Experimental Biology and Medicine

Following oral administration of either saccharin or galactose, no evidence was obtained of a reflex hyprglycemia.

A simplified purification procedure, based on acid precipitation, is described for the preparation of highly potent botulinus toxin.

Previous experiments (external application of sap, perfusion of vacuole with sea water 1 , 2 ) have indicated that the large bio-electric potential (P.D. = 70 to 80 mv. outside +) across the protoplasm of impaled Halicystis cells (coenocytic marine alga) is mostly independent of inorganic ions or other gradients between cell sap and environment. Internal gradients or assymetries in the protoplasmic film itself were suggested. Since the P.D. can produce a measurable current for long periods through a completed circuit, a source of energy, eventually metabolic, must be available. Experiments have therefore been directed toward analyzing the relation between P.D. and metabolism in this organism. The electrical methods are those previously employed, 1 , 2 the cells being kept in the dark to avoid photosynthesis. Manometric measurements of O₂ consumption by the Warburg method were made on groups of several intact cells under comparable conditions (except for impalement). Concurrent P.D. and respiration measurements on identical impaled cells are now under way.

Oxygen tension. Over the range where respiration is unchanged, the P.D. is also unaltered. This holds for pure O₂, air, and for mixtures of O₂ with N₂ down to about 2% O₂, brought into equilibrium with sea water by continued bubbling. If bubbling of the 2% O₂ mixture is stopped, the P.D. drifts downward as O₂ is consumed in the vicinity of the cell, eventually falling to 10 or 15 mv. +. Stirring of the sea water, or rebubbling of 2% O₂ promptly restores the P.D. This downward drift and recovery may be repeated almost indefinitely. Further lowering of O₂ tension, e. g., to 0.2% O₂ in N₂, reduces the P.D. to 10 mv. or less while bubbling or stirring continues. Respiration also falls to low values in this mixture.

In earlier notes 1 we reported observations on the prophylactic action exercised by picric acid and certain other compounds. In this note we are reporting results which indicate that zinc sulphate applied to the nasal mucosa is even more effective than picric acid. These observations are set forth in Table I, in which the relative efficiency of the respective agents is compared in terms of the approximate per cent of animals which were protected for at least one month against repeated intranasal instillations of poliomyelitis virus.

While the number of animals in individual experiments and the mode of treatment and the intensity of the subsequent virus inoculations are not all strictly comparable, certain experiments are sufficiently alike to reveal important differences in the degree of protection, For example, in Experiment 4, in which 8 monkeys were treated with 0.5% picric, and in Experiment 7, in which 9 monkeys were treated with 0.5% zinc sulphate, but otherwise were dealt with in the same way, there is a difference amounting to 88% in favor of ZnSO₄. Furthermore, the addition of zinc sulphate to picric acid (Experiment 6) enhanced its protective value somewhat more than did the addition of alum (Experiment 5). Of significance also is the fact that zinc sulphate when used alone has afforded over 95% protection in a total of 53 animals (Experiments 7 to 13). Thus far, only 2 animals treated with zinc sulphate alone have succumbed to poliomyelitis. Six animals have survived repeated virus instillations for 3 months.

It will be noted that the protection afforded by picric acid is distinctly greater in Experiment I than in Experiments 2, 3, and 4. This is accounted for by the fact that in the latter experiments, virus was instilled intranasally almost daily during the entire month; an exposure which brought down 100% of the controls.

Although most of the attempts to apply the complement fixation test in the field of filterable viruses have either been negative or inconclusive, within recent years a few favorable reports have lent more encouragement. The work of Craigie 1 and his coworkers has been of especial value in suggesting a new method of attack. Based on their reports, work was undertaken to extend the test for the differentiation of strains of equine encephalomyelitic virus, representing the so-called eastern and western American types and that of Moscow No. 2. All 3 of the strains may be differentiated serologically by the neutralization test. 2 The virus of lymphocytic choriomeningitis isolated by Dr. C. Armstrong of the National Institute of Health, was also included in the work.

With some slight modifications the antigens were prepared according to the method of Craigie and Tulloch 1 for vaccinia. The equine viruses were carried in the guinea pig and that of the “l.c.m.'virus in the mouse. The brains were removed aseptically from the respective infected animals, were frozen in an ice-salt mixture and dried in a desiccator evacuated by the Cenco-Hyvac pump. The dried material was weighed and ground with ether in the desired proportions. The mixture was shaken, stored on ice for several hours. Then the ether was removed; the residue was ground with 0.85% saline, left for 6–12 hours on ice, frozen several times in an ice-salt mixture and thawed at 37°C. Finally the material was centrifugated and the supernatant fluid removed, which was then ready for use after a preliminary titration for anticomplementary, hemolytic and antigenic properties.

After a careful titration of the complement with the antigen, the test was set up with 0.1 cc. of serum from guinea pigs hyperimmunized to each strain, 0.2 cc. of antigen and 0.2 cc. of complement containing 2 full hemolytic units were then added and the mixture left for 16 hours at refrigerator temperature.

It is generally conceded that one important function of the growth hormone of the anterior hypophysis is the promotion of protein anabolism. Evidence for this has been obtained from the results of numerous nitrogen balance studies which have been published in recent years, all of which show a marked retention of nitrogen after single or multiple doses of the hormone. The same conclusion has been drawn from several studies of the nitrogen content of animals, in which it has been found that the tissue nitrogen is significantly higher after growth hormone has been administered. So far as we are aware, however, all of these analyses have been made on the entire carcass, the soft tissues, or on the liver. With the exception of liver, therefore, no information is available on the influence of growth hormone on the nitrogen content of individual organs or tissues. It seemed, then, to be of interest to include analyses of skeletal muscles in the course of a general study of the effect of various nutritional conditions on growth and development in rats.

The rats were of standard, pedigreed stock, and were placed on the special diets when 13 weeks old. Seven groups of 7 to 10 rats each were treated as follows:

Group 1. Control, on normal diet only.∗

Group 2. High phosphorus diet: disodium phosphate was added to the normal diet in sufficient quantity to increase the phosphorus content to 1.25%-1.5%.

Group 3. Normal diet with addition of 22.5 units of vitamin B₁ per day in the form of Tiki-tiki extract† during the first 10 weeks, thereafter 45 units per day.

Group 4. High phosphorus diet with the same vitamin B₁ supplement as in group 3.

Group 5. Normal diet and growth hormone (Antuitrin Growth†) given intraperitoneally.

Since there were no observations reported in the literature on the long continued administration of creatine to experimental animals, it seemed worth while to conduct a series of observations on young and old animals in which creatine was fed in doses comparable to those used in certain clinical therapeutic experiments in man. This seemed especially desirable since unfavorable effects of long-time administration might conceivably occur. The series of experiments was further used in order to determine the influence of moderately high ingestion rates of creatine on the amount of that substance stored in various tissues in the body, particularly in muscle. Rats were, therefore, fed on the following stock diet, half of the animals receiving this diet alone, and the other half receiving the same food mixture with 2 gm. of creatine hydrate added to each kg. of food. Assuming an average food intake of 14 gm. per day per rat of 350 gm. weight, the creatine intake was 75 mg. creatine hydrate per kg. per day.

The rats were from 2 sources. One group consisted of 25 animals∗ age 3 years or over at the beginning of the experiment, which had been obtained from the breeding colony of the Wistar Institute. The other group was of 50 Wistar strain animals 15 months of age at the beginning of the experiment. One-half of the animals in each group were placed on the control and the experimental diets for a period of from 4–6 months, at which time those still living were sacrificed and analyses made. The animals were anesthetized with amytal, 50 mg. per kilo, intraperitoneally. The tissues analyzed were the gastrocnemius muscle, the liver, and heart, which were excised in that order and plunged immediately into liquid air on excision.

Since the time of Strecker 1 chemists who have worked with bile have appreciated the difficulty of separating lipoids from bile. The lipoid constituents as given by the text books at the present time are merely repetitions of the early figures given by Strecker, Hammarsten, 2 von Gorup-Besanez, 3 and others. These constituents are given as fatty acids, soaps, phosphatides, fat, and cholesterol. An examination of this early literature does not yield satisfactory evidence for the presence of either neutral fat or lecithin in bile.

Fresh bile may be extracted in 2 ways. One is to deproteinize by means of alcohol, then dry the protein-free bile and dissolve the residue in absolute alcohol, filtering off the inorganic salts, that are precipitated, and finally pouring the alcoholic solution into a large volume of anhydrous ethyl ether. This is the method used by Hammarsten.

The second method is to extract directly with ethyl and petroleum ether in the presence of some alcohol. In this manner a very persistent emulsion is formed that requires time to break. In either method a sticky molasses-like substance is obtained on evaporating the ether, which does not look like fat and contains bile salts, fatty acid, phosphorus, sulphur and nitrogen containing compounds and non-saponifiable material. After as thorough an extraction as can be carried out in this manner, saponification of the non-ether soluble residue with strong alkali will permit the extraction of much fatty acid that could not be extracted in the first place.

When 2,500 cc. of beef gall bladder bile is dried in air, and then taken up in alcohol and precipitated in ether, the ether takes up 11.45 gm. of material or 0.458%.

Suspecting that the drying of bile and solution in alcohol might hydrolyze the fats, bile was extracted directly using a modification of the Roese-Gottlieb 4 method for fat extraction from milk. This method proved very difficult on account of the persistent emulsions that were formed.

According to Evans, 1 the metabolism of endogenous protein is interfered with in the adrenalectomized, rat. In our present studies the metabolism of an orally administered amino acid, alanine, has been investigated in animals with the adrenal gland removed. Butts 2 has shown that this amino acid is converted to glycogen in the organism, the maximum storage being 8 hours after the oral administration of the acid.

To 7 normal male and a like number of normal female rats 1.6 mg. alanine per square centimeter of body surface, was administered by stomach tube. A like amount was given to the paired brothers and sisters of the above animals which had been bilaterally adrenalectomized 6 days previously and had received salt solution since. All animals were fasted 24 hours before the alanine was fed. Three animals of each sex and of each group were given an amount of water by stomach tube equivalent in volume to the alanine solution fed in the other group.

In the groups of normal and adrenalectomized males, blood sugars were determined on blood obtained from the tails just before alanine administration and at the periods after administration shown in the table. At the end of 8 hours the livers of all animals were removed under amytal anesthesia and glycogen determined in the organ. A summary of the results is given in Table I.

Apparently adrenalectomy does interfere with the conversion of alanine to glycogen. The blood sugar changes are also less marked. Not only this but all of the adrenalectomized animals showed a drop in the sugar level after the peak which was reached one hour after alanine administration, while the normal rat showed a high level throughout the 4-hour period.

Ischemia of the dog's kidney, produced by constriction of the renal arteries, results in a chronic hypertension (Goldblatt, Lynch, Hanzal and Summerville 1 ). Destruction of the nerves to the kidney does not prevent the development of this hypertension (Page, 2 Collins 3 ). Hence it may be supposed that we are dealing with a chemical mechanism. A number of workers (Prinzmetal and Friedman 4 ; Harrison, Blalock and Mason 5 ; Govaerts and Dicker 6 ) have reported that extracts from the kidneys of dogs with such experimental renal hypertension exert a greater pressor effect than similar extracts from normal kidneys. This finding leads one to believe that the hypertension is due to the liberation of pressor substances into the blood stream from the ischemic kidney. The theory would be greatly supported if increased amounts of pressor substance could be demonstrated in the blood. Dicker 7 in fact reports that the blood of such dogs contains a hypertensive substance, while that of normal dogs does not. However, Page 8 could not find increased pressor substances. Alcohol extracts of the plasma of such dogs, when injected into cats, showed no more pressor activity than similar extracts made from normal plasma. We have attempted in this investigation to demonstrate increased pressor substances in the blood by massive transfusions from large dogs with renal hypertension into small normal dogs. The results have been entirely negative.

The hypertension in the donor dogs was produced by constriction of the renal arteries by a method described previously (Collins 3 ). The systolic blood pressures of these animals varied from 180 to 230 mm. Hg. These dogs were large (20–30 kg.), and small dogs (4.5–9 kg.) were chosen as recipients in order that large amounts of blood, in relation to the size of the animal, could be injected.

Blood cells are most readily classified when seen in blood smear preparations or dry imprints (smears) of tissues stained with Romanowsky dyes. In most laboratories, however, only paraffin sections are studied when the hematologist or pathologist is interested in the hemopoietic activity of spleen, liver, lymph nodes, etc.

American investigators have studied mammalian embryonic hemopoiesis from paraffin or celloidin sections only. It was deemed worthwhile to study embryonic hemopoietic organs using the dry imprint method, checking our observations with paraffin sections. The embryonic tissue (yolk-sac, liver, spleen, bone marrow) was touched gently to a chemically clean cover glass and waved vigorously until the imprinted material was dry. May-Grünwald-Giensa staining was used immediately as in the staining of blood smears. Preparations made in this way make possible a comparison between embryonic blood cells and elements seen in blood smears and organ imprints of the adult under normal or various physiologic and pathologic states. It is important to know which cells are found normally only in the embryo, and the elements of the embryonic hemopoietic organs which are identical with normal cells of the adult.

The material used for this study consisted of rat embryos from 9–22 days'gestation, plus various stages of rabbit, human, mouse and pig embryos and foetuses. The following were conclusions drawn:

1. The first circulating blood cells of the rat are not lymphocytes (Maximow, Jolly, Jordan), but are immature red cells of the embryonic megaloblastic series.

2. Two generations of red cells are produced in all mammalian embryos, the megaloblastic, formed primarily in the yolk-sac, and the normoblastic which appears in the liver in great numbers. The normoblasts of the liver are cytologically identical with the nucleated red cells of the fetal spleen and fetal and adult bone marrow.

One of the most specific and sensitive tests for acetylcholine in body fluids is the leech muscle suspended in a saline bath containing eserine (Fuehner, Minz 1 , 2 ). When only small quantities of blood are available they must be diluted with sufficient saline to fill the bath. This procedure naturally decreases the sensitiveness of the test. Moreover, in smaller animals, the repeated drawing of several cc. of blood is often detrimental to the carrying out of a series of tests. The method presented here eliminates these disadvantages by suspending the leech in foam from small quantities of blood.

The leech is attached to a hook at the bottom of a glass bath (2×6 cm.), the other end leading to a writing lever. A narrow opening at the funnel-shaped bottom of the bath is connected with an air tank by means of rubber tubing, so that air is allowed to enter at the desired rate. A wire loop serves as a guide to prevent the blood soaked thread from adhering to the wall of the tube. The leech is prepared in the usual manner and is suspended in eserinized saline until it is relaxed. The saline is then drained completely and the blood which is to be tested is placed in the bottom of the tube. The air passing through the blood creates a foam, which passes over the muscle as it is carried upward. If there is acetylcholine present in the blood, the contraction of the muscle then starts immediately. (See Fig. 1.) In order to obtain uniform results, it is necessary to keep the amount of air entering the bath constant. After the foam has exerted its effect on the leech, the bath is washed with saline, with which it is left filled, until the muscle is relaxed and ready for another test.

The writer isolated 1 a “rough'and “smooth'form of a chromogenic acid-fast bacillus which was recovered from the human leprous lesion. The 2 forms differed both morphologically and culturally. A study was made to determine any difference in pathogenicity. Inoculations of rabbits began about a year ago. Large doses of the 2 forms were given at frequent intervals over a considerable length of time. Three rabbits were respectively injected with the “R'form subcutaneously, intraäbdominally and intranasally, and 3 other animals received the “S'form by the same routes. Preliminary to the injections of living bacilli, all 6 rabbits received several injections of a lepra broth-filtrate to produce, if possible, an allergic condition which would enhance the growth of the acid-fast bacilli in vivo. 2

The “S'form produced the most striking results. The rabbits became markedly emaciated after approximately 7 months. Several subcutaneous nodules were noted at the sites of inoculations in the rabbits receiving the subcutaneous injections. Microscopic examination of ulcerated nodules revealed many acid-fast bacilli. The internal organs, especially the liver, spleen and kidneys, showed many large phagocytic cells, simulating lepra cells, containing acid-fast bacilli. Fibrosis and generalized thickening of the blood vessels was marked. Two rabbits showed paralysis of 2 or more extremities which probably was the result of extra- and intraneural fibrosis. Less conspicuous changes were noted in the rabbits injected with the “R'form. Emaciation was present but not to the degree seen in the rabbits that had received the “S'injections. Subcutaneous nodules developed but gradually disappeared without ulceration. Careful microscopic examination of the internal organs of the “R'animals revealed nothing characteristic of leprosy. No acid-fast bacilli were detected in any of the organs.

One of the objects of this investigation was to determine whether it is possible to induce in rabbits. by means of the chromogenic acidfast bacillus isolated from the human leprous nodule, a disease comparable to human leprosy.

Hall, Ettinger and Banting 1 found that long-continued administration of acetylcholine produced myocardial and coronary artery damage, the effects being more severe and extensive in old than in young animals. In pursuing our interests in the problem of myocardial and skeletal muscle disease in relation to the creatine reserve, we attempted to study the effects in the rat. As in the experiments reported from the Banting Institute, acetylcholine bromide (Eastman-Kodak) and acetylcholine iodide (Hoffmann-La Roche) were used. The dose was 10 mg. of acetylcholine daily, administered in a single dose or in 2 divided doses. The rats were 5–6 months old at the beginning of the experiments and the average weight exceeded 300 gm. Fourteen of the 17 rats on which the present report is based received the drug for a period of 85–90 clays, at which time they were sacrificed.

Within a few seconds after each injection, the characteristic effects of the drug referable to autonomic activity, i. e., profuse salivation and lacrimation, accelerated respiration and heart rate, were manifested. These symptoms gradually subsided during the succeeding 10 minutes.

Heart. The changes in the heart were somewhat variable, but in most animals there was present some degree of myocardial degeneration, hyaline change and beginning fibrosis. A striking feature was the patchy distribution of the small areas of degeneration, often verging on necrosis. Infiltration of these areas with inflammatory cells, vacuolization, hydropic change, thickening of the vessel walls due mainly to endothelial proliferation, and scattered small hemorrhages into the myocardium were likewise in evidence. Despite these changes it was remarkable to discover that the creatine concentration was essentially unaffected (159–237 mg., average 206 mg.) in all the animals that survived the period of experimentation (15 out of 17).

The level of production of the “male hormone'in cryptorchid as compared with scrotal testes is of interest on two counts. Failure of the testis to descend is a fairly common defect in development, and an altered hormone-producing capacity of the retained testis may have a bearing on the origin and perhaps even on means of correcting the defect. Moreover, the inhibited differentiation of the seminiferous tubules in the cryptorchid testis, accompanied by evident increase of the interstitial elements, renders it interesting in relation to the question of the site of production of the testis hormone.

Apparently there have been few studies concerned with hormone production in the undescended testis. Early workers merely noted that the secondary sex characters are essentially normal in cryptorchid animals (Bouin and Ancel 1 ). The first quantitative study seems to be that of Moore and Gallagher 2 who found, in testing the cryptorchidized guinea pig by the electric ejaculation method, that hormone production was as great as normal. Jeffries, 3 utilizing the cytological signs in seminal vesicle and prostate, observed no castration changes in rats 60 days after cryptorchidizing, from which it was thought that there was no diminution in hormone production. Nelson, 4 using the same tests, found in rats 240 days or longer after being cryptorchidized normal prostates, but seminal vesicles showing castration changes, an observation indicating reduced hormone production in the operated animals. It is to be noted that these authors used as criteria of hormone production not quantity of hormone itself but hormonally regulated characters in their cryptorchid animals.

The present report is based on a study of 2 lots of mixed cryptorchid and normal testes of swine obtained in New York City through the assistance of Dr. C. A. Slanetz.

The fate of morphine in the animal body is a question that has stimulated much excellent work and the development of many methods for the determination of the drug. We do not have space to enumerate, much less to discuss, these methods, 2 of the more recent examples of which are those described by Wolff, Riegel, and Fry 1 and by Abe and Uchida. 2 Most of these methods were unsuited to our purpose since they were not designed for use with blood, and the others required more material or a more elaborate procedure than we wished to employ.

The method herein described has the advantage of simplicity, takes but a few hours to complete, and gives quantitative results with small amounts of blood. It is based on that of Sanchez, 3 which was designed for the determination of morphine in pure solution. We have modified it for use with blood filtrates, deproteinized by the method of Somogyi, 4 introducing scopolamine to bring down the precipitate and permit the removal of the reacting solutions. The method in detail is as follows: A 1-to-10 filtrate is prepared by laking 1 part of blood with 7 parts of water. To this is added with constant stirring 1 part 10% zinc sulphate and 1 part 0.5 N sodium hydroxide.∗ After standing 10 minutes the precipitate is centrifuged down and the supernatant liquid filtered through No. 2 Whatman paper. Treated in this manner 5 cc. blood yield over 30 cc. filtrate.

To 20 cc. of the filtrate, equivalent to 2 cc. blood, is added 2.5 mg. of scopolamine hydrobromide, from a solution containing 1 mg. per 1 cc. (Proportionally smaller amounts may be used if the concentration of morphine is high.) The morphine is then precipitated by 0.4 cc. of Wavelet's solution.† The material is thoroughly mixed by stirring or shaking, and set in a cold place for about an hour.

Although considerable data have been published on the creatine content of the muscle of laboratory animals, data on the creatine content of human voluntary muscle are somewhat meager. For this reason a study was made on material obtained from 74 human autopsy cases (less than 36 hours post mortem). Creatine was determined on 3 voluntary muscles, namely the psoas major, rectus abdominis and sternocleidomastoid, the average for the respective muscles being 402, 405, and 388 mg. per 100 gm. of fresh muscle.

If the average data secured on muscle obtained at autopsy may be taken as representing the normal creatine content of human voluntary muscle, the concentration is in the neighborhood of 400 mg. of creatine per 100 gm. of muscle. Although in this series there was a fairly even balance between groups of cases showing high and low creatine values, it is possible that the average value for strictly normal individuals is somewhat in excess of 400 mg.

It would appear on the basis of the data obtained on the 3 muscles studied, and also on unpublished data from this laboratory by Linegar and by Mangun for the pectoralis major, that the creatine concentration of various striated muscles in the human is essentially the same. The findings for these muscles may differ somewhat in an individual but the averages for a group are of the same order of magnitude.

The results of this work indicate that relatively high values for muscle creatine, to about 550 mg., may be found in uremia, pneumonia, tuberculosis, early malignancy, and in some cases with circulatory involvement, while low values (as low as 250 mg.) may be encountered in late malignancy, acute inflammatory diseases, uremia plus heart failure, and in some cases with circulatory involvement.

During the last few years increasing attention has been given to the electrolyte content of tissues (particularly heart tissue). Some time ago we began a study with the purpose of comparing the electrolyte content of tissues from cases with renal disease with that of tissues from patients who had died with other diseases. At present our series does not include a sufficient number of renal cases to make this comparison significant. That is, since the electrolyte content of a given tissue may vary quite widely, a fairly large number of cases must be obtained. Therefore, the purpose of the present report is to present a summary of the results which have been obtained on a series of miscellaneous cases.

The tissues (right ventricle, left ventricle, skeletal muscle, kidney, liver and spleen) were analyzed for water, chloride, phosphorus, sodium, potassium, calcium and magnesium in a manner similar to that outlined by Cullen and Wilkins, 1 Table I presents the maximum, minimum and average values found. The parentheses indicate the number of cases used in computing the average. It will be seen that in addition to the analyses made by Cullen, Wilkins, and Harrison 2 and Wilkins and Cullen, 3 the present study includes the determination of the electrolyte concentration of spleen, the determination of the calcium and magnesium concentrations of kidney and liver and the determination of the sodium concentration of muscle, kidney and liver. The difference between the electrolyte content of right ventricle and left ventricle has already been discussed by the above workers. The data of the present series are in agreement with their findings with the exception that the average magnesium content of the right ventricle is slightly greater than that of the left ventricle.

It was recently reported by us 1 that following ether anesthesia in dogs, rats and guinea pigs the urinary excretion of ascorbic acid is increased. This excess excretion, which amounts to a 10 to 15 fold increase in the case of the dog but which is somewhat less in the case of rats and guinea pigs, is only of short duration. That is, the excretion falls to the normal level or slightly below in the second 24-hour urine collection following the anesthesia.

It was naturally of interest to determine whether similar periods of ether anesthesia as employed in the above studies are followed by changes of the ascorbic acid content of the tissues. Therefore, rats and guinea pigs, after being maintained on a constant diet for some time, were subjected to 2-hour periods of ether anesthesia.

The animals were killed by a blow on the head either immediately after the anesthesia or 2 to 4 hours later. The ascorbic acid content of the various tissues was determined by titration with 2,6 dichlorophenol indophenol according to the procedure outlined by Birch, Harris and Ray. 2

Table I presents the results obtained. In the case of rats which were killed immediately following the anesthesia, the average ascorbic acid content of kidney and liver is definitely higher than that found in the controls. On the other hand, the average ascorbic acid content of the adrenals is reduced. When the rats were killed 4 hours following the anesthesia, the ascorbic acid content of kidneys and liver, though less than that observed in the animals killed immediately following the anesthesia, is still definitely above the control level. At the same time the ascorbic acid content of the adrenals shows a continued decrease.

Most prominent in the records of the electrical activity from the brain in intact human subjects are large and fairly rhythmic oscillations of potential called alpha waves. In a recent study of brain potentials in children and adults by Lindsley 1 the average frequency of the alpha rhythm in 54 adults was found to be 10.4 per second with a range of variation from 8 to 12 per second. Under normal conditions the frequency for any one individual was remarkably constant, often varying by less than 1 cycle per second over a period of months and only occasionally by as much as 2 cycles per second. In children the rhythmic alpha waves first appeared at about 3 months of age and at a frequency of 3 to 4 per second. The frequency increased with age until the adult average was reached at 8 to 10 years of age. A slight rise in frequency above the adult level occurred between 10 to 12 years of age.

The present study is concerned mainly with the frequency variations observed in adults, since the various processes of growth and development make difficult certain comparisons in children. An attempt has been made to determine the relationship between the frequency of the alpha waves and some other physiological variables such as metabolic rate, heart rate, blood pressure, rectal temperature and respiration.

Thirteen adults, 12 women and 1 man, were used as subjects. Of these, 4 women medical students ranging in age from 21 to 31 years were studied every morning for 32 or 34 consecutive days. All records were obtained early in the morning under basal conditions. Rectal temperatures were taken by the women on awakening and before getting out of bed.

Chromogenic members of the colon-group are not unknown. MacConkey 1 listed yellow colon-group liquefiers from horse feces, pond water, rain water, roof washings, oats, beans, malt and ears of corn, and he reported a yellow B. coli communis from rain water. Rogers, Clark, and Lubs 2 reported that but few grain cultures are without pigment, many being decidedly chromogenic. They stated that this property is correlated with other characters and consequently is of value in classification. In a collection of colon-bacteria from human feces they found chromogenesis almost entirely absent. All their fecal cultures gave a faint yellow color but this was so slight and showed so little variation that it was of no value in differentiation. They did state, however, that there were a few exceptions to this rule. Wood 3 reported that 7 of 20 colon-group strains isolated from grains, hay and dried eggs and milk produced yellow pigment. In his Pocomoke river series Perry 4 encountered 5 cultures of aërobic, non-sporulating bacteria producing gas from lactose which produced a distinct yellow pigment. He excluded these chromogenic strains from consideration as fecal coli.

On January 14, 1936, a fecal specimen was received for study. Although the patient complained of certain general symptoms none referred to the gastro-intestinal tract and the analysis was under-taken as part of a routine rather than as an indicated procedure. A suitable saline suspension was at once prepared and adequately plated on citrated agar, blood agar, and Endo's agar. A small bit of the stool was placed in a large tube containing 30 cc. of lactose-indicator-broth. In this enriched medium typical acid- and gas-formation was observed in 24 hours. No colonies appeared on the citrated agar although it was heavily inoculated.

In the investigation of the problem of experimental neurosis in animals, one of the possible causative factors to be considered is restriction through habituation of the animal's freedom of movement. As an approach to this part of the problem, a study of the animal's spontaneous activity previous to any restriction becomes pertinent.

Experimental neurosis in the sheep leads to a permanent state of hyperexcitability with loss of formerly established discriminations. 1 In the dog the neurosis may manifest itself in chronic somnolence or in hyperexcitability during the conditioning tests. 2 It is reasonable to suppose that the absence of the enduring somnolent or inhibitory experimental neurosis in the sheep may be related to the fact that the sheep does not sleep as the dog does. The pig is a sound sleeper and for this reason it was decided to compare the diurnal cycles of spontaneous activity in these sleeping and non-sleeping animals.

The subjects for investigation were 10 sheep, of both sexes, ranging in age from 1 1/2 to 8 years, and 5 pigs from the same litter, 14 months old, 3 females and 2 males, one castrate. The sheep and male pigs ran in a 15-acre pasture while the sows were limited to a one-acre field. Each of the animals wore a standard New Haven pedometer attached to a harness. They had all become thoroughly habituated to the harness before any records were taken. The pedometers were read twice daily, at 6 A. M. and 6 P. M. Readings were continued for a period of 16 days, from October 15 to October 31. At this season of the year, the intervals between readings corresponded closely to the actual hours of daylight and darkness.

Ten lion-aniine catechol derivatives cause a rise in the blood pressure of the intact cat. Acetylation of catecfiol and the alpha (3,4-dihydroxyphenyl) ethanone derivatives does not destroy the vasopressor ability. The physiological effects of these drugs on other organs and the effects on the blood pressure of the pithed, decerebratecl and tleiiiedullated cat are now being studied and will be reported in the near tuture.

We 1 reported the discovery of a new drug, a vasopressor local anesthetic: alpha (3,4-dihydroxyphenyl) beta (paraaminobenzoylbetadiethylaminoethanol) alphaethanonehydrochloride, designated for brevity as epicaine.

Procaine and its known relatives cause a pronounced fall in blood pressure, as does cocaine, in the ordinary concentrations. 2 - 5 Epicaine raises the blood pressure in the intact cat.

We 6 have previously pointed out that biochemorphologically the following factors are contributory to the formation of a typically sympatheticomimetic drug: the 3,4-position of the phenyl hydroxyls, a 2-carbon sidechain, a beta-carbon hydroxylated, an alpha-carbon hydrogen substituted by some indifferent radical, preferably an amine (but not necessarily so), and the laevorotatory isomer. The chemical structural configuration of epicaine is compatible with these criteria.

The drug was injected into intact cats anesthetized with pentobarbital (40 mg. per kilo). Ten cats were used; in 8 both vagi were cut and the animal given atropine sulfate intramuscularly (2 mg. per kilo) at the start of the experiment. In 2 animals ether was used and the cats were neither atropinized nor vagotomized. Thirty-eight injections of the drug were given, and each was always controlled by epinephrine. The cannula was washed after each injection until no effect was obtained from 2 successive washes before the next injection of a drug.

Immediately upon intravenous injection of epicaine in doses from 2 to 10 mg. there is an abrupt rise in blood pressure ranging from about 20 to 40 mm. of mercury. This is illustrated in Fig. 1. Concomitant with the increase in pressure there is a dilatation of the pupil and a retraction of the nictitating membrane. Subsequent doses continue to elicit the same effect.

We' have stated elsewhere that in the intact animal ergot reversal and cocaine synergy of a blood pressure rise indicate that the impulses eliciting the rise reach the blood vessels by way of the sympathetic nerves; they do not indicate the site of origin of the impulses. Therefore these invalid criteria were not employed; further experiments are noiv being carried out on decapitated, decerebrated, and demedullated. as well as pithed, animals.

Studies of the pituitary following thyroidectomy in young rats 1 have been reported, describing the appearance of large numbers of basophilic cells containing hyaline material, as a constant feature in all rats in which thyroidectomy was adequate, as evidenced by retardation of body growth, of kidney growth and alterations in the ratios of gonads and adrenals to kidney growth. 2 During these experiments a puzzling feature was the variation in the acidophile loss. In some cretin pituitaries, all acidophiles had disappeared, while in others there was a moderate or slight reduction in number. It was thought that perhaps a factor in the disappearance of the acidophiles might be some incidental trauma to nerves in the course of the thyroidectomy, 3 as recent work had suggested a nervous control of the anterior pituitary through the cervical sympathetic nerves. Collin and Hennequin 4 had found in male adult rabbits that ablation of one of the superior cervical sympathetic ganglia resulted in vasodilation of the pituitary and a rapid loss of acidophiles in 5 hours to 7 days. On the 7th day the pituitary was composed chiefly of chromophobes. Stimulation of the central end of the cervical sympathetic caused an increase in the granules and numbers of acidophiles in one-half to three-quarter hours. That nervous stimulation of the pituitary may occur is indicated by the fact that Friedgood and Pincus 5 found that stimulation of the cervical sympathetic nerves in adult female rabbits resulted in extensive maturation of the ova and in some cases ovulation.

In performing thyroidectomy in young rats the cervical sympathetic fibres could be traumatized inadvertently, as the thyroid is bound laterally closely to the carotid artery region.

The important discovery of Masugi 1 that in rabbits and rats glomerular nephritis can be produced by intravenous injections of anti-kidney serum has been confirmed by Fahr 2 and his pupils Hemprich 3 and Weiss, 4 in rabbits; by Arnott, Kellar and Matthew, 5 in rabbits; and by Farr and Smadel, 6 , 7 in rats. It has been shown that the animals develop albuminuria (up to 30%, Esbach), hematuria, cylindruria, lipuria, moderate oliguria or occasionally anuria (in acute stages), moderate edema, rise in blood urea (up to 224 mg. %), rise in blood pressure, cardiac hypertrophy, and anemia, and that at autopsy the kidneys exhibit extensive glomerular lesions which closely resemble those of human glomerular nephritis.

Masugi injected 10 cc. of a 10 to 30% suspension of blood-free kidney juice of rabbits in saline solution into the peritoneal cavity of ducks, and that 18 to 30 times at intervals of about 5 days. Five to 8 days after the last injection serum was prepared, and after inactivation by heating to 56°C. for 30 minutes, was injected intravenously once or repeatedly into rabbits in doses of 5 to 15 cc. Most rabbits received more than 15 cc. of serum. Hemprich suspended the juice of 2 kidneys in 100 cc. of saline solution. Of this suspension, 10 cc. were injected into ducks 9 to 22 times at 4 to 8 days'intervals. The serum of these animals was injected into rabbits in doses of 3.5 to 26 cc. Most rabbits received more than 10 cc. of serum. Weiss improved the method by thoroughly squeezing the remnants of the ground up kidneys while preparing the juice. He produced glomerular nephritis already with doses of 3 to 7 cc. of ducks'serum, in most cases with 4 to 5 cc. Arnott, Kellar and Matthews, using the original method of Masugi, injected the ducks 25 to 40 times.

The operating box herein described was developed for use in 2 studies upon the sculpin (Myoxocephalus octodecimspinosus), a common marine teleost. Grafflin and Ennis 1 reported experiments upon the blockage of the gastrointestinal tract, and Grafflin and Eisenberg 2 upon the direct observation of the living kidney. There is obviously a wide field for comparative studies in the fishes in which abdominal surgical procedures and direct observation of living organs in situ would play an important part. The sculpin is readily available for experimental work; and the operating box which we have developed has been so satisfactory in our experience that it is described here in the belief that it will prove useful to other workers. The box is illustrated in Figures 1 and 2.

With the use of water outlets C and C', the water level in the box is usually maintained at a sufficiently high level. With some larger specimens a higher level is necessary to insure complete covering of the head, and in these cases the rubber tubing is switched from C to D, and the opening at C is plugged. The rather tight fit of the animal between the clamps, and the upward projection of the pectoral fins, suffice to maintain a higher level in the anterior than in the posterior chamber under these conditions.

A constant stream of sea water is supplied through the inlet B. In the experiments previously reported urethane anesthesia was used. Anesthesia was rapidly induced by immersing the fish in a 1% solution of urethane in sea water in a separate chamber. The animal was then transferred to the box, and anesthesia was maintained throughout the experiment by a 0.25% solution of urethane in sea water.

Although the literature is full of reports on nerve degeneration in avian polyneuritis and B deficiency in the rat and other mammals, the most recent contributions in this field reveal conflicting evidence. 1 In order to circumvent uncertainties in the interpretation of Marchi preparations and any possible artefacts that may be introduced during fixation and staining, Sutton, Setterfield and Krauss 2 have recently applied the polarized light technique for the study of degeneration of myelinated nerves in vitamin A deficiency. This technique was found by Setterfield and Baird 3 to have advantages over the Marchi method of staining with osmic acid, since it does not depend upon fixation and staining but on the chemical structure of the myelin sheath; moreover, it is more rapid, more sensitive and constant.

The polarized light technique, however, as employed by Sutton and associates requires previous fixation of tissues in formalin, which procedure may introduce artefacts. We have applied the technique of Sutton and co-workers in studies of vitamin B deficiency and have checked the formalin procedure by elimination of all fixatives as well as all staining operations. Small pieces of spinal cord, trigeminal, sciatic and optic nerves from avitaminotic and litter mate controls on the same plane of nutrition, were fixed in 95% alcohol in liquid air, and dehydrated in vacuo, at −32°C., by the Altmann-Gersh-freezing-drying technique, as modified by Scott and Williams. 4 Frozen sections were made and compared under polarized light with those fixed in 10% formalin. Twelve groups of rats were used for this comparative study. The pathological animals of 9 groups were on diets deficient only in vitamin B₁ and had varying degrees of polyneuritis, ranging from slight to marked paralysis.

Bisulphite binding substances (B.B.S.) in the blood have been reported increased in vitamin B₁ deficiencies both in experimental animals 1 and in clinical “wet beriberi”. 2 The present study of the B.B.S. in the blood was undertaken to determine its value in distinguishing vitamin B₁ deficiencies from other disease states.

Oxalated blood samples were taken fasting and at rest from 30 healthy controls, and from 110 patients. The method of Clift and Cook 3 was modified to adapt its use to trichloracetic acid filtrates of whole blood. Five milliliters of oxalated blood were precipitated with 20 ml. of 10% trichloracetic acid, allowed to stand 30 minutes and centrifuged. Five milliliter aliquots of the supernatant liquid were adjusted to pH 2 by the addition of 1.5 ml. of normal sodium hydroxide and allowed to react with 0.2 ml. of saturated sodium bisulphite solution for 15 minutes. Twenty-five milliliters of distilled water and 2 ml. of fresh starch solution were added and the excess of bisulphite titrated out with normal and N/10 iodine solutions adjusting the end point with N/200 iodine solution and N/100 sodium thiosulphate solution. The bound bisulphite was released by the addition of 2 gm. of solid disodium phosphate (Na₂HPO₄· 12 H₂O) and titrated within 5 minutes with N/200 iodine solution. The values were expressed in milligrams of pyruvic acid per 100 ml. of blood, using the expression: 1 ml. N/200 iodine = 0.22 mg. pyruvic acid. In most cases the non-protein nitrogen, the plasma carbon dioxide capacity and blood sugar were measured on the same samples. In a smaller group the urine was tested for sugar, acetone, acetoacetic acid and pyruvic acid.

Results. Thirty normal subjects had values for B.B.S. in the blood ranging between 3.66 and 5.75 mg. with an average of 4.74 mg. per 100 ml. Values for B.B.S. above 6.0 mg. are accepted as elevated. Normal values were found in the following conditions: arteriosclerosis, compensated heart disease, carcinomatosis, glomerulonephritis, hepatitis with jaundice, severe anemia, toxemia of pregnancy, acute alcoholism, chronic alcoholism without vitamin deficiency, chronic alcoholism with vitamin (B₁) deficiency after treatment.

I. It is shown that the inhibiting effect on pneumolysin by cholesterol and certain related sterols is apparently determined primarily by the presence of the OH group in the sterol structure and secondarily by the double bond. Possible peroxide foimation would seem to account for only a small portion of the cholesterol effect. 2. Active pneumolysin is inhibited promptly, while inactive (air-oxidized) lysin is affected slowly if at all by cholesterol. Likewise, active lysin is adsorbed by red cells, while the iiiactive form is not. That is, the state of oxidation of the lysin conditions its reactivity with the sterol and with its adsorption on red cells. 3. The free thiol grouping on the active lysin molecule remains free after treatment with excess cholesterol. The lytic activity, therefore, is associated with at least 2 functional groupings, one reversibly oxidizable, and the other more or less specific for a certain sterol grouping and configuration.

Schoenheimer and Rittenberg 1 have proposed that if non-labile deuterium is found in the molecule of a fatty acid isolated from tissues of mice which were receiving heavy water, this finding is then indicative of the synthesis of the fatty acid in the animal body. In connection with the study of the synthesis of protein and amino acids in animals, we were interested to ascertain whether or not a similar criterion, as used by Schoenheimer and Rittenberg, is applicable to protein and amino acid synthesis. Barbour, et al., 2 on the assumption that one hour after a single injection of D₂O into mice a maximum of exchangeable deuterium will be fixed in the tissue, conclude that inasmuch as “mice drinking 15% D₂O for 2 months have 3 times the concentration of deuterium (relative to body water deuterium) in the tissue as mice receiving a single injection of D₂O,'the difference in the deuterium content of the tissue indicates the fixation of deuterium in tissue in stable form. It occurred to us that the isolation of tissue protein and of amino acids derived therefrom and the analysis of the isolated products from which all deuterium in labile position has been removed would constitute more direct evidence for the fixation of deuterium in the protein in stable form than the one offered by Barbour. 2 Twenty adult and 20 growing mice were used in a preliminary study. The animals were kept, each group separately, in metabolism cages and 2% D₂O and food consisting of 70 parts of oats, 15 of yeast powder and 15 of “Klim'milk powder were allowed ad libitum. A record of food and water consumption of the young mice was taken daily.

It has been shown 1 , 2 that saliva added to shed blood has a remarkable accelerating action both on normal and haemophilic bloods. It was thought desirable to see if saliva, when swallowed, had any effect on the coagulating time of blood or contributed in any way to its coagulability. Mills 3 , 4 has shown that the ingestion of a meal rich in protein has an accelerating effect on the coagulation of blood but the ingestion of fats or carbohydrates has little effect.

The results of this work may be briefly stated. It is easy to confirm Mills observations. Thus a meal of 600 gm. of meat caused a reduction of clotting time from 3 1/4 minutes to 1 minute 54 seconds two hours after the meal. No, or at most a few seconds, reduction in clotting time resulted from carbohydrate or fat meals and this is true also for the ingestion of large amounts of saliva. Paraffin or gum was chewed for 15 to 30 minutes and the saliva swallowed as formed. Samples of blood were taken every 15 minutes and in no case was any material difference in clotting time noted. Thus the average of 6 experiments carried out on 6 different individuals shows a clotting time before of 3 minutes 14 seconds, one hour after the saliva had been swallowed it was 3 minutes 15 seconds. The capillary tube method of Mills and Petersen 5 was used for obtaining the coagulation times.

In one dog all the salivary glands were removed aseptically and although the animal was kept a number of months the coagulating time of its blood remained normal.

Thus no effect on the coagulating time of blood could be made out either from an increase or a decrease in the amount of saliva swallowed.

After the first extensive internal use of naturally radioactive compounds in man by Proescher and others, 1 , 5 Seil, et al., 6 estimated that from 40% to 60% was lost from the body, chiefly through the feces. A progressive diminution in the rate of excretion was indicated by Schlundt and his group, 7 who found that patients retained 4.3% 6 months after administration and 1.9% 6 months later. Sehlundt and Failla 8 found 24 μgm. and 14 μgm. respectively in 2 women 12 years after oral ingestion of radium, with respective coefficients of excretion of .005% and .0025%. Prolonged retention has been reported in many cases of industrial radium poisoning. 9 , 10

Following the announcement by Curie and Joliot 11 of the preparation of an artificial radioactive isotope of nitrogen in 1934, Lawrence and his co-workers 12 produced relatively large amounts of radioactive isotopes of many elements using their magnetic resonance accelerator. Radio-sodium became available for clinical study at the University of California Hospital in the Spring of 1936. It was felt that many of the disadvantages of internal radium therapy could be avoided by the use of radio-sodium, since this latter substance does not tend to become fixed in the body tissues and the duration of its effect is limited by its short half-life of only 14.8 hours.

Initial investigations were made of the clinical effect and the rate of excretion of the radio-sodium following its intravenous administration to 2 human leukemic subjects.∗ An approximately isotonic solution of sodium chloride was used in each experiment.

Each sample was measured with an electroscope just prior to administration and periodic determinations of the degree of activity of the patient's body was carried out. At the same time all the stool and urine samples were collected and their activities measured. The activity of 100 cc. of blood from the second patient was determined. The first patient received 13 mc.e. (milli-Curie equivalents) of radio-sodium and in Fig. 1 are shown the measured and theoretical decay curves in the upper portion of the chart. The theoretical values were computed from the first measurement of the patient's activity.

Beef-blood serum-albumin, when suspeiidecl in Locke's solution or in plasma, prolonged the sedimentation time of human erythrocytes. A solution of human-blood serum-albumin suspended in Locke's solution and in plasma prolonged the sedi mentat ion time. A sample of powdered human-blood serum-albumin shortened the sedimentation time when it was suspended in Locke's solution, and prolonged it when suspended in plasma. A solution of bleef -blood serum-fibrinogen suspended in Locke's solution and in plasma did not significantly affect the sedimentation time. Witte's peptone suspended in Locke's solution or in plasma prolonged the sedimentation time insignificantly. Talc or kaolin were without effect on the sedimentation time, Dog-bile suspended in plasma prolonged the Sedimentation time.

The increasing use of propylene glycol as a solvent for various pharmaceutical preparations 1 and coloring extracts, 2 and its possible use as a food, indicates the need for a better understanding of the actions of this alcohol. The known production of oxalic acid in the metabolism of ethylene glycol 3 , 4 has been responsible for the substitution of propylene glycol for the former, since oxalic acid is not a possible metabolite of the latter. 5 The properties, general actions, and acute and chronic toxicities of propylene glycol have been investigated by Seidenfeld and Hanzlik 5 ; toxicity was unusually low.

The concentration of propylene glycol in the blood was determined by treating the protein-free filtrate with a solution of potassium dichrornate in strong sulphuric acid, and estimating the amount of reduced dichromate iodometrically. From this value was subtracted the amount of oxidizable material normally present in the blood, a variable which remains relatively constant in fasting animals. In urine, the glycol was estimated directly by using an aliquot portion of a 1:100 dilution, and subtracting the blank value for normal urine from the final result.

The concentration of propylene glycol in the bloods of 2 dogs and 10 rabbits was followed for variable periods after giving doses from 1 to 12 cc. per kilogram body weight, intravenously or orally; the dogs were used repeatedly.

Figure 1 shows the rate of disappearance of propylene glycol from the blood of dogs following the administration of 2 cc. and 4 cc. per kilo intravenously, and 8 cc. gastrically; the intravenous doses are compared with similar doses of ethyl alcohol.

The rate of disappearance of propylene glycol from the blood is proportional to its concentration in the body, as contrasted with alcohol, which disappears at a constant rate 6 .

We have described a micrometric method of representing the thyrotropic effect on the thyroid gland of test animals. Since each increased dosage of the hormone caused a characteristic curve, this procedure affords a quantitative method of determining the effects of the thyrotropic hornioiie on the thyroid of the guinea pig.

Under the conditions of this experiment colloidal sulfur was not utilized by rats either for production of cystine or for growth.

In the hearts of rats recovering from hyperthyroidism restoration of the creatine concentration occurs in advance of any marked anatomic improvement and it is therefore not inconsistent to find normal values in the presence of moderate myocardial damage. Reparative processes occur somewhat more gradually in skeletal than in cardiac muscle and the restoration of creatine to normal more nearly parallels the degree of tissue regeneration.

Since Meyerhof and Lipmann, 1 and Meyerhof, Moehle and Schulz 2 studied the exchange of CO₂ in frogs'muscle during fatigue, we know that under anaerobic conditions the pH of the muscle, when subjected to periodic stimulations, will first increase and then gradually decrease. However, the method applied by these authors does not show the pH changes during the activity itself, i. e., contraction and relaxation of the muscle. Margaria and Pulcher 3 attempted to show the pH change during contraction itself by coloring the muscle with bromocresol purple. They found a change in color during the contraction corresponding to an increase in pH of about 0.5. According to Margaria, 4 however, the same changes occur when the muscle is mechanically stretched. Later Margaria and von Muralt 5 described a photo-electric method with which it would be possible to record those changes of color. On summarizing, as far as I know, no records showing the pH changes during and after contraction have been published. I succeeded in obtaining such records during tetanus by an entirely different method. Its principle is to cover a part of the surface of the muscle with a glass electrode and to record the pH changes occurring on stimulation.

The glass electrode consists of a membrane of suitable glass sealed to the end of a tube of ordinary glass. This is the first of the two types of glass electrode described by MacInnes. 6 The glass membrane is brought into contact with the wet external surface of the muscle. Glass electrode, muscle, and a potentiometer are mounted in series in the grid circuit of a Pliotron tube F.P. 54 7 .

A coniplernent-fixation reaction is described, with extracts or filtrates of papillonias containing infective Shope virus and antisera effective against the latter. The implications of the work are being studied.

A new capillary fragility test based on intraderimal injections of titrated moccasin snake venom is described. This test was found helpful in the niaiiagement of thrombocytopenic purpura heniorrhagica. Its applicability as a guide to local, general and toxic capillary changes is discussed.

The subneural gland of the ascidian has long been the subject of controversy in regard to its probable function. On developmental grounds it has been denied admission to phylogenetic series dealing with the pituitary and until quite recently there has been no physiological evidence which might justify including it in such a series on a functional basis.

Butcher 1 demonstrated the presence of the oxytocic principle in the gland of Molgula and this result suggested the possibility of obtaining other pituitary effects such as the ovarian stimulating action in immature animals. The present work deals with an attempt to demonstrate such an effect using the gland of the large solitary ascidian Polycar pa tecta common in Bermuda waters.

In 1934, through the courtesy of the Bermuda Biological Station for Research, 70 fresh specimens of Polycarpa tecta were obtained and the ganglion, subneural gland and dorsal tubercle removed and desiccated in acetone. The dry weight was found to be 45 mg. and from a study of serial sections it was apparent that not more than 20–30 mg. of this weight could be considered glandular substance. Assuming a potency equal to that of mammalian whole gland it was not thought advisable to divide the material among more than 3 test animals. Accordingly, after pulverization and suspension in sterile isotonic saline equal amounts were injected subcutaneously into 3 nineteen-day-old mice. Three control animals received subcutaneous saline. None of the animals died and all were killed and autopsied on the fifth day.

Grossly the ovaries of the test animals were larger and appeared more vascular. The collective weights to the nearest milligram compared as follows: Test, 25 mg.; control, 8 mg.

Microscopic examination of the ovaries showed an increase in vascularity arid in the nuniber and size of the follicles in the test specimens. (Fig. 1.)

The inaccessibility of ovaries and uterus no longer handicaps the study of ovarian function in the human female since Papanicolaou and his associates 1 established the diagnostic value of vaginal smears. The smear technique, however, is necessarily surrounded with such precautions as to make it too elaborate for general use. In our effort to find a simpler but nevertheless reliable substitute we have correlated body temperature with changes in the vaginal smears on the ground that body temperature is known to vary in a regular manner during the menstrual cycle. 2

The cycle of vaginal smears can be conveniently divided into 6 phases: (1) menstrual (3–7 day), (2) post-menstrual (3–6 day), (3) preovulative (1–5 day), (4) ovulative (1–3 day), (5) postovulative (5–8 day), (6) premenstrual (3–7 day). Rectal temperatures, taken before rising in the morning, i. e., between 6 A. M. and 7 A. M., and vaginal smears were obtained daily during 5 complete menstrual periods of 4 young adult women without pelvic abnormality.

Table I summarizes the data. The lowest temperatures occur in the ovulative phase, the highest in the premenstrual phase. The rank-correlation coefficients of body temperatures with vaginal smears arranged in the order (1) ovulative, (2) preovulative, (3) post-menstrual, (4) menstrual, (5) post-ovulative, (6) premenstrual are statistically significant, the values ranging from +0.833 to 0.912.

The negative results obtained from the feeding of niassive doses of charcoal adsorblate, prepared from bleef adrenal cortex may indicate that the sulrstance in the adrenal cortex, which stiniulates the reproductive system, is a different hormone from that demonstrated by Harrop 1 as affecting the metabolism of salt and water and it may not be extracted by the methods at present in use for extracting the latter. The excellent condition of the experimental animals is evidence that the substance is not toxic.

The stimulus of suckling definitely decreases the galactin content of the rat pituitary gland even when no milk is removed.

1. Dogs were fed diets of varying sulfur content and 1.0 gm. doses of bromobenzene were fed on 4 consecutive days on each of the diets. The synthesis of p-brornophenylrnercapturic acid and ethereal sulfates on these diets was estimated. 2. The extent of the synthesis of the mercapturic acid and of the ethereal sulfates is apparently a function of the nutritive state of the animal: when the animal was deprived of dietary sulfur, a decrease in the output of mercapturic acid and an increase in the output of ethereal sulfates was noted. 3. The results offer further support to the previous suggestion that the dietary sulfur is not the immediate source which is used for the detoxication of blromobenzene in dogs.

1. Parahydiroxyphenylisopropylamine, when adminnstered by niouth, is effective in the prevention of cardiac arrest. 2. This substance is more active than ephedrine on induced cardiac arrest. 3. When administered in a dosage effective in preventing cardiac staiidstill, parahydroxyphenylisopropylamine does not produce a central nervous stimulation with resultant unpleasant side effects.

Very dilute concentrations of cocaine HC1 depress slowly conducting nerve fibres; in higher concentration, a similar depression is produced by ephedrine HCI, atropine sulfate or by nicotine base.

In previous work 1 we found that the ability of the hypophysectomized rat to remove excess sugar from the blood stream decreased with time after operation. This gradual decrease in ability to remove excess sugar indicated that the effect must be due to the progressive atrophy or derangement of some other organ following removal of the pituitary body. We gave our first attention to the rôle of the adrenal gland in this effect since Leloir 2 had shown a decrease in the rate of removal of glucose from the blood in adrenalectomized dogs, associated with decreased glycogen formation.

To test this possible connection the following groups of male rats were given 0.125 gm. glucose per 100 gm. body weight by injection of 50% solution into the saphenous vein:

a. Normal control animals.

b. Normal animals injected with 0.2 cc. Wilson's adrenal cortex extract per day.

c. Young adult animals adrenalectomized 6 days previously.

d. Similar adrenalectomized animals injected with 0.2 cc. extract per day for the previous 4 days.

e. Hypophysectomized animals, operated on 17 days previously.

f. Similar hypophysectomized animals injected with 0.2 cc. extract per day for the previous 6 days.

All animals were fasted for 24 hours before receiving the glucose injections. Blood sugar samples were taken from the tail vein, the first few drops being discarded. Samples were taken fasting and at 10, 20, 30, 60, and 120 minutes after injection. Table I gives the average values for the various groups.

The defect in ability to remove excess sugar from ithe ldooci in the hypophysectoniizecl iiiale rat is not relieved by cortical extract, while that observed in the atlrenalectornized animal disappears. It seem then that the deficiency brought on by renioval of the pituitary glaiitl is not solely, ii at all. a result of atrophy of the adrenal cortex. Since the normal rats injected with cortical extract showed no greater rate of g-lucose removal than the uninjected controls. the action of the cortical extract in adrenalectoniized rats on the tolerance must be due to its specific action in relieving cortical insufficiency.

Sheep-pituitary extracts, which had produced gonadotropic antihorniones in several species of animals, were injected into 2 sheep for 6 months, during which time no gonadotropic antihormom was found in the sheep-sera.

Daily injections of Jieep-pituitary extract into a dog produced an antihormone which inactivated in guinea pigs substantial test doses of thyrotropic hormone from sheep and bovine pituitary glands and f rom human urine oi myxoedenia. Injections of the serum alone into guinea pigs produced upon their thyroids an effect which resenibled hypophysectomy.

Injections of a gonadotropic extract of sheep-pituitary glands into a horse arid 2 dogs induced the formation in their sera of a factor which augiiientetl three-fold the gonadotropic activity of the extract when tested in immature rats. The augmenting factor had no gonadotropic action in liypophysectomized immature rats, but produced in immature normal rats an effect resembling F.S.H. or synergist. The auginentitig factor, possibly an antihormone to the pituitary gonadtropic antagonist, was present only in the pseudo-globulin fraction of the sera, which fraction also carries the immune bodies and other antihormones.

A niethod has been described for extracting mammotropic hormone from sheep pituitaries involving its solubility in HC1-acetone and NH₃-acetone, and its precipitation with isoelectric protein at pH 5.5. A fraction made by the same procedure hut precipitated at pH 6.5 has heen shown to contain relatively little mammotropin, and is identified in the following paper as the adrenocorticotropic hormone. The “local” intradermal squab test has several advantages over the “systemic” tests of Riddle, et nl., when only mammotropic hormone is to be tested for, but the latter test has the very great advantage of allowing one to assay for the gonadotropic, thyrotropic, adrenotropic and mammotropic hormones on the same bird.

Adrenocorticotropic extracts† were tested in normal 21-day-old male rats and in hypophysectomized rats. The amount of adrenocorticotropic hormone necessary to cause an increase of 50% over the adrenal weight of controls when injected into 21-day-old male rats in 3 doses over a period of 3 clays was defined as one normal rat unit. A total dose of 20 mg. was usually found to be adrnal to one unit.

The injections of large amounts of beef brain (1000 mg.) produced no significant change in adrenal weights of normal 21-day-old male rats. Adrenocorticotropic preparations from beef anterior pituitaries showed the same activity as adrenocorticotropic preparations from whole sheep pituitaries, indicating that posterior lobe substances were not causing the hypertrophy of the adrenal cortex in the normal male rats.

Male rats between 40 and 50 days old were hypophysectomized,† and at least 30 days were allowed for atrophy of the adrenals before injections were begun. The completeness of the operation was checked by examination of the sella turcica under dissecting binoculars. Table I shows the effect of adrenocorticotropic extracts in hypophysectomized rats.

From the results obtained in hypophysectomized rats, it can be seen that the adrenocorticotropic preparations contained no growth hormone at the level tested. The thyroids showed no histological evidence of stimulation.

Adrenocorticotropic extracts when injected subcutaneously into squabs for 4 days showed no stimulation of the thyroids or of the testes. The crop glands were stimulated.

Adrenocorticotropic and mammotropic fractions from the same extract were compared at various levels for their adrenocorticotropic potencies in normal 21-day-old male rats. The results are given in Graph 1.

The adrenal cortices of nornial inimature rats in jectecl with aclrenocorticotropic hormones% (Fig. 2) differ niarkedly from the untreated controls (Fig. 1). There is an increased fat content throughout the whole adrenal cortex. In untreated controls there is an intermediate zone between the glonierulosa and the fasciculata in which there is very little or no fat.

Considerable difficulty has been experienced in estimating the abundance of marine anaërobic bacteria although they have been demonstrated 1 in nearly all samples of water or mud examined. Most of the conventional procedures 2 such as the incubation of plates in anaërobic jars have failed to yield reproducible results and moreover, the use of such complicated, space- and time-consuming apparatus is entirely impracticable aboard a rolling boat at sea. The application of oval tubes as described by Anderson 3 for the enumeration of anaërobes has exceeded expectations.

Ordinary round glass tubes with sealed ends as used by Roux 4 and Burri 5 are satisfactory for the cultivation of anaërobes but the curvature of the glass makes it virtually impossible to count the colonies which develop, particularly when it is necessary to use a hand lens. The Kimball Glass Company fabricated special oval tubes for us with flat, parallel sides, thereby obviating this difficulty. The oval tubes are 6×14 mm. in cross-section and 380 mm. long with one end permanently sealed and the other end flared to facilitate the introduction of the medium. The tubes are sterilized in a pipet can. A test-tube containing 10 cc. of nutrient agar recently heated to nearly 100°C. to expel oxygen and cooled to 45° is inoculated with the proper dilution of the sample to be analyzed for anaërobes. Without undue agitation (shaking has been found to be unnecessary to insure an even distribution of bacteria) the inoculated medium is poured into the oval tube. As soon as the medium has solidified, it is covered with a deep layer of reduced methylene-blue agar. The seal excludes oxygen and indicates the degree of anaërobiosis, Hall 6 having shown that conditions are suitable for the growth of anaërobes in an environment in which methylene blue remains colorless.

Interest in the effect of seasonal variations in the diet of guinea pigs upon their susceptibility to bacterial toxins led to an extensive study 1 of the metabolism of vitamin A in these animals. The results indicated the participation of some other factor or factors in the increased variations in susceptibility which occur during the winter months. Published reports 2 , 3 directed our attention to vitamin C.

The guinea pigs used in the experiments were bred by this laboratory and weighed from 230 to 280 gm. They had been fed the routine winter diet: alfalfa hay, oats, barley, commercial rabbit pellets, cabbage, carrots and mangels, and sodium chloride and water ad libitum, During May, occasional supplements of grass were fed.

The adrenals of 10 guinea pigs injected early in December with a uniform lethal dose of diphtheria toxin were examined for their vitamin C content by the method of Bessey and King. 4 One of 10 similar animals which had received one-half the dose of toxin, and one uninjected control were destroyed as each in the first group died. The average vitamin C content of the adrenals of the guinea pigs which had died from the effects of diphtheria toxin was reduced to less than 15% of that of the control animals. The change was much less in the adrenals of the 10 animals that received half the lethal dose of toxin. The average amount of vitamin C was 85.5% of that of the normal controls. In fact, the adrenals of the 5 which were destroyed approximately 48 hours after injection showed an increase in the vitamin C content, and it was not until 24 hours later that a diminution became apparent.

(1) The cholesterol content of rabbit liver, increased by cholesterol feeding, tends to return spontaneously to a normal value when the feeding is stopped. KI apparently does not accelerate this fall. (2) The normal decrease in adrenal weights and the prompt fall in the blood cholesterol occurring when cholesterol feeding is stopped are both inhibited by the administration of KI at the conclusion of the feeding period. (3) The high incidence of males among rabbits failing to develop a hypercholesterolemia with cholesterol feeding warrants further study.

The vitamins may be generally grouped into two classes, namely: (1) those soluble in fats and (2) those soluble in aqueous solutions. Vitamins A, D, and E are found nearly exclusively in association with fats, and vitamins B and C with substances soluble in water.

The reason for the distribution of vitamins in certain vehicles has not been definitely explained, although it may be readily surmised that specific vitamins probably deteriorate in solutions other than those in which they are found in nature. We have encountered such deterioration of vitamin D when emulsified in water.

Shelling and Tidwell 1 prepared oil-in-water emulsions of viosterol, which were miscible in water and in milk. They suggested that such emulsions offered a means by which larger amounts of vitamin D might be added to milk than was possible to impart by direct irradiation of the milk or by feeding vitamin D preparations to the cow. Subsequent clinical experiments 2 revealed that the emulsified oily solutions of vitamin D, when added to milk, were more than 10 times as potent as viosterol in oil, not so treated. Thus, in a study of 134 cases of severe rickets studied by Shelling and Hopper, 3 many of which were partially or entirely refractory to ordinary doses of cod liver oil, at least 20 to 30 drops of viosterol were required to heal the rickets completely in an average time of 3.7 months. On the other hand, with emulsions of viosterol, severe forms of rickets may be healed with as little as 1.5 drops of viosterol (125 Steenbock or 350 International units) in an average time of about 3 months.

In the first few months of the experiment, several rachitic children were given the emulsion and healing took place as expected, but after that time healing occurred very slowly and, in some instances, not at all.

Chlorazol-fast-pink, one of the azodyes, 1 , 2 , 3 has been found an effective anticoagulant in vivo. In the effort to avoid clotting in arterial cannulae in some pharmacological studies of the circulation of the dog fish, the author has attempted to use this substance. Huggett and Rowe give its M.L.D. for rabbits as 300 mg. per kilo and Feldberg has found that in dogs 80–100 mg. per kilo in 8% solution given intravenously did not exert any deleterious action on heart or vessels. However, the author in some 20 experiments found that the intravenous injection of as little as 20–30 mg. per kilo of this dye in 3% solution regularly depressed the heart and the blood pressure markedly and that about 100 mg. per kilo was a lethal dose.∗

This report is made in the belief that it may be of value to any one contemplating the use of this dye in fishes.