Experimental Biology and Medicine

The consumption of oxygen by minced tissue and by tissue slices in the presence of sodium benzoate was studied in order to determine whether this experimental procedure might be used in the investigation of the in vitro synthesis of hippuric acid. The oxygen uptake of the tissue was measured at 38°C. in an atmosphere of oxygen using the Barcroft differential manometer. The tissue was suspended in the isotonic salt solution of Krebs and Henseleit 1 with and without the addition of extra phosphate. Sodium benzoate or hippurate, equal to 5 mg. of benzoic acid per cc, replaced an equivalent amount of sodium chloride in the experimental solutions. In most cases glucose was added as a substrate.

Sodium benzoate decreased the oxygen uptake of the following tissues of the rat: liver and kidney slices, diaphragm, and minced brain, liver and kidney. A similar effect was obtained with minced pigeon brain. The inhibition of respiration varied with the concentration of benzoate. It was always more marked if glucose was used as a substrate in a poorly buffered medium due to the production of acid and the resulting decrease in pH. By using tissues, such as minced brain or washed minced liver, which possessed low respiratory activity in the absence of added substrate, it was possible to demonstrate that the inhibitory effect was on the glucose or lactate respiratory mechanism rather than on the succinate mechanism. With minced brain the inhibition was only slightly evident in the presence of succinate and marked in the presence of glucose, lactate or pyruvate. Benzoate toxicity in the presence of pyruvate was unaffected by the addition of vitamin B. Jowett and Quastel 2 have reported that concentrations of benzoate smaller than those used in these experiments affected that respiratory process in liver concerned with the oxidation of fatty acids to aceto acetic acid.

While injecting a test-meal into the duodenum of guinea pig Higgins and Mann 1 observed that exceptionally some of the material entered into the common bile duct. Burget and Brocklehurst 2 report that they were not able to drive duodenal contents into the intact ampulla of the guinea pig. Neither group of authors stated the degree of pressure, although Higgins and Mann note that the pressure employed was not unusual.

In 23 out of 26 guinea pigs, we were successful in driving air or dilute India ink into the gall-bladder and biliary passages and section of the liver lobes frequently revealed air in the finer bile channels. The effective duodenal pressures ranged between 10 and 20 mm. Hg; in 2 pigs pressures of 30 and 40 mm. Hg were necessary. In 10 experiments, an intravenous injection of 0.5 to 2 cc. of epine-phrin solution (1:10,000) caused generally the entry of air or dilute ink into the gall bladder and bile passages at duodenal pressure-levels that had been ineffective in the same animal before the injection of epinephrin.

The main details of procedure were as follows: The guinea pigs were chiefly males, weighing an average of 700 gm.; sodium barbital, 300 mg. per kg., subcutaneously; abdomen opened in mid-line from the ensiform cartilage to pubis; the antrum of the stomach was ligated; a cannula was tied into the duodenum and connected with a mercury manometer; a compressible bulb in the manometer-circuit permitted accurate variations in pressure.

The pressure that a normal duodenum could exert when stimulated to contraction by faradic currents or by BaCl₂ solutions was 20 mm. of Hg.

Tel ford 1 and Smithwick 2 have described operative procedures for sympathetic denervation of the upper extremity by means of which the preganglionic fibers in question from the second thoracic nerve downward are interrupted but the inferior cervical and upper thoracic sympathetic ganglia are left in situ and the preganglionic fibers of the first thoracic nerve and the gray communicating rami joining the nerves of the brachial plexus are left intact. The advantages claimed for these procedures are avoidance of the unwanted effects of extirpation of the inferior cervical and upper thoracic sympathetic ganglia, particularly Horner's syndrome and sensitization of the vascular musculature in the affected area to adrenin.

The assumption that preganglionic components of the first thoracic nerve play no part in the sympathetic innervation of the upper extremity, although in accord with certain early experimental data recorded by Langley, 3 is not supported by certain more recent experimental and clinical observations. In view of the importance of complete functional sympathetic denervation of the upper extremity in the treatment of peripheral vascular disease it has seemed desirable to obtain additional data regarding the distribution of the preganglionic components of the upper thoracic nerves by means of anatomical and physiological experimentation. The anatomical experiments have been carried out on cats; the physiological experiments on both cats and dogs.

In the anatomical experiments, some cats were subjected to unilateral section of the roots of the second and third thoracic nerves proximal to the communicating rami and division of the sympathetic trunk below the level of the third thoracic nerve; others were subjected to unilateral section of the roots of the first thoracic nerve, leaving the sympathetic trunk intact. Preparations of the inferior cervical ganglia, taken after degeneration of the divided fibers, in the first series, showed extensive degeneration of the intercellular

During the present summer a recurrence of epidemic encephalitis has taken place in St. Louis and St. Louis County. Clinically the disease appears similar to that seen in the same area in the summer of 1933. On September 21, 1937, we reported before the St. Louis Medical Society 1 the successful recovery of a strain of the virus of the present outbreak, the original inoculation being made on August 28, 1937. This had been successfully carried through 4 series of white mice. At that time the first virus neutralization test was in progress but the final report was not available. This virus has now been carried through 8 transplantations in mice. Its virulence for mice is practically identical with that of the virus of 1933. Two strains of the 1933 virus were recovered and studied by two of us in the laboratories of Firmin Desloge Hospital, 2 subsequent to the isolation of the virus by Webster and Fite. 3

We are now able to report the completion of the 3 virus neutralization tests demonstrating the degree of protection afforded by the serum of 3 patients who had acute attacks of encephalitis in the summer of 1933 against the virus of the 1937 outbreak.

Virus neutralization tests were carried out on the sera of these same patients during 1935 and 1936 against the 1933 virus. These tests are included for comparison with the present tests.

The results are summarized in Table I.

The significance of these results is to indicate the identity of the virus causing the 1937 summer outbreak of encephalitis with that which caused the epidemic of 1933. It also points to the existence of an endemic focus of this infection in the St.

Numerous observations have been made on the fall in serum protein immediately following hemorrhage (particularly plasmapheresis), and its subsequent rise. According to one view, this rise is ascribed to regeneration of new serum protein, presumably from amino-acids which were absorbed from the intestinal tract or mobilized from various tissues, notably the liver and muscle. According to another view, the rise of serum protein is due to the direct entrance of protein from a “body store”. This “body store” is apparently different from that contained in the various tissue fluids and lymph which furnish a ready source of fluid, but contain only 1% of protein, which is but one-seventh of its concentration in normal blood plasma.

There is general agreement regarding the fall in serum protein which follows a severe hemorrhage. Accompanied as it is by a fall in the red cell count, the process is obviously one of dilution whereby the fluid stores of the body, poor in protein, enter the blood stream in the attempt to restore blood volume back to normal. Once this dilution has occurred, further increases in serum protein concentration are presumably due to the regeneration of new protein or the direct entrance of new protein from some immediately available source, as mentioned above. However, no inferences regarding the concentration of serum protein should overlook the influence of changes in plasma volume, which occur particularly after severe hemorrhage. Such blood volume changes are due to the fact that the vasomotor system is especially active after severe hemorrhage, resulting in rapid, and often marked, changes in the size of the blood bed. Thus, actual protein may enter the blood stream and yet be masked as far as its concentration is concerned by a coincident vasodilation; conversely, a vasoconstriction may diminish blood volume and result in an increase in the concentration of serum protein, which, in fact, is unchanged as far as the total amount circulating in the plasma is concerned.

In the interval between the 1933 encephalitis epidemic and the present outbreak of the disease, neutralization tests have been made to determine the presence of virucidal antibodies in serum, obtained during convalescence, from 10 patients whose illness had been diagnosed as acute encephalitis. In none of these instances was the test positive. Efforts have also been made to isolate the virus from human brain material obtained at autopsy from 6 fatal cases in which encephalitis was regarded as the cause of death. Intracerebral injections in mice∗ 1 2 which proved susceptible in 1933, failed to demonstrate the virus in any of these 6 attempts. Pathological lesions consistent with encephalitis were observed in 3 of these fatal cases, 2 of them being in children.

The clinical diagnosis in the cases without brain lesions might be regarded as incorrect. Concerning those which showed pathological lesions, however, another possibility is that these sporadic cases of the disease were produced by a virus of altered virulence which was not infective for mice in the dilutions employed.

Recalling this interepidemic experience, it was surprising to note the ease with which the virus was isolated from the fatal cases occurring this summer. Using again the same method of intracerebral injection of human brain emulsion into mice, 7 different strains of the virus have already been obtained from 19 different fatal cases. This incidence of the infectivity of brain tissue is almost the same as that observed in 1933 when the virus was first isolated. At that time 7 out of 15 brains were shown to contain active virus by producing the disease when injected into Macacus rhesus monkeys. 1 Webster 2 obtained 5 strains out of 11 trials in mice.

Advantage has been taken of the fact that the opacity of tissues to light varies with the blood content, to record photoelectrically the changes in the blood content of the nasal septum.

The arrangement used (shown schematically in Fig. 1) is essentially an adaptation of the photoelectric plethysmograph for the finger, previously reported. 1 Illumination of the septum is provided by a small ophthalmoscope bulb inserted in one nares. Local heating is largely prevented by a heavy metal cap which, placed over the bulb, conducts the heat away to the lamp carrier. Local heating may be practically eliminated by using a mirror arrangement which permits the light source to be placed some distance from the nares. The latter method of providing illumination has the additional advantage of offering opportunity to control amplification, independent of septal luminosity, through predetermined decrements in light intensity by means of suitable filters. The light transmitted by the septum is reflected out the other nares by the mirror to the photoelectric cell. The entire assembly is mounted on a dental impression plate which, carried between the teeth, not only provides a rigid mount but also guarantees constancy of alignment with the nasal septum. The mirror and light tubes may be varied in diameter to fit varying nares. Any desired penetration is readily provided. The apparatus is light and comfortable and may be worn for hours without discomfort. Breathing through either the mouth or nose is equally feasible. The photoelectric oscillations are recorded galvanometric-ally on the photokymograph after amplification.

Preliminary observations indicate the feasibility of using the same arrangement for photoelectric plethysmography of the skin of the nose, employing either transmitted or reflected light.

Fig. 2-A shows the volume pulse of the septum so recorded, contrasted with the finger volume pulse (B) of the same subject.

Due to the general influence of the pituitary gland on body function it is important to increase our knowledge of those factors which alter pituitary secretion. Marrian and Parkes 1 reported that vitamin B deficiency, causing loss of body weight or partial inanition, brought about the cessation of estrus and atrophic changes in the ovary. The administration of anterior pituitary substances during the experimental anestrus resulted in the immediate appearance of estrous changes and in ovulation. Evans and Simpson 2 showed that there was a decreased amount of the gonad-stimulating hormone present in the anterior pituitaries taken from animals fed a diet deficient in the antineuritic factor. Mason and Wolfe 3 found an increase in the gonad-stimulating capacity of the anterior pituitaries of male rats which had been fed a diet deficient in vitamin A and which induced xerophthalmia. Furthermore, vitamin E deficiency seemed to have the same effect. Nelson 4 assayed pituitary glands from rats which had been on vitamin E deficient rations and found these glands more potent for the gonad-stimulating hormone than pituitaries from normal animals, but less potent than glands from castrates. Female pituitaries were comparable to glands obtained from normal males. However, Rowlands and Singer 5 reported that vitamin E deficiency caused a definite decrease in the capacity of the pituitary body of the non-pregnant rat to cause ovulation in the estrous rabbit and therefore, a decrease in the content of luteinizing or ovulation-producing substance.

We were interested in determining the lactogen† content of pituitary glands from rats on vitamin deficient rations.

This report is presented in an effort to show that determinations for gonadotropic hormone content of the urine are frequently erroneous due to the precipitation of contaminating amounts of fat-insoluble or combined estrogen.

Concerning urinary gonadotropins there is no doubt that pregnant women excrete a substance capable of producing corpora lutea in the ovaries of normal infantile animals. Some women in the menopause excrete a gonadotropic hormone capable of provoking follicle growth in the ovaries of hypophysectomized rats or mice. A gonadotropic hormone prepared from the pituitary and capable of interstitial cell stimulation has been described, but its detection if present in the urine, depends also upon the use of hypophysectomized animals. For studying the urine of non-pregnant women most workers have resorted to the use of intact animals and depend upon more cursory and unreliable criteria for the estimation of gonadotropic hormone. Speaking collectively, their criteria have been the appearance, in infantile rats or mice as old as 32 days and within periods up to as long as 130 hours after injection, of follicle growth, interstitial cell hypertrophy, increase in uterine and ovarian size and weight, establishment of vaginal introitus, and estrus. The reactions in the lower tubular tract have been interpreted as gonadotropic responses in some instances, in the absence of demonstrable changes in the gonads.

The uterus and its endometrium depend upon the ovaries for changes in histologic structure. If the uterus of an infantile rodent were stimulated directly without the medium of the ovaries but in their presence, by an estrogenic substance that does not in any way depress ovarian activity, then an independent activation of the ovaries by the animal's own pituitary might occur. Hypothetically this pituitary activity can be construed as being either independent and spontaneous, or as a result of stimulation by an estrogen.

This is a brief preliminary report of results indicating that we have been successful in sedimenting poliomyelitis virus, one of the smallest of the ultramicroscopic viruses, 1 from clear aqueous suspensions by means of ultracentrifugation in vacuum. The machine employed is similar in design to that recently described by Bauer and Pickels, 2

Thus far we have obtained complete results on 2 experiments. In both, the material subjected to ultracentrifugation consisted of a 25% suspension of glycerinated pooled virus cords in physiological saline. The lipoids in these suspensions were removed by ether extraction and the aqueous fraction was centrifuged in an Angle centrifuge at 3000 r.p.m. for at least an hour. In the first experiment the resultant fluid was water-clear to the eye; in the second, very slightly opalescent. Eight cc. of the clear suspension were placed in each of a series of celluloid tubes seated in the rotor.

In the first experiment, the rotor was in motion for a total of 4 hours. Stroboscopic determinations indicated that for 2 hours of this period, the speed was between 27,500 and 30,000 r.p.m. After centrifugation, all of the tubes contained a small membranous type of sediment which could not be completely dispersed by repeated pipetting. The thicker central portion of the sediment presented a pinkish tinge apparently due to sedimentation of hemoglobin present in the original cord suspension.

The supernatant was removed in 2 portions, the upper 6 cc. comprising the “top supernatant,” and the pooled bottom 1 cc. portions being the “bottom supernatant.” The sediment was resuspended in 6 cc. of saline. Monkeys were injected intracerebrally with 1 cc quantities of each of these fractions in varying dilutions. The results.

That some annelids exhibit peculiarities in the behavior of their phosphagen complex has been demonstrated by Arnold and Luck 1 and by Needham, Needham, Baldwin and Yudkin. 2 This note extends previous observations.

Early in our work on annelid muscle extracts clarified with basic lead acetate we noted positive Jaffe reactions in the lead-free filtrates. More important was the steady increase in the amount of color produced as the filtrates were evaporated at pH 6.0 at temperatures below 100°C. Thus in Nereis brandti muscle extract the total “apparent creatinine” values increased 59% when the volume of the extract was reduced to one-fourth the original. Extracts of Audouinia spirabranchus muscle gave smaller increases in color production. That the substance responsible for the positive Jaffe reaction was not creatinine itself was shown by negative Weyl and Salkowski tests. The substance could, however, be precipitated by phosphotungstic acid and recovered in the fraction insoluble in absolute methanol. Arginine phosphotungstate is fairly soluble in this reagent, while creatinine phosphotungstate is but slightly soluble. 3 The methanol insoluble phosphotungstate on removal of the precipitant yielded a filtrate giving positive Jaffe and Sakaguchi reactions; attempts to isolate the substance or substances responsible were not successful.

Determinations of labile phosphate in Audouinia spirabranchus, Nereis brand ti, Glycera rugosa, and also in Urechis caupo indicated a slowly-hydrolyzable phosphate, unstable in acid at room temperature. Glycera rugosa, the most active species, gave the highest values for this labile phosphorus.

A new apparatus whose advantages are increased speed and uniformity of column has been described. A new method of working with colorless, non-fluorescent compounds by use of a color reaction has also been reported.

During the course of a comparative study of media for isolation of Mycobacterium tuberculosis from sputum and other infectious material, it was decided to attempt culture of this organism in eggs. This was prompted by the observation that cultural methods now used for this purpose prescribe media containing whole eggs or egg yolk and because of the successful cultivation of viruses and bacteria on the chorioallantoic membrane of the developing egg. The developing egg has been used by Brandly and Graham 1 and Bradford and Tittsler 2 for the culture of bacteria. The former authors obtained growth of Brucella, Salmonella and Pasteurella species upon the chorioallantoic membrane of developing eggs and the latter cultured Neisseria gonorrhoea in the developing egg. Although these authors were studying the invasive powers or pathogenicity of the inoculated organisms their observations are of interest. Bradford and Tittsler suggest the method as a means of culturing Neisseria gonorrhoeα from sources such as joint fluid.

In the present study the primary object was cultivation of the organism from infected material in the simplest and quickest manner possible. The egg of the chicken was used with no attempt to obtain a developing embryo. As a preliminary experiment, a suspension of a pure culture of freshly isolated Mycobacterium tuberculosis was injected into the yolks of fresh eggs. The egg shell was sterilized with 5% phenol followed with 95% alcohol and then pierced with a sterile pin. The suspension of Mycobacterium tuberculosis was then inoculated (1/2 cc.) into the yolk of the egg and the pin hole covered with a drop of paraffin. The egg was then placed in the 37°C. incubator and incubated for 20 days. After incubation the egg was broken and the contents were placed in a sterile Petri dish.

In 1931 an investigation showed that, in many of the hatcheries of New York State, practically every trout 6 months of age or older had distinct opacities in both lenses (bilateral cataract). The lenses were not the only organs in the fish that were affected. The different fins, including the tail fin, were abnormal and showed signs of alternating periods of partial degeneration followed by partial regeneration. The iris of the eyes in the older fish showed degenerative changes. The scales likewise showed degenerative changes in these older fish. All fish that had well developed cataracts were dark colored, a condition known to occur in fish that are blind.

This investigation was undertaken to determine whether the cataract in these fish was due to a contagious infection or whether such other factors as diet, light and heredity were the cause.

The methods employed here were much the same as those used in previous work (Hess 1 ). Sixteen standard size rearing troughs were used, and were operated at the Caledonia fish hatchery under standard conditions. Fish of 2 different age groups were used, approximately 2 1/2 and 4 months old respectively. There were 2 groups of the younger fish. One (Exps. 4 and 5) were hatched from trout seined from Seneca Lake. The other (Exps. 6, 7, and 8) as well as the older fish used in Exps. 1, 2, and 3 were from hatchery stock. Each trough contained 5000 fish at the beginning of the experiment. Only rainbow trout (Salmo irideus) were used.

To test the possibility of contagion 200 trout with cataract were placed for 4 months in a small pond with 400 trout without cataract and fed the control diet of liver and heart.

The diffuse distribution of the pancreas in the rabbit and the attending problems of surgical removal have prevented the study of pancreatic diabetes in this animal. In fact, it has been pointed out that “in rabbits the operation itself is impossible, because of the spread-out condition of the pancreas.” 1 However, a method for complete pancreatectomy is presented here together with data pertaining to insulin requirements, glycosuria, blood sugar levels, etc.

The pancreas in general, is a thin sheet of tissue lying in the meso-duodenum with one large mass on the right between the portal vein and the vena cava and another mass in the mesentery of the spleen.

At operation it is essential to use a Beebe binocular loupe to pick up the small fragments of pancreas which break off and to prevent injury to small intestinal blood vessels. For rabbits of about 2 kilos the anesthetic used is 1 cc. of nembutal intravenously, 0.2 cc. of 1% atropine subcutaneously and sufficient ether at the beginning to allow opening of the abdomen.

Complete removal of the pancreas at one operation usually results in a very high mortality in from 24 to 60 hours from severe diarrhea, gastric detension, surgical shock, or unknown causes. A 3-stage operation with 3-4 weeks intervening between operations gives very satisfactory results. Three different abdominal incisions are used; for the first stage, about 1 inch to the left of the mid-line; for the second, a mid-line, and for the third about 1 inch to the right of the mid-line. Twenty-day chromic catgut size 00 is used for closing the abdomen.

In the first stage, all pancreas in a pocket formed roughly by the transverse colon, stomach and duodenum is removed. A splenectomy may be done by ligating and sectioning the larger vessels, or the pancreas can be gently pulled off from the splenic vessels.

Hitherto all scaly dermatoses produced in rats by means of diets have been broadly characterized as pellagra or pellagra-like. Failure properly and exactly to describe the scaly diseases of the rat and to differentiate one disease from another has led to confusion and controversy in the field of nutritional investigation. At least 3 scaly dermatoses can be produced in the rat and prevented or cured by nutritional means. They are due to (1) vitamin H deficiency (egg-white injury), (2) vitamin B₆ deficiency, and (3) lactoflavin deficiency. (1) Vitamin H 1 deficiency disease is the name given to the general disorder experimentally produced in the rat by inclusion of a high proportion (15 to 40%) of egg-white in an otherwise well-balanced diet which contains all the well-known vitamins. Administration of vitamin H cures this condition. Rats kept on a diet of this kind exhibit in from 4 to 6 weeks typical symptoms of sebor-rheid 2 dermatitis, such as erythema, intertrigo particularly around the neck and genitalia, a brown crusting chiefly over the back that is similar to “cradle cap,” and scaling that progresses from the areas of intertrigo to involve the entire surface of the body and that leads finally to a generalized exfoliative dermatitis. Generalized alopecia and exquisite pruritus are also manifest. The hind legs and the ears escape involvement. Excoriations heal slowly. Skin abscesses are rare; when present they are ecthyma-like. Mild sublingual ulcers are fairly common.

The microscopic picture reveals that in the earliest stages of the disease there is edema in the upper portion of the corium; there is also acanthosis with intercellular and intracellular edema. Altération cavitaire and spongiosis of the stratum spinosum precede the formation of intra-epithelial vesicles.

The anti-streptococcal effects of prontosil on oral administration are somewhat superior to those noted after subcutaneous injection. This confirms Rosenthal. 8 The chemotherapeutic efficiency of prontylin by the subcutaneous route is superior to that noted after oral medication. This confirms Proom 9 and Rosenthal. 8 The observed differences in efficiency by these 2 routes of administration were not due to the greater volume of fluid administered by the subcutaneous route, in that normal saline injections alone were of no therapeutic value. However, the superiority of the parcnteral route from a therapeutic standpoint is open to question, when due consideration is given to the low solubility of the compound and the close similarity between the time intervals, at which the peak! blood concentration occurs, after medication with equal dosages of prontylin by these 2. routes. (Marshall, et al. 12 )

Prontylin orally appears to be 1.8 times more effective than prontosil for low-grade infections and 1.4 times more effective for high-grade infections. The therapeutic margin of safety of prontosil administered orally, however, is quite superior to that of prontylin. Disulon orally in dosages of 40 gm. per kilo does not produce symptoms referable to the central nervous system. This compound, due to its lower toxicity, better tolerance and the greater protective efficiency of unit dosages in the presence of infections, has a therapeutic margin of safety quite superior to that of prontylin. The observed superior therapeutic efficiency of disulon as compared with prontylin confirms similar observations of Rosenthal. 10

Additional evidence is presented to support the Bancroft colloidal coagulation theory of disinfectant action. A new measure of the efficiency of antiseptic agents, the Toxicity Coefficient, is defined on the basis of the Ostwald toxicity equation.

Additional evidence is presented to support the Bancroft colloidal coagulation theory of disinfectant action. A new measure of the efficiency of antiseptic agents, the Toxicity Coefficient, is defined on the basis of the Ostwald toxicity equation.

The frequent symmetrical distribution of the cutaneous manifestations of Pemphigus vulgarisand their resemblance to trophic lesions as well as the limitation of the pathologic changes to the skin and nervous system have suggested a neurotropic etiologic agent of the disease. Carol 1 has summarized the work of earlier investigators in this field and has recorded his own results following intracerbral inoculation of pemphigus material into rabbits, guinea pigs, monkeys, and mice. He concluded that a specific virus of pemphigus could not be demonstrated by the methods hitherto employed. We, however, felt that there was more likelihood of the transmission of an etiologic agent of pemphigus to animals if the resistance of the animals was previously lowered by irradiation. The results obtained by the inoculation of blister fluid and spinal fluid from 3 cases of Pemphigus vulgaris into the brains of irradiated mice are recorded here.

Case I, D. R., an adult female, had been sick for 6 months when bacteriologically sterile blister fluid was obtained 23 days before death. Five mice which received a generalized x-ray dose of 400 r. were inoculated intracerebrally with 0.03 cc. of fluid. Three weeks later all were sick. One was killed on the 22nd day, another died on the 94th day, and the remainder recovered. A 40% bacteriologically sterile emulsion in physiologic saline of the brain of the killed animal was used for passage. All of the animals which received the brain emulsion became sick in the second week after inoculation and died by the 27th day. The active agent has since been transmitted for 46 passages and at present 0.03 cc. of a 5% emulsion of bacteriologically sterile infectious brain material kills 90 to 100% of the mice in 7 days.

Since the report of Sakel 1 on the successful treatment of certain cases of schizophrenia by producing a hypoglycemic shock with the use of insulin, there has been increasing interest in the study of the physiological, psychological and chemical changes that occur during the treatment.

Our previous studies on mental and nervous diseases have been concerned mainly with the physicochemical changes that occur in the blood serum during the different stages of acute insanities. With this previous experience we thought it of interest to study the changes that may occur during the shock treatment with insulin.

The method of treatment is, with slight modifications, essentially that suggested by Sakel. 2

In the present communication we shall not be concerned with the clinical results or changes in the individuals treated, but exclusively with the changes observed at different periods of the treatment. This report includes the result of 23 different days of treatment of 8 patients. The bloods were taken during fasting (i. e., before the insulin was given), during the coma or deep shock, and finally 15 to 20 minutes after the administration of glucose intravenously, given to terminate the shock or coma. At the same time that blood was taken for the study of the proteins, a sample of oxalated blood was also taken to determine the amount of glucose.

The methods used in this study are the same as those reported previously. 3

We notice a change in the specific gravity at the time of the coma, which continues after termination. This increase we attribute to dehydration, which is probably due to the marked perspiration before the patient gets into shock. This loss of water can easily account for this change in the specific gravity.

1. There appears to be a significant relationship between nutritional deficiency and alcoholic cirrhosis of the liver. 2. It is believed that patients with alcoholic cirrhosis of the liver are benefitted by high vitamin therapy.

In a previous publication 1 one of us (Mellon) reported a poten-tiative effect of sulfanilamide on hemolytic streptococci when the organisms had been exposed to physiological salt solution during the process of dilution preliminary to seeding the test cultures. If this exposure was omitted and dilution in broth substituted, the remarkable bacteriostatic effect of sulfanilamide in low concentrations was not noted. In the presence of normal human serum the effect is enhanced. In other words, a sterilizing effect is obtained from the combined action of these minimal factors which exceeds many times a simple summation effect.

Kenny, Johnston, and von Haebler 2 have recently reported a very favorable series of clinical cases of E. coli infection of the urinary tract which were successfully treated with sulfanilamide. They showed that with oral medication of 1.5 gm. a day for 5-7 days sterilization of the urine was obtained during the period of treatment in all of 46 cases of infection with a typical E. coli. The concentration of free sulfanilamide obtained in the urine of treated cases ranged from less than 1:100,000 to 1:1,000. It was shown experimentally that the static or bactericidal action of the compound in vitro was roughly proportional to its concentration.

It appeared to us that a potentiation similar to that displayed by saline for hemolytic streptococci might be anticipated in this instance with the culture medium itself (the urine) playing the role of the potentiating agent. Bacteriostatic tests were accordingly made on a strain of E. coli freshly isolated from a case of cystitis which had had no sulfanilamide therapy. Normal pooled urine was adjusted to pH 7.2 and sterilized by Seitz filtration. The broth employed in the following experiments was a 2% proteose-peptone beef infusion of the same pH.

Strain-differences in Group A hemolytic streptococci evoke marked differences in the inflammatory cell-response in mice undergoing treatment of experimental peritonitis with sulfanilamide.

The reaction of the intestinal contents and its control has assumed increasing importance during recent years with the growing realization that acidity and alkalinity are important factors in the digestion and absorption of food and in the progress of various pathological conditions of the intestinal tract. With this knowledge has come an increased interest in the factors influencing the reaction and the methods for their control. One of us 1 has published data indicating that there is a certain constancy about the reactions of the various sections of the intestine which have the appearance of physiological constants and which the body strives to maintain. Unpublished results have confirmed this and demonstrate a rather efficient mechanism for the accomplishment of this end. Apparently changes of long duration are difficult to maintain. The only method whose effectiveness has been adequately demonstrated is that of increasing the acidity by the administration of large amounts of lactose. Several studies have been made of this process, 2 3

Presumably it depends on the alteration of the intestinal flora in such a way that the acid-producing organisms predominate and by the production of lactic acid produce their effects. Lactose, and to a lesser extent dextrine, are the only 2 substances which accomplish this. Most of the results have been secured on carnivorous or on omnivorous animals like the dog, cat and rat. The ordinary alpha lactose has always been used.

It occurred to us that in an herbivorous animal with a longer intestinal tract the effects of lactose feeding should be emphasized by the longer time required for the passage of material through the gut. It was also thought that beta lactose, whose nutritional status has become of interest since processes for its manufacture have made it available might, because of its greater solubility, be less effective than the alpha form.

Cutaneous absorption of insulin occurs in rabbits and in patients independent of abrasions produced on the skin. This absorption is, however, very inconstant.

Weinstein and Weiss 1 reported that banana, apple and raisins when fed to white rats subsisting on a high-protein diet consisting solely of raw chopped lean beef are able to bring about a change in the intestinal flora from a type in which non-aciduric bacteria predominate to one in which L. acidophilus is the outstanding organism. It was our purpose in the work presented here to further enlarge the group of fruits studied and to determine to what extent the ability to alter the intestinal flora is a general property of the fruit family or whether it is limited to certain members.

The bacteriological methods and feeding procedures used in the experiments to be described here are the same as those reported previously by Weinstein and Weiss. All of the animals were fed 12 gm. of lean chopped beef daily until repeated stool examinations revealed the absence of L. acidophilus from the intestine. At this time the fruit to be tested was added to the meat diet of one group of animals in 4 gm. amounts daily and 6 gm. to the diet of another group. Periodic fecal examinations were made and the L. acidophilus content of the feces determined.

The fruits used were cranberry, strawberry and tomato, supplied to us in the dehydrated form by a commercial concern engaged in their preparation.

Ten rats were fed cranberry-powder. One group of 5 rats received 12 gm. of meat plus 4 gm. of the powder; another was fed 12 gm. of meat with 6 gm. of the dried fruit. Two rats were used as controls; they subsisted on a diet of lean chopped beef with no added carbohydrate. Four of the 5 rats receiving 4 gm. of dried fruit underwent a change in their intestinal flora after 2 weeks of feeding.

In order to explain the mechanism of the antigonadotropic function we thought it necessary to examine the following questions: Is prolan irrevocably destroyed by antiprolan or is the process reversible? While investigating the chemical properties 1 of antiprolan we found that prolan and antiprolan differ from each other as follows: Prolan is more sensitive to HCl than antiprolan but less sensitive to NaOH. n/10 up to n/15 HCl destroys prolan, but not antiprolan; n/10 up to n/15 NaOH destroys antiprolan, but not prolan. If the prolan-antiprolan effect is reversible it must be possible to destroy antiprolan by means of NaOH and thereby to release the prolan and render it biologically active once more. By using HCl we should be in a position to destroy the prolan, to liberate the antiprolan once more and to render it capable of again inactivating freshly added prolan. If the process is irreversible the prolan-antiprolan-complex cannot be changed in any way whatsoever either by NaOH or by HCl.

The following experiments make it evident that the prolan-antiprolan effect is reversible.

The reactivation experiments can be divided into two groups. I. Reactivation of the prolan out of the prolan-antiprolan complex by selective destruction of the antiprolan by means of NaOH. II Reactivation of the antiprolan out of the prolan-antiprolan complex by selective destruction of prolan by means of HCl.

I. Reactivation of Prolan. Thirty mg. of antiprolan-acetonedry powder (corresponding to 30 PAU∗) was dissolved in 1.2 cc. of aqua dest. 0.8 cc. = 20 PAU of this solution was mixed with 20 RU of prolan (dissolved in 0.4 cc. aqua dest.). This neutral prolan-antiprolan mixture (= 1.2 cc.) was now divided into 2 parts (0.6 cc. each) with which Experiments 1 and 2 were carried out.

The inactivation of prolan by antiprolan is a reversible process, since prolan and antiprolan may be released from a neutral prolan-antiprolan mixture and thereby reactivated.

The assumption is made that antiprolan is neither an (anti)hormone strictzi saw nor a ferment, but is possibly a new kind of factor approaching very closely the immunebodies to which it is in some respects quite similar.

Crystalline vitamin D (100-400 units) when added to Proskauer and Beck's medium did not change the virulence or growth characteristics of either the Ra or Rv variants of strain Ha37 M. tuberculosis.

It has been reported 1 that in the normal dog a marked decrease in the excretion of Na and usually an increase in the excretion of K occurs during a 6-hour period following the intravenous injection of 20-40 cat units of cortin; and that with repeated injections (at intervals of several days), the effect becomes progressively less, until there is little change in electrolyte excretion following administration of the extract. It has since been found that NH₃ excretion is also increased following cortin and that with repeated injections this response is decreased. Of 9 animals injected with 20-40 cat units of cortin, all gave the characteristic electrolyte response described (Table I). Four different cortical extracts prepared in this laboratory were used. The response elicited by a given extract in different dogs was very similar (Table I). Five of these animals were injected until they became completely refractory.

Failure of these animals to respond to further injections suggested the possibility that some antagonistic substance was present in the blood. Using sterile precautions, blood was taken from 2 dogs (I and 111), which had received 7 and 11 injections respectively over periods of 5 and 9 weeks. The serum was separated and stored at 4°C. Five untreated animals were given 10-25 cc. of this serum intravenously about 15 minutes previous to the injection of the usual amount of cortin. In every case in which cortin and serum were given, the Na and the NH, did not change beyond the range of the controls, whereas cortin alone always reduced the Na to a level well below that of the lowest control and increased the NH, well above the highest control value (Table 11). Decreases in Na of 86-890/, in 3 animals (I, 111, X) were shown when 0.5 cc.

Despite the importance of an understanding of amino-acid metabolism, there has been no trustworthy method of amino-nitrogen determination that could be easily applied as a routine in a clinical laboratory. The chief difficulty has been in the preparation of filtrates of blood and urine for the actual titrations. In this note a method is described for the removal of NH₃ and CO₂ from urine filtrates enabling one to determine amino-nitrogen expeditiously by the Sörensen titration. Many determinations may be carried out in parallel with little supervision by the analyst.

The Sörensen 1 titration as described by Northrop 2 is used. The preparation of urine filtrates follows the procedure of Van Slyke and Kirk. 3 Instead of distilling the NH₃ and CO₂ successively in vacuo, the filtrates are exposed in shallow layers in vacuo in a desiccator over dilute H₂SO₄. This means of collecting or removing NH₃ has been known for a long time and occasional reference is made to it in the current literature. It requires no further effort on the part of the analyst, in contrast to the individual distillations in vacco as described by Van Slyke and Kirk or as further improved by Kirk. 4 Quantitative transfer of solutions from the large inaccessible surface of the flask is likewise avoided.

In preliminary trials it was found that NH, could be removed from ammonium chloride in about 3 hours. With urine filtrates, however, 5-7 hours was required for constant titration figures. The alkaline filtrate remains unchanged in the refrigerator for several days so that analyses may be repeated.

Urine filtrates after Van Slyke and Kirk (5 ml.) are placed in 50 ml. beakers with as little exposure to air as possible. The lips of the beakers may be paraffined to facilitate pouring.

The correlation between total solids and pH of dog' gall bladder bile, normal or ox bile fed dogs, is high indicating with a certainty of approximately 451% greater than sheer chance that concentrated biles will possess a greater hydrogenion concentration than those which are more diluted.

The wide use of metrazol in the treatment of schizophrenia has made it advisable to study the physiological changes produced by metrazol convulsions. 1 Twelve observations were made on 7 patients with schizophrenia. As seen in Table I, 7 samples of blood were collected from the femoral artery during various stages of the convulsions, 4 pairs of samples were collected simultaneously by 2 observers from the femoral artery and internal jugular vein immediately after the seizure had ceased, as was one additional sample of arterial blood. Breathing was greatly diminished during the convulsions and this was reflected in the analyses of the arterial blood, which disclosed a retention of CO₂ as well as a diminished O₂ content. Even during the first part of the seizure, as seen in Wi, 9/13, the O₂ content was diminished so that the Hb saturation was reduced from a theoretical normal of 95% to 84%. As the convulsions progress, the Hb saturation continues to fall so that towards the end of the seizure the saturation of Hb may be below 50% (M., 9/7). During these convulsions the patient's face is at first a dark red color. When the convulsion is completed, the anoxemia is evidenced by a leaden cyanosis, Neyertheless, as soon as unimpeded breathing is reëstablished the Hb saturation, though still reduced, is found rapidly rising towards a normal value (last 5 observations of Table I). The anoxemia, as well as the severe muscular effort, combine to increase blood lactic acid and blood sugar.

These convulsions, grand mal in character, cause a temporary but marked depression of cerebral functions as evidenced by amnesia, confusion, disorientation, and the elicitation of various abnormal reflexes, such as the Babinski and ankle clonus.

Grollman and Firor 1 have demonstrated that chronic adrenal insufficiency, induced by various methods, is primarily a disturbance of pituitary origin. The syndrome which follows, cessation of growth, failure of reproductive activity and subnormal body temperature is, according to the above workers, relieved by administration of pituitary extracts and not by cortin. Long 2 has shown that acute adrenal insufficiency caused by the removal of the adrenal gland, though accompanied by injections of cortin, ameliorates experimental pancreatic diabetes. The object of the present experiments is to determine the effects of chronic adrenal insufficiency on the course of experimental pancreatic diabetes.

Eight cats were anesthetized with sodium pento1)arl)ital and both lumboadrenal veins were ligated distally and proximally to the gland. L4t the same time the entire pancreas was removed. The animals received neither insulin nor cortin. Fluid was given ad lib. along with a Lveighetl amount of food (Bovex) each day. The periods of survival were 11, 11, 12, 15, 17, 18, 20, and 98 days. The life span of these animals was definitely prolonged by the ligation, for depancreatized cats usually succumb before 8 days. At the present time we shall present a brief description of the cat which survived for 98 days. After the operation the cat exhibited a pro found glycosuria. hut 2 iveeks later the urine ivas free of sugar and remained so until death. There was a marked (501%) loss in body weight. - At autopsy careful examination revealed no traces of pancreatic tissue. The adrenal glands appeared degenerated. The kidney cortex Was ivhite due to increased deposition of fat, 14.8% on analysis, although the liver Ivas free of fatty infiltration and contained the normal amount of fat, 4.5%. Two large and several small ulcers were found on both sides of the pyloric orifice of the stomach.

The male hormone, testosterone propionate, inhibited the oestrous rhythm of the rabbit uterus. This was manifested by a disturbed rhythm, a lowered height of contraction or complete quiescence. Such inhibitions appeared in animals whose uteri showed but little or no progestational response. During the period of quiescence, pituitrin failed to elicit a strong contraction. Testosterone prevented castration atrophy in the rabbit's uterus with or without minimal progestational changes.

1. In 15 monkeys and 8 humans testosterone propionate effected changes in specialized nasal areas. These changes produced by androgens occurred in both male and female monkeys. 2. In monkeys and in humans congestion, swelling and fluid formation were observed grossly. Histologically, perivascular edema was pronounced in the monkeys. 3. The nasal areas affected are similar to those which exhibit vicarious menstruation. Discussion is given as to possible physiological significance of gonad-controlled function of these areas with regard to warming of inspired air, the prevention of infection, and therapeutic use of gonadal substance in conditions of atrophic rhinitis and nasal hyposecretion.

Sodium chloride therapy in suprarenalectoniized rats during our period of oliservation was effective not only in prolonging life hut also in restoring normal hypophyseal-ovarian activity in 55% of the cases.

Webster and Clow 1 and Kanazawa 2 reported the first success in cultivating rabies virus. The former accomplished this with a medium consisting of Tyrode solution containing normal monkey serum and minced mouse embryo brain, while the latter propagated the virus in Tyrode solution in the presence of rabbit embryo brain tissue, but without the addition of serum.

We wish to report our confirmation of the observations of Webster and Clow, since by the procedure which they describe, we have been able to carry the virus through 16 subcultures. Material from the final culture has proved infectious for Swiss mice in dilution of 10 2 . In addition a series of passages carried out on the same medium, but with an equivalent amount of normal rabbit serum in place of normal monkey serum yielded results which were similar in all respects to those obtained with normal monkey serum.

Attempts to propagate the virus on media containing a larger ratio of serum to Tyrode and considerably smaller amounts of tissue proved unsuccessful. The fact that Kanazawa propagated the virus in a medium containing rabbit embryo brain, without the addition of serum, suggests that possibly the concentration of serum may have been a factor and that a low concentration of serum or no serum at all, may be more favorable to growth.

Twelve separate attempts were made to propagate the virus on the chorio-allantoic membranes of developing chicks. All yielded entirely negative results. This is in accord with the experience of Waldhecker. 3

In a study of the skin of guinea pigs, it has been found that the tuberculous animal reacts differently from the normal in regard to the spread of dye which has been injected intradermally.

We have made tests to see if differences could be detected in the spread of dye in the skin around the zone of primary inoculation as contrasted with other areas. The work was started in the course of completing a study of the Koch phenomenon which had been begun by the late Henry Sewall. He had obtained evidence that there are differences in sensitivity in different areas of the skin of the tuberculous guinea pig.

The dye used was pontamine sky blue which we obtained through the courtesy of Dr. P. D. McMaster, and in general we have followed his technique. 1 We have used the dye as a 2.5%, approximately isotonic solution. The amount of solution injected was 0.025 cc, given with a Dewitt and Herz syringe which has an automatic guard on the plunger, assuring exact dosage. The injections were made under a binocular dissecting microscope under brilliant light, the animal having been anesthetized with sodium amytal.

It was found that there are some differences in the rate of spread of dye, according to varying thickness of the dermis. In the guinea pig the thickness of the dermis in dorsal, lateral, and ventral zones is in the proportion of 2:l.S :1, that is to say, the skin on the back is twice as thick as that on the aldonien. Hence, to be comparable, injections must Ix restricted to one of these 3 zones. Usually 3 injections. 2 on each sidewere made in each animal but in the case of some of the dorsal injections, only 2 were made, each in the mid-line. IVhen the injections Ivere thus limited, the spread in a given animal 11 as practically identical. provided that in the tuberculous animal the indurated Imrder of the primary ulcer was avoided.

Flemming Miller and Gudjonsson 1 called attention to a peculiar form of generalized osteosclerosis, hitherto unknown, occurring in Danish workers exposed to dust of cryolite, a fluorine compound (Na₃AlF₆). The condition is characterized 1 2 3 4 5 by a diffuse increase in density of the bones, particularly of the spine, pelvis and ribs, and by calcification of ligaments and tendinous muscle attachments. Because of marked periosteal proliferation and the calcification of contiguous fibrous tissues, the sclerotic bones are often irregular in contour. There may be considerable impingement also upon the medullary cavity. The spine and thoracic cage ultimately become rigid, resulting in loss of mobility and vague “rheumatic” pains; but clinical signs and symptoms are otherwise remarkably few, even in advanced cases. At necropsy, 2 ; 3 4 5 6 the sclerosed bones are found to be heavy (up to 3 times the normal weight) with rough surface and relatively brittle texture. Histologically, the peculiar if not pathonogmonic osseous structure is characterized by the partial deposition of lime salts in the form of irregular granules. The fluorine content of the bone ash 2 3 4 5 is increased to about 10 times the normal values.

While distinctly less common than abnormalities of dentition, (“mottled enamel”), 19 skeletal changes due to chronic fluorine intoxication probably occur more widely in man than is generally appreciated. A number of cases of osteosclerosis have been reported from North Africa 7 and from India. 8 In the United States, Bishop 5 recently described typical skeletal changes in a man who for 18

We are presenting a summary of observations on the renal excretion of inulin, creatinine, xylose, and urea in a single normal sheep. These observations were made in 1934 and since the results were concordant with similar observations on the dog, 1 further investigation seemed unnecessary. The observations were made on an unan-esthetized animal, restrained upon an animal board. The experimental procedure and chemical methods were similar to those used in observations on dogs. 1 The urine flow at which these observations were made varied from 0.82 to 2.20 cc. per minute.

That the spinal ganglion cells and sheath cells of Schwann have a common origin in the neural crest, his been accepted generally since Harrison's original experiments in 1904. When the dorsal portion of spinal cords of anuran embryos were removed (elimination of the ganglionic crest), the larvae lacked spinal ganglia and sensory nerves, and the motor nerves present were devoid of sheath cells. Recently Raven employing a different experimental approach assigns a cord origin to the sheath cells and claims that Harrison's conclusions were not justified, since in eliminating the neural crest he also destroyed the dorsal part of the spinal cord, thus not only removing such presumptive sheath cells as may originate there, but obstructing by deformation of the cord the dorsal migration of these elements which are supposed to lie in the ventral portion of this structure. Raven also concludes from his experimental results that sympathetic elements arise from both neural crest cells and from the ventral portion of the spinal cord. His findings do not support those of Müller and Ingvar and Van Campenhoutwho claim a neural crest origin exclusively for sympathetic ganglion cells, but they do support in part those of Kuntz and Batson, and Kuntz, and Jones, who assign a neural tube origin for the sympathetic elements.

Raven interchanged trunk neural crest and also median part of medullary plate (future ventral portion of spinal cord) between embryos of Anzblystoma mexicanum (axolotl) and Triton tuaniatus (xenoplastic grafts). Axolotl (donor) cells in Triton embryos could be recognized by their larger nuclear sizes. Employing variation curves for nuclear size difference in both donor and host cells, Raven employed statistical methods in the analysis of his results. Although he obtained evidence for a dual origin of sympathetic ganglion cells, he says that the sheath cells of Schwann are derived exclusively from the tube.

Poliomyelitis virus was isolated from the nasopharyngeal secretions of a bulbar case on the 9th day of illness.

The authors wish to acknowledge their appreciation to Dr. Morris Schaeffer of the Bureau of Laboratories, New York City Department of Health, for his valuable suggestions during this work.

Although pedigree isolation cultures of protozoa have been carried on during the last 50 years, relatively few investigators have experimented with carnivorous forms. Woodruff and Spencer 1 and Woodruff and Moore 2 kept Spathidium spathula alive for long periods without degeneration, conjugation or endomixis on a diet of Colpidium colpoda. Beers, 3 feeding Didinium nasutum on Para-mecium caudatum succeeded in attaining 1384 generations in about a year's time, without any degeneration or internal reorganization of any kind. With these results in mind it was decided to attempt to find a suitable diet for the carnivorous ciliate, Dileptus gigas, and to ascertain its division rate on such diet in isolation pedigree cultures.

In 1936 a number of individuals of Dileptus gigas were isolated from some of the writer's stock cultures. These cultures were originally collected from Van Cortlandt Park Pond, New York. By preliminary feeding experiments it was soon ascertained that the large, blue Stentor coeruleus made an excellent food basis for Dileptus and this readily cultured organism 4 was therefore selected as the standard food supply for the experiment. Visscher 5 has described the interesting and unusual manner in which Dileptus manages to attack, paralyze, and cytolyze by means of the powerful trichocysts (toxicysts), stentors much larger than itself and feast upon its prey. It is interesting to add that Stentor, itself predominantly carnivorous, was grown on Belepharisma undulans, 4 the latter being supplied with a mixed diet.

On Nov. 21, 1936, two series, consisting of 4 pure lines each, were established. All of these 8 lines were the progeny of a single Dileptus isolated from stock culture on Nov. 18. The organisms were kept in Maximow culture dishes in a moist chamber and were isolated daily (occasionally every other day) by capillary pipette under a binocular dissecting microscope.

Of the several methods employed in assays of thyrotropic hormone that utilizing the structure and weight responses of the thyroids of guinea pigs is the most commonly used. 1 2 Day-old white leghorn chicks have been found not only to respond more sensitively than guinea pigs but also to give a relatively greater weight increase and to exhibit a more uniform thyroid structure.

The preparation used was a partially purified alkaline extract of beef anterior pituitary. 3 Although a response is elicited by a single dose in 24 hours, 5 daily injections were found to give a maximal reaction. Further division of doses into 2 a day for 5 days gave a somewhat greater response.

One hundred and seventy-five injected chicks and 36 uninjected controls furnish the basis for the assay curve. Each sample of extract was tested on 5 chicks. At autopsy the thyroids, dissected under a binocular microscope, were weighed and body weight and sex recorded. The total dose was divided into 5 daily subcutaneous injections. It was found advisable to do the autopsy 24 hours after the last injection. Table I gives a representative series of the mean thyroid weights and total dose of extract used. Most dosages were repeated on different hatchings, with separate batches of extract. Total doses varied from 0.1 to 20.0 mg. Increases in thyroid weight were produced by increased dosages up to 10.0 mg. but beyond this there was no further increase in thyroid weight. Thus, a maximum thyroid weight is produced with 100 times the minimum stimulating dose. The larger doses produced thyroids averaging 6 to 7 times the mean weights of controls.

Mice and rabbits receiving large doses of estrogens develop pyometra. 1 2 3 4 5 6 Estrogenic hormones may induce physiological changes favoring a leucocytic invasion of genital tissues. 2 3 4 It has been suggested that this inflammatory condition may be related to the leucocytic infiltration occurring during normal post-estrus. We have tried to determine whether bacteria are present in the uteri of normal mice and whether estrogenic hormones induce changes resulting in bacterial invasion of the uterus.

Mice from several strains and of varying ages were used. All that were treated with estrogens (hydroxyestrin benzoate) were young, sexually mature, virgin females. The estrogen, in oily solution, was injected subcutaneously, usually in a single dose of 500 I.U. (1/20 cc). The mice were killed after intervals of 2, 4, 7, 10, 14, and 15 days following the initial injection (Table II). Bacteriological studies were made on the uteri of immature, mature virgin, multiparous and castrated females; of mice receiving injections of oil alone and of the blood of injected and untreated mice (Table I). In a third series one uterine horn was cut from the cervix and the cut end fixed to the abdominal wall. One month later 500 I.U. of estrogen were injected subcutaneously and 7 days later bacteriological examination of the ligated and intact horns was made.

The mice were kilIed with illuminating gas and opened under aseptic conditions. The uterine horns were removed well above the cervix, ground in 0.5 cc. sterile saline and one loopful of the resulting suspension was streaked on heart-infusion blood agar and another inoculated into heart-infusion broth. Growth, if any, appeared in 12 to 24 hours. Negative cultures were examined for 5 days. Frequently colonies growing on the plates were subcultured and identified.

In recent publications 1 2 it was shown: (a) that during one phase of the reaction between phage and bacteria in the presence of 0.25M NaCl phage is formed without concomitant bacterial growth; (b) that when pH and temperature are properly adjusted the bacterial substrate can be maintained in the resting state while [phage] rises logarithmically with time at the rate of a ten-fold increase per hour.

The obvious inference of these experiments is that bacterial growth, long held to be the prime conditioning factor for phage-formation, is actually not essential at all. Instead it would appear quite possible that resting cells may produce some sort of phage-precursor which in the presence of phage is promptly converted into more phage. This hypothesis is supported to a considerable extent by our observations during the past 3 years on the increase in phage-titer that occurs when cell-free ultrafiltrates of bacterial suspensions are added to known quantities of phage. Young cultures of susceptible organisms are washed in saline solution to free them from phage-inhibitor. 3 4 Dense suspensions of the washed cells are placed in infusion-broth of pH 7.4 at 36°C. for 2 hours. The fluid portion of the mixture is then separated from the cells by ultrafiltration through a 2% or 2.5% acetic-collodion membrane. 5 When equal quantities of the ultrafiltrate and known phage-preparations are mixed an increase in phage-titer occurs. For example when 1 ml. of ultrafiltrate is added to 1 ml. of phage containing 1×10 activity-units/ml.₆ the final titer remains 1 × 10 activity-units/ml. Such yields, 100% increases in the amount of phage originally added, are quantitatively significant inasmuch as the method of titration is capable of detecting differences in [phage] of ± M5%. Furthermore, when a solution of phage is subjected to serial dilution, using the ultrafiltrate as diluent, the actual titers are consistently higher than the concentrations calculated from the original amount of phage added and the difference between these values becomes progressively greater as the range of dilution is extended.

This note summarizes the results of an investigation of the possible glandular nature of the parenchymatous cells of the pars nervosa of the hypophysis. Functional histological analysis in rats of various ages under experimental conditions leads to the conclusion that these cells produce and secrete anti-diuretic substance for the control of the water metabolism of the animal.

This “glandular” cell has been seen in the pars nervosa of almost every class of mammals, and of pigeons and chickens. The cell is distinguished by the presence of granules or of lipoid droplets which were first seen in fresh mounts. The inclusions fill the cytoplasm, and extend out into the cell processes. These intracellular elements are of approximately the same size in any one cell, though they may vary somewhat in size in adjacent cells. Histochemical analysis of the droplets or granules shows that in the rat they are rich in neutral unsaturated fats; no other lipoids have been identified. In some other animals, the granules contain no visible lipoids. The inclusions are preserved best by Maximow's fluid in the mouse and white rat.

The characteristic features of the glandular cell appear very early in the rat's embryonic life, being easily recognizable in a 23 mm. embryo. The number and size of the cells and of their inclusion bodies increase throughout life to become most prominent in rats 2 1/2 years old. The cells are distributed more or less evenly in the pars nervosa except at its junction with the hypophyseal stalk; there they are closer together. They are more widely separated and smaller in the remaining portion of the stalk than in the glandular portion.

1. The variation of reticulocytes in normal pigeons has been determined. 2. Concentrated liver extract, injected intramuscularly, produced the usual rise in reticulocytes in normal pigeons, while Congo red, injected intravenously in the same and other pigeons for days, did not. This does not mean that Congored can not act beneficially in pernicious anemia. The difficulties of bioassaying antipernicious anemia agents in normal animals are real and not always appreciated.

In recent years much experimental investigation of agranuloeyto-sis has given rise to the view that “chemicals containing the benzene ring, may so depress the bone marrow that leucopenia results, and bacterial infection of the mouth may develop in consequence.” 1 It has been the contention of Madison and Squier 2 that agranulocyto-sis is the result of drug hypersensitivity (to amidopyrine).

Landsteiner and Jacobs, 3 reported sensitization of guinea pigs to simple benzene ring compounds injected into, or spread on the skin of those animals for a number of consecutive days. Among those was 2:4 dinitro parachlor benzene. Accordingly, this procedure was repeated here, and blood studies made.

An alcoholic stock solution of 2:4 dinitro parachlor benzene was made, which when diluted with normal saline solution contained 1/500 mg. of the compound per 0.1 cc. Blood counts (r.b.c, w.b.c, and differentials) were made on all guinea pigs before, during, and after the injection period. Eight animals were used, 4 of which remained alive throughout the entire experimental period. Ffteen daily injections of 0.1 cc. each were made intracutaneously. The compound was found to be a slight irritant. Skin reactions, however, were read at all times by comparison with a previously uninjected albino guinea pig control. One month after the beginning of the injections each animal received 5 separate intracutaneous doses of 0.1 cc. each, and an albino control was similarly treated. The previously injected animals responded with marked zones of inflammatory edema at each injection site, a slight eosinophile rise, no significant change in the white blood cell count, no change in the red cell count, an increase in the lymphocyte percentage in three of the injected animals, and a decrease in the polymorphonuclear percentage in the same animals.

Jusatz 1 reported that oral administration of vitamin A, B, C, or D is without appreciable effect on the bactericidal titer of the blood serum of rabbits or on specific-antibody production in this animal species. However, intravenous injection of a massive dose of vitamin C (sodium salt of ascorbic acid) increased the bactericidal index about two-fold and specific-precipitin production about fivefold. We have attempted to repeat his experiments with ascorbic acid and to extend his antibody-stimulating studies to include other enzyme-activators, other animal species and other types of antigens. The present paper summarizes our initial confirmatory results with ascorbic acid introduced parenterally into rabbits during the process of active immunization against horse-serum proteins.

A total of twenty 2000 gm. control rabbits were each injected intravenously with 0.5 cc. of horse serum. An equal number of rabbits of the same size and weight were each injected intravenously with 0.5 cc. of horse serum plus 100 mg. of crystalline synthetic ascorbic acid (Merck). Each injected animal was bled from the ear vein at frequent intervals during the next 50 days and the resulting antiserums were titrated for antihorse precipitins. Composite data from the two groups are recorded in Fig. 1.

Ascorbic acid plus horse-serum proteins caused a prompter formation of specific precipitins than occurred in the control group injected with undenatured horse serum. The antibody-stimulating ratio for ascorbic acid when thus used is about 30:1 during the earlier stages of active immunization, falling to about a 12:1 ratio during the height of active immunity (14-21 days). Specific pre-cipitins disappear from the control or non-vitaminized group by the fiftieth day, at which time the vitaminized group has a residual titer nearly equal to that of the control group at the height of active immunity.

Improvement in 4 patients with pellagra following administration of nicotinic acid was as satisfactory as that following administration of liver filtrate except for an increase in time required for complete disappearance of dermatitis.

The study of the physiological regulation of blood-sugar concentration embraces a complexity of independently variable factors whose individual influences are still incompletely understood. Many experimental substances and conditions will cause hyperglycemia, but none besides insulin are known which will cause hypoglycemia.

Alloxan will produce hypoglycemia in normal rabbits in a very characteristic fashion. If upwards of 70 mg. of alloxan monohydrate per kg. of body weight is injected intravenously the blood sugar level of the animal will fall below the normal value (less than 70 mg. %) in about 3-4 hours, and will continue to fall steadily during the next 2-4 hours until the convulsive level (less than 35 mg. %) is reached. It will keep falling further during the convulsive stage until the animal expires, and very low (less than 15 mg. %) values may be observed terminally. All normal rabbits respond in the same way, although some delay in the time of onset of convulsions is observed if the animals have not fasted for 12-24 hours before the experiment. The larger dosages of alloxan (150-200 mg./kg.) will not hasten the appearance of convulsions, but will very definitely cause the recurrence of convulsions after remissions induced by glucose.

Both the convulsions and the hypoglycemia are promptly relieved by glucose intravenously administered. Animals in violent convulsions will return to a conscious and quite normal state within one minute after glucose has been given. After 5-10 minutes they will eat and drink in a normal manner. If the dosage of alloxan has been large hypoglycemia and convulsions may recur several times at intervals of 2-3 hours provided that each attack is treated with glucose. Large dosages of glucose or sufficient food intake tend to forestall subsequent seizures.

In an experiment in which the effect of pituitary removal on the rate of growth of Walker tumor No. 256 was compared with the effect of caloric restriction in intact tumor-bearing male rats, samples of liver, muscle, and tumor tissue were secured for glycogen determinations.

All animals had been fed by stomach tube with a formula composed of

The hypophysectomized animals received twice daily throughout the experiment 1 cc. of this mixture per 40 gm. of body weight, calculated at the beginning of the experiment when tumor weight was negligible. The amount of food received by the controls was varied to maintain the total weight (somatic weight plus tumor weight) roughly equivalent to that of the hypophysectomized rats. This resulted in a restriction to approximately ¾ of the normal food intake in the intact tumorbearing animals, while the hypophysectomized animals were given more food than they would have voluntarily consumed. At the time the glycogen samples were secured the hypophysectomized group had been deprived of pituitary tissue for 21 days. Liver, muscle, and tumor samples were taken in the order mentioned under amytal anesthesia 4 hours after the last feeding, and were frozen, weighed, and digested in the usual manner. Analyses were made according to the method of Good, Kramer and Somogyi 1 except that, in addition, the precipitate was washed once with 60% alcohol after being drained. The wash alcohol was also carefully drained off. The sugar in the final hydrolysate was determined by the Shaffer-Hartmann-Somogyi method. The percentage of glycogen present in the sample was calculated from this figure. For the tumors, glycogen was expressed as percentage of the total weight, or as mg. per sq. cm. of surface.

Korányi and Szent-Györgyi 1 have reported recently that succinic acid given as the calcium salt in daily amounts of 5 to 10 gm. or less is able to decrease the ketosis in diabetics. In the present investigations we have compared the ketolytic action of succinic acid with that of glucose when administered twice daily to fasting male rats weighing approximately 200 gm., in which the endogenous ketonuria was induced by the previous administration of a high butter-fat diet low in protein, as reported earlier. 2 The studies on ketonuria were made on the second to fifth fast days inclusive during which sodium chloride solution (fasting controls), sodium succinate in doses from 9.95 to 149.2 mg. per 100 gm. rat daily, or glucose in equivalent amounts (7.55 to 113.2 mg. per 100 gm. rat) was fed. Urine collections were made daily. The ketonuria of the fasting controls usually rises to a maximum on the third fast day, after which it diminishes rapidly. In Table I a summary of the data is given. No ketolytic effect obtained when as much as 149.2 mg. of succinic acid per 100 gm. rat was given although glucose at a comparable level gave a marked decrease in the acetonuria. This amount of succinic acid on a surface area basis is 50% greater than the largest dose employed by Korányi and Szent-Györgyi. As slight decrease in ketonuria appears when only 7.55 mg. of glucose is given while a dose of 37.8 mg. caused an average decrease of acetone-bodies in the urine from 33.7 mg. per 100 gm. rat in the control tests to 19.4 mg. There was no evidence of diarrhea in any experiments nor of other toxic effects ascribable to the succinic acid.

In an earlier paper by one of us 1 it was shown that the eviscerated rabbit utilizes glucose at a rate that is quite definite and fairly constant for a given animal. The rate varies, however, between different rabbits. The suggestion was made in that paper that this variation was due to the differences in the degree of fasting to which the different animals had been subjected prior to operation and that the effect of fasting is to reduce the utilization rate of the eviscerated animal. The work here reported was planned to investigate this relationship. The same technique was used as in the previous work. Essentially this consisted of measuring the rate at which glucose had to be administered to the eviscerated rabbit to maintain the blood sugar at a constant normal level.

Frequent blood sugar determinations served as a guide to the injection rate; if the blood sugar level rose the rate would be diminished, and vice versa. The determination was started 3 hours after completion of the operation and continued for 4 hours thereafter. Only those animals that sat up in normal posture and had normal righting reflexes after operation were used. The animals were also tested for kidney function by injecting phenol red after the operation. Any animals not secreting this dye were eliminated from the series. Ten rabbits were used in each of the two groups—one group fed to the time of operation, and the other fasted 4 to 6 days prior to operation. The glucose utilization rates are given in Table I. The rates are given for the period between the 3rd and the 7th hours after operation, In this way we could be sure that we were not getting any anesthetic effect.

The purpose of this investigation was to ascertain whether the restoration to normal of the clotting time of chicks on a diet deficient in Vitamin K was accompanied by a cure of the existing anemia. This anemia has been observed by Dam, 1 and Hoist and Halbrook, 2 but thus far no extensive investigations on the regeneration of hemoglobin have been made. The changes toward normal produced by the administration of minute quantities of a Vitamin K concentrate were so rapid that it seemed desirable to report our results.

The chicks used in our experiments were one-day-old white Leghorns hatched from eggs laid by hens which had been fed a diet relatively free from Vitamin K. They were raised in battery brooders and had free access to food and water. The basal diet used in these experiments was that of Almquist 3 with only minor modifications. Our active extract was prepared from alfalfa meal by an adaptation of the methods of Dam and Sch⊘Hnheyder 4 and of Almquist. 3

Blood was drawn from the brachial vein, samples being taken for the determination of clotting time, hemoglobin, and the erythrocyte count. The clotting time (Sch⊘nheyder 5 ) was determined by allowing the blood to flow directly into a small porcelain dish. Zero time was taken as the time when the first drop fell into the dish. When the blood no longer flowed upon tilting the dish it was considered to be clotted. The blood was observed for 300 minutes, if it failed to clot in a shorter time. The hemoglobin was determined by the method of Palmer 6 in which the hemoglobin is converted to carbon monoxide hemoglobin and compared with a standard solution of carbon monoxide hemoglobin in a colorimeter.

The interest in the effect of zinc on the action of insulin and various other hormones stimulated a study of its effect on the activity of adrenalin. The subjects were patients at rest in bed and in the post-absorptive state. Capillary blood sugar and blood pressure were determined before and for four hours after subcutaneous injection of 0.5 mg. of adrenalin hydrochloride (1-1000' solution). Curves for comparison were made with and without the added zinc, using symmetrical areas for injection and intervals of 2 to 3 days between examinations.

In the first series equal amounts of zinc and adrenalin were used. The zinc was added, immediately before injection, as 0.1 molar solution of zinc sulphate. Correction was made for the small increase in volume. The average blood sugar curves for 9 subjects are shown in Chart 1. The marked decrease in response is evident.

In the second series the effect of one-tenth as much zinc as adrenalin was studied. (0.05 mg. of zinc was given with 0.5 mg. of adrenalin. This is about the same order of magnitude as the concentration of zinc in commercial protamine zinc insulin.) The zinc was added as 0.01 molar zinc sulphate solution. The average blood sugar curves for 6 subjects are shown in Chart 2. The decrease in response to adrenalin is again evident.

Average blood pressure curves were similar to the blood sugar curves but, in individual cases, more irregular and not always consistent.

With small amounts of zinc the total activity of adrenalin appears to be definitely diminished. Since adrenalin is oxidized rapidly in the body, it seems probable that, under these conditions, it has been absorbed more slowly and therefore a greater proportion oxidized in a given interval of time.

The high unitage of gonadotropin in the blood 1 and especially the placenta 2 of the mare during the second, third and fourth months of pregnancy when the fetus is growing from about 2 to 20 cm. (crown-rump length) would seem to be responsible for the secondary, multiple luteal bodies of the maternal ovaries, 3 and the remarkable overgrowth of the fetal gonads. 4 The primary corpus of pregnancy regresses at about the end of the first month, and from then until about the end of the fourth month multiple corpora are found in both ovaries. Cole and others 3 found rupture points in some of the secondary corpora, leading them to the belief that these bodies followed a true ovulation as normally, but histologic studies have led us to believe that the secondary corpora may also be formed by hyperplasia of the theca interna with accompanying degeneration of the ovum and granulosa (Fig. 1). Such thecaluteal bodies may be produced in rodents by injections of pregnant mare's gonadotropin, and appear similar to the aberrant corpora of rhesus monkeys. 5

The secondary corpora usually regress by mid-pregnancy, and the maternal ovaries become atrophic and fibrotic. It was of interest, therefore, not only to determine the progestin-content of the corpora lutea, but also to investigate other possible sites of production or storage of that hormone, especially the placenta in the latter half of pregnancy, since some workers 6 7 8 have found progestin in the human placenta. Unfortunately the work on the detection of progestational hornione in the atlreiial cortex 9 10 had not come to our attention until this study 11 as completed.

The experiments here reported represent an attempt to determine whether a relation exists between the growth rate and first estrus in the rat.

It has been common knowledge for a decade that rats and mice may be retarded in growth rate and in size at weaning by the amount of available nourishment. Many breeders have found that optimal conditions of growth and size in an animal colony may be attained by limiting the size of the nursing litter to 5 or 6 animals.

Litters of rats were selected for this study over a period of more than a year. They were taken from every month in the year, but few were studied during July and August. Records were obtained on 286 rats which lived to first estrus. These represented 63 litters. To indicate the random selection of litters for study, the serial numbers of the animals reported range from 13,992, born Sept. 27, 1935, to 16,721, born Oct. 4, 1936. Those of Group D were studied between January and May of 1937.

The sex of the young was noted at birth, and females otlly were left in each litter. The rats were originally divided into 3 categories : those lvith 2 or 3 in a litter (Group A), those with 4 or 5 (Group B), and those Ivith 10 or 11 (Group C). The latter represent in each instance 2 litters Imrn the same night, and reared by the mother of one of the litters. Thus half of this group were foster rats. Because of the high mortality of infantile rats under such conditions of crowding, there were in Group C only 7 litters with 10 each alive at weaning and 3 litters with 11 alive at weaning. Sulxequent losses reduced this number to 91 at first estrus.

From an analysis of the method of measuring blood flow by the Rein 1 2 thermostromuhr it became evident that the passage of the high frequency current through the intact blood vessel, on which a diathermy thermoelement is placed, results essentially in a localized heating of the wall of the blood vessel. Hence it was of interest to construct a thermostromuhr unit similar to the modified type introduced by Baldes, Herrick and Essex 1 in which a direct current heater replaced the platinum electrodes and likewise was spaced midway between the differential thermocouple. This arrangement of the Rein unit differs somewhat from the thermostromuhr described by Schmidt and Walker 4 in which the hot thermojunction is a silver trough interposed between a direct current heater and the blood vessel.

The arrangement of the direct current heater and the thermocouple in the bakelite block is shown in Fig. 1. The heating unit is made of No. 36 or 38 B. & S. gauge nichrome wire rolled to a ribbon 0.75 mm. to 0.5 mm. wide. This unit consists of a folded loop ‘c’ soldered to copper wires ‘b’(2 strands number 38 B. & S. gauge), the resistance of the loop being at least one ohm. The unit is held in place by bakelite lacquer. The thermojunctions at ‘a’ are made by soldering copper wires (0.0016 in.) to a constantan wire (0.002 in.) which is embedded in the groove ‘d.’ The fine copper wires as well as the copper wires attached to the heater are then soldered to braided copper wires (17 strands 0.003 in. tinned copper). All grooves are filled by several applications of bakelite lacquer 3128 or Sterling varnish M-472, each application being followed by suitable air drying and baking.