Experimental Biology and Medicine

It has been pointed out by one of us 1 that pitressin (Vaso-pressin) caused a marked drop in blood pressure and decreased pulse rate in the unanesthetized dog. The decreased pulse rate we then believed to be of vagal origin. The present work is a continuation of these earlier experiments.

The effect of the drug upon the heart was noted by recording the electrical potential changes in the action current by the string galvanometer in leads I and III, although usually only the latter lead was employed. Unanesthetized dogs were used. The pitressin was injected intravenously in small doses 0.1 to 0.5 cc. and in some cases followed by the injection of atropine sulphate in doses of 0.2 mgm. or more per kilo. In other experiments the atropine was injected intravenously before the pitressin and other experiments were performed on animals in which the vagi had been severed under ether anesthesia a sufficient time earlier for the animals to have recovered.

In a series of unanesthetized dogs the effect of pitressin upon the blood pressure and pulse rate was studied by means either of a mercury monometer or of a membrane monometer. The pulse rate was counted before, during and following the fall in blood pressure. Other conditions were similar to the above described experiments.

The effect of pitressin upon coronary flow was studied in excised perfused rabbit's hearts.

Results. The electrocardiographic studies in the normal unanesthetized dog shows the heart slowed, 10 to 15 seconds after the injection of pitressin. This slowing was quickly followed by an acceleration which was subsequently followed by marked slowing of the heart, which persisted for several minutes. During the period of excessive slowing high branching of the T-wave occurred with a marked increase in the height of the wave.

In 1927 Kempner, 1 working in Warburg's Institute, demonstrated the disappearance of oxygen and the development of CO₂ in the plasma of chickens suffering from fowl-plague. He obtained the plasma by adding heparin to whole blood taken at the height of the disease. The plasma was placed in Warburg's manometric apparatus, saturated with a mixture of CO₂ and O₂ and kept at constant temperature. Readings were done for several hours and changes in pressure noted. Since the plasma from normal chickens did not show respiration and the changes observed were sufficiently great to be outside of experimental error, Kempner concluded that the respiration was connected with the metabolism of the virus.

Using Warburg's apparatus and the technic described above, we repeated the experiments using plasma obtained from pigs injected with hog-cholera. It was impossible to obtain virus of fowl-plague either in the United States or in Canada. Plasma from cases of measles was similarly used as there is considerable evidence for its being a virus disease. In addition plasma from miscellaneous affections, including rheumatic fever, was similarly studied.

Our results were as follows: (a) Experiments with normal human or animal plasma did not show a disappearance of oxygen. (b) A suspension of red cells from chickens was similarly used. Respiration was measured and found to occur in agreement with the values obtained by Warburg 2 for goose-blood cells. (c) Hog cholera plasma was obtained from experimentally infected hogs at the height of the disease. The animals were killed by cutting their throats and the blood caught directly in large sterile test-tubes containing heparin. Autopsy in each case showed that the animal had been suffering from hog cholera. Blood from 15 hogs was used and in most instances duplicate experiments were done.

By means of direct quantitative carbohydrate determinations employing the Shaffer-Hartmann blood sugar method I have shown there is a rather wide utilization of carbohydrates by organisms of the genus Mycobacterium.

The reaction changes both in plain broth and carbohydrate broth cultures of the organisms of this genus are toward progressive increase in alkalinity. The reaction change is less rapid in the presence of utilizable carbohydrates. This increase in alkalinity whether in plain broth or carbohydrate broth cultures has been shown to be associated with an increase in the ammonia content of the media. This increase in ammonia is in most cases approximately equivalent to the increase in the titrable alkalinity. In other cases other substances either acid or alkaline in character, which are apparently derived from protein cleavage, are involved to some extent in the causation of the reaction changes and changes in titratable alkalinity. Such changes are accounted for by the ammonia increase equally as well in the carbohydrate broth cultures as in the plain broth cultures.

The ammonia production was found to be distinctly less for the production of a given amount of growth in the presence of utilizable carbohydrates. This diminished ammonia production was associated with a diminution in the degree of reaction change of the media. Also the higher the carbohydrate concentration, so long as it is within the limits that will permit growth, the less the ammonia increase and the less the reaction change of the media during the production of a constant amount of growth. Thus not only the presence, but also the concentration of a carbohydrate, is a factor determining the degree of protein sparing action it exerts.

The presence of utilizable carbohydrate does not directly have any effect upon the reaction change of the media.

In former investigations from this laboratory Loeb, 1 Gray 2 and Rabinovitch 3 have shown that potassium iodide may exert, a stimulating effect on the thyroid gland of the guinea pig. It does not prevent compensatory hypertrophy, but may modify its character; in the normal gland it may increase the mitotic activity as much as 40-60 times, and quite commonly increases it 20 times. Furthermore, it produces a slight increase in the size of the acinus cells and a slight softening of the colloid and a very marked increase in the number of phagocytes in the colloid. Loeb 4 has shown that oral administration of anterior pituitary substance (Armour & Co.) prevents compensatory hypertrophy of the thyroid gland and McCordock 5 has shown that it prevents the hyperplasia caused by potassium iodide. However, if instead of oral administration of anterior pituitary we give daily subcutaneous injections of either acid or alkaline extracts of anterior pituitary, no inhibition of the thyroid gland results, but, on the contrary, a very pronounced stimulation (Loeb and Bassett 6 ). Hyperplasia and hypertrophy of the acinar epithelium, liquefaction and absorption of the colloid proceed very rapidly. After 7 daily injections of such extracts extreme changes are found in the gland resembling in many respects those obtained in typical cases of Graves disease.

We have then in potassium iodide and in acid or alkaline extract of anterior pituitary gland 2 substances which stimulate the thyroid gland of the guinea pig and which produce a very pronounced cell proliferation in the epithelium of the acini. They differ, especially, in that the hypertrophy and softening of the colloid caused by KI is very slight as compared with the pronounced effects of anterior pituitary extracts and secondly, in that, under the influence of anterior pituitary extract, the colloid is rapidly absorbed from the acini, whereas under the influence of KI it is only slightly softened and largely retained in the acini.

A sign usually considered to be pathognomonic of coronary thrombosis is the high branching of the “T” wave of the electrocardiogram. It appears soon after the accident of coronary occlusion and reaches its maximum intensity within a few hours; and disappears after a few days. Although the mechanism of the change is unknown, it has been attributed to a number of factors, among which may be mentioned the influence of the current of injury on the normal change of electric potential of the heart, 1 anoxemia of the heart muscle 2 and asphyxia.

The following experiments enable us to draw certain conclusions concerning the mechanism of the change.

Dogs were anesthetized with amytal and were placed under conditions of anoxemia. To accomplish this, they were connected by means of a tracheal cannula to a large dead space into which the animal rebreathed and from which the carbon dioxide was absorbed with soda lime. They, therefore, breathing air from which the oxygen was gradually reduced, passed into a state of anoxemia, and carried to a point of complete asphyxia at which time the oxygen content of the arterial blood was as low as 20%. When the oxygen saturation of the arterial blood fell below 50% of the normal, the animals showed in their electrocardiogram changes similar to those observed clinically in coronary occlusion. The release of the animal from the anoxemia state caused the electrocardiogram to return to normal.

As was pointed out by Gruber and Kountz, 3 a picture similar to anoxemia was produced by the injection of pitressin in normal unanesthetized animals. They have also shown that one of the primary actions of the drug on the heart was that of coronary constriction. The change in the electrocardiogram produced by the drug could be caused to disappear by vasodilator substances such as sodium nitrite.

The concept that the highest neurological levels are functional parts of the peripheral arcs is becoming more and more widely accepted. Of special importance for this concept is the work of Hoffmann, 1 Travis and Dorsey, 2 , 3 Herren and Haterius, 4 and Coghill. 5 Hoffmann and Travis and Dorsey found that both anatomical and physiological alterations of the higher nerve centers alter reflex time in humans, Herren and Haterius found that functional changes in the higher nerve centers of the rat produce changes in the reflex time. All of these workers used the action current technique for measuring the reflex response latency. Further experiments in which this same technique was utilized furnish additional evidence for the proposition that the highest levels of irradiation are functional parts of the lowest levels of irradiation.

In the first study patellar reflex records were secured on 122 and Achilles reflex records on 119 normal adults. The reflex time in both reflexes regularly correlates higher with standing height than with measures that should more closely approximate the true lengths of the peripheral arcs.

A second study shows that during severe tonic blocks in stuttering the patellar reflex time is reduced, whereas in the normal speech of both stutterers and normal speakers it is not affected. The reductions in 8 severe cases varied from 10 to 50% of the reflex time during silence. There appears to be some relationship between the severity of the block and the amount of reduction of the reflex time.

A third study shows that alcohol decreases the patellar reflex time in dogs. In general each of 5 dogs ranging in weights from 31 to 37 pounds was given intravenously rapidly 20 cc. of 97% alcohol in 80 cc. of normal salt solutionX.

The data herein presented include only a part of an investigation of the disturbances in respiration due to submersion in water of various temperatures.

Wasserman, 1 Bazett, 2 and Hill and Flack 3 made observations on the effect of bath temperature changes on respiration, but we have failed to find a complete picture, beginning with cold and ending with hot water. However, the results reported by these investigators parallel our findings.

In securing the data, use was made of the constant temperature baths provided by the Psychopathic Hospital. Each subject was placed in the tub in a prone position during which time a record of normal respiration was taken. Following this the tub was filled, temperature changes made and data recorded as indicated by Fig. 1. Time is recorded in 5 sec. intervals.

The data presented in Fig. 1 show that there are 3 distinct changes in the respiratory movements when a subject is placed in a water bath varying from 65°F to 116°F. During the filling of the tub the movements are irregular, becoming slightly faster and much deeper while the bath remains at 65°F. As the temperature is increased, the respiratory movements tend to become more quiet and much shallower. At 80°F the movements are practically normal as to rate but are very shallow. The shallow characteristic begins to disappear at 110°F. When 115°F is reached, the respiratory movements again become deep and irregular, assuming a form somewhat similar to those recorded in the cold bath.

In most cases, very little discomfort was experienced in the cold bath. However, when the temperature reached 105°F, the subjects perspired profusely. As the temperature was increased still more, symptoms such as nausea, dizziness and extreme irritability were observed.

The metabolism of blood cells can be estimated from their glycolytic activity in vitro. This rate of glycolysis has been studied in the blood of patients with pernicious anemia during a remission induced by liver extract. In all of 5 cases the amount of sugar glycolyzed increased abruptly at the beginning of the remission. In 2 of the 5 cases the increased glycolysis during the reticulocyte crisis could he explained by an increase in erythrocytes and leucocytes. In the other 3 cases some of the increased glycolysis seemed to be attributable to the increase in the reticulocytes per se. This might indicate that young blood cells have a more active metabolism than adult cells.

The larval nictitating membrane, as it occurs in the frog tadpoles prior to the onset of metamorphosis, consists of a small mass of tissue imbedded within the integument bordering the anterior end of the eye socket. During metamorphosis this mass of tissue undergoes rapid growth which results in the formation of the nictitating membrane.

It seemed likely that extirpation and transplantation experiments with this mass of tissue before its transformation might throw some light upon the nature of the development of the nictitating membrane. By means of operative technique parts of it were removed and transplanted to other locations on the body. The animals, used were normal tadpoles of the species, Rana pipiens.

The total extirpation of the integument in the region anterior to the eye socket in which this tissue is imbedded resulted in the failure of the nictitating membrane to be formed during metamorphosis. Removal of any part (i. e., anterior half, posterior half, dorsal or ventral half) of this mass of tissue in the larvae, failed to hinder in any way the formation of a complete and well developed nictitating membrane during metamorphosis.

Autoplastic transplants to the back, of a section of the integument surrounding the eye and including the conjunctiva and the undifferentiated mass of tissue, resulted, during metamorphosis, in the formation of a mass of tissue resembling a partially formed nictitating membrane.

Extirpation of the integument along the ventral border of the eye which forms the lower eyelid and upon which the nictitating membrane normally forms, resulted in the partial regeneration of this area and the formation of a perfect nictitating membrane during metamorphosis.

It may be concluded that the small mass of tissue imbedded within the integument at the anterior border of the eye is undifferentiated in the larval stages of the frog, and that growth and differentiation are due primarily to hormonic influences released into the blood stream during metamorphosis.

That the incidence of gangrene in a healthy extremity, whose main arterial supply has been suddenly interrupted, is reduced by the simultaneous or early obstruction of the concomitant or proximal venous return has been demonstrated repeatedly by clinical and experimental observers.

The physiological explanation of this phenomenon, however, is in part still the subject of controversy. It is recognized by all recent workers that ligation of the venous return causes an increase in the blood pressure of the affected extremity. Opinions as to its effect upon the flow of blood, however, differ widely. Brooks and Martin 1 believe that this procedure decreases the blood flow, while Holman and Edwards 2 maintain that the blood supply is increased. Neither of these 2 groups of workers measured the flow of blood directly.

Recently there has been devised in our laboratory a direct, continuous volume flow apparatus, a modification of the Ludwig stromuhr and of Stolnikow's double inlet-outlet apparatus, which is controlled by an electrically regulated valve. 3 By means of this instrument we have measured directly the changes in blood flow to an extremity, resulting from ligation of its principal peripheral arterial supply and venous return. A preliminary report on this work forms the basis of this paper.

Male dogs, weighing 13 to 17 kg. were anesthetized by the intravenous injection of 200 mg. per kg. of sodium barbital. Coagulation was prevented by 30 mg. of heparin per kg. The volume flow apparatus was placed in the right iliac artery, thus measuring the blood which entered the right femoral artery as well as the chief collateral arterial supply to the right leg. The systemic blood pressure was taken from one of the carotid arteries. The right superficial femoral artery and the right common iliac vein were exposed to permit ligation.

Carrell 1 by the injection of chick embryo pulp in conjunction with dilute arsenious acid obtained in fowls a tumor of sarcomatous type. The result was the same whether the embryo was mixed before injection with the arsenious acid or the two injected simultaneously at different places. White 2 repeated these experiments and confirmed the results. Askanazy 3 transplanted rat embryo into grown rats and added Fowler's solution to their drinking water. In each of 2 series in which arsenic was used one rat was found to have a tumor of a malignant character. In one, the examination was made 13 and in the other 15 months after the embryo transplant. In the experiments of Carrell and of White the tumors developed as early as the ninth day and rather frequently in the fowls receiving the injections. From the results obtained in these investigations it appears that the response in the rat as compared with fowls is slower and less certain. The aim of our experiments was the investigation in rats of some of the variable factors, especially sites of injection and ages of embryos, which might explain slow and uncertain results.

Experiment I. The usual procedure with minor variations has been to draw much of the embryo into a syringe fitted with a short needle having 1 mm. lumen and at once to draw dilute arsenious acid to the 2 cc. mark. Of this suspension 0.5 to 1 cc. was injected. The time elapsing from removal of living embryo to injection was under 5 minutes. One of us (McJunkin) injected the testicles of 7 rats with a suspension of 3 mm. embryos in 1-150,000 arsenious acid. Six weeks later there was found in the testicle of one rat a minute opaque focus which microscopically consisted chiefly of cysts filled with cornified material.

In the experimental study of the cause of jejunal ulcer following gastro-enterostomy, 3 factors have become quite evident as playing an etiological rôle; first, the irritating and digestive properties of the gastric contents; second, the motor drive of the stomach, and third, the possible greater susceptibility of the jejunal mucosa as compared to the duodenal mucosa to the first 2 factors.

We have devised an operation in the dog for the purpose of analysing the relative rôle played by these 3 factors. The operation consists in (1) dividing the duodenum about one inch below the pyloric sphincter and the jejunum about 12 inches below the ligament of Treitz; (2) then the distal end of the jejunum is anastomosed to the proximal end of the duodenum; (3) the distal end of the duodenum is closed and the proximal end of the jejunum is anastomosed (end-to-side) to the distal ileum about 15 inches from the ileocecal valve. The gastric chyme on being ejected strikes the first inch of the duodenum and then passes into the jejunum. There is no pancreatic juice or bile to play a rôle in neutralization, since these secretions are diverted to the lower ileum. The principle of the operation is that the acid factor is constant for both the duodenal and jejunal mucosa, and the motor drive of the stomach affects chiefly the duodenal mucosa. If an ulcer occurs only in the duodenal mucosa, it would mean that it was caused by the acid plus the motor drive factor. If an ulcer occurs in both the duodenal and jejunal mucosa, it would mean that the acid factor was the chief cause. If an ulcer occurs in the jejunal mucosa only, it would mean that its mucosa is more susceptible to the action of the gastric contents than the duodenal mucosa.

In view of the existence of both humoral and nervous mechanisms for the regulation of the secretory activity of the stomach and pancreas, it was decided to investigate the possibility of such dual control of the activity of the salivary glands. Although it has been tacitly assumed that no hormone mechanism for salivary secretion exists, the literature contains no definite statement on the point. Babkin 1 makes no mention of it in his monograph on the secretory activity of the digestive glands. Langley 2 and Malloizel 3 found that substances which caused a flow of saliva when applied to the tongue were without effect on the paralytic secretion by the submaxillary resulting from section of the chorda tympani nerve.

Two methods were used in this work. The mucous membrane of the mouth and tongue of several dogs was extracted with 0.4% HCl. The application of this reagent to the tongue normally produces a marked flow of saliva. The extract was injected intravenously into dogs under barbital-ether anesthesia in which Wharton's duct had been cannulated. Quantities of extract sufficient to lower the blood pressure from 60 to 80 mm. of Hg were injected without any evidence of increased flow from the cannula. In each case stimulation of the chorda tympani nerve gave a marked response.

The second method was a study of the effect of the application of 0.4% HCl to the tongue of a dog in which one submaxillary gland had been denervated. A fistula of Wharton's duct was prepared in each of 2 dogs. After recovery from the operation, the dogs were placed in the stocks daily, and 0.4% HCl was applied to the tongue. A copious flow of saliva resulted. Three weeks after the first operation, the chorda tympani and cervical sympathetic nerves were sectioned, and the daily applications of acid to the tongue resumed on the second day after the operation.

In previous work 1 we were unsuccessful in attempts to record a gall-bladder contraction in the rabbit on the injection of cholecystokinin. It was reasonable to suppose by analogy from Graham's and Cole's 2 work, that we would be able to study the problem roentgenologically, after the administration of sodium tetraiodophenolphthalein. Graham, Cole and Copher 3 report that they have visualized the gall-bladders of rabbits after the subcutaneous injection of sodium tetrabromphenolphthalein, but add that these shadows were faint and inconsistent.

We hoped to get more consistent results using intravenous injections of tetraiodophenolphthalein. Because of our early failure in 4 rabbits to get shadows with the usual doses of 0.2 gm. per kilo, we increased the amount. Four rabbits were given 0.3 gm. per kilo, one of which showed toxic symptoms. One of 2 rabbits given 0.4 gm. died in 4 hours. Twenty-four rabbits were given the usual dose of 0.2 gm. per kilo, which proved fatal in 2 cases. This is in accord with Whitaker and Milliken, 4 who studied the toxicity of tetraiodophenolphthalein and tetrabromphenolphthalein, finding that 0.24 gm. is the largest single non-toxic dose for rabbits, Because of the possibility of missing the time of maximum concentration, which in dog and man is after 14 hours, the dye was given to fasting animals, and pictures taken every 2 hours until 40 hours after the injections. We were uniformly unable to obtain shadows of the rabbit's gallbladder. As control experiments we injected the same dye, in the same dose (0.2 gm. per kilo) in dogs and sharply defined shadows were obtained.

Because of this lack of concentration of the dye in the rabbit, we took specific gravity determinations. We found the rabbit's gallbladder bile to have a specific gravity of approximately 1.048, which is within the lower range of the specific gravity of gall-bladder bile of man and dog.

After Bayliss and Starling found that extracts of the upper intestinal mucosa on intravenous injection would cause the pancreas to secrete, numerous investigators have reported that such an active principle was present in various animal and plant tissues. Such an active principle has been reported to be present in Witte's peptone, stomach and colon mucosa, muscle tissue, brain, thyroid, liver, parathyroids, pineal gland, mammary gland, spinach, nettle, and hydrolized egg-white. All these extracts contain a blood pressure reducing substance; and since histamine increases pancreatic secretion, it may be that the augmentation of pancreatic secretion caused by these various tissue extracts may be due to vaso-dilation and not to a specific substance or secretin.

Having at hand the method of Weaver, Luckhardt and Koch 1 which yields secretin from the upper intestinal mucosa of dogs, hogs, sheep, cattle and man free of vaso-depressor substances, we decided to apply this method to various body tissues and to spinach. The tissues were collected from freshly killed animals, and were cut into small pieces, and 0.4% HCl was added in the proportion of 2000 cc. per kilo of tissue. The acid was allowed to remain in contact with the tissue for 1/2 to 1 hour, with frequent stirring. The acid extract was then strained off through gauze, and immediately saturated with NaCl (30 gm. per 100 cc.), as soon as the salt was dissolved the precipitate was filtered off, the filtrate being called B. The precipitate was then taken up in water and the insoluble residue filtered off and discarded. The soluble portion was called A. The extract A was used for injection. The extracts from the various tissues, brain, bone, liver, lung, kidney, pancreas, thyroid, stomach, intestine, colon, muscle, heart and spinach were injected in doses varying from 5 to 50 cc. No increase in the secretion of the pancreas was noted with any of these tissues except the extract of intestine and stomach.

This investigation was begun in the hope that the intravenous administration of ergosterol might throw some light on the mechanism of its action, if it could be shown to be effective by this method. Normal dogs of 10 to 12 kilos weight were selected and confined to a stock diet of ground beef heart and Puppy Meal, 3:1. Ten animals have now been studied. Routine determinations of calcium and inorganic phosphorus were made. Figure 1 shows the results on one animal, which is fairly typical of the series. After a short period of preliminary observation, injections were begun of ergosterol in corn oil. This oil alone, in amounts up to 15 cc. does not produce any of the observed effects. In the illustrative case. approximately 20 mg. were administered daily during 2 weeks. The calcium concentration was increased, but there were pronounced fluctuations for which no explanation can be offered at present. During 10 days, the animal gained weight rapidly and became extraordinarily active. During this time also, there were pronounced fluctuations in the concentration of inorganic phosphorus, but the average level was unchanged. At the end of this period it became apparent that the calcium concentration was decreased almost to the original level; so the daily dose was increased to 30 mg. The result was a second increase in calcium to almost 16 mg. at the end of another week. The dose was now increased to 50 mg. On the 25th day after the first administration, the calcium concentration was increased to almost 19 mg.; at this time the phosphorus had fallen to 4.7 mg. The dog had now begun to lose weight and had become less active. At the termination of the observation, post mortem examination of various organs, particularly of the parathyroids, will be made.

In order to study the physiological effects of certain alkaloids upon some of the lower organisms, and to determine, if possible, whether there was any evidence of specificity of action in these forms, the following experiments were planned: (a) A study of the effects of caffein on some of the physiological and morphological processes of the young and adult forms of Planaria dorotocephala, an organism with relatively labile tissue; (b) a study of the effects of caffein, atropine, and pilocarpine on cleavage and on the embryonic development of one of the lower organisms in which differentiation is rapid enough to permit of modification under the influence of agents acting only for a short period of time.

The first series of experiments, (a), concerned themselves with a study of the effects of caffein upon oxygen consumption, carbon dioxide production, and head frequency in Planaria dorotocephala. A record of differential disintegration in high concentrations of caffein also indicated a differential susceptibility to this alkaloid. No specificity of action was found for caffein in this form, and both stimulation and inhibition were recorded in the studies made with head frequency and with respiration, the effect depending upon the concentration used, and the length of the period of exposure. 1

The experiments on early cleavage will be reported in this paper, and studies on differential modification of development in Arbacia punctuation will be reported in the following paper. In the study of the effects of various concentrations of alkaloids on the rate of early cleavage of Arbacia eggs, the following procedure was observed: Stock solutions of a 0.5% concentration were made up for each alkaloid. From these, further dilutions were made up such that concentrations ranged from 0.1 cc. to 10 cc. of stock solution per 100 cc. of sea water.

In order further to test the effects of the alkaloids, Pilocarpine, Atropine, and Caffein upon development of Arbacia embryos, the following experiments were made: Fertilized eggs were allowed to develop in the various concentrations of alkaloids, as in previous experiments reported in the preceding paper, and in a paper by Mathews. 1 A 0.5% solution was used as a stock solution, from which dilutions were made from 0.1 to 10 cc. per 100 cc. of sea water.

The eggs were allowed to develop for 48 hours, after which time micrometer measurements were made of arm length, body length, arm width across the tips, width of the base, and the base and hypotenuse of a triangle which would include the oral lobe in optical section. Twenty-five embryos were measured from eggs of each of 3 females, for each concentration studied, and compared with the controls, measurement for measurement. On the basis of these calculations, it was possible to obtain percentage values for each dimension which indicated increase or decrease in growth rate relative to the same dimension in the control.

It was found that there may be either stimulation or inhibition of development in Arbacia larvae, and that the effect is dependent on the concentration of alkaloid used. As previously observed, Atropine is more strongly inhibitory than either Pilocarpine or Caffein, and Pilocarpine is more strongly stimulative than either Atropine or Caffein. Also, it was obvious that the effect in any case was differential. The regions of most rapid growth, i. e., the oral lobe region, and the aboral arms and median region between them, are most rapidly and most completely modified in their rates of growth. Both differential stimulation and inhibition were obtained with each alkaloid, depending on the concentration used.

Eight normal, 13 phlorizinized, and 9 depancreatized dogs under amytal anesthesia were studied during rest and exercise. Blood entering and leaving muscle and liver was analyzed for fat, using the method of Stewart and White. 1 The experimental error was ±20 mg. %, and differences of 40 mg. % or more were considered significant. The differences of the arterial and venous fat contents of the blood of the muscles of the normal post-absorptive dogs varied, since the muscles liberated fat 5 times and removed it on 3 occasions. Of greater interest are the observations of the diabetic dogs, since in 10 of 13 significant determinations on the phlorizinized dogs, and 12 or 15 on the depancreatized animals, the muscles of the lower extremities removed fat from the blood passing through them.

The results obtained on the blood of the liver were not the same in the phlorizinized and depancreatized dogs. The livers of the phlorizinized dogs usually removed fat from the blood, while those of the depancreatized dogs added fat.

Thus these results obtained by the method of Stewart and White 2 indicate the utilization of the fat of the blood by the muscles of diabetic dogs.

Collens and Murlin¹ have recently reported that the portal injection of insulin into dogs, in dosage of 0.05 to 0.1 unit per kilo weight, results in an immediate sharp rise of blood sugar of 20 to 80 mg. The rise occurs within 5 minutes and is then followed by a rapid decline. No such rise occurs following the systemic injection of the same dose of insulin. BÜBurger and Kramer 2 at about the same time reported that the injection of 10 to 20 units of insulin into the cubical vein of human beings produces a rise of blood sugar averaging 11.5%; intra jugular injection of 40 units into dogs of about 20 kilograms causes a rise averaging 28%; intraportal injection results in a rise averaging 46%. The rises occur within 5 minutes and are followed in 10 to 30 minutes by a rapid fall. In both of these reports, the results are interpreted as showing that insulin has a glycogenolytic action on the liver and that the hyperglycemia is a physiological or normal response to this action. Since such an action of insulin would have a bearing on work we were carrying out, we have gone into the matter in order to have a clearer understanding of its significance.

We have carried out experiments on dogs, corresponding to those described in the reports cited. We employed the Lilly insulin, as did Collens and Murlin, whereas the Burroughs Wellcome product was used by BÜBurger and Kramer. Using small doses, 0.1 unit per kilo, we obtained no rise in blood sugar on intrajugular injection, but a rise followed intraportal injection. Our maximum rise, however, was 15 mg. as compared to the 20 to 80 mg. rise of Collens and Murlin.

Lack of accord between infant feeding practice and metabolic principles has led to the present investigation. The effects of natural acid- and base-forming diets on the growth and metabolism of young rats have been studied from the physical, chemical and pathological standpoints.

Comparative studies were made on 2 groups of 22 and 25 young albino rats weighing between 30 and 35 gm. Litters of the same stock and age were maintained on acid- and base-forming dietaries, in sunlight and in darkness, during all the seasons.

At the end of 28 weeks the dietary acidity and alkalinity showed marked effect on the animals. The acid-forming diet depressed on the average the serum phosphorus from 6.1 to 5.7, the alkaline reserve from 48 to 44 vol. per cent, and total base from, 148 to 137 with a compensatory rise in the chloride from 280 to 305 and cholesterol from 140 to 150. The base-forming diet produced the converse changes, serum phosphorus from 6.1 to 7.5, alkaline reserve from 48 to 56 vol. per cent, total base from 148 to 154 vol. per cent. The albumin-globulin ratios, percentage fat and iodine numbers of the fat were all normal. The hemotological data were also unaltered in both groups.

The animals maintained on acid-forming diets showed rickets clinically and histologically while those fed on base-forming diets showed normal bones. The animals on the base-forming diet in particular showed the greater percentage of ash, 61%, and had heavier bones while those on the acid-forming diet showed 54% of ash according to analyses of the femora. The animals that had been exposed to the light showed a higher ash content, 54% compared with 49% of those in darkness. The rats on base-forming diets showed a more marked gain in weight, 17 gm. per week as compared with 7 gm. per week for the animals on the acid-forming diets.

In a study of the neuro-muscular mechanism of respiration, carried out on cats, it has been observed that, in many cases, division of the vagi is followed not only by a marked decrease of the respiratory rate, but also by a diminution in amplitude of diaphragmatic movements, while costal movements increase in depth. Subsequent section of the dorsal cervical (phrenic) nerve roots in these cats is attended by very little further change in respiratory rate or movement.

When, on reversing the order of experimental procedure, the dorsal roots of the cervical nerves (iii, iv, v, vi) are divided first, both a diminution in amplitude of diaphragmatic contractions and slowing in rate ensues, and subsequent division of the vagi brings about almost no further change in respiratory rate.

That respiratory impulses to the diaphragm may still bring about increased depth of diaphragmatic movement after section of the dorsal roots of the cervicals, has been shown by division of the dorsal spinal nerve roots of the intercostals in the thoracic region with the resulting diminution in costal movement. Under such conditions, there is an increase in the magnitude of diaphragmatic movement. This is similar to the increase in costal movements observed by us some years ago when, after division of the dorsal spinal nerve roots in the thoracic region, the phrenics were excised and an increase in the magnitude of costal movement was observed. 1

The fact that diaphragmatic movement and not costal, is- diminished in magnitude by division of the vagi, appears to indicate a closer relationship of the vagi to the phrenics than to the thoracic roots of the nerves concerned in the control of costal respiratory movements. These latter appear to have central stations as high as the posterior corpora quadrigemina, section behind which affects costal respiratory movement to a much greater extent than diaphragmatic. 2

Following the intravenous injection of concentrated, anti-pneumococcus horse serum, there frequently occurs a systemic reaction of varying intensity. The reaction comes on usually in from 30 to 60 minutes after the injection, and begins almost always with a chill, which is accompanied and followed by an elevation of temperature.

It seems that this reaction is not an essential factor in the beneficial effects of the serum, indeed it is considered by many to be a distinct disadvantage and to be responsible for some limitation of a more general employment of the serum therapy. Unfortunately there has been no method devised to determine, in advance of its use, whether the serum to be employed contains chill-producing properties, and so this knowledge is obtained only after the occurrence of the reaction in a treated patient. The present communication is a report of a successful imitation of the reaction in dogs, thus affording a method of determining before its clinical employment, the presence or absence of the chill-producing factor in any given sample of serum.

In the first experiments, it was found that a serum which had produced a typical reaction in a patient, produced a corresponding reaction when injected into the jugular vein of a dog. On the other hand, a serum which had failed to give a reaction in a patient, also failed to give one in a dog. Following these initial experiments, a considerable number of serums, which had been or subsequently were used in the pneumonia wards at Harlem Hospital, were tested. The experimental results were in striking agreement with the clinical ones.

The procedure in the animal test is as follows: The dogs selected are of the short-haired variety, of a weight between 5 and 10 kilos; the temperature is taken by rectum before and at 20 minute intervals after the serum injection; the thermometer is inserted the same distance for each reading; the serum is injected into the jugular vein, no local anesthesia or operative exposure being necessary.

Changes in teeth as a result of deficient diets have been reported by a number of investigators. McCollum, Simmonds and Kinney 1 produced a number of defects in the teeth of rats by feeding rachitic diets. Orban, 2 by means of histological examination, found poor calcification in the incisor teeth of rats on a number of deficient diets. Mellanby 3 , 4 reported poor calcification in the teeth of dogs and rabbits on rachitic diets. Toverud 5 obtained a small reduction in the ash, calcium, and phosphorus in the teeth of rats on a diet deficient in calcium. Perlzweig 6 found decreases in calcium and phosphorus in the incisor teeth of rats on diets low in either calcium or phosphorus, the low calcium diet giving a greater decrease than the low phosphorus diet.

The object of this investigation was to determine whether the calcium, phosphorus, and ash of the incisor teeth of rats would undergo changes similar to those produced in bone by a rachitic diet known to produce marked changes in bone. The Steenbock diet as modified by Epstein 7 was employed. This consists of the following and will be referred to as the basal diet:

Yellow corn meal 66%, wheat gluten 20%, egg albumin 10%, calcium carbonate 3%, sodium chloride 1%, spinach 10 gm. per rat per day, water ad lib.

Excluding the spinach, this diet contains 1.22 gm. Ca and 0.09 gm. P. (ratio 13.5 :1) in 100 gm. of the ration and produces marked rickets accompanied by good growth. 7 The addition of 2% KH₂PO₄ or 2% cod liver oil to this diet prevents rickets. 7

The teeth and bones (femur) were compared on 2 groups of diets as follows:

Group I—(1) Basal; (2) Basal + 2% KH₂PO₄; (3) Basal + 2% KH₂PO₄ + 2% cod liver oil.

The therapeutic effect of liver extract on splenectomized anemic rats was investigated with the principal object of inquiring into the possibility of developing a biological assay for liver extracts. Great similarity between this severe rat anemia and human pernicious anemia had been noted by several observers, Lauda going so far as to name the disease “perniziöse Anämie” in rats. 1 At the time of our research we were unaware of the recently completed work of Vedder, who found that liver extract therapy was of no value in the treatment of this anemia. 2

Our experiments confirmed this finding. In a series of 40 splenectomized rats (averaging 40 gm. in weight, the experimental animals being fed from 0.0125 gm. to 0.4 gm. of Lilly's liver extract No. 343 per day) no effects of the extract were noted as judged by the mortality rate, length of life after splenetic, and total erythrocyte count. Fifty-seven per cent of the experimental animals died in a period of from 6 to 26 days after splenectomy, average 15 days; 60% of the controls died in a period of from 5 to 25 days after splenectomy, average 13 days. No significant differences were noted in the erythrocyte counts of the control and experimental animals.

The results of previous work by one of us 1 indicated that the pyrimidines uracil and thymine, when fed in small amounts to dogs, are to a large extent metabolized. In the case of cytosine it was found that this substance was not utilized. It was partly excreted unchanged, partly deaminized and excreted in the form of uracil.

These experiments were undertaken to study the behavior of the pyrimidine nucleosides, uridine and cytidine. The substances were fed to dogs maintained on a nitrogen equilibrium. Our results show that these nucleosides are metabolized in the animal body, being broken down predominantly to urea. The behavior of cytosine in the nucleoside molecule is therefore in marked contrast to that of the free base, which is not utilized. Pentose determinations in the urine showed that the pentose sugar in the nucleoside molecule is practically completely burned; certainly not more than a trace is excreted in the urine.

Working with rabbits and man Wilson 2 previously concluded that when uracil, in the form of a nucleoside or nucleotide, is administered, only a small portion of the uracil is excreted.

Gigantism has been produced by Evans and Long 1 by the injections of an anterior pituitary extract in immature rats. The same result was obtained by Putnam, Teel and Benedict 2 , 3 when the extract was administered to a bulldog. Normal growth was induced by injections of this fluid in hypophysectomized rats although there was no evident repair of the thyroid, suprarenals or gonads. 4 Putnam, Teel and Benedict 2 state that “in hypophysectomized dogs and rats restoration of growth has been produced in preliminary experiments.”

The effect of the anterior pituitary extract upon a hypophysectomized puppy has been studied over a period of 2 1/2 months. A sterile bovine extract of the anterior lobe, the preparation of which is given elsewhere, 5 was kindly furnished by Dr. H. M. Evans of the University of California. This extract has been found active for the growth hormone when administered to rats.

By means of the intracranial approach of Dandy and Reichert 6 a total hypophysectomy was performed on an 8 weeks old female puppy. That a total extirpation was accomplished was indicated when the animal remained infantile and failed to grow over a period of 4 weeks. During this period the increase in length of both the femur and tibia, measured by skiagrams, was 6 mm. and of the skull 2 mm., while in the litter mate control the femur and tibia had lengthened 33 mm. and the skull 12 mm. The weight of the operated puppy increased 32 gm. while the control gained 88 gm.

Daily intraperitoneal injections (25 cc.) of the anterior pituitary extract were given during the following 2 1/2 months. At the end of this period the operated puppy although quite thin appeared in size the same as the control.

Fourth generation of subcultures of B. typhosus, S. viridans, and certain other microorganisms grown in 10% horse serum (Ringer's solution) are agglutinated in dilutions of 1:20 to 1:160 by antihorse rabbit precipitin, controls cultures (nutrient broth) showing no agglutination.

With eighth to twelfth generation subcultures of the same microorganisms, definite agglutination takes place with 1:1000 anti-horse rabbit precipitin. The susceptibility of eighth to twelfth generation subcultures to precipitin-agglutination is not materially reduced by repeated washings with Ringer's solution. No agglutination of these subcultures takes place with any heterologous precipitin thus far tested (e. g., anti-egg rabbit precipitin).

During the past year a careful systematic bacteriological study has been made upon the organs and body-fluids that were obtained from 168 necropsies. This communication is a preliminary statement concerning the results obtained and the technique employed.

The technique for the collection of the material from the organs and body-fluids consists of carefully searing the surface of the organ or vessel and removing a portion of the tissue. The material is immediately placed in a sterile receptacle and removed to the laboratory where various kinds of media are inoculated. The media used routinely for every organ and fluid is (1) a surface infusion agar plate that contains 5% rabbit blood; (2) infusion broth; (3) Holman's Meat Media and (4) Sabouraud's agar. In addition, for each fluid specimen obtained, 2 pour agar plates are prepared for the purpose of a quantitative estimation of the organisms present. Direct smears that have been stained by a special bacteriological stain have recently been added as a routine procedure for every organ or fluid cultured. This is to serve as a microscopic check for the cultured findings.

The bacteriological studies were made upon necropsies from many different pathological conditions, including deaths from acute and chronic causes and several deaths due to accidents. There was, in 75% of necropsies, an average of one to 5 hours elapsing between the time of death and the time of culturing. Very few necropsies had an interval of 24 to 48 hours before culturing. Of these, it is surprising to find that the greater portion were free of organisms. However, it was found that if there were 7 to 9 hours elapsing between time of death and time of culturing there would be an increase in the frequency of the occurrence of anaerobic organisms within the tissues.

Intravenously injected horse proteins are retained quantitatively in the normal canine circulation for at least 4 days, by which time they are so far denatured as to call forth no recognizable anaphylactic reaction on massive blood transfusion into horse-protein-hypersensitive recipients. 1

No anaphylactic denaturization is demonstrable at the end of 4 days in endothelial blockaded (India ink) dogs. Approximately half of the routine protein dose remains anaphylactically active as late as the ninth day in these animals, and about an eighth as late as the fourteenth day.

This paper summarizes the results from 20 transfusion tests. The technique has been previously described. 1

The diets shown in Chart 1 were designed for the purpose of determining the influence of large quantities of “roughage” upon the rate of growth. Ration 1 contains 10% cellulose while numbers 2 and 3 contain 20%. Diet 4 contains 20% of agar-agar. Nos. 1 and 2 contain moderate amounts of fat while numbers 3 and 4 have 45% of their total weight in the form of lard and cod liver oil. All rations were designed to contain adequate amounts of protein, fats, carbohydrates, mineral matter and vitamin supplements. The levels for protein and mineral matter were increased for rations 3 and 4 in accordance with the procedure of Smith and Carey. 1 The salt mixture employed was that of Osborne and Mendel. Cellophane was selected as a source of cellulose since it represents a very pure form. In addition to the mixed diet all animals were fed separately a daily allowance of 200 mg. of vitavose and 3 drops of cod liver oil.

Ten male rats were reared from the time of weaning upon rations 1 and 2. These showed a marked decreased rate of growth when compared with a control group whose growth rate is shown by curve C while representative rates for the other groups are shown by typical curves properly numbered. The control ration was identical with number 1 except that the cellulose was replaced by the same per cent of sucrose. Curves S are those for males and females reared upon our stock ration. These are described elsewhere by Maynard. 2 Since we obtained subnormal growth with rations 1 and 2 containing 10 and 20% cellulose we felt that this might be due to the inability of the rat to consume sufficient calories.

A fairly large number of persons show a marked urinary alkalinity which persists through the morning period. This alkalinity may be independent of diseases of the kidneys and urinary tract, and of the type of diet preceding the test. It may not be present during the afternoon period. It probably represents an unusual adjustment to waking conditions, exaggerated in most instances by the secretion of hydrochloric acid by the stomach.

The reason why such an adjustment is different in these cases from that more commonly observed did not appear in a study of the type of patient upon whom the results were obtained. It seems to the authors that the following general statement covers the fact: there are 2 methods of compensating for a tendency towards alkalosisthrough the urine and through the lungs. In these subjects the compensation is accomplished through the kidneys to a greater extent than is the case in most persons.

As the result of investigations carried out by Francis, it has become customary to test for the presence of Bact. tularense in animals dead from tularemia by the injection of a suspension of liver or spleen into guinea pigs. Francis 1 has pointed out that Bact. tularense gradually disappears from the liver and spleen of animals dead from tularemia. He 2 has demonstrated that Bact. tularense in spleen tissue may be kept viable 30 days or longer by storage in 50% glycerine at ice-box temperature. He states that liver is inimical to the life of the infection and, when stored in glycerine with spleen, will destroy the infectivity of the spleen tissue. In his investigation of rabbit livers from the Washington Market, Francis 3 found Bact. tularense viable in rabbits shipped from Tennessee, but the lapse of time after death was unknown.

We have investigated the longevity of Bact. tularense in the liver and spleen of animals dead from experimental tularemia and have gathered comparative data with respect to the longevity of the organism in muscle tissue. Tissues from one guinea pig and 2 rabbits were injected at intervals into 142 guinea pigs used as test animals.

Test No. 1. The carcass of a guinea pig dead from experimental tularemia 4 days after inoculation was kept at room temperature (ca. 20°C). The spleen and liver were thickly studded with necrotic areas. At intervals portions of the liver and spleen of approximately the same size were removed and emulsified in normal saline solution. The suspensions were immediately injected subcutaneously into guinea pigs. The results are shown in Table I.

The work of Stockard in varying the normal environment of developing eggs with resulting malformations suggested carrying out similar experiments on a warm blooded animal, such as the chick. The eggs were placed in an incubator for 16 to 18 hours prior to operation. A piece of shell was removed, and in some instances 3 drops of 1/1000 solution of 95% alcohol were added to the side of the embryo, in others, 3 drops of normal saline (both at room and incubator temperatures), while a few eggs were carried along as controls.

At various times, chiefly 48 and 72 hours later, the eggs were reopened and where embryos existed, they were fixed. Photographs were made and then the specimens were sectioned. The control eggs showed no abnormality.

Results: Total eggs treated with saline—–18

Total eggs treated with alcohol—–27

Of these 5 were completed for microscopic study; of them all fail to show a pituitary gland; 4 showed optic defects; 2 showed failure of closure of the head folds and in one no olfactory pits appeared. It made no apparent difference in the results whether the environment was changed with weak alcohol or normal saline solution. The treated embryos exhibited malformations of the nervous system. The abnormalities were chiefly (1) an absence of the pituitary gland, (2) in some an absence of an eye, (3) failure of the head folds to close, (4) in one, an absence of the olfactory pits.

In a previous communication 1 we reported the results of splanchnic analgesia in the treatment of experimental ileus, and concluded that this form of treatment is undoubtedly of great value in reestablishing motility in the intestinal wall. The present communication concerns the relative value of splanchnic analgesia and spinal analgesia. Our results are based upon a series of 70 dogs. In 50 animals novocain splanchnic analgesia was produced, and records of the blood pressure, intestinal motility, and respirations were obtained on the kymographic drum. In most of the cases a single tracing of intestinal motility was obtained, and this from the terminal ileum. In some cases, however, additional tracings were obtained from the duodenum and also from the colon. In 9 animals instead of using novocain an aqueous solution of nicotine was used, as advocated by Rosenstein and KÜhler. 2 Nicotine is apparently an unsatisfactory substance when used in this way, since it increases the blood pressure unduly, and fails to increase the intestinal motility to a satisfactory degree. The range of blood pressure rise in all but 3 animals was greater than 90 mm. of mercury, and, while in 5 cases the tone in the ileum was raised from 10 to 50 mm., as recorded by the writing point on the kymographic drum, the other 4 animals showed insignificant effect on both tone and motility, and in the case of the duodenum and the colon the tone of the intestine was actually decreased.

The results of the present investigation, with respect to novocain, show that both the tone and amplitude of intestinal movement are markedly increased in the ileum, the average figure for the tone being an increase of 29.5 mm. and in the amplitude a value of 11.2 mm. The induction of novocain splanchnic analgesia, however, reduces the blood pressure, but not unduly, the average figure being 20 mm. of mercury.

Experiments were undertaken to throw light on the controversial question of the relative value of local applications of heat and cold on the course of peritonitis. They are based on 119 animals. Of these, 9 were sacrificed in obtaining cultures.

Experimental peritonitis was produced in dogs by the intraperitoneal injection of combined anaerobic and aerobic cultures obtained from animals in which a primary peritonitis occurred as a result of artificially produced obstruction of the cecal appendage. Peritoneal fluid was obtained with hypodermic syringe and needle and inoculated into tubes of meat digest broth, one test tube being incubated under anaerobic and the other under aerobic conditions. The 24 hour cultures of the 2 tubes were then mixed, and equal quantities of the resultant mixture introduced into the peritoneal cavity by hypodermic needles, the skin having previously been shaved and sterilized. The injection was uniformly made into the left lower quadrant at a point approximately midway between the umbilicus and anterior superior spine of the ilium. The animals were then divided into 3 groups. One group was used as control, and received no treatment except the administration of hypodermoclysis. A second group received, in addition to hypodermoclysis, a Leiter coil over the area of injection, through which a continual stream of cold brine flowed. In the third group a similar application to the abdominal wall was made, but hot water was passed through the coil. Observations every 3 hours included rectal temperatures and temperatures of the coil, the latter being obtained by a thermometer inserted between the lower surface of the coil and the abdominal wall. Hemoglobin estimation and leucocyte and differential blood count were made every 24 hours during the survival of the animal.

The following observations have been made in the course of preliminary studies with 48 cultures of the brucella group on 74 rhesus and 14 cynomolgus monkeys:

A single oral administration of 21 different Br. abortus strains produced in 24 Macacus rhesus and 1 M. cynomolgus monkeys nonfebrile infections, followed by the formation of specific agglutinins of moderately high value. The dosage varied from 7 to 400 million and in some experiments it consisted of many billions. The strains identified serologically as abortus or para-abortus varieties and in the dye test as “bovis” or “melitensis” types had been isolated from bovine pathological specimens in the United States, Germany, Hungary, Italy and Switzerland.

Blood cultures have not been successful. The value of the serum agglutinins and their persistence depends on the feeding dose. Rapid disappearance of the agglutinative power to a low titer or to the zero point is worthy of note. A cutaneous application of approximately 20,000 bacteria has induced an infection. The incubation period as indicated by the appearance of the serum reaction varied from 9 to 30 days and is influenced by the infective dose.

The absolute evidence of infection has been secured through the recovery of the organisms from the tissues of 4 monkeys which have been sacrificed on the 34th to 52nd day. Three animals killed on the 43rd, 56th, 199th day furnished sterile cultures. Probably every Br. abortus strain when fed in sufficiently large dosage is pathogenic provided susceptible monkeys are used.

By feeding 100 million Br. abortus type “suis” of bovine, but in all probability of porcine origin which has retained its characteristics through the passage, a febrile disease with anatomical lesions indistinguishable from those of a Br. melitensis infection has been produced.

As shown previously 1 he addition of a water insoluble acid to a fat mixture leads to basophilic staining and a relative positive potential. The addition of a water insoluble base has the opposite effect on both properties. In tissues, basophilic staining is also associated with a positive potential, acidophilic staining with a negative potential. Water immiscibility of the acid or of an added base is essential. By the addition of a water-soluble acid or base both stain ability and e. m. f . are influenced in opposite directions.

An effect of this type is observed in Loeb's gelatin systems,† e. g.,

In this case also the basophilic substance is on the side of the positive pole; as a general rule we may consider that almost any system—

produces an e.m.f. as indicated. A few exceptions to this rule have been observed in certain artificial systems which, however, are composed of substances not likely to occur in tissues in general and hence have a minor biological importance (details about these observations will be reported in a later publication).

According to a recent observation of R. Chambers 2 the nucleus has a higher pH value than the protoplasm indicating a slight alkalinity. This alkalinity, as well as the nuclear content of water-insoluble acids, probably bound to proteins as nucleo-proteins, would explain its basophilic stainability. Consequently, according to our experiments, the nucleus should be electrically positive as expected by G. W. Crile some time ago.

Whether the concomitant changes of stainability and e.m.f. are brought about in tissues chiefly by variations of its water-soluble or of its water-insoluble constituents can not yet be decided.

To prove this point new experiments have been performed. A mixture of equivalent parts of a fatty acid and an oil-soluble amine in a neutral fat or other similar solvent was used. This mixture when shaken with aqueous buffer solutions of varying pH shows the same type of stainability as does gelatin, namely: staining acidophilic below a certain pH value; and basophilic above a certain pH value, forming the following cell system:

It has been found 1 that eosin, an acidophilic dye, is taken up by a fat mixture containing an oil soluble base, while a basophilic dye, methylene blue, dissolves in a fat mixture with an oil soluble acid. The fat mixture with a base is electrically negative against the mixture with an acid.

Considering the general character of these findings, it seems probable that the same conditions hold also in tissues; hence our model experiments are suitable for explaining that relation of stainability to electromotive forces which has thus far been found in tissues. To corroborate this finding we have performed the following comparative experiments. Forty-three different dyes were used for testing the differential stainability of artificial mixtures such as those mentioned which contain either oleic acid or an amine, and this was compared with the stainability of white blood cells. It was found in each case that a dye which was taken up, exclusively or preferably, by a non-aqueous solution of a higher fatty acid, produced a differential stain on the nucleus of white blood cells. On the other hand, any dye with a preference for a mixture containing a fat soluble base preferably stains the cytoplasm and leaves the nucleus unstained, or but slightly stained.

Many nuclear stains like methylene blue are water-insoluble bases. Evidently, they are taken up by a solution of oleic acid in a suitable solvent, forming, e. g., methylene blue oleate, which is oil-soluble and hence stains. However, there are also nuclear stains which are not bases, e. g., hematoxylin, a widely used nuclear stain which has all the characteristics of an acid. It was of special importance to test the type of oil solubility in this case.

The results strongly support the probability that changes in pH can determine the directional action of the reversible ferment, urease. Toward the alkaline side of the isoelectric range the tendency to convert urea into (NH₄)₂C0₃ predominates; toward the acid side the tendency to synthesize urea is more pronounced.

In conclusion the writer wishes to express his gratitude to Dr. M. Garcia Banus for his advice and supervision of the work.

Of the substances normally present in the diet, structural relationships point to arginine as the most logical precursor of creatine and creatinine. In view of the recent work of Benedict and Osterberg, 1 who fed creatine over long periods of time, and the confirmation of their work in man by Rose, Helming and Ellis, 2 it seemed possible that earlier attempts to demonstrate that creatine may originate from exogenous arginine failed because the amino acid was administered over too short a period to observe any significant change in the excretion of creatine or creatinine. Therefore it was decided to study the effect of arginine on the creatine-creatinine elimination, when the arginine was fed daily to a dog over a period of several weeks. After the completion of the experiment, Hyde and Rose 3 published the results of a similar study of prolonged administration of arginine to a normal man and woman. Although their results showed no evidence of the conversion of exogenous arginine to creatine or creatinine, they have suggested that other species, particularly the pig, 4 may differ in their response to arginine feeding. Our results presented in Table I, with another species, the dog, confirm those of Hyde and Rose, in that oral administration of arginine for a period of 35 days failed to influence the excretion of urinary creatine or creatinine, although exogenous creatine (Periods XIII and XIV) in small amounts resulted in prompt increases in both these catabolites, thus confirming the earlier work of Benedict and Osterberg.

Our experiment was carried out with a trained metabolism dog, female, of about 14 kg. weight. Urine was collected by catheterization daily and analyzed for creatine and creatinine by the standard micromethods of Folin.

It has been previously demonstrated by us 1 that an exclusive diet of egg albumin is not capable of promoting growth in Planaria maculata. Although the food is well taken, the worms show progressive decrease in size throughout the experimental period. Upon an exclusive diet of egg yolk the worms may maintain or increase their size for a period of 2 or 3 weeks, but after that time their length becomes progressively less.

We have confirmed these results repeatedly with the species of planarian worm (Planaria agilis?) which we are at present using, and have found in addition that a combination of egg yolk with egg albumin produces a diet decidedly superior to either substance used alone.

Each group in an experimental series consisted of 30 worms of equivalent size which were fed ad lib. twice a week on the experimental diets. The total length of the worms was found at the beginning of the experiment and at its conclusion and the proportional increase in length was determined. In one experiment of 2 weeks duration kept at room temperature, the group of 30 worms fed upon egg yolk made a gain in total length of 3.9%, while those fed upon a combination of equal parts of egg yolk and albumin gained 20.2% in total length. In another series incubated at a temperature of 27° C. over a 2 week period, egg yolk diet produced an increase in total length of 32%, while a diet of blended egg yolk and albumin in equal parts gave an increase in total length of 51.5%. In a third series pastes were made of 15% liver pulp and 45% starch paste (10%) with the addition of 40% egg yolk or egg albumin or a mixture of equal parts egg yolk and albumin.

The exudates in the peritoneal cavity of the frog and guinea pig have recently been used 1 as culture media for cultures in vitro. In the present experiments, various tissues of the albino rat have been successfully cultivated in the peritoneal cavity exudate of the same animal. However, the growth of certain tissues, which it was particularly desired to cultivate was not so vigorous as hoped for. Consequently an endeavor was made to find a culture medium better adapted for the cultivation in vitro of these tissues.

Attention was centered upon a medium containing embryonic tissue juice because of the favorable results obtained by various investigators with this type of medium. It was felt, however, that the usual method of obtaining the embryonic tissue juice by mincing the embryonic tissues in a saline solution might be improved upon by using body fluids normally present in the embryo. There was the possibility that these fluids might contain the growth promoting substances and thus obviate the necessity of adding embryonic tissue juice to them.

In working with newborn albino rats it was observed that a clear fluid would flow from little cuts in the skin, and it was decided to try this exudate. However, there was considerable difficulty in getting a sufficient supply for use as a culture medium. It was found that a much more abundant supply of body fluid could be obtained from the embryo by inserting the point of a fine bore pipette through the skin and frontal bone into the underlying cranial cavity. Care should be taken not to insert the point of the pipette too deeply or the brain tissue may be disturbed and bits of it be taken up into the pipette.

In a previous communication we 1 have reported that the total loss of gastric juice from the body causes death in the dog in from 5 to 10 days, with symptoms of depression and marked changes in the blood chemistry. These may be summarized as follows: a progressive decrease in the concentration of blood chloride, an increase in the CO₂ capacity, an increase in the pH, and a late increase in the NPN and Urea N. Water and salts are not absorbed to any appreciable extent in the stomach or duodenum and it is therefore clear that any factor such as obstruction at the pylorus or in the duodenum, gastric or duodenal fistula, or profuse persistent vomiting, will lead to this loss of gastric juice constituents through failure of reabsorption in the lower intestine. It is likely that the property of the gastric mucosa, by virtue of which it can continue to separate the elements for its secretion from the altered blood plasma until death is produced, is of major importance in the pathogenesis of these disorders. In the present communication we wish to report a case of acute post-operative dilatation of the stomach together with evidence which indicates that here also the failure of reabsorption of gastric juice is the cause of death.

The patient, a female of 70, was brought to the hospital with a strangulated femoral hernia. The findings are unimportant for the purpose of this paper except for the blood chemistry report, which is as follows: Chloride 196 mg., CO₂ 60 cc.; Urea N 14.0 mg., and NPN 31 mg.

There had been no vomiting. A blood transfusion (500 cc. citrated blood) was given and a section of gangrenous ileum, imprisoned in the hernial sac, resected.

Administration of theophylline ethylenediamine intramuscularly to rabbits calls forth an acute and profuse diuresis. In an extensive physico-chemical study of the initiating events, as well as of the subsequent diuresis, it was found that a slight but constant rise in the whole blood chloride occurs relatively soon after the administration, and as a rule preced es the maximal diuresis. The resulting diuresis may exceed 50 times the normal urinary output. The specific electric conductivity of the urine and particularly the concentration of the urinary chloride rise, even throughout the periods of increasing and maximum diuresis. Consequently so much as one third the amount of the total blood chloride may be excreted within an hour. However, at the end of the hour the whole blood chloride is even higher than during the control preadministration period. It follows that during the hour chlorides pass rapidly from the tissues into the blood stream, and thence through the kidney into the urine.

A consideration of these findings, the rise in blood chloride and the profuse excretion of urinary chloride, led to a consideration of the importance of the chloride in the mechanism of the induced diuresis. Cohnheim 1 had found that chloride soon passes into sugar solutions injected intraperitoneally. The intraperitoneal injection of sugar solutions results in a fall in the blood chloride, since chloride leaves the blood stream and enters the artificial transudate up to a concentration of 0.4%. 2 Other electrolytes are likewise deviated from the blood stream, after the laws governing permeability.

In addition to rickets arising from diets deficient in vitamin D or salts of calcium or phosphorus, bone dystrophy has been noted in various disturbances of mineral metabolism which lead to imperfect deposition of the mineral constituents of bone, or to their excessive mobilization. Because of the similarity of clinical picture and the mechanics of production, the term rickets may justifiably be applied to the whole of this diverse group of bone dystrophies. Those cases arising from deficient absorption of inorganic bone constituents should be considered of exogenous origin, whether the deficient absorption be due to inadequate intake of minerals or of vitamin D, to inadequate ultraviolet irradiation, or to other cause. Those instances in which the metabolic disturbance is not dependent upon inadequate absorption are secondary to some endogenous disturbance of metabolism.

Several patients with endogenous rickets have been studied intensively in this clinic, and while their bone lesions were very similar, the metabolic data indicate that their anomalies were not dependent upon a common metabolic defect. The nature of the metabolic data indicated that in one group of patients the condition was secondary to chronic acidosis, due in one instance to persistent ketosis, and in another to constant reabsorption of urine. In a second group the bone changes were dependent upon hyperparathyroidism. The remaining group must be termed idiopathic, until further studies serve to establish the etiology of their condition. This third group is very similar in some respects to infantile rickets of the usual, or exogenous, type; it is distinguished from it in its failure to respond satisfactorily to adequate calcium and phosphorus ingestion, to vitamin D in the form of cod liver oil or viosterol, or to ultraviolet irradiation.