Experimental Biology and Medicine

Since Fisher 1 and Allan, et al., 2 first reported fatty infiltration of the livers in depancreatized dogs maintained on insulin, the observation has been confirmed repeatedly. 3 Recently, it has been shown in this 4 and other 5 , 6 laboratories that ligation of the external pancreatic ducts results in similar fatty changes in the liver. In all of these studies the diet fed to the dogs consisted largely of raw whole meat and glucose.

In the experiments reported here a dried meat powder preparation,∗ from which the extractives had been removed, was substituted for the whole meat. The diet contained meat powder, cracker meal, milk powder, salts, brewer's yeast, bone ash, cod liver and corn oil. As shown in Table I, little or no fatty infiltration of the liver occurred in 6 depancreatized and in 3 ductligated dogs which were fed the diet for periods varying from 6.5 to 19 weeks after operation. The fatty acid content of the whole livers, determined as previously described, 7 averaged 6.2% with a range of 3.12 to 10.5% (Table I). When the meat powder diet was supplemented with 15 ml of a concentrated meat extract∗ (the combination of meat powder plus extract being equivalent to about 250 g of raw lean beef) the livers of one ductligated and 2 depancreatized dogs contained 18.3 to 19.8% fatty acids after 5 to 9 weeks (Table I). It is therefore concluded that the fatty liver of these dogs is due in large measure to some substance or substances present in the extractive fraction of meat.

Studies in this laboratory in recent years have been concerned with the hydrogenion concentration of the bile of the guinea pig 1 and dog 2 with respect to the dissolution of human gall stones. Having available a series of Capuchin monkeys (Cebus fatuellus) upon which metabolism studies had been conducted, it was deemed of interest to study the bile of this species of monkey. The animals were narcotized with sodium amytal; the bile was drawn from the gall bladder without exposure to air. The hydrogenion concentration was determined by means of a glass electrode at 25° and an analysis of the principal constituents was carried out according to the Douglas-Sauermann 3 method. The results on 10 animals are shown in Table I.

The average pH of the bile and the percentage of various constituents present in the Capuchin monkey are similar to these data on the gall-bladder bile of dogs. 2 Also as with the bile of the dog it was observed that the correlation between a high pH and high total solids content is great.

A pooled urine collection from 2 male castrates contained 6 I.U. per day of androgenic activity. Capon assay of the results of digitonin fractionation indicated an androsterone to dehydroisoandrosterone ratio of 4:1 compared with the approximately 1:1 ratio found in normal male urine. In the absence of the testis smaller amounts of the same or similar androgens are excreted in slightly different proportions.

1. Beginning at the age of 2 weeks, 122 non-breeding male strain C3H mice were treated with estrogen for varying periods of time. At the end of the specified course of injections the mice received no further treatment until they died or were sacrificed. 2. Four weekly doses of 100 rat units of estrogen produced no mammary gland carcinomas; 8 and 12 weekly doses of 100 rat units produced a low incidence of these tumors. 3. After 16 weekly doses of 100 rat units of estrogen had been given a relatively high percentage of carcinoma of the mammary gland developed; the administration of 20 weekly doses or an average of 32.7 weekly doses of 100 rat units did not raise the incidence of tumors above this level. 4. Three thousand rat units of estrogen administered over a period of 3 days at the age of 2 weeks produced no tumors.

A transient, small but measurable and significant decrease in limitation to motion was observed in 10 persons with atrophic arthritis following inhalation of 10 to 15% CO₂ (in O₂) for a length of time (2 to 5 min) sufficient to induce a feeling of warmth and breathlessness. An equivalently definite and regularly elicited effect was not observed in 10 additional persons with limitations due to conditions (osteoarthritis, fibrositis, etc.) not classifiable as atrophic arthritis. Transient decrease in sensitivity to cold and in susceptibility to histamine was observed in rabbits during inhalation of CO₂ in O₂.

1. Human plasma administered intravenously is retained and utilized. A patient to whom a daily average of 450 cc of plasma was given over a 5-day period was maintained in positive nitrogen balance. 2. Human plasma appears to be a better agent than blood to employ to maintain nitrogen equilibrium in starvation states. 3. Bovine plasma can be given intravenously to man in fairly large quantities; it is retained and apparently utilized.

Discussion. An exhaustive pharmacological study of this drug is being carried out at the present time and the results will be reported in the immediate future. It has been suggested 2 that the activity of the drug is dependent on its breakdown with a resultant liberation of sulfanilamide. Our present direct evidence shows that no such breakdown occurs in the gastrointestinal tract. Indirect evidence from studies on therapeutic efficacy 3 indicates that this N 1 -acyl compound is more active than sulfanilamide on a molecular basis.

Results. 1. N 1 -dodecanoylsulfanilamide, when administered in oil to mice, shows marked therapeutic efficacy against beta hemolytic streptococci. 2. This therapeutic effect is lost when the drug is administered in an aqueous medium.

1. N 1 -dodecanoylsulfanilamide inhibits the growth of tubercle bacilli in vitro at a concentration of 10 mg/100 cc in beef infusion-dextrose-glycerine media over a period of 90 days. 2. N 1 -dodecanoylsulfanilamide inhibits the development of the tuberculous process in guinea pigs infected subcutaneously with a human strain of tubercle bacilli.

Rake 1 demonstrated that pneumonia could be produced in mice when the pneumococci were introduced by the intravenous route. The important factors were the strain and dose of the organism, and the breed of mice. Twelve of 87 mice had macroscopic lesions; 6 of the 87 failed to show any microscopic lesions. The pathology of the various stages encountered was clearly described.

Prior to this work, with a few exceptions, intravenous injection of pneumococci has failed to give rise to pneumonia. Although it has long been postulated that the origin of the infection might be by way of the blood stream, there has been little evidence to support it.

Employing the Schwartzman phenomenon, Witebsky, Neter, and Ward 2 were able to localize pneumococci injected intravenously in dermal lesions of rabbits.

In our experiments, rats were inoculated intrabronchially with 0.1 cc of sterile mucin according to a technic previously described; 3 immediately thereafter, the animals were injected intravenously (using the tail vein) with various amounts of culture. The data are presented in Table I.

In interpreting these results it must be remembered that these animals received only one inoculation of organisms, many of which were distributed throughout the body and removed by the fixed phagocytic cells. It is noteworthy that in at least one rat, the disease was produced with as small an inoculum as 10^-7 cc of culture.

Although in the past it has been generally impossible to produce pneumonia in experimental animals by intravenous injection of pneumococci, this may be accomplished in rats following intrabron-chial inoculation of sterile mucin. The presence of mucus material in the lung or some comparable condition is a necessary predisposing factor for the localization of the pneumococci.

Physical exercise, maximal anaerobic in type and constant as to the rate of working and speed, was performed to the point of exhaustion by 6 healthy young adult women. After a period of preliminary training varying in length in the different subjects, gelatin was added to the diet and its influence upon the time of onset of muscular fatigue was noted. The evidence presented substantiates the following conclusions. Gelatin has no effect

Evidence has been presented that demonstrates quite convincingly that the growth rate and fate of spontaneous carcinoma of the mammary gland in mice may be significantly influenced by heptyl aldehyde. 1 The effect of liquefaction (both partial and in some cases complete) together with a slower growth rate and complete disappearance of the tumor mass could be brought about by the introduction into the organism of the drug either by the diet or by subcutaneous injection (at areas remote to the spontaneous tumor). One of the limiting factors in the introduction of the material through the diet is the fact that the resulting mixed food becomes so bitter with the larger effective doses that the normal food habits of the mouse may be interfered with. The greatest limiting factor of the injection technic is that local ulceration is sometimes quite pronounced.

In a recent paper, it was pointed out that in order to influence malignancy the heptyl aldehyde must be used fresh. It was found that as soon as the heptyl aldehyde-normal diet mix had “aged” even for a few days the efficacy of the drug was greatly diminished. One way of “protecting” the precocious deterioration of the heptyl aldehyde was the addition to it of a small amount of methyl salicylate —that is, to approach more or less the chemical situation in the original true or natural oil of Gaultheria. 2 Since, however, the heptyl aldehyde undergoes oxidation readily, one may prohibit or delay, to a certain extent, this change by either of two methods: (1) the addition of a known anti-oxidant, such as salicylic aldehyde to it, or (2) the combining of it with such a chemical as sodium bisulphite.

During the course of a study on the maintenance of the completely depancreatized dog, it was observed that cirrhosis of the liver developed in 8 of the 16 animals that had been kept alive with insulin for periods between 2.6 and 5.5 years. 1 Infection and obstruction of the extrahepatic bile passages were ruled out as factors that might have induced the scarring of the liver. The animals received a diet containing lean meat, sucrose, bone ash and vitamin supplements and despite its completeness with respect to calories, proteins, vitamins and salts it has been demonstrated repeatedly that this diet induces fatty livers in depancreatized dogs. Although it was recognized that the absence per se of the pancreas might be involved, it was nevertheless suggested that an important causative factor in the production of the hepatic fibrosis was the excessive deposition of fat which appeared in the liver during the first few months after pancreatectomy and remained there for long periods.

In view of the importance of the above findings, it seemed highly desirable to determine whether cirrhosis could be induced in the fatty liver of the normal dog. The observations recorded below establish for the first time that the continued presence of excessively high amounts of fat in the liver for several months stimulates hepatic fibrosis even in normal dogs to the extent that it may be called a true cirrhosis, and cause the death of the animals.

Experimental. Well nourished dogs were selected for use in this experiment. Throughout the periods of observation they were maintained on a high fat diet that contained 10 g of lard and 7 g of lean meat per kilo per day. They were fed twice daily by stomach tube when they would not eat, at 7 :00 a.m. and at 2.30 p.m.

Immediate generalized skin-reactions following contact with or ingestion of allergens have been observed in humans but not hitherto in animals, although Dienes and Simon 1 reported a generalized delayed reaction in guinea pigs treated with human serum. No observations of generalized skin-reactions in animals, due to simple chemical substances, have been noted in the literature. It seemed of interest, therefore, to report recent observations of such effects in a number of guinea pigs sensitized to citraconic anhydride, together with the sensitization of guinea pigs to new anhydrides having similar properties.

The animals were rendered hypersensitive to citraconic anhydride by a method previously described 2 and were tested by scratching the skin through a 25% dioxane solution of the same substance, usually at intervals of 2 weeks.

It was regularly observed that the more hypersensitive the guinea pig the quicker the onset of the reaction. Animals developing generalized reactions showed within a very few minutes a large local wheal and erythema surrounding the scratch, often several centimeters in diameter. At the same time in both contiguous and remote parts of the skin some pigs showed a diffuse pinkishness apparently accompanied by slight thickening, while others exhibited discrete, urticaria-like wheals with distinct elevation and pinkness. Often some of the old injection-sites on the back flared up at the same time, with marked wheal-formation. The erythema reached a maximum 20 to 30 minutes after the test, following which it gradually faded. Edema associated with reactivated sites and other lesions distant from the original scratch increased for about 2 hours and then also diminished. By the next day all such subsidiary reactions had completely disappeared. In striking, possibly significant contrast to this, the reaction in the vicinity of the scratch after receding to a minimal color and edema in 4 to 5 hours, gradually increased in pinkishness and induration to a second maximum reached about 24 hours after the test.

The presence of fluorine in large amounts may decrease the solubility of the dental hard tissues. Small amounts of fluorine show no demonstrable reduction in enamel solubility. It seems doubtful that the amounts of fluorine present in slightly fluorosed teeth is sufficient to alter their acid solubility.

Aqueous solutions of 3 times recrystallized Ea were allowed to stand at room temperature for 1 to 2 days with HC1 between pH 1.5 and 2.3. Depending upon the acidity, from 25 to 75% of the Ea was denatured. The DnEa was precipitated by adding NaOH until the isoelectric range, pH 5.2 to 5.4, was reached. Three additional reprecipitations usually sufficed to remove unchanged Ea. The precipitate was dispersed in water, dissolved at pH 7, and allowed to stand in the cold for 4 to 5 days after which it was more stable to physiological concentrations of NaCl. Reversion to Ea did not take place.

Three preparations which differed in appearance and solubility in water at pH 7 were found to remove, per mg of DnEa N, the same amount of N from a calibrated anti-DnEa serum.

The reaction between DnEa and its homologous antibody is similar to that between Ea and anti-Ea. 1 The cross-reactions between anti-Ea and DnEa and between anti-DnEa and Ea differ greatly from both homologous reactions and from each other, as shown in Fig. 1.

A study of the chemical and physical properties of Ea denatured in various ways and a correlation of these properties with the immuno-logical findings is now in progress. Previous work in this field appears to have been confined to a preliminary note by Wu, TenBroeck, and Li, 2 the studies of Flosdorff and Chambers 3 and Blumenthal, 4 and the work of Porter and Pappenheimer, 5 and Landsteiner and Rothen 6 on surface-films.

The procedure described here for purification of equine encephalomyelitis virus protein 1 yields consistently a product of high molecular homogeneity. Advantage is taken chiefly of the following principles: (1) prolonged extraction to aid in eliminating the normal chick tissue component; 2 (2) extraction, fractionation and solution of the protein in a balanced salt solution, mammalian Ringer, 3 instead of 0.9% NaCl or buffer salt solutions; (3) filtration of crude extracts with celite to remove mucoid and colloid materials; and (4) aggregation or partial precipitation of the protein in slightly acid medium prior to the first ultracentrifugal cycle.

Diseased embryo tissue ground in the cold in the usual way, 2 is suspended in 4 times its volume of normal Ringer's solution made to pH 9.0 with NH₄OH without buffer salts. After extraction at 5°C for 72 to 96 hours, gross material is eliminated in the angle centrifuge. To each 100 cc of the turbid supernatant fluid, 5 g of No. 512 Celite Filter Aid (Johns-Manville Co., N. Y.) are added and the suspension is filtered with suction through a 1 to 2 mm mat of No. 503 Celite. Standard celite then added to the filtrate, 2 g per 100 cc, is filtered off through a mat also of standard celite. Sometimes the latter step is repeated to obtain an entirely clear filtrate.

The filtrate is acidified to pH 6.5 with 0.2 N HC1, and 120 cc of it distributed immediately in 8 collodion tubes is spun at 17,000 g for 45 minutes. The pellets are taken up in 30 cc of Ringer's solution. In 2 tubes, the solution is spun at 17,000 g for 5 minutes. The supernatant fluid diluted to 60 cc is ultracentrifuged 30 minutes at 67,000 g and the resulting pellets are taken up in 15.0 cc of Ringer fluid.

Previous ultracentrifugal studies 1 of crude equine encephalomyelitis chick vaccines 2 have failed to yield definite evidence of the character of the immunizing principle. Information concerning its probable nature has been sought in the present work by study of the purified virus protein treated with formaldehyde.

Purified protein (Eastern strain), 3 dissolved in Ringer's solution, pH 8-9, 2.0 mg per cc, was treated with various concentrations of CH₂O. Before exposure to CH₂O, one m.i.u. 4 of protein was 10^-13.5 g. When the concentration of CH₂O was less than 0.01 M, inactivation was not always complete in 2 weeks, and tests for immunizing capacity were not made. With other concentrations of CH₂O used, inactivation was complete in the time shown in Table I, as judged by the failure of 10^-5.5 g of protein to infect mice.

The effect of CH₂O on the protein molecules was somewhat similar to its action on tobacco mosaic virus protein. 5 With CH₂O 0.01 M or less, the protein remained in solution but lost slightly in homogeneity, showing a slightly diffuse boundary with a sedimentation pattern similar to that of the untreated protein 3 persisting with little change for more than 3 weeks. In 0.02 M and higher concentrations of CH₂O, the protein rapidly became quite inhomogeneous and increasingly insoluble, losing the definite boundary in 2-4 days. In no instance was there evidence of molecular disruption to small soluble protein fragments of uniform size, nor was there evidence of the component S₂₀° = ca 60 × 10^-13 cm sec^-1 dynes^-1 previously described. 6

The non-infectious, soluble though slightly inhomogeneous protein derivative with 0.01 M CH₂O has immunized all guinea pigs receiving it, as shown in the typical experiment in Table I.

Methylnaphthoquinone or methylnaphthohydroqui-none is the most active vitamin K compound known. It is as effective intravenously in an aqueous medium as orally in oil solution. Per os its potency is greater in water than in oil. The phosphate derivative is considerably less active. The sulfate is not as potent and not as rapidly absorbed by the organism as the phosphate. A new water-soluble vitamin K-active compound is described.

Comparing the gonadotropic response of the organism to subcutaneous or intraperitoneal administration of pregnancy urine prolan the following results were secured: (1) If the minimum dose is split up into 6 portions intraperitoneal and subcutaneous administration are equally effective. (2) If the minimum dose is split up into 3 portions the intraperitoneal route is approximately half as effective as the subcutaneous one. (3) If the minimum dose is given in one injection the intraperitoneal administration is about 1/10 as effective as subcutaneous. (4) Using the subcutaneous standardization method with minute doses as e.g., 1 RU (estrous effect) the gonadotropic effect of 6 subthreshold doses is less marked than that of one single threshold dose.

Aykroyd and Krishnam 1 observed that the incidence of angular stomatitis (sores in the corners of the mouth, perleche, cheilosis) coincided with a deficiency of some factor or factors of the vitamin B₂ complex.

More recently Sebrell and Butler 2 produced these lesions in 10 out of 18 women subsisting on a riboflavin-deficient diet. The lesions developed in a period of 94 to 130 days. They failed to respond to nicotinic acid but responded to riboflavin, the complete healing requiring from 5 to 58 days. Sebrell and Butler 3 added more evidence to their previous findings and these have been confirmed by others. 4 , 5 , 6

In view of these facts, it seems important to report our observations on 4 consecutive cases of cheilosis occurring spontaneously in association with other deficiency syndromes in patients admitted to this hospital and treated with synthetic vitamin B₆ [2 methyl, 3 hydroxy, 4.5 di(hydroxymethyl) pyridine].∗

The first patient treated was a 7-year-old colored girl admitted to the hospital with a diagnosis of pellagra. Pellagrous lesions were present on the hands and feet. A dietary history revealed a marked deficiency of the vitamin B-complex. There were typical cheilosis lesions of the lips, characterized by maceration and fissuring at the angles. (Fig. 1a.) There was an associated severe anemia. The patient was placed on the basic diet of Smith and Ruffin which is known to be low in the B-complex and especially so in riboflavin. 7 Treatment with synthetic vitamin B₆ hydrochloride† was started with 20 mg intravenously 24 hours after admission. Within 5 hours slight but definite objective improvement could be observed in the mouth lesion.

Sulfathiazole is an effective bacteriostatic and bactericidal agent against S. aureus when added to defibrinated whole blood in vitro. Concentrations between 2.5 and 5 mg/100 cc are necessary to obtain the maximal effect. Comparison of this drug with sulfamethylthiazole, sulfanilamide, and sulfapyridine show it to be somewhat superior in the experiments reported.

We wish to thank The Squibb Institute of Medical Research for the supply of sulfathiazole and The Department of Medical Research of the Winthrop Chemical Company for the supply of sulfamethylthiazole used in this investigation.

We wish to thank Miss Marjorie Jewell and Miss Eleanor Fleming for technical assistance.

In studying the metabolism of vitamin C in normal and depan-creatized dogs, the urinary excretion of the vitamin was found to be strikingly reduced in the diabetic animal. The diets in both groups of animals were totally devoid of vitamin C; dried meat powder was used in place of raw beef. The dogs were kept in individual metabolism cages and the urines were collected by catheter at the start and end of each 24-hour period. Urines voided between catheterizations were collected in dark bottles containing sulphuric acid and hydroxyquinoline to prevent the destruction of the urinary ascorbic acid. Fasting bloods were taken for the determination of the plasma vitamin C. 1 Urinary vitamin C was determined by the method of Evelyn. 2 The depancreatized dogs received insulin twice daily in amounts which allowed a moderate daily excretion of sugar.

The 6 normal dogs excreted an average of 20.6 mg of vitamin C per kg of body weight in 24 hours. The fasting plasma levels of vitamin C varied from 0.55 to 0.94 mg %. The 7 diabetic dogs maintained on insulin excreted on an average only 3.6 mg of vitamin C per kg of body weight in 24 hours. The fasting plasma levels varied from 0.33 to 0.71 mg %.

The influence of injected insulin on the plasma level and excretion of vitamin C was studied in a series of acute experiments in both the normal and depancreatized dog. Control experiments were run on each dog to ascertain the hourly plasma variation and urinary excretion of vitamin C. Over a period of 8 hours the plasma levels of vitamin C in the normal dog varied from 0.58 to 0.66 mg % and the urinary excretion varied from 2.16 to 4.92 mg per hour (Chart 1).

Standard tests for the sterility of finished vaccines or serums consist of the inoculation of 0.25 cc and. 1.0 cc from the proper number of samples of the product to Smith fermentation-tubes containing infusion-broth. The tubes are heated within 5 hours before inoculation to drive off dissolved oxygen. They are then inoculated and are incubated for 7 days. The presence of weak points in the standard procedure has long been suspected.

The inherent weaknesses of the standard test are, in part, dependent upon (1) the frequent use of merthiolate which, because of its bacteriostatic effect, prevents the growth of contaminants, if any, in many instances; and (2) standard Smith tubes are not well adapted to the cultivation of anaerobes. The use of a second transfer, after 7 days, from optically clear Smith tubes to fresh Smith tubes in an endeavor to overcome the bacteriostatic action of the preservative is definitely irrational and thus unsatisfactory. 1

The use of Brewer's medium 2 containing thioglycollate overcomes the objections given because it permits the growth of organisms in the presence of merthiolate and because it is suited to the cultivation of anaerobes. The results reported here support these views.

Tests were made with vaccines (bacterins) and serums, preserved with merthiolate, 1:5000 or 1:10,000, or with phenol, 0.5%. As contaminants 24-hour cultures of Staph. aureus, Staph. albus, Ps. pyocyanea, B. subtilis and C. xerosis were used. 3 In addition, there were available dried spores of Cl. tetani in sand, 10 years old, prepared by Coleman. 4 Vaccines and serums were inoculated lightly with these organisms, singly and in mixtures. From these products 0.25 cc and 1.0 cc respectively, containing from 5 to 500 organisms, were transferred to standard Smith tubes and also to tubes containing 12 cc of thioglycollate medium.∗ Incubation at 37 °C for 7 days followed, with frequent observations.

The known biological incompleteness of the protein fraction of the bean has received considerable attention in interpretations of the malnutrition caused by a raw bean diet. Belák and Szathmáry, 1 , 2 and Zselyonka and Illényi 3 were the first to point out that, together with this loss of weight, there was also observable an inhibition of the estrus of mice during the period of feeding with a diet containing 15% of raw bean. They isolated a globulin fraction from the bean flour, which they referred to as “phaseolin.” Together with a loss of weight, the inhibition of the estrus of the mice could be observed when 5% of the basic food of the mice was replaced by this protein fraction. The fat-soluble fraction of the bean was found by the same authors to be ineffective. Selye and Collip, 4 in replacing 30 to 50% of the basic food of the rat with bean flour, observed a certain atrophy of the ovaries and interpreted their findings as a consequence of a “malnutrition.”

In our feeding experiments, first using rats as test animals, and following the course of the estrous cycles by means of the vaginal smear method of Long and Evans, 5 we were able to establish the following :

1. Complete inhibition of the normal estrous cycle could be observed when 15% of the basic food was replaced by bean flour. Controls, on a diet without bean flour, failed to show any change in their regular cycles, whether that diet was offered to them (a) ad libitum, or (b) in an amount 15% less by weight than that regularly taken by these animals.

2. The pure globulin fraction of the bean, as prepared by Osborne, 6 given in 5 % by weight to the basic food did not inhibit the estrus of the rats.

A method is devised for the quantitative determination of antithrombic activity of serum and plasma. One unit of antithrombin is defined as that amount which will inactivate or neutralize 1 unit of thrombin in 4 minutes' incubation at 28°C. There is little or no quantitative difference in the antithrombin of serum and plasma. The antithrombic activity of normal serum and plasma is considerably greater than has been previously described.

Antitoxin-formation in the rabbit is more rapid and abundant after intravenous than after subcutaneous injections of 25 L_f alum-precipitated diphtheric toxoid.

1. Embryonic chicks are apparently not susceptible to infection with this avian virus, although birds just hatched are. This is a strikingly reversed state from that which prevails in the case of certain other viruses, 5 which multiply readily in the undifferentiated tissues of the developing embryo but are inactive in the hatched chick. Whatever the reason may be, a fact of epidemiological significance emerges: the disease-agent is probably not transmissible by way of the egg, thus supporting the prior finding of Jones. 1

2. The virus multiplied in minced whole-embryo tissue-cultures in vitro only under certain indicated conditions. The method in its present state, however, is not favorable for obtaining large yields of highly potent virus for use in immunizing procedures. No multiplication of virus was noted in this medium when chick-embryo brain was used instead of whole-embryo tissue. It is of interest in this connection that mammalian embryo-brain cultures have been found suitable for the multiplication of the neurotropic viruses of poliomyelitis 6 and of rabies. 7

1. Leech extracts contain a powerful mucolytic enzyme, as shown by its effect on the viscosity of chicken tumor I extracts. 2. Leech head extracts exhibit a mucolytic activity considerably greater than that of similar extracts obtained from the rest of the leech body. A comparable quantitative relationship is found to exist between head and body extracts when tested for another property, e. g., their power to spread in the rabbit skin.

3. The parallelism in the strength of various extracts, as regards both mucolytic activity and spreading power, supports the view that the “mucinase” and the leech spreading factor may be identical.

4. A mucoprotein has been prepared from normal rabbit skin. 5. The viscosity of a skin mucoprotein solution is rapidly and considerably reduced by the action of leech extracts. 6. The effect of leech extracts on the skin mucoprotein in vitro suggests that their ability to spread through the skin may be due, at least in part, to their power to cause hydrolysis or depolymerisation of the same or a similar compound in vivo.

Three synthetic vitamin K compounds were administered to 26 patients with hypoprothrombinemia. Following treatment the prothrombin concentration: (1) was markedly elevated in 11 patients with obstructive jaundice, (2) was not elevated in 8 and only slightly elevated in 3 patients with chronic diseases of the liver; (3) was not elevated in 1 patient, and after an initial delay was elevated in 1 patient with acute diseases of the liver; (4) was moderately elevated in 2 patients, one with non-tropical sprue, and the other with gastro-colic fistula. When considered in terms of the dosages employed, there were no significant qualitative differences in the relative effectiveness of the three compounds. No untoward reactions were observed except that the patients receiving large doses of 4-amino-2-methyl-naphthol-hydrochloride complained of slight burning pain at the site of injection.

The finding that the osmotic concentration, as revealed by the depression of the freezing point, remains constant during the action of pituitary extract on the diuresis induced by rapid intravenous infusion of sucrose solutions, lends support to the view that the absorption capacity of the tubules has been reached under these conditions of experiment.

It is generally agreed that the hypertension which develops in experimental animals as a result of partial 1 or complete 2 ischemia of the kidneys is of humoral and not of nervous origin.

Although renin, a pressor extract prepared from the renal cortex, has been implicated by some observers, 3 , 4 neither this product nor any other has to date been proved to be the substance responsible for ischemic hypertension.

In this communication we wish to report the rinding of a per-fusate of ischemic kidneys which causes the elevation of blood pressure following the release of complete renal ischemia. Until the precise nature of the active principle has been determined, we propose the name ischemin, which may be defined as the pressor substance contained in perfusates of completely ischemic kidneys and which is responsible for the hypertension resulting from the release of total renal ischemia. For convenience, the renal per-fusate containing the pressor substance will also be termed ischemin.

The results of the observations made thus far are summarized briefly.

In 8 experiments, cats were anesthetized with ether and the renal pedicle of one kidney clamped. After 4 to 6 hours the animal was reanesthetized with nembutal, the blood pressure recorded from the carotid artery, and both the normal and ischemic kidneys removed. Within 5 minutes following the removal of the kidneys, both renal arteries were cannulated and the kidneys perfused with 1 cc of warm saline per gram of kidney. The perfusates were then injected intravenously into the same animal. The perfusates of the ischemic kidneys uniformly gave a marked and prolonged rise in blood pressure, amounting to as much as 100 mm of Hg, whereas those obtained from the normal kidneys had no pressor effect.

A method of obtaining virulent, encapsulated group A beta hemolytic streptococci, sensitive to the reaction of capsular swelling with homologous antisera, has been described. Essentially this consists of the injection of living streptococci into mice simultaneously treated with unrefined scarlet fever antitoxin.

Small secondary colonies, corresponding morphologically to young colonies of the L1 strain isolated by Klieneberger from cultures of Streptobacillus moniliformis, have been observed occasionally in cultures of various Gram negative bacteria. 1 In all cultures in which such secondary colonies developed a large number of bacteria swelled up into large deeply stained, spherical or fusiform bodies. It was first seen in a culture of Bacillus influenzæ that occasionally L type of colonies developed from the large bodies. Later this process was observed repeatedly in the cultures of 2 colon bacillus strains.

After 24 hours of growth the colonies of these strains consisted mainly of large spherical bodies. In order to observe the development of these large bodies in these 2 strains different procedures proved to be helpful. The colonies of one strain were very tenacious and when an impression of the colonies was made on blood agar plates only the large bodies, with very few bacteria, were transferred. After 6 to 12 hours fine curved filaments grew from the large bodies into the agar and after 24 hours about two-thirds of them developed into tiny L type colonies. With the other strain a similar process was observed when a broth culture was transferred on blood agar plates after varying time intervals. In the transplant made after 24 hours' growth many large bodies were visible on the agar surface. Only a few bacterial colonies developed in these plates but after 6 to 12 hours the large bodies began to germinate and produced L type colonies. Transplants made from the broth culture after 48 hours, gave only abundant bacterial growth. With either strain, L type colonies developed only at the places where large bodies were present.

Although pneumococci can be typed directly from sputum, it has been recognized that the results are sometimes unsatisfactory. Taplin, Meneely and Hettig 1 have outlined an improved method using fresh sputum to obtain the Quellung phenomenon. Essentially, this method consists of breaking up and suspending the sample in saline, centrifuging, and using the sediment for typing. The authors suggested that the water-soluble capsular polysaccharide might well be the interfering substance, which was removed by their technic.

In work published elsewhere, 2 it has been determined that in undiluted samples of ground lung from pneumonic rats, pneumococci present did not give the Quellung reaction with specific antiserum. The reaction did occur if the material were sufficiently diluted with PSS. Heidelberger 3 has shown that excess of antigen prevents visible evidence of antigen-antibody reaction, and has ably discussed the reversibility of the precipitin and agglutinin reactions. Since the above findings indicated that excess antigen prevented the occurrence of the Quellung reaction, it seemed that reversibility of this phenomenon might likewise take place. Hence, the following experiments were performed.

In each of 4 serological tubes were placed 0.1 cc of a broth culture of Type I pneumococcus and 0.1 cc of undiluted type-specific rabbit antiserum; after a short interval, the pneumococci were examined for the presence of swollen capsules. These were uniformly present. Then 0.1 cc of dilutions of Type I polysaccharides were added to each tube to give final amounts of 1.0 mg, 0.1 mg, 0.01 mg, and 0.001 mg in 0.3 cc of fluid, respectively. Five minutes later, the preparations were reëxamined. In the first 2 tubes, the swollen capsules had disappeared. In the last 2 tubes, no change was observed.

Sulfathiazole and sulfamethylthiazole, administered as 1% of the diet showed a distinct and equal therapeutic value in prolonging the lives of mice heavily infected with Staphylococcus aureus. Sulfathiazole proved to be somewhat more efficient in this respect than sulfapyridine.

We are indebted to E. R. Squibb and Sons and the Calco Chemical Division of the American Cyanamid Company for the sulfathiazole used in these experiments, and to the Department of Medical Research of the Winthrop Chemical Company for the sulfamethylthiazole.

It appears from this study that Δ⁴ pregnen-in-20-on-3-ol-17 exhibits bisexual properties when administered subcutaneously in oil to immature, female rats, having an estrogen-like action on the uterus and vagina and an androgen-like action on the preputial glands and clitoris. The trophic effect on the genital tract appears to be a direct one and not mediated through the ovaries, as shown by the elicitation of similar effects in ovariec-tomized immature animals. Unlike testosterone, however, preg-neninolone appears not to have a stimulating effect upon the gonado-tropic activity of the hypophysis, as indicated by the absence of corpora lutea formation or evidence of follicle stimulation in the ovaries.

It is interesting to note that this compound possesses a unique variety of biological properties. In addition to its progesterone-like action on the endometrium, Courrier and Jost 14 have found that pregneninolone will maintain pregnancy in spayed rabbits and is androgenic in castrated rats and chicks. Furthermore, Emmens and Parkes 15 have shown that this compound has estrogen-like properties when administered to adult, female rats, as well as androgenic activity, as indicated by the capon comb growth. It is worthy of note that the introduction of the ethinyl group at the 17th carbon atom -of testosterone produced a compound possessing properties that are characteristic of estrogens, androgens and progesterone and at the same time has resulted in a loss of hypophyseotropic potency—the power to stimulate gonadotropic hormone secretion by the hypophysis.

The local tissue response to implanted crystals and pellets is a typical non-specific foreign body reaction. Following the implantation of crystals of a-estradiol and a-estradiol benzoate, the tissue surrounding the crystals responds by the formation of tiny nodules. In contrast to this type of reaction, a thick avascular capsule forms about the pellets of the implanted hormone. It is important to note that the epidermis overlying the implants showed no evidence of cellular atypism and, similarly, in no instance did the subcutaneous tissues in contact with, and adjacent to, the implanted hormone show any abnormal cellular proliferation.

A significant observation which emerges from this study is the probable effect that the thick avascular capsule has on the absorption rate of the estrogenic substances employed. It was noted that the crystals had a strikingly more prolonged therapeutic and physiologic effect than the pellets composed of the same estrogenic substance and of comparable weight. It would appear, therefore, that the capsule might have a marked retarding effect on the rate of absorption of the hormone. Apparently, with the passage of time, the absorption rate is progressively diminished by the growing thickness of the capsule around the pellet. After a period of approximately 3 months, absorption is either completely stopped or so reduced as to have no demonstrable physiologic or therapeutic effect. It seems, therefore, that for purposes of implantation, pellets of a-estradiol and a-estradiol benzoate are not as efficient as crystals of the same chemical constitution.

Typical poliomyelitis has been produced in 4 chimpanzees by the intranasal, intragastric, and oral inoculation of untreated human stools. In 2 instances the olfactory portal was ruled out by section of both olfactory tracts.

Extracts of kidney and muscle effectively reduce arterial pressure in hypertensive human beings, dogs and rats for prolonged periods and simultaneously reduce the ability of plasma to activate renin and angiotonin.

An antiprothrombin serum may be prepared in the guinea pig by injecting it with fibrinogen-free prothrombin isolated from rabbit plasma. Intravenous injections of this antiserum in fasting rabbits produce a hypoprothrombinemia of a transient and moderate character.

It is felt that on the basis of the reports quoted and on the above data, thymectomy by surgical methods has no apparent effect on the growth, development, or endocrine organ differentiation of the rat when performed at the age of 1-2 days and studied past the onset of puberty. Since surgical thymectomy results in no apparent effects as studied above, it is probable that irradiation thymectomy can have no further effect, and that the cause of the effects described 1 , 2 must be sought elsewhere.

During insulin shock induced therapeutically there is a progressive depression of nervous functions, in a gradient manner, from the highest (cortical) levels to lower subcortical levels (striatum, mid-brain, medullary) which is temporary and reversible. 1 , 2 It was decided to demonstrate this gradient effect clinically with the production of animal preparations showing permanent, irreversible lesions at progressively lower levels in the nervous system by prolonging the period of hypoglycemia.

Cats, fasted 18 hours, were given 15 to 20 units of insulin per kilo body weight. When clinical signs indicated early medullary decompensation, small amounts of glucose were given intraperitoneally to prevent death and still maintain a marked degree of hypoglycemia. Hypoglycemia was terminated after 12 to 20 hours. Persistent brain damage was observed in 17 animals. Group I consisted of 5 animals with variable degrees of cortical damage. Group 2 totaled 8 animals with cortical plus subcortical injury (“thalamic preparations”?). Group 3 included 4 cats with loss of function at the midbrain level or lower.

The decorticate preparations (Group 1) showed impairment of vision, impairment of placing and hopping reactions (Bard), slight impairment of righting reactions, difficulty in feeding self, impairment of cleaning habits, restlessness and absence of rage reaction when confronted by a dog. They were able to walk and maintained normal body temperature. These preparations were kept alive from 6 to 90 days.

Preparations in Group 2 showed in addition to the above, inability to walk, decerebrate rigidity, increased tonic neck reflexes and mock rage. These animals were also able to maintain body temperature. These signs point to loss of function at the “thalamic level.” The animals lived 19 to 48 hours after the hypoglycemia was terminated.

Animals in Group 3 were not studied as carefully as the above, but are added because they showed inability to maintain body temperature.

Several years ago, soil bacteria capable of producing adaptive enzymes which specifically decompose creatine and creatinine were isolated. 1 , 2 This report presents preliminary observations on the manner of preparation and properties of soluble enzymes prepared from one species (NC) of these bacteria.

When the washed cells are desiccated by various methods a portion of the enzymic system which decomposes creatinine is rendered soluble. This soluble portion can be obtained by adding water to the dried suspension, centrifuging, and collecting the clear supernatant solution. The methods that have been used include grinding with abrasive materials, such as pumice-stone, desiccation with phosphorus pentoxide and sulphuric acid, and desiccation with alcohol-ether, or acetone-ether.

The method of assay employs 0.5 mg of neutral creatinine-hydro-chloride as substrate, 0.5 cc of 0.5 M phosphate buffer pH 7.0, the substance being tested, and water to 5 cc. The suspension is incubated at 38° to 40°C for 30 minutes. The undecomposed creatinine is determined colorimetrically after the addition of alkaline picrate.

The activity of the preparation obtained under the above conditions represents about 10% of the activity of the original cells when assayed under the same conditions. The properties of the extract differ in certain respects from the original cells. Sodium cyanide 10^-3 M, and thymol inhibit the cells but not the extract. Toluene and formaldehyde depress the activity of the cells and extract. Sodium fluoride, 10^-3 M, does not inhibit the cells or extract.

Studies on the purification of the enzymes are in progress.

Testosterone propionate is capable of stimulating the thyroid and parathyroid glands of the intact immature female rat. This stimulation is manifested by increased mitotic activity and by histological evidence of functional activity.

It is known that heparin does not prevent the coagulation of a fibrinogen solution by purified thrombin. 1 It likewise is unable to prevent the conversion of isolated prothrombin to thrombin. 2 , 3 To exert its powerful anticoagulant action, heparin requires an additional factor which is known to be present in blood, plasma, and serum. 1 , 4 , 5 This factor has been shown by Quick 4 to be contained in the serum albumin fraction.

We have investigated the activity of various components of the albumin fraction in producing the anticoagulant effect of heparin on the fibrinogen-thrombin system.

The thrombin was obtained by the method of Eagle, 6 and the fibrinogen was prepared in the usual manner by repeated precipitations with sodium chloride. Heparin was used in the form of the pure sodium salt.†

The activities of the albumin components studied are shown in Table I, which reproduces a typical experiment. The letters in the first column of this table stand for the following compounds: A, crystalline albumin from human serum, prepared by Dr. F. E. Kendall of the Research Division for Chronic Diseases, Welfare Island. We are greatly indebted to Dr. Kendall for different specimens of this substance. B, albumin from sheep plasma, prepared according to Howe. 7 C, fraction from human plasma insoluble at 55% saturation with ammonium sulfate, presumably containing the globoglycoid of Hewitt. 8 D, fraction from human plasma insoluble at 75% saturation with ammonium sulfate. E, fraction from human plasma insoluble at 100% saturation with ammonium sulfate. This fraction presumably contained the seroglycoid of Hewitt. 9 F, the supernatant solution after removal of E, dialyzed and concentrated by ultrafiltration.

Six patients with primary hypothyroidism, in whom chloride excretion was studied during periods of sodium chloride restriction, showed no evidences of limitation of adrenocortical function.

Type I pneumococcus could be dissociated by gamma ray radiation. The rough forms appeared suddenly, becoming totally avirulent and irreversible. This dissociative change could be achieved in a shorter period of irradiation by the addition of immune serum.

Antigen was recovered from Type I Pneumococcus immune precipitate from which some antibody had been removed by treatment with acid. This experiment substantiates our previous finding that the recovery of antibody from immune precipitate by the action of acid or alkali is due to a shift in the antigen-antibody equilibrium. The present finding also suggests a possible method for the isolation of pure antigen which may be useful when it cannot be obtained otherwise.

From the observations made on the chemical and immunological studies of the polysaccharide, lipoid, and protein fractions obtained from representative serological and cultural types it is justifiable to conclude that the alkalisoluble protein is the cellular constituent responsible for type-specificity in C. diphtheria.

That the feeding of liver to animals produces an enlargement of the thyroid gland was reported by Hunt, 1 who found that the thyroid of mice fed on a diet of oatmeal and liver was larger and more vascular than of those on a diet of eggs, crackers and milk. Marine 2 produced a hyperplasia of the thyroid in brook trout by feeding them with liver and heart. Burget 3 found that rats kept under hygienic conditions and fed a high protein diet, consisting of fresh liver and lean beef mixed with a little oatmeal or bread crumbs, developed a hyperplasia of the thyroid. All the above reports of enlargement of the thyroid were interpreted as due to the high protein diet.

Recently, Remington 4 reported that in a low iodine goiter-producing diet, the replacement of a part of the wheat gluten by dried pig liver aggravated the degree of goiter, while purified casein prevented goiter formation, and yeast had no effect. This led Remington to believe that the liver in his goiter-producing diet was responsible for the enlargement of the thyroid. The writer (Hou 5 ) in the same year reported that rats fed on a soybean-millet diet with dried liver powder as the only source of animal protein developed a marked hypertrophy of the thyroid gland. The enlargement was over 10 times when the animal was fed on the millet and liver diet for over a year. It becomes of great interest to know which constituent or constituents of the diet is or are responsible for the enlargement of the thyroid. The following series of experiments were accordingly carried out.

Albino rats of the Wistar strain were selected with reference to litter mates and sex when about one month old.

Although the virus of encephalitis of St. Louis type has been successfully cultivated by Maitland technic and on the chorio-allantoic membrane of developing chick 1-4 the titer obtained in these cultures has been invariably low. None of the cultures showed a titer as high as 10^-6 even after repeated passages. Recently Kawakita 5 has reported successful cultivation of Japanese encephalitis virus in a special medium containing chick allantoic fluid. It has occurred to us to attempt to cultivate the virus of St. Louis type on the agar-tissue medium which has been found to be satisfactory for the growth of various types of rickettsiae. 6 , 7 The results of such a study together with a preliminary study on the immunizing value of vaccine prepared from these cultures are hereby communicated.

The virus, Webster No. 3. which has full virulence for mice, was propagated in the following fashion: Infected mouse brain, diluted with serum-Tyrode solution mixture (1:5) to 10% by weight was filtered through Seitz E. K. pad after brief centrifugalization. A small amount of the filtrate was inoculated on to a small piece of young mouse embryonic tissue in a sterile Petri dish. These were minced together, and after being allowed to stand for 15 to 20 minutes at room temperature, the finely cut tissue bits were carefully laid on the surface of agar slants employed for the cultivation of rickettsiae. These cultures were incubated at 37°C, and transferred every 5-8 days. By this method, the virus has been carried for over 40 generations in a period for more than 10 months. The activity of the virus for Swiss mice by intracerebral injections of different generations was found to be as follows: 10^-6 for the sixth, 10^-6 for the sixteenth, 10^-7 for the twenty-first, and 10^-6 for the thirty-fourth generations of cultures.

Dixon, Hill and Keilin 1 published the ultraviolet absorption curve of cytochrome c. A sharp maximum was found at about 4150 Å. Other than a plateau in the region 2600-2900 Å no other structure was detected. Roche and Benevent 2 also found a similar curve.

The cytochrome c used was prepared from fresh beef heart by the method of Keilin and Hartree. 3 Tetrapyrrolic iron was determined by the dipyridyl technic of Hill, 4 following the treatment of cytochrome c with alkali and hydrogen peroxide. Several lots were assayed for the hemin iron content with an average value of 0.39%. The theoretical value is around 0.4%. 5

Since it is known that the absorption curves obtained with the line source and photometer may fail to show all the details of the bands, 6 photographs were taken with the continuous light of the hydrogen discharge tube and a small Hilger quartz spectrograph. The spectrum obtained is drawn in Fig. la. The band at 4150 Å is seen, also other bands not clearly indicated in the curve. The band in the region of 3500Å is rather diffuse as are the other bands in the protein region. Not shown in the illustration are bands in the region 2550-2600 Å. These are of such intensity that it is difficult to obtain their exact wave length. 5

When cytochrome c is treated with sodium formaldehyde sulf-oxylate (CH₂OHOSONa) the band at 3500 Å disappears and a new band appears at about 3100 Å, as depicted in Fig. 1b. At the same time the well characterized band of reduced cytochrome c becomes apparent in the visible at 5500 Å. Experiments are now in progress to determine the possible reversibility of this process and the complete significance of the above data is being further investigated.

Investigation of the various factors which control the placental circulation has necessarily been limited owing to the experimental difficulties involved. It is suggested that the following technic may offer a line of approach to some of the problems involved.

Methods. The experimental animals used were cats or bitches which were in an advanced stage of pregnancy. Rabbits were found to be unsuitable owing to the friability of the placental vessels. Anesthesia was induced with open ether, and maintained with chloralose given intravenously in a dose of 100 mg/kilo body weight. Both vagi were cut, and the carotid blood pressure recorded by means of a mercury manometer. The abdomen was opened with as short an incision as was compatible with adequate access, and a part of the uterus containing one foetus brought up into the wound. A small cork platform approximately 5 cm square was approximated to the section of uterus exposed, and the uterine peritoneum loosely stitched to the two nearest corners of the platform with silk ligatures. An incision about 2 cm long was then made through a part of the uterine wall which was free of placental attachments, the membranes ruptured, and the hind-quarters of the foetus delivered until the umbilical cord came into view. If the foetus was lying in a favorable position, it was possible to expose the umbilicus with the umbilical vessels without delivering any part of the foetus. The umbilical cord was then ligatured in 2 places as close to the umbilicus as possible, divided, and the placental end delivered through the wound in the uterine wall. Thereafter the foetus was replaced within the uterus and the uterine wall stitched up again, leaving only the free end of the umbilical cord protruding.

In animals metrazol caused cerebral vasodilation. This was unrelated to changes in blood pressure or in respiration, and showed no constant relation to convulsive seizures. No cerebral vasoconstriction was observed.

The exact relationship of leukemia to neoplastic disease has not been determined. The following types of human neoplasm, when examined by the procedure used by Kögl and Erxleben, 1 have been reported to yield partly racemized glutamic acid : 2 carcinoma of the ovary, carcinoma of the breast, adenocarcinoma of the colon, and sarcoma of the thigh. Normal human tissues (ovaries, colon) have been reported to yield only 1 (+)glutamic acid. 2

We have isolated glutamic acid from the liver and spleen of a patient who died of subacute lymphatic leukemia. Microscopic examination revealed that at least 20% of the liver, and 30% of the spleen, were composed of leukemic areas. Major portions of these organs removed at autopsy were placed in alcohol. Following transport to the laboratory, the tissues were finely minced, after which they were covered with 0.1 N hydrochloric acid and heated several hours on the steam bath until a majority of the material had gone into solution. Sufficient trichloracetic acid then was added to make a concentration of 10%. The precipitated proteins were filtered off, washed and extracted several times with hot alcohol in order to remove trichloracetic acid. The proteins were dried at 110° and lipids partly were extracted with boiling ether. Hydrolysis and isolation of glutamic acid were then carried out as described by Kogl, Erxleben, and Akkerman. 3 The results obtained are recorded in Table I. No racemic acid was isolated from the leukemic tissues.

As a control, glutamic acid was isolated from protein obtained from a kidney neoplasm (dog). We are indebted to Dr. E. T. Bell, Head of the Department of Pathology, University of Minnesota Medical School, who examined sections of this tumor and found it to be an undifferentiated sarcoma.

Proteins obtained from the embryonic tissues of chicks and pigs have been hydrolyzed, and the glutamic acid hydrochloride obtained from the hydrolysates examined in the polarimeter. The glutamic acid hydrochloride isolated was entirely the 1(+) variety.

Proteases secreted by the embryos of numerous species of fish and amphibia are believed to be important factors in bringing about hatching by digestion of the egg membranes. It has been asserted that in teleosts this protease is much like trypsin and so powerful that it attacks the embryo itself. 1 This statement conflicts with the dictum that living organisms are resistant to digestive enzymes. A striking confirmation of the latter view is Northrup's demonstration of the immunity of a wide variety of organisms to concentrated trypsin. 2

These conflicting statements suggested the desirability of investigating the action of trypsin on the embryologically useful amphibian Rana pipiens. Since this anuran appears to possess a hatching enzyme the effect of trypsin on both membranes and embryonic viability was studied. The eggs were stripped of excess jelly on paper toweling and immersed in a 10% solution of dialyzed trypsin made up in 10% Ringer solution. Each experiment involved 25-50 eggs. Table I gives some typical results.

These results are in agreement with Northrup's work in their clear distinction between the susceptibility of the membrane proteins and the resistance of the living organism to trypsin. The possibility, suggested by work on teleosts, of a general embryonic sensitivity to digestive enzymes is not substantiated. While this may still be the case in teleosts, it seems more likely that the injurious action of the egg contents on the embryos was due to digestion products.

The parallelism between the effects of trypsin and normal hatching in Rana pipicns is in agreement with the theory that normal hatching is due to a digestive enzyme. It should be pointed out in this connection that the power of trypsin to mimic normal hatching makes it useful in large scale chemical work with early stages of anurans where the presence of the difficultly removable membranes may prove a hindrance to analysis or penetration.

Saline suspensions of virus-induced rabbit papillomas may stimulate the production of specific antiviral antibody when injected in-traperitoneally into normal rabbits, as Shope found, even though they contain no pathogenic virus demonstrable by the ordinary test. 1 In experiments of the same sort we found that saline extracts containing infectious papilloma virus in quantity elicited the antibody in much higher titer than extracts in which little or none was present. 2 Other studies already reported from this laboratory have shown that the antibody often extravasates into the large, disorderly papillomas of cottontail rabbits in such quantity as to “mask” the causative virus, 3 and that the antibody can be identified as such in extracts of the growths. 4 With these findings in mind, experiments were undertaken to determine whether the antibody, which accumulates in the papillomas in various amounts depending upon the titer of it in the blood and upon the local vascular conditions determining its extravasation, may not influence the anti-genicity of extracts of the growths.

The antigenicity of the papilloma virus, as determined by its capacity to elicit antibody upon intraperitoneal injection into normal rabbits, was found to be markedly reduced when antibody was mixed with it in vitro in amounts sufficient to neutralize it. When an excess of antibody was added to a filtrate containing highly infectious virus, the mixture elicited no antibody upon repeated intraperitoneal injections into normal rabbits, although the control mixture containing saline and the same amount of virus (approximately 20,000 infectious doses for each animal) proved highly antigenic.

To procure virus-induced papillomas that were certain to contain the extravasated antibody in quantity, a number of cottontail and domestic rabbits carrying vigorous confluent growths of 2 to 4 weeks' duration were injected intraperitoneally with large quantities of active virus.

All smooth strains of the 3 species of Brucella have been found to possess a definite capsule.

The technic for demonstrating the presence of a capsule on Brucella is as follows: A dilute suspension of organisms is prepared in physiological salt solution from a 48-hour liver agar slant growth. A drop of the suspension is drawn across a clean glass slide similar to the method of making a blood film. Allow the film to dry in air. Place a small drop of Kesso India ink near one end of the slide and draw it over the film of organisms by placing the end edge of another slide at such an angle that the India ink spread becomes thinned out. After air drying, the spread is flooded with an aqueous solution of crystal violet (5 cc of saturated alcoholic solution of crystal violet in 95 cc distilled water). The excess of stain is washed off the slide with distilled water at the end of 2 minutes. The stained spreads are dried in air.

When examined under the microscope, magnification 1000X, the organisms appear as dark purple cocci or rods located in the center or near the edge of a clear area surrounded by India ink. The clear area, which is the capsule, varies in size from 2.5 to 4. The size of the capsule varies with the size of the stained cell.

The writer has met with no success in staining the capsule on Brucella cells.

The rough form of Brucella also shows a capsule. A large proportion of the cells of this form emerge from the capsule when suspended and allowed to stand in distilled water at 37°C. As the cell leaves the capsule, the cell becomes swollen and more intensely stained.

At the present time it is not as yet possible to predict from the chemical composition of various sulfanilamide derivatives their chemotherapeutic activity toward different microorganisms that are more or less susceptible to the action of sulfanilamide. Thus, it is necessary to test the efficacy of new compounds both in vitro and in vivo. It is generally agreed that sulfanilamide in therapeutic doses has little or no effect on enterococci. Recently Helmholz¹ reported that sulfathiazol and sulfamethylthiazol in urine are bactericidal toward enterococci. Hill² found that sulfanilamide, sulfapyridine and sulfathiazol in a concentration of 100 mg % have a marked bacteriostatic or germicidal action upon relatively small numbers of alpha-Streptococcus fecalis in urine. This author made the important observation that sulfathiazol as present in urine after administration of the drug exerts greater bacteriostatic activity than the same amount added to normal voided urine. Long and Bliss³ observed that sulfanilamide, sulfapyridine and sulfathiazol in concentrations of 10 mg % only slightly inhibited the growth of hemo-lytic streptococcus zymogenes (Group D) in beef infusion, 2% Neo-peptone, 0.075% dextrose broth. Clinically, Pool and Cook⁴ reported sterilization of urine of patients harboring Streptococcus fecalis following the administration of sulfathiazol and sulfamethylthiazol, respectively. The concentration of the drug in the urine of these cases exceeded 150 mg %. The following in vitro experiments were carried out in order to determine the relative bacteriostatic activity of various sulfanilamide derivatives toward hemolytic and non-hemolytic enterococci.

As culture medium 1/4% maltose phenol red broth base (Difco), containing tryptose (1%), sodium chloride (0.5%), dipotassium phosphate (0.1%), and phenol red, was used. Sulfanilamide, sulfapyridine, sulfanilyl-sulfanilamide (Disulon), sulfathiazol (2-sulfanilamidothiazol) and sulfamethylthiazol (2-sulfanilamidomethyl-thiazol) were added to this culture medium.

Employing the technic of Wiggert, et al., 1 an enzyme-preparation has been extracted from cells of Aerobacter aërogenes, with apparently no hydroclastic activity, nevertheless, capable of converting pyruvic acid into CO₂ and acetylmethylcarbinol. Typical fermentation-balances are shown in Table I. The ratio of pyruvate utilized to CO₂ and acetylmethylcarbinol formed is approximately 2:2:1.

Attempts to increase the carbinol yield by the addition of acetaldehyde were unsuccessful. The added aldehyde was recovered unchanged. If acetaldehyde is an intermediate, only the biologically nascent form is utilized by this enzyme-preparation.

As indicated in Table II no appreciable production of CO₂ and consequently no carbinol occurs at pH levels above 6.8. There is a direct relationship between CO₂ and carbinol production.

The relationship between pH and the direct cleavage of CO₂ from pyruvic acid by Aerobacter will assist in elucidating the mechanism of pyruvate-breakdown. From the investigations of Mickelson and Werkman 2 and from Table II, the carbinol is not formed at alkaline reactions and the predominating mechanism involves reaction I. Under acid conditions both reactions occur. Mickelson 3 has shown that the products of the alkaline fermentation of pyruvate by Aero-bacter are acetic and formic acids in equimolar proportions.