Experimental Biology and Medicine

Synthetic vitamin B₆ relieves the hypochromic microcytic anemia produced in dogs deficient in this factor. An adequate supply of the non-adsorbable fraction of the vitamin B complex is necessary for the complete disappearance of this anemia.

Goss and Cole 1 have shown that extracts of mare serum can be prepared testing 4000 to 7000 R.U. per mg total solids (40,000 to 70,000 R.U. per mg nitrogen). The question arises regarding the nature of the hormone present in these extracts as compared to that in untreated mare serum. Evans, et al., 2 and Hellbaum 3 have reported that more than one gonadotropic hormone is present in mare serum though these reports have not been confirmed. In the first mentioned paper the male rats showing only a Leydig tissue response were possibly sacrificed too soon to expect an effect upon the seminiferous epithelium. Further, had the time of autopsy of the females been delayed beyond 72 hours corpora lutea might have been encountered following the treatment with so-called FSH fractions. The extracts prepared by these authors were relatively crude preparations. We were interested, therefore, in determining whether or not highly purified materials would give similar evidence of fractionation of gonadotropic hormones. A fraction giving pure follicular stimulation in the female with little or no effect on the interstitial tissue of the male would give evidence of a purified follicle stimulating hormone whereas a converse relationship would indicate the presence of a luteinizing hormone.

Experimental. Two extracts were used: one, No. 15-19-2, tested 35,000 and the other, No. 17-76-4, 40,000 R.U. per mg nitrogen.∗ Four to 6 female rats, hypophysectomized on the 23rd day of age, were used at a given level for each extract. The intraperitoneal administration of a total of 30 R.U. over a 3-day period beginning on the 30th day of age produced ovaries averaging 116 mg for No. 15-19-2 and 127 mg for No. 17-76-4, on the 5th day after the initial injection.

The extracts were also tested in hypophysectomized male rats. The results on one extract, No. 15-19-2, are shown in Table I. There was a strong interstitial cell response as is indicated by the response of the accessory organs. Two rats treated similarly with No. 17-76-4 gave like results.

Thus it is seen that these extracts containing much less inert material than any previously reported for mare serum give a good follicular response in the hypophysectomized female and a strong interstitial cell response in the hypophysectomized male. In other words, in spite of the high degree of purification attained in the present extracts, no evidence was secured to support the view that mare serum contains 2 distinct hormones, one specifically affecting the interstitial tissue of the ovary and the Leydig tissue of the testis, and a second causing follicular growth in the female and germinal tissue development in the male.

By means of a titration method for estimation of bacterial growth it was found that growth is proportional to the quantity of nicotinamide present. Nicotinamide is more active than an equivalent amount of either pyridine-containing coenzyme. Hydrolysis increases the activity of the latter, indicating that the function of nicotinamide is not based entirely on synthesis to either of the known coenzymes. A method has been developed for determining nicotinamide and related substances in blood. The values obtained are higher if autoclaved blood is used.

The foregoing observations show that “tagged” chloride ions when injected into the general circulation are identified in the acid gastric juice of the stimulated stomach in dog and in man as quickly as 60 to 120 seconds after injection. Presumably at least some of these ions in the juice were derived from the HC1 present. Tagged chloride ions continued to circulate in the blood for more than one hour after intravenous injection and were transported into the gastric juice continuously during this period. Such ions did appear in the urine but were detected at irregular intervals and in relatively low concentration.

After intramuscular injection of staphylococcal antitoxin the rate of absorption is quite uniform in normal monkeys and humans. The antibody available in the total plasma-volume during a given period of days after injection of a specified dose (per kg weight) may be determined from the area under the curve, formed with time intervals as abscissae and daily concentrations of antibody per cc as ordinates, multiplied by the plasma volume. This new method of calculating available circulating antibody has also been applied successfully to the data of Glenny and Hopkins 3 on diphtheric antitoxin and hence may be of general im-munological interest.

A serial study of 11 parturient women and their infants by the serum volume index confirmed the findings of others, by other tests, that a definite tendency to hemorrhage exists in infants for a short time shortly after birth.

The method of natural transmission of the dog heartworm, Dirofilaria immitis, is still incompletely elucidated. The larvae of this parasite have been shown to develop in many species of mosquitoes and, thus, mosquitoes have been believed to be incriminated in the natural transmission, but substantial proof of this is lacking. The possibility that other blood-sucking arthropods serve as transmitters was overlooked until 1921, when Breinl 1 reported finding the micro-filarial embryos of Dirofilaria immitis in the fleas, Ctenocephalides canis and Ctenocephalides felis, collected from heartworm infected dogs in Australia. He also observed the advanced stages of development of this parasite in the Malpighian tubules of the fleas and the infective stage larvae in the hemocelic cavity. Recently, Brown 2 has observed the microfilariae and partially developed larvae of this parasite in Ctenocephalides canis collected from heartworm-infected dogs in the eastern United States.

In November of 1938, the writer, in examining dogs for heart-worm infection in New Orleans, observed the typical microfilariae of Dirofilaria immitis in Ctenocephalides canis. The early developmental stages and the infective larvae were also seen in the hemocelic cavity of fleas collected from a dog having a heavy infection. On other dogs harboring Dirofilaria immitis Ctenocephalides felis and Pulex irritans were found similarly infected. The female fleas of the 3 species were much more frequently infected and were more heavily infected with all the larval stages of the parasite than were the males. From some dogs infected with heartworm as many as 100% of the female fleas examined contained one or more infective-stage larvae and many of the earlier stages of development. The males harbored these stages only in small numbers and rarely contained the infective stage larvae.

1. Using a standard diet, with yeast of a known vitamin content, the liver functions of dogs was studied at two levels of thyroid feeding. 2. The production of the abnormal liver function in hyperthyroid dogs bears a causal relationship to the yeast in the diet.

Since little attention has been given to the primordial regions of plants as influenced by X-radiation and chemical growth-promoting substances the author has carried out experiments to determine effects of these agencies on growing points. Snow 1 observed that heteroauxin applied to the growing regions of certain plants caused union of leaf primordia, and Bausor 2 describes the development of root primordia on stems, petioles, and apical meristem when these parts are treated with beta-naphthoxyacetic acid.

In the present study, seedlings of sunflower (Helianthus annuus), zinnia (Zinnia elcgans), and tomato (Lycopersicum esculentum) were given moderate X-ray doses (2300 to 2500 r-units for sunflower and zinnia and up to 3000 r-units for tomato). Plants were stunted, and showed rough, warty, and abnormally-shaped leaves, with fasciation of leaves and of stems and frequent fusion of leaf primordia, as previously reported by Johnson. 3 Often, a few weeks after treatment, the meristematic region was divided into 2 or 3 parts, each with a separate group of primordial leaves, showing that the stem was about to branch. Indole-3-acetic acid (0.5 to 1.0%) applied in lanolin paste to seedlings of the above-named species caused increased growth, and with higher concentrations (2%), a fusion of leaves. The meristematic area either resembles that of untreated controls, or various leaf primordia may become joined, or else displaced from normal position. Colchicine (0.5%) applied also in lanolin paste, was found to retard growth, and it induced development of rough, warty, and misshapen leaves just as X-radiation does, while short,

The question of infection of air by droplets given off in coughing and sneezing has received considerable attention during the last few years. In particular, investigation has been directed towards determining the role of the air-borne droplet nuclei which result from the evaporation of droplets proper. The bacteriological and epidemiolog-ical aspects have recently been discussed by Wells, Wells, and Mudd. 1 Experimentally, little is known of certain of the characteristics of such droplets—their number, size, velocity, settling rate, and rate of evaporation—although these factors are concerned in their dissemination and the production of droplet nuclei. Wells 2 has calculated theoretical settling times and distances of fall before evaporation, for plain water droplets of various sizes suspended in still air of different humidities. These figures are only roughly applicable to actual conditions of air infection, since mouth spray and naso-pharynx droplets will contain some dissolved solids or be mucus-like, and the air will not be still. Rooks 3 has shown experimentally that droplet size is an important factor in nasal filtration, although he knew only the theoretical differences in average size of the droplets with which he worked. Weyrauch and Rzymkowski 4 have photographed the tracks of moving droplets given off in sneezing and talking. The paths shown indicate that most of the actual droplets travel only relatively short horizontal distances.

We have been able, by means of high-speed photography, to “stop” the motion of droplets given off in coughing and sneezing, thereby permitting measurements of droplet size, velocity, etc. The technic utilizes the light source and control instruments developed by Edger-ton, et a/., 5 for stroboscopic illumination and high-speed photography. The light source, which is placed at one side of the subject's face, consists of a 9-inch specular reflector with a spiral argon-filled tube through which a 56 microfarad condenser (charged to 2500 volts) discharges.

Large quantities of cobra venom were injected in a series of rabbits for periods varying from 2 to 21 weeks. Morphological and biochemical studies on the blood revealed no striking pathological change and no specific effect on the blood picture of the animals as compared with normal controls.

A study has been made of the toxicity for dogs of a protein-free preparation of the bactericidal substance described by Dubos when injected by the intravenous route. Seven of the 8 animals which received 0.4 mg/kg or more daily died as a result of the injections, and in 6 of these death occurred before the course of 10 daily injections was completed. All animals receiving 0.3 mg/kg or more snowed well-marked acute or chronic changes in the liver, spleen, kidneys, heart and lungs. Animals which received daily 0.2 mg/kg or less for 10 days showed only minor evidence of toxicity.

Neither the administration of narcotic doses of luminal nor the production of systemic shock by means of insulin or metrazol were capable of influencing the course of experimental poliomyelitis. Moreover, the extent and severity of the lesions in the spinal cord showed no significant difference between treated monkeys and untreated control animals. Even though essentially negative, the above results are considered important in demonstrating that propagation of the virus of poliomyelitis in the central nervous system is not affected by profound cytological and metabolic changes in the nerve tissue, as were produced by the methods employed in this work.

Dealing first with normal (control) urines, the amount of glucuronic acid in 24-hour samples varied from 350-650 nig, with an occasional lower or higher figure. These results agree well with those obtained by Maughan, Evelyn, and Browne 5 and by Roe and Hall. 7 Next, with regard to the effect of ingesting 5 g of phenylacetic acid, we find that this results in an average increase of 535 mg, an amount which corresponds to a detoxication by glucuronic acid of 7.5% of the phenylacetic acid ingested.

Two patients with dermatomyositis showed a decreased excretion of urinary calcium and an apparent impaired ability to store phosphorus. The amounts of urinary creatinine were diminished proportionally to the amounts of muscular wasting. Both patients had considerable creatinuria and an impaired creatine tolerance. Prolonged administration of large amounts of wheat germ was followed by decrease in creatinuria, increase in urinary creatinine, and definite clinical improvement.

While carrying on a clinical evaluation of the effects of feeding dried whole bile to patients suffering from a variety of lesions it was noted that the symptom of anorexia was often relieved. 1 Sensations of hunger occurred within a period of a few minutes to a few hours although in an occasional instance anorexia has been increased. Because of these observations we conceived the possibility that gastric contractions might be affected, or even called forth from a quiescent stomach. Accordingly, we determined to investigate the response to dried bile of the stomach of fasting dogs.

Method. Three dogs were prepared by operation with gastric fis-tulae after the method of Carlson 2 and after training and conditioning the animals, the gastric tonus and motility was measured by the balloon-manometer method. A condom balloon 8-10 cm in length was attached to the end of a No. 10 catheter and a No. 8 catheter attached alongside for gastric injection. The catheters and balloon Were inserted through the gastrostomy opening into the fundus of the dog's stomach and then 50 cc of air was injected into the balloon and the catheter connected to a bromof orm manometer. The 50 cc of air usually created a pressure of 3-6 cm.

The gastric tonus and motility were then recorded by Patterson's kymographic ink recording method. 3

The dried whole bile used in the experiments was swine gallbladder bile prepared by vacuum distillation at low temperature.∗As sodium a-glycohyodesoxycholate is the principal bile salt in swine bile, 4 it was decided to test the effect of this salt as well as whole bile.

From analysis of the results of the foregoing preliminary study it would seem that when either dried whole swine bile or sodium a-glycohyodesoxycholate dissolved in water are placed in a dog's stomach during the quiescent phase gastric hunger contractions are produced (Figs. 1, 2).

The technic described, while similar to that used with fair results for the isolation of the virus from stools and naso-pharyngeal washings, 1 failed to detect the agent in extraneural tissues of poliomyelitis patients. Lennette 14 has suggested that the virus may be present outside the CNS, but unidentifiable because of an attachment with antibody. Definite conclusions regarding the presence or absence of virus in extraneural tissues must await the development of more precise methods.

The results reported in this paper leave no doubt that the Eastern cotton rat is markedly susceptible to diphtheric infection and intoxication. While not equalling the extraordinary susceptibility of the guinea pig, the cotton rat appears to be at least many times more susceptible than the highly resistant albino rat. The significance of this observation in relation to the zoological classification of the cotton rat among the group of rodents remains to be determined.

The nature of the mucopolysaccharide of the cornea has been much debated. A number of investigators have considered it to be a mucoitin sulfuric acid, 1 while others have found no sulfuric acid. 2 It was reported recently, on the basis of colorimetric analysis, 3 that the carbohydrate contained an amino sugar and sulfuric acid, but galactose instead of uronic acid. Most of the reports have been based on analyses of protein complexes (“mucoids”) or of digests prepared by strong alkali.

In the past we have prepared this mucopolysaccharide acid in high yields both by alkaline digestion and by more gentle methods which avoid the use of strong alkali. 4 Our preparations contained one mol each of hexosamine, acetyl, uronic acid, and sulfuric acid, and had the same composition and general properties as those of the mucoitin-sulfuric acid obtained from gastric mucosa. 5 Good yields of glucos-amine were isolated from both acids. In several important respects, however, the two compounds were found to differ: (1) the acid of cornea always formed quite viscous aqueous solutions, while that of gastric mucosa did not; (2) the specific rotation of the acid from cornea was about −50°, while that of the gastric mucosa was around 0 (+2° to −8°); and (3) the acid from gastric mucosa was completely refractory to the specific enzyme from pneumococcus, 6 while the polysaccharide from cornea was hydrolyzed by this enzyme at about half the rate of hyaluronic acid. From this latter finding and from the fact that the rotation of hyaluronic acid is similar to that of the cornea polysaccharide, we conclude that cornea polysaccharide is the naturally-occurring mono-sulfuric acid ester of hyaluronic acid.

In a recent report by Zwemer and Lowenstein it was suggested that adrenalectomized cats can be maintained in good condition by the administration of strophanthin. 1 This substance was administered daily in the concentration of 15 μg per kg of body weight.

We have attempted to extend these findings to the mouse. For these experiments 21-day-old mice were bilaterally adrenalectomized. A total of 140 operated animals were employed, 40 serving as unin-jected controls, while the remaining 100 animals received various concentrations of strophanthin. The strophanthin was dissolved in olive oil so that the daily dose was contained in 0.1 cc of oil. Injections were made subcutaneously, beginning 24 hours after the operation and continued for 8 days.

Table I summarizes the results of the administration of strophanthin in daily concentrations of 0.02, 0.2, 2.0, 5.0, 10.0, 20.0, 50.0, and 100.0 μg, respectively. Of 40 uninjected control animals, only 4 were alive on the 10th day after adrenalectomy. The average survival of the remaining 36 animals was 4.7 days. The administration of desoxycorticosterone acetate† in a concentration of 0.2 mg per day protected 15 of a group of 17 adrenalectomized mice.

Strophanthin proved to be ineffective in the protection of the adrenalectomized mice in any of the 8 concentrations tested. No significant difference could be noted between the strophanthin injected and uninjected animals on either of the two criteria used; that is, number of animals alive 10 days post-operative, or the average survival of those animals dying before the tenth day. Strophanthin proved to be toxic in doses of 50 and 100 μg, respectively. All the animals were dead within 24 hours after the first injection of strophanthin at these concentrations.

N⁴-n-valeryl-, N⁴-n-caproyl-, and N⁴-n-heptanoyl-sulfanilylhydroxamide possess approximately the same therapeutic activity as siilfanilamide against sepsis in mice produced by 2 strains of hemolytic streptococci and one strain of Type II pneumococci.

Since Grassman 1 first showed that papain and kathepsins can be activated by cysteine or by reduced glutathione many papers have appeared in the literature dealing with the influence of sulfhydryl compounds on the activity of hydrolyzing and other enzyme systems. A search of literature has not revealed any researches concerned with the effect of these compounds on carboxylase. This paper is a preliminary report on the effect of sulfhydryl and other reducing compounds on the activity of carboxylase.

The procedure used was a modification of the method of Ochoa and Peters. 2 As a source of carboxylase commercial baker's yeast which had been air-dried with the aid of an electric fan for 3-4 hours or desiccated over concentrated H₂SO₄ for several days was employed. It was freed from cocarboxylase by rapid washing of the yeast 2-3 times with M/10 Na₂HPO₄ (25-30 cc for 0.5 g yeast), and once with distilled water. The washed yeast was suspended in 5 cc M/10 phosphate buffer pH 6.2. Formation of CO₂ from pyruvic acid was measured manometrically at 28° in air with conventional Warburg manometers attached to 15 cc vessels. Each vessel contained 0.5 cc washed yeast suspension, 0.1 mg Mg as MgSO₄ in 0.1 cc of solution, 0.2 cc sodium pyruvate solution adjusted to pH 6.2 and containing 5 mg pyruvic acid. In each case reagents to be tested or phosphate buffer pH 6.2 were added to bring the total volume of fluid to 2 cc. The pyruvate solution was tipped into the vessel after 8-12 minutes aeration. Merck's synthetic cocarboxylase and crystalline vitamin B₁, were used throughout the experiments.†

The results of 5 experiments dealing with the activation of alkaline washed yeast by cysteine hydrochloride, reduced glutathione, sodium bisulfite and phenylhydrazine hydrochloride are listed in Table I.

A method is described of demonstrating the toxicity of urine by its effect on the growth rate of cell colonies in vitro. Normal human urine in a dilution 1:5 completely inhibits the growth of fibroblasts; urine in a dilution of 1:50 still perceptibly inhibits their growth. The cells growing in media containing urine even in small concentrations exhibit signs of degeneration. Heat does not destroy this property of urine. The toxic substance is nondialysable.

Of the changes in the vaginal secretion during the menstrual cycle 1 those occurring in the first half concurrent with the growth and ripening of the ovarian follicle are most clearly defined and best understood. The demonstration in this laboratory 2 that they are similar to the smear changes induced in menopause and amenorrhea by the administration of estrogens, indicates that during the first half of the cycle the vaginal epithelium and secretion are largely, if not entirely, under the control of the estrogenic hormones. The peak effect of the estrogens as seen in the fully expressed “follicular picture” induced after menopause can, therefore, serve as an index of the extent of spontaneous follicular activity during the normal cycle, and affords a simple histological measure of ovarian function. It can also be employed as a guide for replacement therapy with estrogens 2 and as a measure of ovarian stimulation by gonadotropic agents. 3

The changes in vaginal smear during the second half of the cycle from the time of ovulation up to the next menstruation are less clear cut and more difficult to evaluate. 1 The lack of uniformity nossibly arises from the more complicated hormonal pattern of this phase of the cycle. The estrogenic titer falls from its mid-menstrual peak and, except for a brief moderate rise about a week premenstrually, gradually sinks to the low levels characteristic of the premenstruum and the menstrual phase. The progestational hormone now appears for the first time, is elaborated for a period of about 10 days and then disappears one to 3 days before the flow. 4 If, as is likely, the structure of the vaginal epithelium is a resultant of the interaction of both groups of hormones, any variation in the tempo or the extent of their production could be expected to vary the cytology of the vaginal secretion.

Among other reasons for regarding the causative agent of trachoma as a virus is the cytoplasmic inclusion of the epithelial cells of the conjunctiva, and occasionally even of the cornea. Marshalling together evidence that is essentially morphological, a number of investigators beginning with Halberstaedter and Prowazek 1 have attempted to prove that the inclusion represents a mass or “colony” of infectious units. While conducting a broader study† on the inclusion of trachoma and clinically allied diseases in this laboratory, observations have been made by indirect approach which are sufficiently interesting on this phase of the subject to merit their publication. 2

In testing out the infective capacity of conjunctival tissues from different patients with trachoma, monkeys in one group were inoculated with individual tissues, all of which were found to contain inclusions. In a second group, monkeys were inoculated in a similar manner with tissues lacking inclusions. In a third group, monkeys were inoculated with pooled tissues, some of which were inclusion-bearing and others inclusion-deficient. A study of the data pertinent to these experiments reveals several interesting and suggestive results. Thus, 70 monkeys (Macacus rhesus) were inoculated with individual tissues from 37 patients, all containing inclusions. Typical experimental trachoma occurred in 35, or 50%, of the animals tested. On the other hand, 158 monkeys were inoculated with separate tissues from 112 patients in whom inclusions were not demonstrated. Of these animals 35, or 22%, were specifically infected. The pooled tissues which were derived from 89 patients, 44 with and 45 without inclusions, were inoculated into 106 monkeys and of these, 41, or 38%, were infected. If the figures for monkeys infected in each group are plotted in the form of a curve, the result is practically a straight line, with inclusion-bearing tissues inducing most infections, the inclusion-lacking tissues inducing least or approximately half as many infections, and the tissues pooled as indicated occupying an almost mathematically half-way position between the two extremes.

In certain well established localities the American newt, Triturus viridescens, shows two well-defined habitats in its life cycle. The larval and adult phases live in water while the immature phase, representing some 3 to 5 years of the cycle, lives on land. In this interesting life-cycle 2 migrations occur, that from water to land following metamorphosis and that from land to water as maturity is reached. In an attempt to determine whether or not these migrations—particularly that from land to water—-are brought about by the endocrine glands Reinke and Chadwick 1 were able to induce the land phases to assume a water habitat, long before they would do so normally, by giving them intramuscular implants of pituitaries from the adults. By appropriate tests the water drive-inducing factor was shown to be a product of the anterior lobe of the pituitary. They were able to show later that this action of the pituitary occurs in the absence of both the thyroids and gonads. 2

Is the factor which induces the water drive peculiar to Triturus, or is it a hormone distributed widely among the vertebrates but with a specific effect in Triturus? If generally present in the vertebrates, with what known pituitary principles is it to be identified? The nature of its effect in Triturus is such that one is led to believe that the same pituitary principle which is responsible for growth in the body generally also induces migration to water as maturity is reached. In order to test the presence of the water drive factor in at least one other vertebrate class and at the same time to test the growth hormone as possibly being the cause of it, an attempt has been made to induce the water drive with a commercial extract of sheep pituitary which contains the growth-promoting factor (Antuitrin G).

It has been possible for some time to grow the entire life cycle of Neoaplectana glaseri, 1 a nematode parasite of the Japanese beetle, in cultures in which bacterial and fungous growths have been inhibited in various ways but not eliminated. 2 These contaminants undoubtedly introduced a high degree of variability into the results obtained. It therefore seemed advisable to attempt to rear this parasite in cultures free from bacteria.

Lapage 3 and Glaser and Stoll 4 developed technics whereby the second ecdysis of strongyloid nematode larvae was easily and consistently obtained in quantity under sterile conditions. It was found necessary to modify one of these technics slightly for work with Neoaplectana. Cultures prepared in the routine manner were permitted to develop for 10 to 15 days, at which time the majority of the parasites were second-stage larval forms in their third or fourth generation.∗ These were removed from the surface of the solid medium and washed with water until free of much debris. To remove the dead larvae they were then filtered through 2 layers of lens paper supported by fine gauze. The larvae were ensheathed by aerating them in 25 cc of water for 3 to 4 days with a change of clean water 3 times daily. They were then washed in 25 cc lots of sterile water 3 times each day for 3 days, after which they were treated for 30 minutes to 1 hour with Labarraque's solution (sodium hypo-chlorite) at a dilution of 1 part to 40 or 50 parts of water. The nemas were again washed 3 times in sterile water, and then placed in water about 5 mm deep for from 15 to 20 hours.† The next day the nemas were again treated with Labarraque's solution and then washed 3 times, followed by another 15 to 20 hours' sojourn in a small amount of water.

The marked fall in plasma volume observed in dogs subjected to continuous distention of the small intestine is at least partly prevented by the intravenous administration of desoxycortico-sterone.

The use of localized cutaneous stimulation in the treatment of visceral and other deeply located lesions has been based mainly on empirical considerations. Certain investigators, particularly Boas 1 and Freude and Ruhmann, 2 advanced data which support the assumption that localized cutaneous stimulation by means of warm applications results in vasodilatation in the corresponding segments of the gastro-intestinal tract. Certain data also support the assumption that localized cooling of the skin results in vasoconstriction in the viscera in the corresponding segments. There is no general agreement regarding the vascular reactions in the viscera elicited by localized cutaneous stimulation or the mechanisms through which such reactions are brought about.

The present series of experiments has been carried out to determine more accurately than has been indicated by previous studies whether appreciable circulatory changes in visceral organs can be brought about by loocalized cutaneous stimulation and whether the changes which occur represent direct effects of the stimulation employed or reflex phenomena.

Decerebrate preparations of the cat have been used in order to avoid the vitiating effects of anesthesia. The stimulating agents employed have been warm and cold applications and vacuum cups applied to the skin of the back and lateral surfaces of the trunk from which the hair had been removed. The circulatory changes brought about in the stomach and intestine were observed with the viscus exposed through a midventral incision, and recorded by means of photography and plethysmograph records.

Moderate cooling of the skin of the back or lateral surface by means of cold applications from the fifth or sixth thoracic segment caudalward consistently resulted in vasoconstriction in the stomach and intestine. Moderate warming of the skin in the same areas by means of warm applications at approximately 45°C consistently resulted in vasodilatation in the stomach and intestine.

The presented data indicate that estrogens produce sex reversal in male salamanders, while testosterone propionate exerts no corresponding influence upon genetical females. As in adult animals, the secondary sex characters respond also in the larval salamanders to the administration of sex hormones. The female hormone stimulates slightly the larval Mullerian ducts, and the male hormone causes an extensive and very precocious stimulation of the larval Wolffian ducts and cloacal glands. It is remarkable that the estrogens affect most profoundly the male gonads and the testosterone the male secondary sex characters. The hormones do not induce original formation of gonoducts; they stimulate only the secondary (functional) enlargement of parts of ducts already present. It has been shown by means of parabiosis that the testes of males release some inductive substance which inhibits the development of the ovaries of female cotwins and indirectly may cause some genetic females to continue development in a male direction. 6 On the other hand, in the male-female parabiotic combinations there is no precocious stimulation of either gonoducts or cloacal glands. These fundamental differences in the observed reactions prove that the crystalline sex hormones used in this experiment cannot be identical with the substances which normally act as inductors of sex differentiation.

It has been shown that the oral administration of non-lipoid foods, glycerol, sucrose, glucose and proteose-peptone exert no demonstrable effect on the serum esterase of rats. The administration of neutral fats, oleic and palmitic acids leads to a marked increase which lasts for several hours.

The authors gratefully acknowledge their great indebtedness to the John and Mary R. Markle Foundation for financial assistance in this research.

An antigenic substance has been isolated from cultures of E. typhosa grown in a synthetic medium. Appropriate amounts kill mice and rabbits and smaller quantities induce a leucopenia and fever in rabbits.

The author wishes to acknowledge with gratitude the advice of Drs. J. F. Enders, J. H. Mueller, Y. Subbarow and M. A. Logan of the Harvard University Medical School.

1. The skin sensitivity of man to bovine plasma and to its albumin and globulin fractions has been investigated. 2. The skin sensitivity of man to the three preparations tested is least marked with the albumin fraction of bovine plasma.

This is the third of a series of studies analyzing the rate of emptying of the gall bladder in patients with lesions of the stomach.

In the first series 1 it was observed that peptic ulcer patients displayed a significantly faster rate of emptying than controls of comparable age. Since the mean curve of emptying for this group could be virtually reproduced in normal individuals merely by injecting one egg-yolk directly into the duodenum (Fig. 1, left) and since the initial discharge of food into the duodenum is known to be more rapid in iiiclividuals having gastric and duodenal ulcer, the faster rate of emptying of the gall bladder in such patients was attributed to a greater food stimulus rather than to an increased production of gastric juice.

This interpretation was strengthened by the second study. 2 This dealt with patients having carcinoma of the stomach. In this group, marked reduction of the amount of free HCl failed to retard the emptying of the gall bladder.

The present report is based upon cholecystographic studies of 22 consecutive, unselected male patients for whom a clinical as well as hematological diagnosis of pernicious anemia had been established. All of the patients were receiving treatment with liver extract. The hemoglobin content of the blood had either reached normal levels or was responding to treatment. The histamine test had disclosed complete absence of free acid in all patients, and only minimal amounts of total acids.

Of special interest was the fact that the gall bladder could not be visualized in 40% of these individuals, notwithstanding the use of the intravenous method of introducing the dye and the absence of any history suggesting gall bladder disease. In the other groups visualization had failed in only 9% of ulcer patients and in 22.7% of those with carcinoma.

The Lansing strain of virus isolated by Armstrong easily produces disease in Sigmodon hispidus littoralis rats. Thus far, we have been unable to produce poliomyelitis with any of the 9 stock virus strains carried in our laboratory and named in this paper.

Previous reports have shown that increasing the amount of hypophyseal tissue in an amphibian embryo by grafting gives rise to an increase in the thickness of the basement membrane of the renal glomerulus. 1 It has been pointed out that this thickening is similar in appearance to the thickened membrane in human cases of hypertension. 1 , 2 The size of the heart, vasoconstriction, reduced heart rate and other conditions in these test animals simulate a hypertensive state. 3 The hypophysectomized individuals show the opposite results of hypotension and may now be stated to have correlated with this a thin basement membrane.

The arrangement of the loops of the capillary tuft indicates that there is a simplification in these hypertensive glomeruli. 4 This is stated by McGregor 2 to be true in human hypertension. She also states that there is contraction of glomeruli although no measurements or volume determinations were made. Goormaghtigh 5 mentions contraction of glomeruli in ischemic kidneys.

In our test animals there appeared to be a contraction in many glomeruli with an increase in the capsular space. How much this increase in space is due to a shrinkage of the glomerulus and how much to an increase in capsular volume had to be determined. The present observations deal with quantitative determinations of volume for the total renal corpuscle, the glomerulus and the capsular space of hypophysectomized, control, and triple pituitary animals. This has been done by the paper cut-out method using every serial section. Two groups of animals were employed. In one, values for every fifth corpuscle were computed individually in 3 sets each consisting of an hypophysectomized, a control and a triple pituitary animal.

Using the completely denervated iris of the cat as an indicator of circulating autonomic substances, it was found that constriction of the pupil ensued from 35 to 70 seconds following the intravenous administration of 1.0 cc of whole human blood. The blood of rabbit, guinea pig, mouse, monkey, hog and hemolyzed erythrocytes of cat also produced a constriction of the completely denervated iris of the cat. Identical amounts of blood from dog, sheep and ox were without significant effect on the same preparation. The most conspicuous reactions were obtained with rabbit's blood. Large quantities of dog, ox, or sheep blood sometimes produced a slight and slow constriction of the cat's denervated iris.

Other effects caused by the intravenous injection of blood into the cat were a fall in blood pressure, as shown by Abramson, et al., 1 and Fuzii, 2 decrease in heart rate, relaxation of anal and urinary sphincters and hypersalivation, all parasympathomimetic manifestations.

The vaso depressor and miotic propertiesof the blood were located chiefly in the erythrocytes. Laked red blood cells also exhibited these properties. Intravenous injection of serum never produced a visible change in the iris.

The washed red blood cells caused a pupillary constriction from 35 to 80 seconds after their intravenous injection, whereas the laked erythrocytes produced a much quicker response, the miosis appearing from 12 to 30 seconds after the administration. The rabbit's erythrocytes showed two types of depressor effects on the cat's blood pressure, an early transient drop from 6 to 10 seconds after the injection of the cells and a later prolonged fall from 45 to 70 seconds after the administration. The hemolyzed red blood cells showed only the early depressor effect.

In a previous communication 1 the serial passage of the human influenzal virus in the European hamster (Cricetus cricetus) was reported. While this hamster was shown to be susceptible to a recently isolated virus strain, it was not demonstrated, because of lack of material at the time, whether or not infection could be transferred directly from humans.

This present report deals with the infection of the Syrian hamster (Cricetus auratus) from throat-washings of persons ill with epidemic influenza.

Material. The washings were collected in buffered broth during the early months of 1939 from persons acutely ill with influenza. The presence of virus originally in these specimens had been demonstrated by the inoculation of ferrets. 2 The washings had been preserved for 9 to 10 months in a low-temperature cabinet at −76°C when the inoculation of the hamsters was made. The hamsters employed were from 3 to 5 months of age and weighed approximately 100 g each.

Method. Preceding inoculation, 1 cc of blood was withdrawn by cardiac puncture from each hamster. From 0.3 to 0.4 cc of unfiltered throat-washing was administered intranasally while the animal was under light ether-anesthesia. Twelve to 14 days after the intranasal inoculation a second blood sample was taken. The blood serum obtained before and following inoculation was then titrated for neutralizing antibodies against the PR8 strain of influenzal virus.

Results. So far, 4 preserved washings obtained during the 1939 epidemic, but from separate localities, have been tested. All of these specimens were known to contain virus originally.

None of the blood samples taken before inoculation showed any neutralizing effect upon the virus in a 1:2 serum-mixture, but samples taken 12 to 14 days after inoculation of the hamsters with each of these 4 washings all neutralized 1000 M.L.D. of the virus in dilutions above 1:32. Despite the immune response, these animals manifested no symptoms or gross pathological lesions which could be used as diagnostic criteria.

In the course of some ultra-centrifuging experiments there arose the need of rearing the parthenogenetic eggs of Cladocera in vitro. A review of the literature revealed no record of the successful growth of eggs of this group of animals outside the brood-chamber of the mother. We have succeeded in rearing the eggs of Daphnia magna in tissue culture slides and inasmuch as the embryos are transparent, this enabled us to make observations on some of the sequence of events in the course of the development of the embryos without the necessity of staining and sectioning.

A number of different media were used in an endeavor to grow Daphnia magna eggs in vitro. A modified Ringer's solution utilized successfully for physiological work on isolated organs of Cladocera by Levy, 1 proved injurious to the eggs. The growth of eggs in a medium consisting of the fluid obtained from the brood-chamber of animals in which there were developing eggs showed no advantage over a simpler medium consisting of sterile pond water. This is of considerable interest in view of the fact that it has been held (Dearborn, 2 Birge 3 ) that the brood-chamber of Cladocera secretes a fluid which serves as nourishment in the course of the development of the embryos. Careful controlled experiments revealed that the parthenogenetic eggs in the brood-chamber of Daphnia magna completed their development in about 46 hours at 25 °C. When the parthenogenetic eggs of this animal were reared in sterile pond water on tissue culture slides, they too completed their embryonic history from eggs to free swimming independent organisms in the same period of time. This demonstrates very clearly that the eggs of this animal are self-sufficient with regard to the nutritive material already stored in them at the time of their deposition.

The changes of the various structural components of the human body in development are well illustrated by volumetric histograms. These are shown in Figs. 1 and 2. They are based upon data collected in this laboratory as well as a series of records collated from the literature.

In this presentation the structural components of the body considered are: skin and superficial fat (paniculus adiposus), the visceral mass (including the heart), the nervous tissues (of both the peripheral and the central nervous systems), the voluntary musculature, and the fresh Iigamentous skeleton.

The periods represented are: 6 lunar or fetal months, birth, and full maturity. Both sexes are included in each of the periods.

Figure 1 illustrates the differences in composition of the body at 6 lunar months and at birth. During this interval the net weight of the body increases approximately five-fold, from 500 to 2500 g.

The relative changes in the distribution of the body components are: the skin and superficial fat increase from about one-sixth to over one-fourth of the body mass. The nervous tissue component and the skeletal component both show a marked relative decrease. The visceral and the voluntary muscle components remain almost constant.

Fig. 2 shows the changes in components between birth and maturity. During this interval the net body weight increases almost twenty-fold. The postnatal changes in the relative distribution of components consist of: a marked increase in the voluntary muscle from about one-fourth to over two-fifths of the body mass. But the visceral component decreases from about one-sixth to about one-tenth of the body mass; and the nervous tissue component from about one-seventh to about one-thirtieth. The skin and superficial fat component and the component of the skeleton remain practically unchanged.

(1) Keto-reacting substances are present in dogs' bile and can be quantitatively determined. (2) The output of keto-reacting substances is increased slightly when unoxidized bile acids are fed. (3) When oxidized bile acids or ketocholanic acids are administered orally, the output of keto-reacting substances in the bile is markedly increased. By assuming that the increase over the control output is due to the ketocholanic acid fed, one may calculate the recovery in the bile of the ketocholanic acid administered.

After the injection of the water-soluble potassium salt of dicarboxy-benzanthracene into Swiss mice there is a marked decrease in the number of polymorphonuclear neutrophils, a slower, and moderate decrease in the number of monocytes, with but slight changes in the number of lymphocytes, eosinophils, and basophils. Recovery is complete 7 weeks after the last injection. Data on the blood of normal Swiss mice are given.

The concentration of capsular polysaccharide appearing in blood-broth culture filtrates following the growth of a highly virulent, standardized strain of Pneumococcus III, was determined photronreflectometrically and correlated with the growth rate of the organism. An initial inoculum of 165 organisms per ml yielded a very small quantity of polysaccharide (0.5 mg %). An inoculum of 9,000 organisms per ml yielded 18 mg % of capsular polysaccharide in 48 hours. The greatest increases in concentration of polysaccharide occurred in two stages after the completion of the logarithmic phase of growth.

In 8 out of 33 treated mice complete regressions of spontaneous breast adenocarcinomas were effected with intravenous and subcutaneous injections of yeast extract.

In in vitro experiments with Staphylococcus aureus sulfamethylthiazole has shown greater bacteriostatic activity than sulfathiazole; the activity of the latter was in turn greater than that of sulfapyridine. In in vivo experiments with the same organism sulfapyridine has had little, if any, activity. Both sulfathiazole and sulfamethylthiazole have protected mice and the former drug has been slightly but consistently more active than the latter.

The large blood pressure rise due to anoxia, observed during hypoglycemia, may be caused by a severe depression by the combined effect of narcosis and hypoglycemia of those reactions which counteract a marked rise in blood pressure, the reactions causing this rise in blood pressure being less severely depressed.

With estrone bleeding may be induced in the uterine mucosa of the rabbit. The blood penetrates through the lifted epithelial areas and eventually enters the uterine cavity and vagina. This effect may be obtained by two injections of 500 to 750 IU of estrone given intravenously during 12 or 24 hours. Bleeding appears after 4-5 days.

A number of anterior pituitary and anterior pituitary-like preparations were found to have no effect during 5 hours after their injection into frogs in water either on normal body water or upon the Brunn reaction.

Four calves were placed on a semipurified ration which analyzed 0.10-0.12% potassium. Four control calves were given the same ration except that potassium was added to bring the level of this constituent to 0.35%. The animals were placed on this ration at 160 days of age. Previous to this they received whole milk and a small amount of hay and grain.

Electrocardiograms were usually taken at monthly intervals, but for some periods more frequently. The electrodes, which were of the German silver plate type, were placed on the upper part of the 2 fore limbs and just above the hock on the left hind leg. The areas on which the electrodes were placed were clipped, cleaned and covered with electrode paste applied with vigorous rubbing.

Serum potassium determinations were made on all calves at 2-week intervals, more often when deemed necessary, by a modification of the titrimetric method of Shohl and Bennett. 1

Plasma calcium, phosphorus, and magnesium determinations were carried out at weekly intervals in order to make certain that the ration was not deficient in these elements.

Blood Changes. Calf C 394. On the 270th day of the experimental period this calf had a serum potassium value of 17.0 mg %. Seventy days later this value had fallen to 10.2 mg % and for a period of about 60 days it remained below 13.5 mg %. On the 350th day of the experiment the potassium in the ration was increased to 0.20% for a period of 130 days after which the potassium was again reduced. During this 130-day period the serum potassium level increased to 18.0 mg %, and it has since varied between 15 and 18 mg % in spite of the reduction of potassium in the ration.

Changes in the duration and form of QRS after section of the branches of the Hisbundle are much less pronounced in calves than in dogs. The difference between these two species is attributed to differences in the distribution of the intraventricular conducting system.

The authors wish to express their thanks for the assistance and advice of Dr. F. N. Wilson, University Hospital, Ann Arbor, Michigan, and Dr. E. T. Hallman, Michigan State College, East Lansing. The experimental animals were generously donated by the Dairy Department of Michigan State College.

It has been reported 1 that the fermentation of glucose by Types A and B Clostridium botulinum differs from the majority of bacterial fermentations in that ethyl alcohol and carbon dioxide are the main products of the fermentation, only small amounts of acetic and lactic acids together with traces of hydrogen being formed. This study has been extended to include the fermentation of pyruvic acid, which may be an intermediate compound in the fermentation of glucose and also in the degradation of amino acids such as alanine, by Cl. botulinum. 2

The experiments reported below were carried out with washed suspensions of Type A (E-43) Cl. botulinum although studies with Type B gave essentially the same results. Glucose-broth (800 ml) containing 0.1% Difco yeast extract was inoculated with 1.0 ml of a beef-brain culture of the organism and incubated for 20 hours at 37°C in a McIntosh and Fildes anaerobic jar. The culture was then centrifuged and the cells suspended in distilled water. The suspension was diluted with an equal volume of M/7.5 phosphate buffer and placed in the central chamber of Warburg vessels. A rapid stream of O₂-free H₂ was passed through the vessels for 10 minutes and they were then equilibrated 10 minutes before tipping in the sodium pyruvate from the side-arm. CO₂ production was determined at 37°C by the Warburg technic, the initial and final bound CO₂ being determined following the addition of 10% sulfuric acid to the contents of separate Warburg vessels. In the semi-macro experiments, Warburg vessels of 40 ml capacity were employed with Clerici fluid in the manometers.

It was observed that pyruvic acid is rapidly decarboxylated by washed suspensions of Cl. botulinum, Qco₂ values of 25-30 being observed under optimal conditions.

A number of amino acids are utilized by washed suspensions of Clostridium botulinum, for the most part in coupled reactions between pairs of different amino acids. 1 2 For example, alanine is deaminatively oxidized to CO₂ and acetic acid by glycine or proline which are reduced to acetic acid or δamino-n-valeric acid, respectively. However, amino acids such as leucine and particularly serine appeared to be deaminated when present singly in washed suspensions of this organism. The utilization of serine has been studied in detail by the technic already described, 3 ammonia being determined by the method of Parnas as described by Niederl and Niederl. 4 Serine is decarboxylated in the presence of washed suspensions of Types A or B Cl. botulinum at a rate (Qco₂ of 7-10) approximately one-third of that observed with pyruvic acid (Qco₂ of 25-30) as the substrate at optimal pH values for both reactions. Typical results of CO₂ production are presented in Fig. 1.

It is apparent that the pH optimum for the utilization of serine by Cl. botulinum lies on the alkaline side of neutrality, probably near pH 7.5. This is in marked contrast with the pH optimum of 6.0 observed with pyruvic acid as the substrate. In further studies it was observed that apparently only one optical form of serine is attacked at an appreciable rate, deamination and decarboxylation of dl-serine proceeding only approximately 50% to completion.

In semimacro experiments in Warburg vessels of 40 ml capacity with Clerici fluid in the manometers the fermentation was studied in more detail. Ammonia, CO₂, ethyl alcohol and acetic acid were identified as the chief products of degradation of serine. In these experiments the reaction was not allowed to go to completion due to the slow rate observed by the time approximately one-half of the serine had been utilized.

The generally accepted method for the bacteriological diagnosis of pertussis is the cough-plate culture, originally described by Chievitz and Meyer, 1 or some modification of their procedure. The percentage of positive cultures obtained by this technic varies considerably in the hands of different workers and under various conditions. 2

We have found the cough-plate method satisfactory for older children, but in infants we have obtained positive cultures in only about 25%, even when the cultures were made during the catarrhal period of the disease. This low incidence seems to be due to the fact that the young infant does not cough as vigorously as does the older child, thereby resulting in an inadequate inoculation of the medium.

During the course of a comparative study of various technics for making cultures, we were impressed by the favorable results obtained from culturing the nasopharynx. Recent reports 3 , 4 5 have described the advantages of the nasopharyngeal culture in the bacteriological diagnosis of pneumonia. This present report describes the results obtained by making simultaneous nasopharyngeal, throat and cough-plate cultures in a series of 25 consecutive cases of pertussis observed in the pediatric clinic during the past 4 months. The procedure follows.

The medium was prepared as previously described 6 and poured into ordinary Petri dishes. Medium more than 3 days old was discarded. The cough-plate cultures were made by exposing the medium during a paroxysm at a point about 6 inches in front of the patient's mouth. The nasopharyngeal cultures were taken by passing a sterile swab, consisting of a small bit of cotton tightly wrapped about the end of a thin, flexible copper wire, through the nose until it touched the posterior wall of the pharynx.

The acyl aminobenzenesulfonhydroxamides have strong anti-catalase activity and, when present in broth cultures of the pneumococcus, cause inhibition of growth associated with increased accumulation of hydrogen peroxide. When the caproyl compound is added to growing cultures, inhibition of growth is detectable almost immediately but reaches a maximum only after time for accumulation of peroxide has elapsed. Inhibition by sulfanilamide, on the other hand, is detectable somewhat later and approaches a maximum more slowly.

The gonadotropic potency of the pituitary of spayed and castrated rats was determined in the immature female rat before and after tryptic digestion. There was a great reduction of potency of the pituitary following digestion by trypsin in both the male and female.

I am indebted to Dr. Graham Chen for suggestions in these experiments and for supplying me with a sample of follicle-stimulating hormone.