 antibodies have been found in 75 to 85% of normal adults regardless of previous poliomyelitis; 4 neither resistance nor susceptibility to the disease in man or monkey presents any close correlation to the presence or absence of demonstrable antibodies. 5 In consequence of these findings, some authors have attempted to seek an answer to the problem of immunity in this disease on an hormonal basis, 6 with conflicting results, others have raised the query of nutritional deficiencies, 7 while still others have employed the term “tissue immunity” 8 to indicate the resistance, due to obscure factors, apparently independent of humoral antibodies. In attempting to unravel the varying reports on this type of resistance, one must bear in mind the primary cellular basis of immunity and anaphylaxis, the many instances of interference phenomena 9 sometimes entirely non-specific, as well as the possibility of closely bound antibodies such as those demonstrated with the Shope rabbit papilloma virus. 10

Recently Howe and Bodian 11 have summarized their extensive investigations on neural mechanisms in poliomyelitis in a telling monograph. Closely related to the problem of our paper is their demonstration that a second attack of poliomyelitis can be induced in rhesus monkeys either with homologous or heterologous strains of virus provided a different portal of entry is employed, thus bringing the virus in contact with nervous tissue not previously involved with the virus.

There is present in joint fluid a polysaccharide which Meyer 1 had named hyaluronic acid. This polysaccharide is believed to be the substance which is responsible for the increased viscosity of synovial fluid. One may obtain a rough estimate of the amount of polysaccharide present in the joint fluid from the relative viscosity of the fluid. A specific enzyme which hydrolyzes this polysaccharide has been recovered from bacterial filtrates and from various animal organs. 2 The enzyme has been shown to have many properties, similar to the spreading factor of Duran-Reynals. 3 , 4 The enzyme has been found to be active in vitro and so an attempt was made to determine its activity in vivo. In the experiments to be reported here the enzyme was injected directly into the joint space.

Methods. Four patients were taken by chance as they were seen in the clinic or hospital. The enzyme was concentrated by Dr. Karl Meyer, who kindly furnished us with the material. It was derived from bulls' testes. An aqueous solution of the enzyme was made up in small volume. The solution was then passed through a Seitz filter. Fluid was removed from the knee, and the enzyme was immediately injected into the joint space. At varying times thereafter, fluid was again removed from the knee. The joint fluid was cooled, the clot separated by slow centrifugation and the viscosity determined on the supernatant fluid, using an Ostwald viscosimeter under standard conditions. Viscosity values are expressed as multiples of the viscosity of water. The total protein content of the synovial fluid was determined by a specific gravity-gradient method. 5

Results. As shown in Table I, in all instances in which a potent enzyme was injected, the viscosity of the joint fluid was markedly reduced.

1. Four protein fractions were observed in 4 normal and one pathological semen specimens subjected to electrophoretic analysis. 2. The electrophoretic mobilities of these protein fractions after dialysis are identical with those of the albumin, alpha, beta, and gamma globulins of normal serum. 3. Average values for these components as percentages of the total nondialyzable semen protein are: beta globulin, 39.6; alpha globulin, 21.3; albumin, 19.9; and gamma globulin, 19.1. 4. The protein composition of the one pathological semen studied did not differ from the 4 normal semens.

A marked difference in the sensitivity of three different strains of rats to the synergistic action of certain gonadotropic hormones has been shown. The three strains, Sprague-Dawley, Wistar and Sherman, give comparable responses to the gonadotropins of pituitary, pregnant mare serum and pregnancy urine when administered singly. Only the Sprague-Dawley and Wistar strains show any augmentative response when purified pituitary follicle-stimulating hormone is given in combination with either chorionic gonadotropin (prolan) or pregnant mare serum hormone.

An augmentative effect was observed when pregnant mare serum gonadotropin was administered in combination with a pituitary follicle-stimulating preparation. On the other hand, the addition of chorionic gonadotropin under the same experimental conditions, did not potentiate the gonadotropic effect of the pregnant mare serum preparations.

Serum from domestic rabbits bearing the growths of infectious papillomatosis 1 and those injected with extracts of domestic or cottontail rabbit growths or with the purified papilloma virus protein may contain immune bodies demonstrable by complement fixation, neutralization or precipitation. 2 Recently, studies have been made in this laboratory to learn whether these immunological characters are paralleled by abnormal electrophoretic behavior of such serum.

Sera from individuals of 6 groups of rabbits were examined: (1) domestic rabbits injected intraperitoneally with 0.5 mg of purified papilloma virus protein, once a week for 6 weeks, and serum taken for study every 7 days (for a total of 51 days); (2) domestic rabbits injected intravenously with 0.5 mg of purified papilloma virus protein, twice a week for 6 weeks, and the serum examined after 21 and 50 days; (3) domestic rabbits injected intraperitoneally with 1.0 cc of a 7.5% suspension of domestic rabbit warts, twice weekly for 6 weeks; (4) domestic rabbits bearing experimentally-induced growths for 39 to 72 days; (5) domestic rabbits carrying transplanted growths 3 in abdominal organs for 115 days; (6) cottontail rabbits bearing experimentally-induced growths for 37 and 40 days. The serum was diluted with 2 volumes of buffer solution of pH 7.8 containing 8.7675 g NaCl, 2.6274 g Na₂HPO₄, and 0.2071 g NaH₂PO₄ · H₂O per liter and dialyzed for 4 to 6 days at 2 to 9°C. For electrophoresis the moving boundary method of Tiselius was used and the boundary gradients were recorded as previously described. 4 The index of serological change was complement-fixing titer.

In the sera of animals receiving the virus intravenously or intra-peritoneally, the complement-fixing titer was 1 :16 to 1 :256.

The effect of certain split products of some carcinogenic azo dyes and related compounds on melanin formation was studied. It was observed that certain non-autoxidizable compounds such as p-aminoacetanilide, B-naphthylamine, p-aminobenzoic acid and sulfanilamide interfered with this reaction. When chemically related autoxidizable compounds (p-aminodimethylaniline, p-phenyl-enecliamine, methyl p-phenylenediamine and p-aminophenol) were used, the color changes in both tyrosine-tyrosinase and control solutions were similar. It is, therefore, difficult to interpret the action of these latter chemicals on melanin formation.

Twenty niale albino rats whicli had been raised to an age of 11 niontlis on an adequate stock diet were fed for 5 weeks on a control diet containing 0.462 % calcium, 0.458% phosphorus and adequate in vitamin D. Ten of the aninials were then transferred to the experimental diet which was deficient in vitamin D and contained 0.0078% calcium and 0.450% phosphorus. Calcium and phosphorus. balances were determined for each animal over 10-day periods for 220 days. The animals of the control group remained in positive calciuni ant 1 phosphorus lxilance throughout the period of observation. The animals fed the dcficieiit diet exhibited a severely negative calcium balance and a positive phosphorus balance.

The humeri and femora and the molar and incisor teeth were dissected free of soft tissue, cleaned, dried and rendered fat-free by continuous extraction with iat solvents. During the removal of the molar teeth from the jaws, it was noted, in the case of experimental rats, that the alveolar hone was soft and friable, the alveolar crest resorbed and the teeth loosened. In the control animals the alveolar bone was well calcified, the crest normal and the teeth firmly fixed in their sockets. The incisor teeth of both groups appeared equally developed and fornied. Tlie average weight of the dry fat-free incisor teeth of the two groups was not significantly different. The awragc percentage of ash in the left femora was not significantly different between the two groups of animals.

The Volume and density of the left humerus of each animal was determined, using a spcially designed pycnometer and mercury as the suspension medium. The average volume of the humeri of the experimental animals did not differ from that of the controls but the average density of the bones of the former animals was 10.8% less than that of the controls. The calcium and phosphorus contents of the left humeri, in terms of percent of the dry fat-free weight, were not significantly different between the two groups of animals.

Functional reorganization in the sympathetic nervous system following preganglionectomy has been demonstrated, using the cat as the experimental animal. Skin resistance change in the forepad, in response to stimulation of the ventral roots, was used as an indicator of preganglionic activity. Following interruption of preganglionic pathways to the forepad it was found that preganglionic pathways develop from roots which normally contribute nothing to the forepad.

This may offer additional explanation for early relapse following preganglionic section for Raynaud's disease in the hands, and for the fact that operations for this disease are generally more successful in the feet than in the hands.

Amounts of benzpyrene ranging between .04 γ and 4 mg dissolved in 0.5 cc of olive oil were injected subcutaneously into mice. The smallest amount producing a sarcoma was 4 γ. The data suggest a linear relationship between the logarithm of the injected dose and the average latent period of tumors, as found by Bryan and Shimkin. A similar relationship was found between dose and average length of survival of the tumor-bearing animals. When dissolved in .125 to 1 cc of oil, 400 γ of benzpyrene produced tumors in 40% or more of the mice. When dissolved in from .0625 to .015 cc of oil, the incidence of tumor production was 25% or less. No relationship was found between average latent period and volume of injection. A technic is described for the study of the rate of re-sorption of benzpyrene and oil.

The diaminocarboxylie acid resulting from the hydrolysis of the urea ring of biotin is capable of stimulating the growth of yeast in a biotin-free medium. The compound possesses about 10% of the activity of biotin. The yeast-growth-promoting activity of the diaminocarboxylic acid is not inhibited by avidin.

Application of the technic described, considering tissue toxicity as well as germicidal power, indicates that in general the chlorine compounds rate the highest, phenolic compounds next, and organic mercurials rate the lowest as efficient germicides. This corroborates certain previous studies. 4 , 8 , 9 Tincture preparations in general are not ideal germicides since the solvents are more toxic to phagocytosis than to bacteria.

The toxicity and germicidal end points found in this study indicate that a large number of germicides in general use are too concentrated. Germicides should be utilized in concentrations which are germicidal but non-toxic to tissue. The efficiency range is defined.

Of the natural barriers to infection, i. e., skin and blood, the blood is more sensitive to the destructive action of germicides. Therefore the phagocytosis inhibition technic may be regarded as a valuable aid in the evaluation of germicides for clinical use.