Experimental Biology and Medicine

A dry form of placental globulin extract could be prepared by means of methyl alcohol precipitation. When dissolved, this preparation was found to be as potent as the preparation from ammonium sulphate precipitation with regard to neutralization of Dick toxin and prophylaxis against measles.

Swabbing alone produces a significant cornification of the vaginal epithelium in a high percentage of ovariectomized rats. The changes thus induced come later than those following injections of estrogenic substance, and are transient, even though swabbing is continued. The reaction to swabbing materially affects the results of biological assay.

The effect of the prolonged use of photodynamically active dyes on a number of bacteria was summarized by Reitz. 1 Workers mixed the dyes and the cultures and let them stand for days exposed or unexposed to light. It was found that some dyes, including methylene blue, were more active in the presence of light than other dyes, such as fluorescein. It was also found that B. diphtheriae was more susceptible to such action than B. typhosus. Since then, however, very few reports have appeared. In view of the recent interest in the study of photodynamic action on viruses, bacteriophage, 2 and toxins, 3 it seems worthwhile to restudy this problem systematically.

We have studied the effect of short exposures to light of different dilutions of methylene blue on a number of Gram-positive and Gram-negative microorganisms. A preliminary report is presented at this time.

Saturated aqueous solution of methylene blue was diluted to different concentrations with normal saline. Twenty-four hour broth cultures of different organisms were mixed with these solutions in Petri dishes. The bacteria-methylene-blue mixtures were exposed to an ordinary electric light of 100 candle power at a distance of 10 cm. for a period of half an hour. These Petri dishes were placed on a cooling machine which prevents the plate from getting too warm. Soon after the exposure, the organisms were plated out and incubated. The other portions of the same mixtures were not exposed and were similarly plated to serve as controls.

The response of nerve, as measured by its heat production or summated action potential changes, has been shown by Bugnard and Hill 1 to decline progressively with increasing frequency of stimulation beyond that which calls forth the maximum response. A similar response-frequency relation in the case of muscle is to be expected, but an attempt to obtain it experimentally with toad's or frog's sartorius stimulated by repetitive condenser charges and discharges from a revolving commutator through a pair of silver wire electrodes lying on the tibial half of the muscle, revealed a very different relation. The curve relating the size of isometric contraction in tetani of any duration from 0.44 to 10 sec. and the frequency of stimulation, was found to be a wavy one with recurrent minima and maxima. In an experiment at 26.3°C. with 0.44 sec. tetani the contraction was at a minimum at the approximate frequencies 380, 750, and 1500 per sec. With longer tetani and lower temperatures, the positions of the minima were shifted to lower frequencies.

Further experiments then showed that this strange result was associated with the fact that the stimulating electrodes were laid on the tibial half of the muscle, and that the muscle was partly stimulated through its intramuscular nerve twigs. For this unexpected result was no longer obtained with curarized muscle or when the muscle was stimulated by electrodes on the nerve-free pelvic end. Conversely it was obtained in a much more striking form when the sartorius was excited indirectly through its nerve. The response-frequency curve of muscle when nerve participation is excluded, is a smooth one, and for short tetani generally very flat after the maximum, unless complicated by fatigue.

There are very few convenient methods for the preservation of viruses. The use of disinfectants is not permissible because of their deleterious effect upon viruses, while the commonly employed glycerol exerts only a feeble antiseptic action and, therefore, cannot be relied upon when freedom from bacterial contamination is important. The preservation of viruses through the process of drying in vacuo at low temperatures is an excellent method but it has the drawback of being somewhat cumbersome.

It has been shown by Ginsburg and Kalinin, 1 Sonnenschein, 2 and others, that 10% solution of sodium chloride is effective in preserving complement for about 4 weeks. This action of sodium chloride upon complement, a substance of known instability, suggested to us that this salt may be a good agent for the preservation of viruses.

We found that saturation of guinea pig complement with sodium chloride preserves it for at least one month, and that various bacteria of the Salmonella group, pneumococcus, meningococcus, gonococcus, Streptococcus hemolyticus and viridans, Staphylococcus aureus, B. influenzae and B. diphtheriae are killed in from a few days' to one month's time when kept in saturated solution of sodium chloride.

The experiments demonstrate that hamsters react much in the same way as guinea pigs to cultures of C. diphtheriae or of diphtheriae-like organisms. There was complete agreement in 95 out of 102 instances. To use hamsters instead of guinea pigs possessed the advantage of giving clear-cut results because these animals invariably died from the virulent organisms and survived the non-virulent ones. Equivocal results from intradermal tests on guinea pigs due to secondary infection from field cultures, or to injections being made too deep into the skin, could thus be avoided.

Other advantages are that 10 hamsters may be purchased for the cost of one guinea pig, and that the control hamsters require much less antitoxin. Objections may be raised in that pure cultures are necessary, thereby causing possible delay. This is only of theoretical interest, because usually it is only in doubtful and convalescent cases of diphtheria that this test is required, and a delay of one day does not limit its usefulness. Furthermore, one must remember that in making the conventional test on guinea pigs a delay of 24 hours is also necessary after the control guinea pig has received antitoxin.

As a result of these studies it has been shown that Chinese hamsters are suitable animals for the routine determination of the virulence of C. diphtheriae. The use of these animals instead of guinea pigs is very much less expensive and in certain respects gives more reliable information.

The effect of photodynamic action of certain dyes on various substances, among which may be mentioned different types of animal cells, viruses 1 , 2 , 3 and soluble toxins of both bacterial 4 and animal origin, 5 is a well-known phenomenon. Workers generally agree as to the extreme susceptibility of these substances to the photodynamic action of dyes, the optimal concentration of the dye employed, and the relatively short time required for exposure, but disagree with regard to the antigenicity of substances so treated. The present study aims to investigate the reaction of diphtheria toxin to the photodynamic action of methylene blue upon which subject little study has been made. Diphtheria toxin is looked upon as a more suitable material than either tetanus toxin or viruses on account of its greater stability and ease with which quantities given can be accurately measured and properly controlled.

The technique employed is essentially similar to those of other workers. 1 , 5 Methylene blue is selected since it has been more thoroughly studied than other dyes. A single batch of toxin containing 400 M.L.D. per cc. for the guinea pig, or 2000 M.L.D per cc. for Chinese hamsters weighing 20-25 gm., was used throughout the experiment, employing as diluent 0.9% salt solution for both dye and toxin. As source of radiation, both direct sunlight during summer and early autumn and electric light of 100 watts, 110 volts at 16.5 inches distance were used. In a preliminary study, the minimal time for exposure was found to lie between 20-30 minutes. In order to obtain full manifestation of combined action of light and dye, it was subsequently fixed at 60 minutes in all instances. Table I shows the maximal M.L.D. for hamsters which may be destroyed by the optimal concentration of methylene blue when exposed to sunlight and to artificial light. The number of animals in each group varied from 3 to 9.

Parabiotic twins of rats exchange daily about 150 cc. of blood. If one member of a female pair is hypophysectomized it exhibits deficiencies which prove that blood received from the normal mate carries not enough hormones to maintain body weight, thyroids, adrenals and ovaries at normal levels. Castration or sterilization of males and females increases the output of follicle-stimulating hormones to the extent that normal or hypophysectomized twin females go into a condition of constant oestrus. The ovaries are visibly overstimulated and contain large follicles but no corpora lutea. Uterus and vagina exhibit also the characteristics of a maximal or even an exaggerated oestrus condition, indicating the release of large amounts of oestrin by the ovaries. Not enough oestrin accumulates in the castrate member to prevent complete atrophy of the secondary sex characters. This indicates rapid disappearance even of high amounts of oestrin from the circulation. Constant oestrus with a moderate degree of ovarian and vaginal stimulation and almost dioestral size of the uterus is observed in females with experimentally masculinized hypophyses. Both types of constant oestrus can persist indefinitely, at any rate over a full year, without loss of sensitivity by either ovaries or genital accessory organs. It is concluded that no specific anti-hormones are formed, though the hypophysis, probably through the intermediary of the thyroid (and adrenals?) checks the excessive growth of overstimulated ovaries. The loss of reactivity in ovaries after prolonged stimulation with gonadotropic hormones of heterogeneous origin is considered as an immunity reaction to foreign proteins.

A thermostromuhr is described which differs principally from that of Rein in substituting storage battery for high frequency current. Its use is thereby vastly simplified. It has been calibrated on arteries and veins of living animals and gives promise of yielding quantitative information as to blood flow in unanesthetized animals.

We are anxious to express our gratitude to Dr. D. W. Bronk for his frequent helpful advice, and to Mr. A. J. Rawson of his department who has made for us the more perfect models of the instrument.

1. The effect of adrenalin on the blood sugar and blood lactate was studied: A. in patients with Addison's disease. B. in adrenalectomized dogs. 2. In patients with Addison's disease, the subcutaneous injection of adrenalin produced a similar increase in the blood sugar to that observed in the controls, and the blood sugar curve showed approximately the same contour in the 2 groups. The blood lactate curves differed, however. Both showed a similar sharp rise, but in the patients with Addison's disease, the curve remained elevated, and had not returned to basal level in 4 hours, whereas in the normal controls, it had reached basal level again within 3 hours. 3. It is suggested that the delay in the disappearance of the accumulated lactate from the blood seen in patients with Addison's disease may be due to a sluggishness on the part of the liver to convert lactic acid to glycogen. 4. The blood sugar and blood lactate curves, following a single intravenous injection of adrenalin in adrenalectomized dogs, are both lower and tend to remain elevated longer than in the same dogs before removal of the second adrenal gland.

An analysis of the effects of 6 different types of globulin, dissolved in Locke's solution, upon the sedimentation time of erythrocytes, reveals that in 12 experiments 4 of the types prolonged the sedimentation time, and that in 12 experiments the remaining 2 types shortened the sedimentation time. In 8 experiments in which 2 of the types of globulin were dissolved in plasma, the sedimentation time was prolonged.

By tarring the ears of domestic and cottontail rabbits tumors have been procured for comparison with others experimentally induced with the Shope virus. 1 The tarred rabbits were strictly isolated.

The growths caused by virus regularly developed from the surface epidermis and were papillomas varying little in structure, whereas not a few of those consequent on tarring originated from the skin appendages, with result in a wider morphological variety. The tar papillomas were scattered, discrete and often punctate in origin; so too were the papillomas due to virus when this had been appropriately inoculated. The growths due to tarring appeared only after it had been repeated often enough to cause general hyperplasia of the epithelium, together with complex connective tissue alterations; and many of them retrogressed after tarring was stopped. The virus tumors, on the other hand, arose on the basis of the slight epidermal trauma incident to inoculation, and their progression was followed, not preceded, by connective tissue changes. The virus evidently needed no help,—though the growths it caused could be stimulated to rapid enlargement by connective tissue disturbances experimentally induced. 2 The unknown cause of the tar papillomas, on the other hand, was effective only when epithelial and connective-tissue disturbances had long existed, becoming independent thereof only after the growths had undergone considerable development, and often not even then. The tar papillomas were structurally much more irregular than early virus papillomas, as would follow from the disturbance of the underlying connective tissue, which was of a sort to bring about corresponding irregularities in the virus tumors. The surface of the tar papillomas often underwent maceration because covered, whereas the exposed surface of the virus growths consisted of dry, keratinized material.

When due allowance had been made for these differing conditions of incidence and growth, the resemblance between the tar and virus papillomas was found to be close.

In the course of investigations on the excretion of gonad stimulating hormones in the urine of mental patients 1 , 2 the following method was developed for the preparation of concentrates of the follicle stimulating hormone (F.S.H.).

The method is based on the observation that the stable aluminum hydroxide C (γ), prepared according to Willstaetter and Kraut 3 is a satisfactory absorbent for F.S.H. from urine. The effectiveness of this adsorption depends markedly on the pH, a definite maximum occurring at pH 4.5. Evans, Gustus and Simpson 4 have reported that solutions of the gonadotropic hormone from acetone-dried pregnant mare's serum are more satisfactorily adsorbed at pH 3.5 by aluminum hydroxides of Types A and B, prepared according to an earlier method of Willstaetter and Kraut, 5 while Type C was considerably less efficient.

The urine is collected in glass jars containing 5 cc. of a saturated alcoholic solution of thymol per liter capacity. Insoluble material is removed by filtration and the filtrate is adjusted to exactly pH 4.5 with 5 N HCl using bromcresol green as external indicator. A suspension containing 20 gm. per liter of aluminum hydroxide C (γ) is slowly poured into the urine until 1 gm. of aluminum hydroxide per liter of urine has been added. For 24-hour specimens of 500-1000 cc. a full 1.0 gm. of aluminum hydroxide is added; for samples of less than 500 cc. only 0.5 gm. is used. The resulting mixture is occasionally stirred by hand (mechanical stirring results in poor yields) during 20 minutes so as to maintain a homogeneous suspension; the solids are allowed to settle during 45 minutes. The supernatant fluid is removed by siphoning and centrifugation, readjusted to pH 4.5 and again treated with aluminum hydroxide by the same procedure.

This investigation was undertaken in an attempt to determine the effect of ultra violet on tissue subjected to potassium excess. For this purpose the heart of the clam, Venus mercenaria, was used.

Records were obtained of the ventricular beat of the heart in situ. A small hook, made of glass capillary tubing, was inserted into the apex of the ventricle and, by means of a thread attached to the hook and a light balanced lever (a drinking straw), records were obtained on a moving kymograph. The clam, on the half shell, was placed in a finger bowl which was filled with sea water. To this the desired amount of potassium chloride was added.

The ultra violet source was a Cooper-Hewitt Uviarc (6-inch tube with reflector and running at 110 volts D. C.) placed at 35 cm. from the preparation. A thermopile, with a blackened couple, at the heart, served as a temperature index. The sensitivity of the galvanometer enabled temperature changes of 0.01°C. to be observed. During all of the experiments the temperature changes were negligible. Room temperature varied from 22 to 27°C. During the course of an experiment the temperature both of the room and of the heart never changed more than one degree Centigrade from the beginning of the experiment to the end. Temperature rise during irradiation never exceeded 0.3°C.

Short ultra violet irradiation (2-3 min.) of the clam heart appears to cause a marked increase in tonus, a definite decrease in amplitude, but does not affect the pace maker.

Experiments were per formed with potassium chloride concentrations up to 2.7% in sea water. When concentrations above 1.36% potassium chloride were used ultra violet (1-2 min.) did not always restore a rhythm.

In view of the increasing frequency with which the Weil-Felix reaction is being used in the United States for the diagnosis of obscure fevers suspected of Rickettsia origin, and especially because of our own interest in the possibility of latent typhus infection in individuals infected a long time ago, we undertook to carry out a considerable number of Weil-Felix reactions on individuals not at the time suffering from typhus or fevers of any kind, for the purpose of establishing the significance of low titre reactions. We are reporting these briefly because they seem to us helpful in appraising occasional doubtful cases.

The reactions were carried out on sera from several groups of subjects. One group was composed of Jewish out-patients furnished us by the Beth Israel Hospital Clinic in Boston, and in these record was made as to whether the individual was born in Russia or in the United States. Since such information had no significant effect on these observations, we abstain from tabulating it. Another group was composed of non-Jewish subjects, another of routine Wassermann sera in which no attention was paid to the race of the patient; and, finally, a small group of 24 Russian-born garment workers. Table I gives the results.

It will be seen that there was no significant difference between the groups, and it is rather fortunate in protecting us against false conclusions that the actual percentage of positive reactions in the Russian-born garment workers was rather lower than in the other groups.

If a stock culture of Streptococcus hemolyticus is grown on 5% rabbit blood-agar plus 10% commercial antistreptococcus serum, the hemolytic zone surrounding each plate colony is at times 100% to 200% greater than the control area of hemolysis on routine blood-agar plus 10% normal horse serum. A typical example of this auxolytic effect is recorded in Table I (serum A).

The percentage increase in average hemolytic area varies (a) with the concentration of the auxolytic serum tested and (b) with the selected strain of S. hemolyticus. Typical variations are recorded in Table II

The above recorded auxohemolytic effects are reminiscent of the paradoxic growth-acceleration of certain bacteria in the presence of homologous specific immune serum. 1

The concentration of potassium in the plasma increases as the circulatory volume decreases. This is a reversible process since the potassium concentration returns to normal if the circulatory volume is increased subsequently by reinjection. Saline is as effective as whole blood in causing the decrease of potassium concentration in this way. The tissue cells are reversibly permeable to potassium ions. The injection of epinephrine has only a transitory effect on the plasma potassium but histamine causes a more lasting increase.

I am very grateful to Dr. W. O. Fenn for his aid with the experiments and in the preparation of this manuscript.

Considerable evidence has pointed to the possibility of the presence in insulin of sulfur other than that attributable to cystine, in which form the major portion of the sulfur is present. That thiolhistidine might account for part of this sulfur was a possibility to be considered. We therefore undertook studies to ascertain whether or not this amino acid is a constituent of the insulin molecule. On the basis of reports in the literature that the sulfur of thiolimidazoles could be split off as sulfate by boiling with ferric chloride and that the sulfur of cysteine was stable, we attempted to test for the presence of thiolhistidine. However, in testing this reaction with cystine, we found that ferric chloride will oxidize the sulfur of cystine to sulfate. 1 This reaction could not, therefore, be utilized to detect thiolhistidine in the presence of cystine in an insulin hydrolysate and some other method of approach was necessary.

In Barger and Ewin's early paper on the betaine of thiolhistidine, ergothioneine it was shown that the sulfur of this compound was oxidized by bromine to inorganic sulfate. 2 In addition, it is a well recognized fact that cystine upon treatment with bromine is converted to cysteic acid and that no inorganic sulfate is formed. 3 It occurred to us that these 2 observations might form the basis of the desired test but before applying this reaction to insulin for the detection of thiolhistidine we felt that it was necessary to test the reaction on the amino acid itself rather than depend on the known behavior of its betaine. This amino acid was, therefore, synthesized by Harington's method, 4 using aspartic acid as starting material. We were able to check Harington's procedure in all essential details and obtained approximately the same yields reported in the various steps recorded by him.

The spider monkey, Ateles ater, like other new world varieties, is naturally refractory to experimental inoculation with poliomyelitis virus (monkey passage).

Nugent and Towle 1 have reported specific gravity values for 33 solutions of beef serum albumin, serum globulin and mixtures of the 2 in the range from 0 to 12% total protein. In all cases the solution contained 0.9% sodium chloride and was adjusted to pH 7.3 to 7.5. Under these conditions, the specific gravity of a solution was shown to be a measure of its total protein content in accord with the finding of Moore and Van Slyke 2 that the specific gravity is a useful measure of total plasma protein in nephritis, more useful in fact than the refractive index, the physical property which has been most widely used in this connection.

At the same time relative viscosity values were obtained for a similar series of solutions which have not previously been reported. The solutions for the relative viscosity measurements were prepared and their specific gravity, salt content and pH value controlled in the same manner as previously described. The relative viscosity measurements were made with a 1 cc. Ostwald viscometer at 25° in a water bath thermostat. Since the relative rates of flow of water and protein solutions through capillary tubes may vary with the applied pressure 3 the values obtained may well be specific for an instrument of the dimensions employed. An instrument of convenient dimensions was therefore indicated since it must serve as a model for others to be employed in checking or utilizing the results. The one chosen for use∗ required a small volume of solution in accord with the amounts of serum usually available in routine clinical work, and was of a type which could be reproduced by an amateur glass blower. (Fig. 1.) The upper and lower bulbs contain respectively 0.6 and 0.9 cc. and their centers are 70 mm. apart.

The virus of equine encephalomyelitis, both the Eastern and the Western strains, was found to pass collodion membranes with an average pore diameter of 66 mμ, but was completely held back by those of 60 mμ. The results indicate that this virus has the same filtration end-point, and consequently the same particle size, as that of St. Louis encephalitis, which was shown by Bauer, Fite, and Webster, 8 and Elford and Perdrau 9 to be 20 to 30mμ.

Normal sera possess a staphylococcal antihemolysin titer of about 1 International Unit or less. The titers of sera from our grow of staphylococcal infections, other than osteomyelitis, vary from 0.5 International Unit or less to 22.7 units, with the great majority falling into the normal range. Sera from 3 cases of superficial infections had titers of 0.5 unit.

In contrast to other reported results, about two-thirds of the sera from cases of osteomyelitis had a titer of 1 International Unit or less. Only one-fifth of the sera had definitely high titers, and these, in our series, averaged 5.1 units; the highest was 17.7 units. From our results, it seems to be necessary to modify the conception that a high antihemolysin titer is characteristic of osteomyelitis.

A new transmissible strain of myelosis that originated in a mouse irradiated by X-rays is described. This strain possesses characteristics which differ from those of the strain previously observed. The immature myeloid cells peculiar to this strain reproduce themselves by mitotic division in vitro and in vivo and like malignant cells, fail to mature.

The following is a preliminary report of a new method of determining linear X-ray absorption coefficients in plano-parallel ground sections of teeth. This coefficient is a precise measure of radiopacity, which, in radiographs of such sections, is usually interpreted as measuring degree of calcification. Variations in calcification of teeth may arise in several ways: (1) With increasing age there may occur a progressive diffuse deposition of calcium salts within the already calcified matrix. (2) Secondary deposition of calcium salts may occur locally in response to some abnormal condition. (3) Certain pathological conditions may cause local rarifactions. (4) Structures in the organic matrix may cause relative variations in calcification merely by spatial displacement of inorganic material.

Such variations in distribution of inorganic material correspond to differences in compactness or density of calcification; i. e., in weight of calcium salt per unit volume. However, the qualitative nature of radiopacity as ordinarily observed in a photographic film limits its value as a criterion of density of calcification for experimental purposes. To offset this limitation, Warren et al. 1 have measured the radiopacity by means of a Capstaff-Purdy densitometer for determining density of silver grains in photographic films. The precision of their method is stated to be of the order of 5% for sections 1 mm. thick and areas 0.5 mm. in diameter. Actually, it is even poorer than this since they used a mean thickness value throughout the section but did not include its precision measure in that of the entire determination.

Our experience with grenz-ray radiography of ground sections of teeth suggested that differences of considerably less than 5% may be significant in studies of normal calcification. In order to measure such differences, a method has been developed for the measurement of radiopacity which is good to 0.5% or better.

The chemical nature of catalase has been studied by a number of authors. The experiments of Zeile 1 and of Stern 2 indicate that the catalase molecule is probably a chromoprotein in which the hemin group is related to that of the natural blood pigments. Recently Agner 3 reported that if horse liver catalase is dialyzed against N/10 or N/100 HCl the enzyme splits into 2 inactive components, one of a low molecular weight which dialyses through the cellophane membrane and which is possibly hemin and another component which remains within the bag and which is a protein. If the 2 neutralized components are mixed, an active preparation is again obtained, according to Agner.

Having the intention of making certain studies on the basis of Agner's report, we first attempted to repeat his experiment. The technique is simple but we were not able to confirm his results. The only difference between his and our technique is in the species of animal from which the liver was derived. Horse liver, which Agner used, is practically unobtainable in our locality. We have, however, tried beef, rabbit, and rat liver, respectively. We followed the experiments of Agner in every detail, dialyzing the purified catalase against N/10 or N/100 HCl, for 10 to 48 hours. No splitting (inactivation) of the catalase could be obtained. With stronger HCl irreversible inactivation of the catalase took place, due to a marked decrease in pH inside the dialyzing bag.

Mercuric iodide is relatively insoluble in water but is easily soluble in an aqùeous solution of potassium iodide. The complex salt potassium mercuric iodide, having the formula K₂HgI₄, is formed by the interaction of one molecule of mercuric iodide (HgI₂) with 2 molecules of potassium iodide (KI). The preparation contains about 25.5% of mercury.

Aqueous solutions of the salt have been extensively employed as disinfectants because they do not possess the corrosive action on instruments and tissues that characterizes aqueous solutions of mercuric chloride. Another advantage is that they do not precipitate albumin.

Potassium mercuric iodide is about one-half as toxic as mercuric chloride when administered to animals. In proportion to the mercury content, however, the 2 salts possess about the same toxicity.

In previous papers of this series 1-6 comparisons were made of the resistance of Staphylococcus aureus and embryonic chick heart tissue to phenol, Merthiolate, Metaphen, Mercurochrome, Hexylresorcinol, iodine, and iodine trichloride. Toxicity indices were determined by dividing the highest dilution of the germicide that killed the tissue by the highest dilution of the chemical showing no growth of the test organism. Theoretically the smaller the toxicity index the more nearly perfect the chemotherapeutic agent.

The methods employed were the same as those given in the first paper of this series. Wide jumps in the dilutions were first prepared to determine approximately the least concentration of the germicide required to destroy the bacteria in 10 minutes but not in 5 minutes. Usually one such preliminary series was sufficient. Occasionally a second series covering higher dilutions was necessary.

About 50 years ago, central stimulants, particularly picrotoxin and coriamyrtin, were used to oppose the depression caused by aliphatic narcotics. Recently, Tatum and his coworkers 1 , 2 tested the antidotal effect of a number of central stimulants with the exception of coriamyrtin in experimental barbiturate poisoning. They produced the poisoning by oral or intraperitoneal administration of the barbiturate and found that picrotoxin was the most effective antidote in both short and long acting barbiturate depressions. Following the barbiturate, picrotoxin was given by diverse routes in doses of 8.8 to 12 mg. per kg. This drug increased the percentage recovery of the animals, e. g., in 7 rabbits given picrotoxin following a dose of 350 mg. of sodium barbital per kg., 4 recovered and 3 died, whereas in 6 controls, 1 recovered and 5 died. Also the average recovery time of the surviving animals was shortened by picrotoxin. However, this dose given by these authors is not in excess of the average fatal dose to any appreciable extent. Gower and van de Erve 3 used a larger dose of sodium barbital and injected the drug intravenously. In one dog, they injected 600 mg. of this drug at 3 different times allowing 5 to 9 days to intervene after the complete recovery of the animal from the preceding dose. In these 3 experiments, the amount of picrotoxin which they used over a period of 4 to 8 hours following the sodium barbital injection varied from 6 to 8.8 mg. per kg. The dog recovered in each case within 32 hours. The dose of sodium barbital used by these workers was at least 50% in excess of the average fatal dose and about 3 times the minimum anesthetic dose.

The Shope rabbit papilloma is an epithelial new growth caused by a filtrable virus 1 and transmissible in series through cottontail (Genus, Sylvilagus ∗) 1 and domestic rabbits (Genus, Oryctolagus). 2 Both in the gross and histologically it is similar to the virus papillomata of man, cattle and dogs. 1 , 3 - 6 Further, it has been found to possess not only the immediate characters of a tumor, 7 but also the potentiality of progressive alteration until finally it may assume the characteristics of a squamous cell carcinoma with metastases. 8 , 9 We have confirmed these findings.

Carcinomatous changes have been reported previously only in experimentally inoculated domestic rabbits. 8 , 9 , 10 It would be highly significant were the host-parasite relationship so perfected in the natural host, the cottontail rabbit, that malignant changes never occurred. The significance of such a finding would apply irrespective of whether the virus per se, or whether secondary factors were responsible for the carcinoma. The purpose of this communication is to report that such is not the case, for we have observed carcinomatous degeneration of a naturally occurring papilloma in a wild cottontail rabbit.

Of 132 cottontail rabbits (Sylvilagus floridanus alacer Bangs) received from southern Kansas, 11 have had spontaneously one or more of the papillomatous growths. These 11 cottontails were kept for observation and for a source of virus. With a single exception, our findings have agreed with those previously described. 1 , 7 The history of the exceptional case follows:

Cottontail rabbit S5-CR 363. Thin, young, adult male. Received March 30, 1935. On lower abdomen 4 pigmented warts were noted: A, 18×22×15 m. in height, B, C and D, 4-6 mm. in diameter x6-8 mm. in height. Rapid gain in weight. Papillomata appeared unchanged for 85 days, when lesion A showed superficial ulceration with thickening of the underlying tissues. Six days later the rabbit died from fracture of the lumbar spine sustained accidentally the previous day.

It has been shown that strains of mice differ in their inherited susceptibility to tumors both spontaneous and tar-induced. 1 Burrows, Hieger and Kennaway 2 and others have demonstrated the effectiveness of 1:2:5:6-dibenzanthracene in inducing sarcoma. The present paper is a preliminary report on the tumors induced by subcutaneous injection of 1:2:5:6-dibenzanthracene into various strains of mice.

Ordinary lard was filtered at 37°C. and the crystalline dibenzanthracene was dissolved in the filtrate by reheating to 140°C. Each mouse received a total dose of 4 mg. of 1:2:5:6-dibenzanthracene in 1 cc. of lard administered in 3 subcutàneous injections of 0.25 cc., 0.25 cc., and 0.5 cc. at biweekly intervals.

Of the mice receiving treatment 2 groups were from our branch of the Bagg albino strain. The 3rd was from our “Yellow” strain which is only pen inbred and probably not very homogeneous genetically but interesting because it produces yellow and brown mice. A 4th was from No. 1194—our highly inbred agouti or wild type strain; a 5th from Strain 5—an imported albino stock from McGill University. The 5th contained males only. The 6th was from Strain 62 which is composed of pink-eyed dilute brown mice, highly inbred brother by sister. Another group of Bagg mice from a previous experiment may be legitimately included for discussion here.

The results are shown in Table I. The presence of lard makes it difficult to determine the time when malignancy supervenes in the injected tissue, and lung tumors are found only at autopsy. Therefore the percentages are based upon the populations surviving at the time the presence of the first tumor of each type was proven by microscopic sections. For the lung tumor, this date was the 66th day after the last injection; for the sarcoma, the 73rd day. No mice lived longer than 376 days after treatment, the majority less than 200 days. The table lists the subcutaneous sarcomas occurring at the site of injection and primary lung tumors of epithelial origin.

(1) Four cases with clinical and pathologic evidence of sarcoid gave skin reaction following the intradermal injection of an antigen made from a sarcoid lesion of the skin. Four normal individuals gave no such reaction. (2) These results suggest that sarcoid is a virus disease and that it is possible to prepare a diagnostic antigen.

The anterior pituitaries of normal adult male rats, when implanted directly over the center of the crop sacs of squabs, evoke a greater response in the crop sacs, hence store more mammotropin, than do the glandules from thyroparathyroidectomized (maternalized) males of the same age.

Acknowledgment is hereby made to Dr. Herbert McLean Evans for his helpful criticism.

Hinrichs and Johnson 1 observed that “short exposures in the region of the sino-auricular node of both frog and turtle produced a noticeable increase in rate.” Also, these investigators found that “it has been possible to stimulate the heart of a frog to regular beat, normal in sequence and in amplitude, by radiation at the sino-auricular node after the heart has been quiescent for an hour or more.” They did not report any amplitude changes after radiation of the normal heart.

The effect of ultraviolet radiation on the exposed heart of the pithed frog, Rana pipiens, and decapitated alligator, A. mississippiensis, immersed in Ringer's solution which was kept at a fairly constant temperature of 12-14°C., was observed during irradiation by a Cooper-Hewitt Quartz Mercury Arc or a General Electric S. 1 lamp at 50 cm. from the preparation. The former was the source employed unless otherwise stated.

Several series of experiments were performed and the following facts were noted:

1 Freshly prepared normal hearts were found to exhibit a slight increase in frequency and amplitude upon irradiation for short intervals (5-10 minutes) after which both indices rapidly fell below the normal and exhibited a delayed and slow recovery. A normal untreated preparation, under the conditions employed during the course of this investigation, exhibits about a 10% fall in amplitude after 3 to 4 hours while the change in frequency may be regarded as negligible.

In order to have adequate controls for further experimental studies upon cardiac hypertrophy we have considered it necessary to accumulate data from a large series of normal rabbits and to treat this data by statistical methods. Many previous investigators have published average heart weight body weight ratios for small series of rabbits, but there are no figures for the ratio of the left to the right ventricular weights. Hasenfeld and Romberg 1 in a control series of 32 normal rabbits found an average of 2.38 gm. of heart muscle per kilo of body weight. Joseph 2 in a series of 38 male and 66 female rabbits found an average ratio of 2.67 in the males with a maximum of 3.42 and a minimum of 2.07, while for the females the average ratio was 2.70 with a maximum of 4.47 and a minimum of 2.0 gm. of heart per kilo body weight. Wassermeyer and Rohrbach 3 quote Kuelb's average figure on a small series of normal domestic rabbits at 2.70 and Hesse's average figure of 2.70 on a similar series and their own figures on 10 rabbits with a maximum of 2.73 and a minimum of 2.03 and an average of 2.38 gm. of heart per kilo of body weight. Sekiguchi 4 used as control his average of 2.04 gm. of heart per kilo of body weight obtained from the study of 10 normal rabbits.

In a series of 168 normal rabbits of Chinchilla, New Zealand, Red, White and Dutch Black strains and mixtures of these, with body weights varying from 900 to 3,000 gm. we have found the arithmetical mean of the HW/BW to be 1.972 gm. heart per kilo body weight, with a standard deviation of 0.299 and a probable error of ±0.015 for the mean and ±0.011 for the standard deviation.

During the progress of studies on the results of digitization on experimental aortic insufficiency hypertrophy it was observed that the gross configuration of the hypertrophied heart appeared to differ. Some were globular in form whereas others were elongated or boot shaped. The hearts were dissected by removal of the auricles and division of the ventricular mass by the midseptal method. It was found that the globular heart was associated with ablation of the posterior leaflet and the right ventricular wall appeared to be considerably thickened along with the increased mass of the left heart. The coeur en sabot type, on the other hand, was found occurring usually in those that had destruction of the left anterior cusp. In the latter group the thickness of the right ventricular wall did not appear to be conspicuously increased in thickness, while the left ventricular wall was strikingly so. In a few hearts with combined lesions the configuration depended upon which leaflet damage was the more extensive. The obvious visual impression was substantiated by the weights of the ventricular masses and the ratios of these to one another as shown in Table I.

The hypertrophy of the auricles was observed to be greater in the hearts with posterior incompetency and the auricular body weight and the auricular heart weight ratios seem to confirm this. The heart weight body weight ratios show that the amount of total hypertrophy is almost uniformly greater in the hearts with posterior lesions. Difference in the intracardiac dynamics of the circulation with the two types of aortic regurgitation is the probable factor. A relative mitral stenosis (Austin Flint phenomenon) is a possible explanation of concomitant auricular and right ventricular hypertrophy in the presence of the regurgitation through the posterior sector.

Following the lead of E. P. Lyon 1 and others, in some previous work I have shown that it is possible by means of a centrifugal force of about 3,000 times gravity to cause separation of heterogeneously dispersed materials inside living cells into distinct layers, depending on their density, without killing the cells, even though they were kept in the centrifuge for a week. In later centrifuge experiments 2 , 3 I separated crystals appearing homogeneous in the polarizing microscope and isolated them for chemical analysis. When these crystals were treated with alcohol they were decomposed into a phospholipin and a vitellin. Cavett, Rice and McClendon 4 reported that thyroglobulin treated with acetone and ether (as a substitute for alcohol, which denatured it) lost weight (increased in iodine percentage), which I interpreted as due to splitting off of some lipin. To test this hypothesis some method of getting thyroglobulin from thyroid without contamination with cell proteins was needed.

Thyroglobulin, the protein of the thyroid gland, is present in the sol or gel known as “colloid”, which is contained in the thyroid follicles. The follicles containing “colloid” are of a spheroidal shape formed by a single layer of flattened or cuboidal cells. Since spaces between these cells have been reported by cytologists it occurred to me that by the use of a sufficiently high centrifugal field, the “colloid” might be removed from the follicle.

The interest in the extent and variability of the cortex of the human brain has led to a number of estimates of the surface area of the cerebrum (generally in the adult) by a variety of ingenious methods. 1 - 7 We have attempted to extend these studies by an experimental investigation of this area through a portion of the developmental period (from the fourth fetal or lunar month of prenatal life to 2 postnatal years) and in maturity. Our technique is described in detail in a forthcoming paper. 8 Briefly stated, the method consists of sectioning formalin fixed brains enclosed in a matrix with a mechanical device into slices 2 to 3.5 mm. in thickness. The area is then determined by measuring the outline of each section with a chartometer and multiplying the reading by the thickness of the section. The surface of the cerebrum is approximated by the sum of the values so obtained. Attempts to improve this technique by computing the sections as segments of cones and taking the means of their anterior and posterior outlines did not increase the accuracy of our determinations. Twenty cerebri were so studied. Figure 1 shows 10 of these drawn to scale (left lateral views) to illustrate the changes in size and form and in the configuration of the sulci in the series.

In measuring these structures figures were obtained for both “total” and “free” surface. “Total” surface indicates the entire cerebral surface including that portion buried in all of the cerebral fissures regardless of their depth. “Free” surface is a term used for the visible or external surface of the cerebrum only. In determining this value the chartometer was passed around the periphery of each section but only dipped into the lips of the sulci to the shallow point where their sides meet.

The estimation of the area of the human cerebrum has become a matter of considerable interest since the mass of the cerebral cortex is closely related to the surface area of the brain.

This subject has been investigated by a quantitative study of 20 brains ranging in age from the fourth (lunar) month of prenatal life to the close of the fifth decade and in volume from about 5 cc. to over 1000 cc. The method of measuring surface area is described in other papers 1 , 2 and the volume was determined by the displacement method. Various indices of the relation of cerebral volume to surface are shown in Table I and in Fig. 1. In both the table and the figure the observations are arranged in order of cerebral volume.

Column (b) of the table and panel (A) of Fig. 1 show the index of “total” surface area divided by cerebral volume (surface in sq. cm., volume in cc.). The index drops slowly at first, until the cerebrum has a volume of nearly 100 cc. (in the eighth lunar month), and shows practically no regular trend of change thereafter.

A better index is the “total” surface of the cerebrum divided by the two-thirds power of the cerebral volume, for this measure considers the factor of dimensionality. The index thus determined [Table I, column (d), Fig. 1, panel (B)] shows no prominent change until the cerebrum attains a volume of nearly 100 cc. (in the eighth fetal or lunar month) and rises abruptly to a new plateau at a period (just before birth) when there is relatively little increase in volume, and thereafter shows little significant change. There could hardly be a better demonstration of the great relative increase in cerebral surface area by fissuration in later fetal life.

The antiscorbutic properties of various substances related structurally to ascorbic acid have been investigated and reported. We have investigated the antiscorbutic properties of methyl-2-keto-gluconate.

Sixteen guinea pigs, between 250 and 300 gm. in weight, were placed on a modified Dutcher scorbutic diet 1 consisting of rolled oats, white wheat flour, and alfalfa flour. They were fed 3 cc. of orange juice daily for 7 days before the start of the test experiment. During this time they showed a constant daily gain in weight. They were divided into 6 groups.

Group 1. Three animals. All showed loss of weight in 10-15 days, followed by the appearance of scorbutic symptoms. One was fed 3 cc. of orange juice at the 23rd day, but died of scurvy at the 26th day. The other 2 were fed 50 mg. and 70 mg. daily of methyl-2-keto-gluconate starting at the 23rd day, after which they quickly gained weight, lost their scorbutic symptoms, and grew steadily. At the 41st day the supplement was removed, and they gradually lost weight, developed scurvy, and died at the 53rd and 55th day, respectively.

Group 2. Two animals. Began to lose weight at the 10th day, and had developed gross scorbutic symptoms by the 15th and 16th days. On the 20th day one animal was started on 100 mg. of supplement daily, blut died on the 23rd day. The other was given 70 mg. of supplement daily starting at the 20th day. At the 27th day this was increased to 100 mg., and the animal began to gain weight. At the 36th day, when the animal was in good condition, the dose was cut down to 50 mg. daily, and finally discontinued at the 41st day. Scorbutic symptoms developed on the 44th day, and the animal died on the 52nd day.

All fats do not bear the same relation to thyroid extract action. Some fats as cod liver oil, olive oil, lard, antagonize to different degrees the action of thyroid extract; others, as cocoanut oil, crisco, do not.

In amphibia, a supernumerary muscle during spontaneous and reflex activity contracts simultaneously with the homologous normal muscle of the same name. 1 Since this is true for each individual muscle, a supernumerary limb (or limb fragment) always exhibits the same movements as the nearby normal limb (or corresponding limb fragment) of the same side. 2 From this phenomenon has been derived the concept of a “resonance-like” principle of communication in the nervous system. 3 The experimental evidence has been confined, thus far, to the amphibia, where supernumerary limbs can be produced at will. In order to test the validity of the principle of homologous (“resonance-like”) response in higher vertebrates and man, where the transplantation of supernumerary limbs has not yet been possible, we must avail ourselves of those cases in which congenital duplications of limbs or of some of their parts present us with a comparable situation. A human case favorable to such study has recently been described by Halverson and Amatruda, 4 and was re-examined by the author as to its bearing on the problem of homologous function.

The case concerns a girl, L. S., 14 years of age, with congenital duplication of the left forearm and hand. While the radial part of the hand, including the thumb, is absent, its place is taken by a mirror image of the normal ulnar part. The resulting double formation is a symmetrical hand with what appears to be the original index as axis of symmetry and 2 groups of 3 fingers each to either side of this index. The order of the supernumerary fingers being reversed, the 7 fingers of the hand can be identified, beginning with the most lateral one, as (n stands for normal, e for extra fingers) : n5, n4, n3, n2, e3, e4, e5.

For the past two years we have been interested in the study of the blood proteins in known or suspected cases of liver injury. That there is an alteration in the blood proteins in liver injury has been known since Glenet 1 showed a reduction of the total protein and numerous studies established that there was an elevation of the serum globulin (Felinski 2 ) and a reversal of the albumin globulin ratio in cirrhosis of the liver with ascites (Abrami and Wallach 3 ). Wiener and Wiener 4 obtained a reversal of the A/G ratio in cirrhosis and a hyperglobulinemia in acute infections. Meyers and Keefer 5 and Snell, 6 in cases of decompensated cirrhosis could not attribute the depletion of the serum albumin to its loss in the ascitic fluid.

Serum albumin, serum globulin and total serum protein determinations were made according to the hypobromite gasometric method of Van Slyke in cases of decompensated cirrhosis, cases with choluric jaundice, both intrahepatic and extrahepatic, without ascites, cases of ascites without clinical cirrhosis, a miscellaneous contrast group as well as a control group of normals. These normals served as a check on our technique and established values comparable to the accepted normal values of Peters and Van Slyke.

In 21 cases of decompensated cirrhosis there was a constant hypoalbuminemia and a hyperglobulinemia with a consequent reversal of the A-G ratio, with an average ratio of 0.55 instead of the normal average of 2.48. In no case was paracentesis performed prior to the serum protein determinations. Of particular interest as evidence of the prognostic significance was the duration of life after the serum proteins had undergone these changes. In one instance, for example, in which the serum albumin was 2.3 g. the globulin 5.46, the A-G ratio 0.4, the duration of life was 5 days. The maximum duration of life with pronounced reversal was 70 days.

The statement has been made that hyperparathyroidism is frequently found as the etiological factor in renal calculi. 1 It becomes of interest to examine this hypothesis by all possible methods at our disposal. Since one may find normal total calcium figures in the presence of hyperparathyroidism the simple determination of the total calcium and phosphorus is not entirely reliable.

McLean and Hastings 2 have recently proposed a simple method for the determination of the ionized calcium in the serum. This they believe is the most sensitive test available for detecting the presence of hyperparathyroidism.

Twenty-four patients with proven calcium stones were referred to the Metabolic Clinic by the Second Surgical Service (Dr. Edwin Beer). The Collip modification of the Tisdall method was used for the determination of the total serum calcium, and the Fiske-Subbarow method for the inorganic phosphorus. The serum proteins, after removal of non-protein nitrogenous substances, were determined by a modification of the Pregl micro-Kjeldahl procedure. The results are shown in Table I with the addition of data from control cases. These controls are used in addition to the normal figures published by McLean and Hastings.

There is no evidence of increased calcium ion concentration in any of the stone cases.

The preparation of extracts from pregnant mare serum presents a problem different from that found in the extraction of pituitary gland material. The main difficulty is found in the solubility of serum proteins which form colloidal solutions and cannot easily be separated from the gonad stimulating factor itself. Such solutions appear opalescent or turbid and pass through a bacteria-proof filter only at a very slow rate. The method as described by Evans, Gustus and Simpson 1 overcomes this difficulty by adsorbing the active principle on aluminum hydroxide. This method yields a highly purified product, but requires special equipment not available in every laboratory.

Following is a rather simple method which gives a product suitable for biological and clinical use.

Serum from pregnant mares was precipitated in the usual way with 2 volumes of acetone, washed with acetone and dried. This precipitate formed the raw material used in the preparation of the extract. The powder was treated with a 6% solution of butyl alcohol in water, as described for pituitary gland extraction. 2 It is doubtful whether the addition of butanol, advantageous in the extraction of the pituitary gland, is of any particular value in this case, since the serum quickly swells up in water and does not present any problem as far as extraction is concerned. However, the slightly antiseptic properties of the butyl alcohol, which prevent putrefaction during the process of extraction at room temperature, made it desirable to apply this method to serum also.

Some time ago in a study in this laboratory 1 dealing with the influence of ether anesthesia on the acid-base balance in dogs it was noted that when silver nitrate was added to urine collected following the anesthesia for the chloride estimation, the silver was reduced and a black precipitate formed before the acid indicator was added. This observation led us to investigate the influence of the ether anesthesia on the excretion of ascorbic acid as determined by titration with 2:6-dichlorophenolindophenol.

Female dogs were employed. The urine was collected by catheterization into a bottle containing a few drops of glacial acetic acid, after which sufficient 20% trichloracetic acid was added to give a 5% trichloracetic acid concentration. This was then titrated into a known amount of 2:6-dichlorophenolindophenol. The dye was standardized with crystalline ascorbic acid (Merck) according to the procedure of Birch, Harris and Ray. 2 It was found, however, that when urine was allowed to stand even for short periods the reducing value decreased appreciably. Consequently it was thought necessary to perform a preliminary step of bubbling hydrogen sulfide through the urine for ten minutes and then allowing it to stand over night under an atmosphere of hydrogen sulfide, after which the H₂S was driven off by bubbling nitrogen through the urine for 15 minutes (or until no nitroprusside test was obtained). The trichloracetic acid was then added and the titration carried out immediately.

Table I presents the results of one of a number of experiments which have been performed.

From these results it is observed that the urinary excretion of ascorbic acid as determined by titration with 2:6-dichlorophenolindophenol is markedly increased in the dog following ether anesthesia.

While investigating the peripheral action of barbiturates it was observed that in all experimental animals where the cardiac vagus response to weak faradic stimulation had been abolished by large doses of most barbiturates, and by moderate doses of amytal and pernoston, the administration of physostigmine salicylate in doses from 0.2 to 0.35 mg. per kilo by vein, caused usually no detectable spontaneous effect upon the heart rate or blood pressure. The injection of comparable doses of acetylcholine and pilocarpine produced in the same animals a marked fall in blood pressure which in many cases was accompanied by a slowing of the heart. 1 If 2 or 3 minutes were allowed to elapse after the intravenous administration of physostigmine and then the peripheral vagus was stimulated electrically, profound cardiac slowing and fall in blood pressure was produced in 6 dogs, 4 cats and 6 rabbits. In 7 experiments these vagus effects outlasted for several minutes the actual stimulation of the nerve. This physostigmine sensitization of the vagus to stimulation of the preganglionic fibers lasted for about a half hour and was antagonized in ten animals by further doses of barbiturates. Since barbiturates abolished the cardiac vagus response to faradic stimulation but not to pilocarpine, it was postulated that their vagus-impairing effects are due to ganglionic depression (see also Kobacker and Rigler, 2 and Garry. 3 ) We have, therefore, employed nicotine and curare, drugs known to produce ganglionic paralysis. After sufficient doses of nicotine (2 to 10 mg. per kilo) or curare (3 to 6 mg. per kilo) had been given intravenously to abolish the peripheral vagus effects in 2 dogs, 3 rabbits and 3 cats, 0.2 to 0.3 mg. of physostigmine salicylate per kilo was injected intravenously.

1. A method for the extraction and assay of male and female hormones from normal urines has been described. 2. This procedure consists in 2-hour acid hydrolysis of the urine, extraction with ten times the volume of benzene using the extractor described, and separation into male and female fractions by alkali. 3. The assay is conducted with standards in parallel and results expressed in international units. 4. An alternative procedure for male hormone alone using an adsorption process is likewise described.

It was shown 1 that preparation of the specific polysaccharides of Types II and III pneumococcus in the cold and without the use of strong acid or alkali gave products forming much more viscous solutions than did the polysaccharides isolated by the older methods, 2 but apparently differing otherwise from the older preparations only in their ability to precipitate more antibody from homologous rabbit antisera. These differences were attributed to the greater chain length of the molecules of the new preparations.

The acetyl polysaccharide from Type I pneumococcus has now been similarly prepared. This product had the chemical and physical properties of the acetyl polysaccharide reported by Avery and Goebel 3 but gave solutions of much higher viscosity than did samples prepared according to Reference 3, and precipitated twice as much antibody nitrogen from a Type I antipneumococcus rabbit serum. Thus a solution of the new preparation, S 120, containing 1 mg. per ml. in 0.9% saline had a relative viscosity, η_r, of 1.69. After 8 hours in a sealed tube at 100° this solution showed η_r = 1.05. After treating with N/2 NaOH for 40 hours at 37° C. η_r was 1.20. A preparation, S91a, isolated from culture filtrate concentrated on the steam bath gave η_r = 1.10.

Table I shows the amount of antibody nitrogen precipitated from rabbit and horse Type I antipneumococcus sera by an excess of the different preparations of Type I pneumococcus specific polysaccharide (SI).

As was found with S II and S III the use of heat as in the initial concentration of S I lowers its power to precipitate rabbit antisera without greatly affecting its reaction with horse antisera.

Prolactin alone, desiccated thyroid alone, or thyreotropic hormone probably unaffected by any contaminating constituent of the preparations used by us, promotes growth in the dwarf mouse. In these mice prolactin and thyreotropic show a synergistic action (32 to 242×) upon body growth. Though the concept of a growth hormone as an individual entity has been useful it does not seem to be true.

Our interest in the above correlation takes origin as follows: For the past 8 or 9 years one of us (Mellon) has studied with encouraging results a new serum approach to the therapeusis of hemolytic streptococcal infections. Treatment of the cases has been conducted exclusively in this hospital. Accompanying the serum's use we have frequently observed an in vivo dissociation of smooth (or smoothmucoid) cultures into more saprophytic colony types—sometimes characterized by rough-colonied hemolytic streptococci, sometimes by typical non-hemolytic diphtheroids, wholly avirulent.

Of special significance is the fact that such dissociation seems to be a direct sequence of a clinical crisis on the part of the severe cases, whose ultimate recovery is thereafter no longer in doubt. Obviously, a serum approach that would transform virulent organisms into their non-virulent phases, is rather different from the conventional bactericidal or antitoxic ones.

Only an occasional observer (Hadfield, et al. 1 ) in the field of fibrinolysis has directed attention to a correlation of virulence (as indicated by colony type) and fibrinolysis. Recently Tunnicliff 2 has shown correlation in a general way between R and S streptococci with respect to the increasing lysis and virulence as one proceeds from R to S. Reich 3 has shown that serial animal passage transformed a fibrinolytic strain of human origin into a non-fibrinolytic one suggesting a bovine origin; (Lancefield classification 4 ), and which on serial subculture returned to its original fibrinolytic state. Accordingly, we have wondered if this correlation might extend to certain variants such as the above; more particularly to diphtheroids whose in vivo dissociation from the streptococci had been confirmed by their occurrence in vitro, under controlled conditions.

Burns and Visscher 1 have shown that an isotonic solution of a mixture of sodium chloride and some other sodium salt with an indiffusible anion when placed in a loop of the small intestine becomes practically chloride-free in 1½ hours. This movement of sodium chloride, against a diffusion gradient into the blood through the intervention of the intestinal epithelium, resembles the removal of chloride by the kidney tubules from the glomerular filtrate. The intestinal preparation affords a convenient method of studying salt movement which may be of general importance.

As a step in elucidating this mechanism it was decided that the specificity of mechanism should be determined. Sodium bromide was chosen as an example of a salt similar chemically to sodium chloride but not a usual constituent of body fluid. It is the purpose of these experiments to see if, in the presence of an indiffusible anion, sodium bromide will diffuse from a certain concentration in the intestinal fluid to the blood where a higher concentration exists.

Dogs were fed 10 gm. of sodium bromide per day for 3 days. At the end of this time about two-thirds of total plasma halides was bromide. Sodium amytal was used as an anesthetic. A loop of the lower portion of the ileum, sufficient in size to hold about 50 cc. of fluid, was tied off, cannulated and flushed with warm saline. The test solution, containing 40 mM/1 sodium chloride, 40 mM/1 sodium bromide and balance to isotonicity of sodium sulphate, was placed in the loop and allowed to remain 1½ hours. Samples were withdrawn at intervals for analysis of chloride and bromide in plasma and intestinal fluid by the electrometric technique of Hastings and Van Dyke. 2

The mammary gland of the human male, though generally dismissed as vestigial, deserves more attention than it has received. Like its counterpart in the female, it is subject to both inflammatory and neoplastic disease. It occasionally increases to such a size that the resulting gynecomastia becomes a medical or surgical problem, and actual lactation has been reported repeatedly. 1 Although the phrase “mastitis adolescentium” occurs in the literature, 2 we have not found any data on the frequency of this condition nor on the behavior of the gland in the normal pubescent boy. Unpublished observations on 49 boys in southern Wisconsin convinced Jung that a certain degree of mammary hypertrophy must be a regular feature of normal pubescence. The present study of a larger series was undertaken partly for the purpose of testing the correctness of this conviction, and was made possible by the generous cooperation of the Department of Public Welfare of the State of Illinois.

One purpose, then, was to obtain data analysis of which would give a chronology of the mammary changes as compared with the other phenomena of puberty. Hence the following observations were made: date of birth; weight; height; bi-iliac diameter; color of hair; amount∗ of hair on the pubes, in the axillae, on the face (i. e., cheeks, upper lip, and chin), and on the chest; diameter of right areola; height of right nipple above surrounding skin; size∗ of the subareolar node on the right; height of left nipple above surrounding skin; size∗ of the subareolar node on the left; pitch of voice; sizes of right and left testicle. The quantities marked ∗ had to be, of necessity, graded in arbitrary units from 0 to ++++. The bi-iliac diameter was taken with an obstetrical caliper.

In the intact animal, ergot reversal and cocaine synergy of a blood pressure rise indicate that the impulses eliciting the rise reached the blood vessels by way of the sympathetic nerves; they do not indicate the site of origin of the impulses.

A review of the published reports of the clinical application of the thyreotropic pituitary hormone is given. Our experience with 24 patients is outlined. In normal patients there is great individual variation in reactiveness to the material, as indicated by the basal metabolic rate. This is also true in castrate women. Some patients with low metabolic rates respond, whereas others do not. Patients with goiters respond with similar individual variation. In one, a diminution of the goiter was observed. Acute temporary exacerbation of hyperthyroidism has been produced in 2 patients; this has been prevented by iodine in others; in one patient with hyperthyroidism refractoriness to the medication seemed present before treatment, and treatment was followed by a fall in metabolic rate.

A study of the literature on the effect of castration on the thyroid gland has shown many contradictory results. Earlier work seems to indicate a reduced thyroid activity in castrate animals. A decreased metabolic rate after castration has been reported by Loewy and Richter 1 and by Savage, Sherwood and Hall. 2 Histological studies by Korenchevsky, 3 Constantini 4 and Chouke 5 show a decreased activity of the gland and a decrease in the number of mitotic figures. A decrease in the weight of the thyroid and an atrophy of the gland have been reported by Anderson and Kennedy 6 and Bokelman. 7 Others have reported no marked change in the structure or weight of the gland, namely Leonard, Myer and Hisaw 8 and Freudenberger and Billeter. 9 More recent work by Bialet-Laprida 10 and Korenchevsky and Dennison 11 report a slight hypertrophy of the thyroid. Bates, Riddle and Lahr 12 in a quantitative assay of the occurrence of thyreotropic hormone in 7 types of cattle classified as to age, sex and stage of reproductive cycle, found a decreased amount of hormone in the pituitary gland of steers (adult, male castrates).

Contradictory results have been reported independently by Loeser and Aron. Loeser, 13 who has studied the thyreotropic hormone extensively, claims that 18 days after castration the thyroid gland of female guinea pigs is markedly stimulated. Other work by Loeser 14 indicates that the pituitary gland of a castrated animal when implanted into a normal animal will cause an increased activity of the thyroid gland, greater than the stimulation resulting from the implantation of the pituitary of a normal non-castrated animal. Aron 15 claims that castration increases the titrable amount of thyreotropic hormone in the blood.

I. The histology of the thyroid gland of female guinea pigs, 18 to 23 days after castration was determined by a study on 37 animals in 4 groups, with weights varying from 120 to 1030 gm.

During the course of recent experimental studies on the pathology and treatment of burns directed toward reducing the incidence of infection associated with the Davidson tannic acid treatment, the addition of various antiseptics was considered. Aldrich 1 had pointed out the low antiseptic value of tannic acid alone and noted the frequency of infection beneath the heavy eschar. In view of his findings, Aldrich substituted crystal violet for tannic acid in the treatment of burns. Experimentally, burns treated with crystal violet, either alone or in combination with tannic acid, allowed the development of B. pyocyaneus and the colon group. Obviously, the proper antiseptic must be compatible with tannic acid, must have high bactericidal value against both cocci and bacilli, and low toxicity for tissue cells both locally and generally.

The carbocyclic group of compounds seemed to give the most promise of satisfying these criteria, and amyl tricresol (Upjohn and Company) was the first one tried. This material used in 1-1000 concentration with 5% or 10% tannic acid reduced the incidence of infection in experimental burns satisfactorily and at the same time did not interfere with the healing of the wounds.

Bactericidal tests (Food and Drug Administration Method) showed amyl tricresol effective in dilutions of 1-2000 against E. typhi and in dilutions of 1-4000 against S. aureus in 10 minutes. In combination with 5% tannic acid it became effective in dilution of 1-12,000 against E. typhi and 1-8000 against S. aureus in 10 minutes. Thus the effectiveness of amyl tricresol was increased 6 times against E. typhi and 2 times against S. aureus through combination with tannic acid.

Hydrogen ions in suitable concentration, when applied to a localized region of the Fucus egg in sea water, induce formation of the rhizoid protuberance. The polarity and the developmental pattern of the whole embryo are determined.

Austin, Stillman and Van Slyke 1 on the basis of data obtained on a series of normal men, showed that when the urine flow was progressively increased from very low values the urea clearance also increased until the rate of urine excretion reached a value they termed the augmentation limit. At rates of urine excretion above the augmentation limit there was found to be no further increase in urea clearance. Subsequently this description has been accepted for dogs by Jolliffe and Smith 2 and Summerville, Hanzel and Goldblatt, 3 and it has been tacitly assumed by Van Slyke, Rhoads, Hiller and Alving 4 in their study of factors controlling urea excretion. A maximum urea clearance has also been posited by Dominguez 5 in his mathematical treatment of the relation existing between urea clearance and urine flow at rates of urine formation below the augmentation limit. The value fixed for the augmentation limit in the dog has been variously stated to be from 0.2 to 0.4 cc. per minute, depending in part upon the size of the animal, and also upon the level of the maximum clearance, which can be varied by changing protein intake.

Recent evidence has made it possible to accept the creatinine clearance as a measure of the rate of glomerular filtration in the dog. (Shannon, 6 Van Slyke, Hiller and Miller 7 ), even at very high U/P ratios (Shannon 8 ). The present observations were made to examine the relations between the rate of glomerular filtration and the rate of excretion of water, and the effect of these variables upon the urea clearance.

There is no standard of reference by which the clearances of various individual dogs can be converted to a common value. For this reason it was apparent beforehand that the data from each dog should be treated separately.

It is known that the oxygen consumption of fertilized sea urchin eggs (Arbacia punctulata) can be markedly stimulated by the addition of suitable concentrations of nitro and dinitrophenols. 1 , 2 Stimulation of oxygen consumption in these cells can also be effected by certain oxidation-reduction indicators. 5 , 6 In concentrations slightly greater than the optimum for respiration, certain nitrophenols block the cell division of fertilized Arbacia eggs. This division block is fully reversible since the eggs, when returned to sea water after a 3-hour exposure to concentrations many times the optimum for respiration, resume division and develop to swimming larvae. 1 In concentrations which are optimum for respiration, a number of oxidation-reduction indicators also inhibit cell division in fertilized Arbacia eggs, but the eggs so treated do not recover when they are returned to sea water. 5 , 6

Since the chemical and biological properties of the nitro compounds differ in almost every respect from those of the oxidation-reduction indicators, 5 it has been tentatively concluded that the nitro and dinitrophenols do not stimulate respiration or block cell division as a result of any possible oxidation or reduction which they may undergo in the cell. For the production of the respiratory-stimulating and division blocking effects, the unesterified phenolic OH group, accompanied by suitable substituents in the benzene ring, appears essential. 3

The experiments presented here support this view since they show that, in fertilized eggs of Arbacia punctulata, oxygen consumption is stimulated and cell division is reversibly blocked by phenols containing no nitrogen and no substituent group capable of oxidation-reduction. These phenols contain, in addition to the OH group and the benzene ring, only chlorine, bromine, or iodine atoms. Phenols of this type are oxidized irreversibly at high positive potentials. 4

Breathing of oxygen tensions of 5.0% to 12.0% caused a marked diminution in the urine secretion in 45 of 50 experiments upon 12 barbitalized dogs.

Electrocardiograms were taken in a series of experiments on 40 cats, in which nembutal was the anesthetic employed. The procedures were as follows:

1 Stimulation of the right and left peripheral vagus only; (20 cats.)

2 Jugular injection of acetylcholine (2 to 6 mg./kg.). (20 cats.)

Typical electrocardiograms illustrate the following results:

1 Stimulation of the right vagus brings about a decrease in the rate of both P and QRS complexes, i. e., a sinus bradycardia.

2 Stimulation of the left vagus brings about the dropping out of some QRS complexes, i. e., a partial block.

3 Acetylcholine brings about (a) a decrease or temporary cessation. of QRS Complexes; (b) a heavier dosage (sublethal, about 6 mg./kg.) may result in temporary elimination of P-waves also.

Dale 1 has stated that “vagus impulses produce their effect by liberating acetylcholine among the fibers of the muscular wall of the heart.” From these results it would seem that the action of acetylcholine on the heart more closely approximates that of the left, than of the right, vagus. The question is, therefore, raised as to whether some vagus fibers to the heart may not be more “cholinergic” than others, and a further differentiation of their activity on such a basis ultimately attempted.