Experimental Biology and Medicine

Moore and Price 1 presented the hypothesis that the hypophyseal hormones excite gonadal production of germ cells and hormone secretion, and that the gonad hormones of either sex suppress pituitary secretion. This hypothesis is in agreement with the conclusions of Ihrke and D'Amour 2 that injections of male hormone concentrates from bull testis-tissue prepared by Gallagher and Koch caused a suppression of the estrous cycle of female rats as determined by the vaginal smear. This effect on the cycle, immediately abolished by the addition of pituitary substance, was explained by Ihrke and D'Amour on the basis of a diminution of pituitary secretions and a resulting suppression of the normal gametogenic and hormonal activities of the ovaries.

In 1937, however, Duncan, Gallagher and Koch 3 reported that crude male hormone preparations from testis tissue inhibit the normal estrous cycle in rats, but that this response is not due to the inhibitory action of the male hormone on the hypophysis, but solely to the phospholipin fraction free from male hormone. They further stated that crude choline obtained by hydrolyzing the phospholipins, and pure choline prepared synthetically likewise produced the same inhibition, but that pure testosterone in doses 20 times those found in crude testis-tissue concentrate did not inhibit the estrous cycle. The papers by Robson, 4 and Browman, 5 however, show that testosterone does cause a suppression of the estrous cycle in both mice and rats. The present work was undertaken in the attempt to confirm the r'eport of Duncan, Gallagher and Koch 3 that synthetically prepared choline causes an inhibition of the estrous cycle of the white rat.

Cysteine diminishes the hypoglycemic action of insulin.

The above data ofier no evidence that the hypoglossal, lingual or upper cervical nerves mediate proprioceptive impulses from the intrinsic tongue musculature of the cat.

Recent evidence has shown that nicotinic acid is a compound of considerable biologic importance. Current interest in this compound can be attributed primarily to the work of Warburg, 1 Euler 2 and their associates, which demonstrated that the amide of nicotinic acid is a constituent of the coenzyme from horse blood and the cozymase of yeast.

To those interested in the nutritive requirements of microörganisms this work has been of considerable significance as it supplied a clue to the chemical identity of one of the essential substances, or “growth-factors,” needed for development of certain of the more exacting bacteria. In studies of Staphylococcus aureus Knight 3 demonstrated that a combination of nicotinic acid and vitamin B₁ (thiamin chloride) was effective in replacing a concentrate prepared from yeast. Neither substance would suffice in the absence of the other, but when both were supplied the staphylococcus developed in a culture-medium containing only known compounds. For growth of the diphtheria bacillus Mueller 4 found that nicotinic acid could replace one of several fractions obtained from liver. A combination of nicotinic acid, beta-alanine and, for some strains of the organism, pimelic acid was effective in promoting growth in the absence of tissue extract preparations. 5 Nicotinic acid without beta-alanine was relatively ineffective. 5

The work reported here deals with the growth-promoting effect of nicotinic acid upon dysentery bacilli. It is of interest for two reasons: first, through the use of nicotinic acid it is possible to cultivate dysentery bacilli in a solution of known chemical compounds and second, nicotinic acid alone is strikingly effective without the addition of any other “accessory” factor.

The present investigation is concerned with the influence of antipyretics on digitalis. The antipyretics investigated were: the salicylates, acetanilid, phenacetin, aminopyrine, aspirin and antipyrine.

Dogs are quite sensitive and uniform in their reaction to digitalis. The average fatal dose of the tincture of digitalis, injected intravenously at the rate of 0.1 cc. per kilo body weight every 5 minutes, is 1.2 cc. per kilo, when dogs are anesthetized with 35 mg. of pentobarbital per kilo intraperitoneally. In previous work it was shown that high body temperature does not prevent the action of digitalis nor influence its toxicity. The variation from this fatal dose is surprisingly small. Therefore, the effect of the various antipyretics in changing the fatal dose of digitalis was tested.

In this report the point to which we wish to draw attention is the astounding effect on blood pressure which is effected by digitalis after all antipyretics. We select in this case antipyrine as the type.

I. Dog-23.3 kg., female; normal Mood pressure 150 mm. Ten equal doses of 10% antipyrine were injected intravenously, making a total dose of 100 mg. per kg. The blood pressure after the antipyrine had been given was 130. At this point, digitalis was started in doses of 0.1 CC. per kg. The pressure rose gradually to 170 after 6 injections of digitalis. At this point the pressure rose sharply to 210, and, after another injection, it went to 300. The pressure was actually higher than this, as our manometer was incapable of recording a higher figure. After one more injection the dog died. The total dose of digitalis was 0.8 cc. per kg., as compared to a normal lethal dose of 1.2 cc. per kg.

During inhalation of 7-9% oxygen the pressor reflexes of the carotid sinus are greatly reduced. This is shown, (1) by a greater fall of blood pressure in the carotid artery upon changing from the horizontal to the vertical (feet down) position than is obtained under control conditions before and after the administration of low oxygen; (2) by a diminished response of the blood pressure under low oxygen to a given increase in pressure in the carotid sinus.

Iodoacetate, a compound known to form mercaptides with sulphydryls, has been shown to inactivate guinea-pig complement. The inactivated complement was partially regenerated with ascorbic acid and glutathione (SH) but not with saturated H₂S water or KCN by our experimental procedure. This would indicate that the action of the iodoacetate on guinea-pig complement is not wholly upon thiol grouping, and that it may have other effects on the protein molecule. The fact that the iodoacetate-treated complement could be reactivated with two natural occurring reductants points to the possibility of a specific action of these two agents.

From these observations it must be assumed that iodoacetate acts primarily upon the protein and possibly on the 4th component of complement.

Active, dehydrated complement showed no reduction of power when extracted in the cold with absolute alcohol, ether and petroleum ether. It was not possible to remove more than 50% of the total phospholipids of active, dehydrated complement by the methods described. Fresh complement was readily denatured by the solvents employed, namely, absolute alcohol, absolute ether, and petroleum ether. Treatment of active, dehydrated complement with chloroform, benzene and pyridine led to no change in initial titers. No evidence of a 5th component in the sense of Toda and Misuse was found. No relationship was found between the phospholipid contents of the serums of guinea pigs fed a vitamin-C deficient diet and their lowered complementary activities.

In this investigation, the quantitative study of capillaries as initiated by Krogh 1 and by Wearn and collaborators 1 , 2 , 3 has been applied to normal and hypertrophied human hearts. The methods of injection and sectioning were essentially the same as those used on rabbit hearts in an earlier study from this laboratory. 4 Counts were made in 5 areas of each ventricle of the number of capillaries and fibers per square millimeter. Measurements were made to determine also the average diameter of the individual muscle fibers.

In this preliminary report we are able to compare the results of such a study in 10 normal and 10 hypertrophied human hearts. As compared with the normal hearts, the hypertrophied hearts show: (1) A marked increase in the average heart weight/body weight ratio and in the average heart weight, (2) a definite increase correspondingly in the average fiber diameter, and (3) a very evident decrease in the number of capillaries per square millimeter. At the same time, there is, (4) no change in the ratio of capillaries and fibers; that is, the capillaries do not multiply to accompany the increase in muscle mass.

From this study on human material, which is in accord with the experimental study of Shipley, Shipley and Wearn 4 on normal and hypertrophied rabbit hearts, it is evident that there is an increase in the distance from the center of a given myocardial capillary to the periphery of its region of supply, which is approximately proportional to the degree of cardiac hypertrophy. At the present time the oxygen utilization of normal and hypertrophied hearts is being investigated to determine whether or not the diminished anatomical vascularization of hypertrophied hearts results in a physiological impairment to the exchange of metabolic substances.

It is the conclusion of Cruickshank 1 that the isolated heart derives its energy at least in part from the combustion of fat. This deduction is based on the negative evidence that combustion of other fuels will not account for the total metabolism. According to Visscher and Mulder 2 the total oxygen consumption of the heart-lung preparation cannot be accounted for on the basis of the carbohydrate loss from the tissue and blood in that system. They found that in some cases as much as 80% of the total metabolism is non-carbohydrate, presumably fat. Previous studies on fat burning by striated muscle are conflicting. Palazzolo 3 reported a decrease, while Winfield 4 found no change in the fat content of muscle as a result of activity.

In order to elucidate this question further measurements of the total fat content of the mammalian ventricular muscle were made in two series of cases. For the first series hearts were obtained directly from anesthetized dogs, and for the second ventricular muscle was taken after periods of work in the heart-lung preparation. The two series were run concurrently and there was no selection of the animals for the two groups. The ventricular muscle was ground and 20 gm. samples taken for extraction and analysis for total fats by the Bloor 5 technique. The heart-lung preparations were made by the usual method and no additions of any material were made to the blood during the course of the experiments.

Analyses were made on 63 hearts, of which 25 were worked for periods of from one to 6 hours, the average time being 3 hours.

The observed data are shown in Table 1. It will be seen that there is great overlapping of values in the two series, as would he expected from the normal variability in fat content.

Considerable work has been done in an effort to evaluate the effect of digitalis on coronary blood flow. A wide variety of methods have been employed and the results on the whole have been contradictory. Gilbert and Fenn 1 have reviewed the pertinent literature which preceded their report. These workers, after an extensive series of acute experiments in which they studied the effect of a number of preparations of digitalis on the outflow from the coronary sinus of the dog by use of the Morawitz cannula, concluded that digitalis preparations may exert a vasoconstrictor action on the coronary arteries.

The use of the Morawitz cannula requires deep anesthesia, an open thorax and artificial respiration; consequently, the period of observation is necessarily relatively brief. For a number of years we have been studying coronary blood flow in the trained animal. 2 It has been possible to measure the blood flow in one of the coronary arteries of the dog as often as desired, and in some experiments for as long as 2 weeks. Formerly we used the thermostromuhr method of Rein, but recently we have employed the method described by Baldes and Herrick, 3 which makes use of a direct current heater. We have, therefore, been able to digitalize animals by divided doses in a manner comparable to the clinical method and at the same time observe the effect on the coronary blood flow.

Seven dogs were prepared for these experiments. They were trained to lie quietly and subsequently the thermostromuhr unit was placed on the circumflex branch of the left coronary artery under general anesthesia and with sterile technic as described in a previous paper. Three of the animals fulfilled the following requirements sufficiently well to permit their use in this study: 1. The body Item perature and pulse rate of the dog must have remained within normal limits for at least 24 hours following the operation. 2. The coronary blood flow must have remained relatively constant for a like period. 3. Prior to injection of the drug the dog must have taken food without hesitation and behaved in all respects like a relatively healthy dog.

Sulfanilamide produced a slight delay of hemolysis in blood-broth cultures of hemolytic streptococci. This effect was probably attributable to modification of the growth-curve. When used in concentration equal to or greater than that induced in the body-fluids therapeutically, sulfanilamide was without apparent effect upon fibrinolysis or formation of erythrogenic toxin in vitro, and was unable to inactivate small amounts of toxin.

The opportunities for direct study of intestinal peristalsis in the living human are necessarily limited. The usual methods of investigation have consisted of radiographic procedures or of the introduction of various mechanisms into the bowel, the very presence of which in the bowel may create abnormal reactions. Advantage was taken of a case of intestinal prolapse or bowel externalization (involving lower portion of the ileum) in an otherwise healthy male to record daily the activity of circular and longitudinal muscle. The loop of bowel was approximately 12 inches in length with an artificial anus at its summit so that the lower limb of the loop was constantly empty.

The method of recording described by Alvarez 1 was adapted in the following manner: Two Wiggers myocardiographs were fastened to permanent non-absorbable sutures loosely encircling a common mass of circular or longitudinal muscle. Lines joining points of attachment of the myographs to either circular or longitudinal muscle crossed each other at their mid-points. Attachment was made to the loop of bowel being traversed by intestinal contents. The apparatus was so arranged that it could be set up in working order without altering the position of the patient and might be used with the patient in a lying or semi-sitting position. While conducting the experiments, the bowel was kept covered with normal saline, and kept warm by a 75 Watt drop light. A thin rubber dam was placed over all to prevent evaporation.

The following precautions were observed in all experiments: The patient was prepared in no way for the experiment with the exception that any medication he might have been taking was discontinued the night before. To obviate reactions to the mechanical stimulus, tracings were not taken until at least 20 minutes after the hook-up.

In June, 1916, sylvatic plague was first demonstrated in San Mateo County by gross anatomical examinations of squirrels (Citellus beecheyi). During the next 4 years infected rodents were again discovered by dissection. Between 1921 and 1931 no surveys were conducted. In 1932 to 1935 annually, a few hundred squirrels and rats were shot and autopsied; no gross anatomical lesions suggestive of plague were observed. During August and September of 1936, a total of 863 beecheyi squirrels were again examined with negative results. The fleas, which had been killed with chloroform, were removed from the animals and collected in physiological salt solution. A total of 18,729 fleas were thus assembled in 355 pools, which were stored at 3°C. for approximately 10 to 11 months since suitable facilities to examine them were not available. In 1937 the insects collected from the squirrels of the same area were pooled in 155 lots, carefully washed with salt solution, ground in a mortar and then injected subcutaneously into guinea pigs. Of the 155 pools so tested 6 produced fatal plague infections within 6 to 8 days. Two of the locations in the County where infected fleas were found in 1936 were recognized as the same colony or series of burrows proven to harbor diseased squirrels in the summer of 1916.

These and similar observations indicate that sylvatic plague persists probably indefinitely in an area once invaded and that the gross anatomical examinations fail to detect rodent infections. Squirrels, just as rats, may harbor P. pestis without visible lesions. The biology of these latent infections is the subject of further investigations. Although the viability of P. pestis in stock cultures for many years is well known, the fact that the bacteria remained alive in the dead fleas soiled with a variety of microörganisms (B. coli, B. proteus, Cocci, etc.) held at ice box temperature is indeed noteworthy.

Of 29 serums from patients diagnosed as having had acute encephalitis or a polio-encephalitis, respectively, in one region of California none neutralized the virus of lymphocytic choriomeningitis, while 16 or 55.1% gave a positive test with the St. Louis type of encephalitic virus.

Appreciation is extended to Dr. E. R. Zumwalt and his staff at Tulare, to Dr. H. M. Ginsburg and the group at Fresno for their kind coöperation and the many courtesies offered and to Dr. C. Hawley for the collection of many of the serums.

Fish meal and rice bran which have been exposed to the action of microörganisms contain the antihemorrhagic vitamin-K. 1 This factor has also been demonstrated in the feces of chicks on a K-free diet. 2 The present work is a preliminary investigation of the production of the vitamin by bacterial synthesis.

It was again found that the putrefaction of ether-extracted, K-free fish meal was accompanied by the formation of appreciable amounts of a fat-soluble, antihemorrhagic substance. Several different types of bacteria were present in the putrefied material. An organism producing a similar putrefaction was isolated in pure culture∗ and inoculated to wet, sterile, ether-extracted fish meal. As a control, a similarly prepared uninoculated fish meal was used. Following 10 days'incubation, when a strongly putrid odor had been developed in the inoculated sample, the material was extracted while still moist by shaking with ether. The ethereal extract, the extracted residue, and the sterile control fish-meal sample were each added to different portions of K-free chick-ration 3 and each portion was fed to day-old chicks for a period of one week. The ethereal extract of putrid fish meal and the extracted residue each contained sufficient antihemorrhagic factor to maintain an approximately normal blood-clotting time of chicks. 3 The sterile fish-meal control gave no evidence of activity.

The fish-meal organism was then inoculated to beef broth, fish-meal broth, proteose-peptone broth, gelatin, and nutrient agar. Washed bacteria from broth and from nutrient agar were found to be rich sources of the antihemorrhagic factor. Liquid media from which the bacteria had ken removed by filtration, and saline used in washing the bacteria were negative. Sterile media used as controls were all negative.

1. In the 3 types of sarcomas, E, E-2, and E-5, studied here, no significant difference was observed between the growth rate of the original implant and the recurrent tumor. This applies to controls as well as to pregnant animals. 2. The slight correlation existing between the growth rate of sarcoma E and the percentage of time the tumor was acted upon by pregnancy is not significant. 3. The original implants of sarcomas E-2 and E-5, when implanted into pregnant animals, showed a significantly lower growth rate than those implanted into controls. No signifiant differences were observed, however, in the recurrent tumors.

The decrease in total myocardial phosphorus which accompanies heart failure is due to a decrease in the acid-soluble phosphorus compounds.

The intravenous injection of mice may prove to be a more satisfactory procedure than the methods now in use for testing the potential toxicity of rabbit antisera intended for human therapy.

Fractions prepared from pancreas, testis, liver, kidney, brain, placenta, muscle, breast, and uterus were shown to possess provocative Shwartzman phenomenon factors, although the results were irregular in some cases. Similar fractions from spleen gave negative results. Only the fractions from the first 3 of these tissues were also tested for preparatory potency, and were found to elicit positive reactions in some of the rabbits.

We wish to express our appreciation to Dr. Gregory Shwartzman of The Mount Sinai Hospital, New York, for his suggestions and criticism.

Current pharmacological concepts embrace the possibility that drugs acting at junctions, where acetylcholine plays an important though perhaps not essential rôle in transmission, do so by exerting their influence on some step in the cholinergic mechanism.

The consistency of such a view was examined by studying the effects on a sympathetic ganglion of drugs known to act at the parasympathetic neuro-effector junctions, since both are sites where cholinergic systems operate. Pertinent observations may be found in papers by Langley, 1 by Dale and Laidlaw, 2 etc.

Action potentials of the tonic impulses normally coursing in the superior cervical preganglionic trunk of the rabbit (anesthetized with nembutal) and of the response in the postganglionic fibers were recorded by a Matthews oscillograph after amplification. The waves were also spread out for visual observation by a rotating mirror (speed corresponding to 1.8 meters per second) and converted into sound by a loud speaker.

Pilocarpine, representing the parasympathomimetic stimulants, and atropine, the depressants, were injected into the common carotid artery, all branches except to the ganglion having been ligated and cut and the carotid sinus nerve severed. Thus the ganglionic circulation was retained intact while allowing an effective concentration at the synapse before diffusion into the general circulation. This made possible the observation of local effects uncomplicated by distant actions such as on the central nervous system directly, or indirectly through the remaining innervated carotid sinus responding to vascular changes.

The increase in postganglionic impulses (compare records A and B, C and D) illustrates the stimulant effect of small doses of pilocarpine. This increase, seen again in E, F, took place without any change in the corresponding preganglionic records G and H, and consequently must have been due to a truly peripheral action at the synapse.

Butenandt and Kudzus 1 have reported that androgenic substances administered to immature female rats cause premature opening of the vagina. The present study was undertaken to determine whether the androgens produce this effect directly upon the genital tract or indirectly through the ovary.

Eighteen rats (comprising 5 litters), varying in age from 27 to 30 days, were used in this study. Twelve were given a single injection of androgen in sesame oil; 6 controls were given an equal quantity of pure sesame oil. Seven of the animals were injected with testosterone propionate and 5 with androstenediol,† the dosage varying from 1 to 5 mg.

The time of the opening of the vagina was noted in each case. Laparotomies were performed and one ovary removed at intervals varying from 60 to 96 hours after the administration of the androgen. The animals were sacrificed at periods varying from 96 to 228 hours after the injection and the remaining ovary was then removed. At corresponding intervals, the ovaries were removed from the control animals. The ovaries were examined microscopically in serial sections.

Opening of the vagina occurred in all androgen-injected animals within 72 hours after the injection. Follicle stimulation was noted as early as 60 and 72 hours after the injection. Corpora lutea were found as early as 96 hours and as late as 192 hours after androgen administration. The ovaries of all the 12 injected animals exhibited some gonadotropic effect, either follicle stimulation, luteinization or both. The uteri in all these animals were markedly enlarged. In the 6 control rats, the vaginas remained closed and the ovaries were negative.

Eighteen rats (comprising 5 litters), varying in age from 27 to 30 days, were used in this study. Twelve were given a single injection of androgen in sesame oil; 6 controls were given an equal quantity of pure sesame oil.

The data here reported indicate that thyroid insufficiency associated With increased pituitary and androgenic activity (active or passive) are important factors favoring the development of exophthalmos in rabbits. The androgenic factor is believed to play a rôle in the percentile increase in the incidence of post-thyroidectomy exophthalmos in Graves' disease in males.

β d-mannoheptitol unlike the hexahydric alcohol, mannitol, and the heptose, β d-mannoheptulose, is not capable of serving as a precursor of glycogen in the liver of the rat.

Pijoan, Townsend and Wilson 1 noted a considerable loss of vitamin C in blood serum done by the Farmer and Abt 2 method. Pijoan and Klemperer 3 reported that this loss could be prevented by the immediate addition of M/50 KCN to the blood. In order to determine the difference in the concentration of vitamin C in blood with and without KCN, the following experiments were done. Forty cc. of venous blood were drawn and placed immediately in an Erlenmeyer flask containing 8 drops of a 20% solution of potassium oxalate. The specimen was shaken gently and divided into two equal fractions, to one of which (B) were added 8 drops of 10% KCN resulting in an M/26 solution of KCN. Both fractions (A and B) were then immediately centrifuged and the plasma drawn off. The separated plasma from each fraction was divided into four 2 cc. portions. One of each of these samples (₀ and ₀) was precipitated immediately, using 2 cc. of plasma, 4 cc. of redistilled water and 6 cc. of a 10% solution of metaphosphoric acid. These samples were then centrifuged for 5 minutes and the filtrate decanted off. Three cc. samples of the filtrate were used for the titrations, which were done by the Farmer and Abt modification of the Tillman's method. 4 The remaining 6 plasma specimens (3 with KCN and 3 without KCN) were placed in the icebox and determinations of the vitamin C content were done after intervals of approximately 30, 90 and 160 minutes as shown in the chart. The first determinations, in this experiment, were done 45 minutes after the blood was drawn. This represents the maximum time required for preparing the filtrate; the average time taken in other experiments was 25 minutes.

1. The red cell longevity in normal guinea pigs ranges from 22-28 days. 2. This value increases after splenectomy, reaching a maximum of 32-38 days approximately a month following the operation, and then falls off, becoming normal in about 2 1/2 months. 3. The longevity of the circulating reticulocyte ranges from 4 to 6 days in both normal and splenectomized animals.

The choline-esterase activity of the serum in patients with jaundice or biliary tract disease was found to be depressed.

Sulfanilamide when given by mouth in pregnant rabbits has been shown to pass from the maternal to the fetal circulation. The acetylated form is relatively lower in the fetal blood than in the maternal blood. Sulfanilamide can be also detected in the amniotic fluid.

The convulsions produced by the intravenous injection of metrazol (pentamethylentetrazol) as a therapeutic agent in schizophrenia, were recorded by means of normal and ultra-high-speed motion pictures and by electromyograms. The electromyograms of the activity of antagonistic muscles were recorded simultaneously by 2 matched amplifier systems which operated crystal actuated ink-writing recorders.

The typical metrazol seizure in man is composed of 3 stages: the first clonic stage, the tonic stage and the second clonic stage. The nature of the clonus is entirely different in the 2 clonic stages. As seen from the films and the electromyograms the clonus of the first clonic stage is the result of the alternating contraction of agonistic and antagonistic muscle groups; 2 to 4 of these 2-phase movements occurring per second. The second clonic stage is the result of an alternation between simultaneous contraction and relaxation of all muscle groups. It represents an alternating disappearance and reappearance of the continuous innervation which is present in the tonic stage. The change between contraction and relaxation is very rapid at the beginning of the second clonic stage, 12 to 14 contractions occurring per second. The movements gradually become slower and coarser. Towards the end of the second clonic stage only 1 to 2 contraction seizures occur per second and finally complete relaxation follows as is evidenced by a complete absence of muscle action potentials in the electromyogram.

The tonic stage, which in the electromyogram shows simultaneous high potentials in all muscles, represents a state similar to decerebrate rigidity. The movements of the second clonic stage correspond to an intermittent innervation of the same characteristics which were continuously'present during the tonic stage. The second clonic stage, therefore, should be.

In addition to being able to initiate malignant growth, the carcinogenic hydrocarbons have been shown to stimulate cell division of certain microörganisms. Since it has been postulated that cancer may be associated with an abnormal glucose metabolism, it was thought of interest to study the effect of a typical carcinogenic hydrocarbon on the growth and glucose utilization of Fusarium lini. This fungus has been shown to grow well on a medium composed of mineral salts and glucose as the only source of carbon producing a typical alcoholic fermentation. 1

The carcinogenic hydrocarbon selected for this study was 1,2,5,6-dibenzanthracene. The culture medium had the following composition:

Since in preliminary tests the above hydrocarbon had no effect on the growth of Fusarium lini grown on the above medium, it was felt that this might be due to the low solubility of the hydrocarbon in the medium. Therefore in the work here reported a water-soluble oxidation product of the hydrocarbon was used. This was prepared by a method similar to that of Boyland and Boyland 2 as follows:

A solution containing 0.123 gm. of 1, 2, 5, 6-dibenzanthracene, dissolved in a small amount of anhydrous benzene, was exposed for 1 1/4 hours to the rays from a mercury arc ultraviolet light source. The solution was exposed directly to the light by being placed in a thin layer in one-half of a large Petri dish. From time to time benzene was added to replace that lost by evaporation. Finally the benzene was evaporated and the residue irradiated for 15 minutes. This residue was then extracted with several small portions of water, the washings filtered and the volume made up to 100 cc. This extract was used in the subsequent work.

In a previous paper 1 it has been shown that theophylline greatly diminishes the toxicity of Mercurin∗ and Salyrgan at the site of intracutaneous injection. The beneficial action of theophylline in enhancing absorption after intramuscular injection has also been demonstrated 2 and it was found 3 that one mole equivalent (3.88% of theophylline monohydrate) produces the maximum effect. Since this and other data 3 are strongly suggestive of compound formation between theophylline and mercurial diuretics of general structure NaOCO-R^II-HgOH it seemed advisable to determine the limiting amount of theophylline necessary to prevent local toxicity.

The experimental procedure was the same as that described before 1 and consisted in the intradermal injection into the abdomen of the rabbit of 0.05 cc. of Mercurin and Salyrgan to which varying amounts of theophylline had been added. Each solution was prepared to contain 39 mg. of mercury per cc. (about 0.2 M) and its pH was accurately determined with the glass electrode.

In the accompanying photograph, taken 24 hours after injection, the mole equivalents of theophylline and pH values for each of the 7 solutions are shown adjacent to the corresponding sites of injection. In all 5 rabbits were injected in this way for Mercurin and 3 for Salyrgan. The results for the 2 drugs are very much alike and were found to be so consistent that only one photograph is shown. It will be seen that the local toxicity decreases in proportion to the amount of theophylline present and in the case of the solutions containing 1, 1.25, and 1.5 mole equivalents, so little reaction was produced that it is difficult to locate exactly the sites of injection.

1. 4,4'-di- (acetylamino) -diphenylsulfone is more efficacious than sulfanilamide against certain hemolytic streptococcal infections of mice. 2. Against these same infections, the 4,4'-diamino-benzenesulfonanilide is as good as or better than sulfanilamide. 3. The sulfone is less toxic than either the anilide or sulfanilamide. 4. Medication of mice with these drugs produced no demonstrable hepatic or renal lesions.

Experiments were designed to show whether bacteria chemotropically attract leukocytes directly, or only indirectly as the result of injuring cells and tissues and causing these to liberate chemotropic substances. Clumps of Staph. albus on a glass slide were separated from a suspension of rabbit's leukocytes by a zone of cell-free plasma. Leukocytes moved into the inner ring of plasma and made their way to the bacteria, showing strong positive chemotropism. Values of chemotropism obtained before any leukocytes reached the bacteria represent the reaction to bacteria alone, no tissue-cells being present in the clump of bacteria. After a number of leukocytes reached the bacteria and began to phagocytize them, the value of chemotropism was determined for other leukocytes en route to the bacteria. The two values of chemotropism were not significantly different. It is concluded that leukocytes may react chemotropically to substances given off directly by bacteria.

The analeptic drug pentamethylenetetrazol (Metrazol) used in the treatment of cardiovascular conditions has been reported 1 to produce dilatation of the abdominal capillaries. Because of the extensive clinical use of this drug, 2 it was felt desirable to restudy its action on the intact splanchnic vascular bed of the dog.

For this study we used 23 female dogs, the average weight being 7 kg. The anesthetic employed was chloralosane—80 mg. per kg. injected intravenously. Blood pressure and respiration were recorded in the usual manner. Oncometers were placed on the spleen and small intestine or a loop of ileum was exteriorized and kept in a chamber containing normal saline at body temperature. The mesentery of this loop was transilluminated from below and the vessels were observed through a lens system whose magnifying power could be varied at will.

Following a control period with observation on the blood vessels of an intestinal loop, the drug was injected intravenously as a 10% solution. The dosage ranged from 0.25 cc. to 2.0 cc. per injection and each animal received several injections during a period of 2 to 3 hours. In some instances the same dose was employed throughout a single experiment; in others, the dosage was varied. Almost continuous observations were made of the caliber of the large and small arteries, the arterioles and venules and the large and small veins. Any changes noted were correlated with the normal variation as established before the administration of the drug.

In a series of 12 animals, the predominant effect of the drug on the entire splanchnic circulation was constriction in 3 cases and dilatation in 3 others. There was no significant effect in the remaining 6 dogs.

Chronic riboflavin deficiency in rats is often accompanied by pediculosis. This disease has never been observed in rats manifesting symptoms of a deficiency of other factors of the vitamin B₂ complex. Administration of riboflavin by mouth has a curative effect on this type of pediculosis in rats.

Sulfanilamide and prontosil have been used successfully in the treatment of distemper in dogs. Both drugs seem to be equally effective.

We are obliged to the Department of Bacteriology of Michael Reese Hospital for advice and help.

It was possible to induce postpartum lactation in hypophysectomized rats, and to maintain a functional development of their mammary glands by injections of combined mammotropic and adrenocorticotropic hormones. That this treament did not constitute complete substitution therapy was shown, however, by the fact that sufficient milk was not available for sustenance of the young.

We have demonstrated the existence of an ovarian factor in the rat, which inhibits the uterine response to estrin, and which operates in a cyclic manner corresponding to the estrus cycle of the animal. Although the development of uterine inhibition during the cycle is paralleled by the development of corpora lutea in the ovary, we do not believe that the inhibition is due to progestin. This conclusion is based on evidence that progestin, in amounts sufficient to inhibit the effects of estrin on the uterus, always results in progestational phenomena in this organ. 2 , 3 , 4 But, at no time in the present or previous 1 work did we observe progestational changes in the uterus. Furthermore, the antagonism between progestin and estrin also includes the inhibition of vaginal cornification, 2 , 3 while in our work the vaginal mucosa never failed to become cornified after estrin administration.

1. In 8 diabetic dogs there was a definite decrease in the hyperglycemia during the hourly injection of amounts of insulin less than that required for the maintenance of a constant blood sugar level within the normal physiological range. Hourly amounts of insulin which would have allowed the blood sugar to rise when given at a level of 100 mg. % caused the blood sugar to fall when given at hyperglycemic levels. 2. In untreated diabetes mellitus, factors other than the hyperglycemia must be responsible for the severity of the condition, as the high blood sugar per se does not interfere with the ordinary action of insulin.

Hoppert, Webber and Canniff 1 reported a diet which produced dental caries in rats. This diet consisted of coarsely ground yellow corn meal 60%, whole milk powder 30%, linseed meal 6%, alfalfa meal and NaCl 1%. Although these investigators considered this diet “adequate” in every particular, they found that from 90 to 100% of their rats developed dental caries in 16 weeks (112 days).

Dental pathologists do not regard this caries as merely “fracture caries,” and no satisfactory explanation of its cause has been reported. This lack of explanation constitutes a challenge to every theory regarding the cause of dental caries today.

In our studies we have found that 60% of 200 carefully controlled rats developed dental caries in 100 days when fed the Hoppert, Webber and Canniff diet. In an attempt to gain further information regarding the cause of the caries produced by this diet, the following experiment was performed.

Fifty-six rats, 30 days old, from our own inbred stock, were fed the above diet modified by substituting commercial casein for the whole milk powder. Tap water ad lib was allowed, and the feeding was continued for 100 days. The animals were then sacrificed and examined under light and magnification for dental caries. One large and 5 small carious lesions were found (10 2/3%).

The marked reduction of the incidence of dental caries which was observed when commercial casein was substituted for powdered whole milk seems to indicate that casein exerts an inhibitory action on the production of experimental dental caries in rats which are fed a coarse corn meal diet.

Whether this inhibitory action is due to the smaller amount of lactose, the relatively low alkaline reaction, or the relatively high protein of the casein diet is a question which can he answered only by subsequent investigation.

For dependable ascorbic acid values, blood should be centrifuged, the plasma deproteinized, and the plasma-HPO₃ filtrate titrated in immediate sequence after the blood is drawn. Whole blood which stands in a closed small phial, with a minimum air space, may be depended upon to give results of clinical value for 1/2 hour. The higher values obtained with bloods to which KCN has been added represent an enhancement due to the action of KCN upon the 2,6-dichlorophenolindophenol, and in no wise a more accurate determination of their ascorbic acid content. This is particularly true with blood of low ascorbic acid value. KCN does not prevent the loss of ascorbic acid from blood.

It is a pleasure to acknowledge our indebtedness to Mrs. Jessie Maaske for technical assistance, and to Mr. Herman Chinn for checking several titrations.

Goodhart and Jolliffe 1 have emphasized the need for a practical chemical method for studying vitamin B₁ metabolism. The fermentation method recently published by our laboratories^2,3 is, we believe, admirably suited to this purpose.

After, and in conjunction with, considerable work on rat metabolism of vitamin B₁, we have applied our method to a group of human subjects. Five adults and 3 children, all ‘normal’ subjects with the possible exception of Subject E, who was under treatment for nervous breakdown, coöperated with us in this project. The main diet was in all cases normal, i. e., no attempt was made to restrict these individuals to a standard diet. The average daily urinary output of the adults (average of 32 determinations) was 497.7±47 gamma. For the children the urinary average was 333 ± 60 gamma.

Having established the normal urinary output on the average or normal diet, doses of 5 and 10 mg. (5000 and 10,000 gamma) were given once daily per os to the adult subjects. Table I gives the results obtained in this experiment.

The first point to be noted is the non-uniform response to the 5 mg. close. We feel that this variation in response may be related to either the level of body stores or individual rates of absorption.

After sufficient time has elapsed on the 5 mg. level, we find that urinary excretion of the vitamin takes place at a steady rate. This condition we designate as excretion equilibrium. When a subject in excretion equilibrium at 5 mg. per day is placed on the 10 mg. level, we do not obtain a significant rise in urinary output even after 15 days at this level. This phenomenon cannot be explained by body storage and the explanation must be sought elsewhere.

Sulfanilamide, 4,4′-di- (acetylamino)-diphenylsulfone, and 4,4′-diaminobenzenesulfonanilide have no therapeutic value in the treatment of mice infected intraäbdominally with virulent B. pertussis.

The results of recent investigations suggest that connective tissue fibers serve as pathways for the extravascular transport of vital dyes escaping from lymphatics. 1 In unpublished studies on the ear of the mouse, gentle, alternating, external pressures (0 to 2 cm. or 8 cm. of water) caused squeezing of the tissues and bending of these fibers and at the same time the rapidity of spread of vital dyes through the interstitial tissues was increased. That gentle massage increases the lymph flow from a part is well known. 2 The work to be reported now deals with the effect of pulsation of the blood vessels both upon lymph flow and upon the movement of substances through the interstitial tissues.

Warmed, defibrinated, aerated rabbit's blood was perfused for periods of 1 to 1 1/2 hours, through freshly cannulated rabbit's ears, with the use of an apparatus providing pulsatile and non-pulsatile pressures. “Systolic” and “diastolic” pressures of 141/60 mm. of mercury were used in the case of the pulsatile pressure at about 100 “beats” per minute, while for the constant pressure the equivalent of 141 mm. of mercury was employed. It will be seen that a somewhat higher mean pressure was used in the latter instances. Lymph formation and flow were estimated by filling a few peripheral lymphatics near the tip of the ear with a vital dye and noting the movement of the dye colored lymph toward the base of the ear. The method has been previously used in human skin. 2 To study the rate of spread of substances through the tissues, minute amounts (0.01 to 0.02 cu. mm.) of a vital dye (pontamine sky blue) were introduced through micropipettes into the subepidermal connective tissue of the ear by capillarity without utilization of pressure, Camera lucida drawings of the outline of the dye spots were made immediately after placing the dye in the tissues and again half an hour and 1 hour later.

Although they do not offer direct proof our experiments support the view that the reduction in liver fat caused by the pancreas extract which Dragstedt has described as lipocaic is due to the sum of the lipotropic effects of the choline and protein which it contains. The influence of the pancreas extract upon the fat content of the livers of rats on a low protein and low choline but high fat diet is greater than that of a similar amount of protein in other forms. The ketosis which results when rats with such fatty livers are fasting is reduced about equally by the pancreas extract and an equivalent amount of casein. Althought both the choline and the protein contained in the pancreas extract affect the fat content of the liver, choline is without antiketogenic activity under the conditions studied here.

Kabat recently stated 1 that, “arterial anastomosis by means of Payr cannulæ, the method recommended by Heymans and his coworkers, 2 proved unsatisfactory in our laboratory.” Inasmuch as we have been using this method, without anticoagulant, entirely satisfactorily for the past 15 years for such varied cross-circulation experiments as perfusion of the isolated head or brain, isolated carotid sinus and perfusion of acutely transplanted spleen, kidney, leg, intestine, carotid sinus and suprarenal, we wish to describe in detail our procedure in blood vessel anastomosis so that this technique may be successfully applied by other workers.

The Payr cannulæ, made of thin brass tubes with 2 external grooves (Fig. 1, A), vary in size from 2 to 8 mm. in diameter, and from 8 to 16 mm. in length. The cannula, corresponding in diameter to the size of the smaller of the 2 blood vessels to be connected (be they arteries or veins) is selected and fixed in the special cannula holder (Fig. 1, D). The ligated vessel is pulled through the cannula and a “bull dog” clip is placed below the ligature. The vessel is then cleanly cut between the ligature and the clip and the severed edge is carefully grasped by 3 delicate ocular forceps and everted over the edge of the cannula (Fig. 2, A). As soon as the vessel has been pulled down far enough over the cannula one forceps is removed from the edge and is placed about the vessel and cannula to hold it for tying. It is then firmly tied in the second groove (Fig. 1, B, 4).

In the phloridzinized dog the changes in the D:N ratio indicate that small doses of succinic acid are entirely converted to dextrose (2 mols. succinic acid = 1 mol. glucose). When larger doses are given a smaller percentage is excreted in the urine as sugar but anti-ketogenic and nitrogen sparing activity indicate its conversion to dextrose before being burned.

Some time ago publication was made from this laboratory of the rapid atrophy of the thymus on the administration of the gonadotropic hormone in pregnant mare serum. 1 The phenomenon was not seen in males or females after castration and was tentatively referred to the increased production of sex hormones on the part of the gonad. Selye, Browne and Collip 2 have noted atrophy of the thymus following administration of progesterone. Selye, Harlow and Collip, 3 and Schacher, Browne and Selye 4 subsequently produced regression of the thymus by administration of estrogenic, as well as androgenic substances. The preparation of adrenocorticotropic and mammotropic hormones in this laboratory has resulted in their administration under a variety of conditions. It became apparent very soon that invariable prompt reduction in size and almost complete regression of the thymus occurs 5 after administration of adrenocorticotropic hormone, while mammotropic hormone does not produce the atrophy. Although adrenocorticotropic hormone in large doses causes a stunting of growth or a loss in body weight, the doses used here which were effective in reducing thymus weight were not always associated with loss of body weight.

It is now known that an increase in the production of cortin by the adrenal is provoked by adrenocorticotropic hormone,⁶ as well as a demonstrable increase in its secretion of androgenic and estrogenic substances, ^{7, 8, 9} and one may naturally inquire as to which of these adrenal functions we may attribute the above mentioned experimental thymic atrophy. The reduction of the thymus by adrenocorticotropic hormone with equal facility after double ovariectomy would appear to rule out the stimulation of the production of progesterone, or androgenic and estrogenic substances, from the ovary which adrenocorticotropic hormones could conceivably occasion.

Mygind and Dederding 1 associated the pathogenesis of Ménière's disease with a disturbance of water and salt equilibria. From their investigations they concluded that a dehydrating and an acid-forming diet was indicated in the treatment of this condition. Two years later Furstenberg, Lashmet and Lathrop 2 extended these observations, showed evidence which indicated that sodium was the offending ion and recommended a regime with a low sodium diet and intermittent periods of ammonium chloride ingestion. This regime, where employed, has been successful, 3 , 4 but we believe from our data that a lowering of the sodium content of the body fluids is not its mode of action.

In this communication blood studies from 14 patients with Ménière's disease are presented. The diagnosis was confirmed by one or more clinicians and satisfied most of the criteria discussed by Crowe. 5 In all of the patients the concentration of serum total base, sodium and potassium was determined and in 4 the concentration of serum protein and hydrogen ion also. Eight patients were seen during periods of acute symptoms and bloods were taken at such times. In 6 patients, bloods were taken after the institution of a low sodium regime or during periods of freedom from symptoms after admission to the hospital. The results are given in Table I. It is observed that the concentrations of serum sodium and total fixed base are within the range for normals. The average concentration of serum sodium is slightly less in the patients with acute symptoms than in those without acute symptoms. The concentration of potassium is somewhat higher in the patients with acute symptoms.

Porphyrin was found by the present authors 1 in bouillon cultures of C. diphtheriæ, in amounts which vary with the toxicity or flocculative titer of the filtrates. Other investigators 2 - 6 have confirmed the occurrence of this pigment in C. diphtheriæ filtrates, by observations made both in the visible and in the ultraviolet spectrum. We have found a pigment, apparently identical, in Old Tuberculin, and in fluid cultures of several species of Mycobacteria. The chemical composition of the pigment, however, has remained in doubt. Its absorption-spectrum is that of a hemochromogen or metal-porphyrin compound. In ethereal extracts sharp bands are found in the visible at 574 and 538 mμ, and in the near ultraviolet an intense absorption with its maximum at 407.9mμ. The pigment shows an oxidation-reduction change, the oxidized form being colorless. When extracts are treated with strong mineral acid the pigment breaks down, and there appears free coproporphyrin and the copper compound of this substance.

In the effort to identify the pigment we undertook the synthesis∗ of compounds of coproporphyrin with a number of metallic radicles: Fe, Mg, Mn, Co, Ni, Cu, Sn, and Zn. After an interruption this work has been resumed under more favorable conditions. Of the various metallic compounds studied, only those of Cu, Fe, and Zn are of present significance.

The Zn compound is formed readily when a solution of zinc salt is added to coproporphyrin in slightly alkaline aqueous solution. The combination is stable over a rather wide pH range but is disrupted by strong mineral acid. The absorption-spectra in various solvents are very similar.

In ethereal solution sharp bands are present about μ 574 and 539; one of greater intensity is found at μ 407.8. In aqueous solution the pigment exhibits an oxidation-reduction change, the oxidized form king colorless.

Using a capillary glass electrode in human extensor digitorum communis muscles carrying an ergograph load of 600 gm. the pH changes between the muscle fibers were determined before and after ischemic work by the method detailed in a previous publication. 1

With the arm rendered ischemic by a brachial pressure of 160 mm. of mercury work was done until pain appeared. Simultaneously, work was stopped and pressure released, Within 10 seconds pain had disappeared, but maximal intercellular acidity was not reached before 30 to 40 seconds had elapsed. If pH continues to fall after pain ceases intercellular pH fall cannot be the cause of pain.

In another type of trial ischemic work was done until pain appeared, work was stopped and pressure released until acidity reached a plateau. Pressure was then again applied. Intercellular pH again started to fall markedly, far below the previous plateau yet the pain did not reappear. If acidity greater than that present with the pain can be created without pain under the same circumstances, then intercellular pH change cannot be the cause of the pain.

If the sensory endings responsible for ischemic pain lie within the muscle cell pH change might still be the cause of the pain, as there is no proof that intracellular and intercellular pH are identical.

The purpose of the studies reported in this paper is twofold: 1. To extend the systematic observations of Gellhorn 1 and collaborators on the effect of oxygen deficiency on the central nervous system to the autonomic nervous system. 2. To choose a reaction which is determined exclusively or to a large extent by a central inhibitory process in order to study the influence of oxygen deficiency on such processes.

Although the literature on the problem of reflex dilatation is still controversial a number of authors have shown that this reaction is largely due to an inhibition of the parasympathetic tonus of the Edinger-Westphal nucleus. This is particularly clear from the work of Bain, Irving and McSwiney, 2 who find that the stimulation of the central end of the splanchnic fails to elicit pupillary dilatation after sectioning of the third nerve, although “section of the cervical sympathetic nerves has not been found to alter either the rate or the degree of the reaction to stimulation of somatic or visceral afferent fibers.”

The experiments were carried out repeatedly on 12 rabbits, half of which had the cervical sympathetic cut on one side. The sciatic was exposed under light urethane anesthesia and 2-3 hours later the experiment was begun. The threshold reaction for pupillary dilatation was determined with faradic stimulation (Harvard inductorium, shielded electrode) while the animals inhaled air or a known O₂-N₂-mixture from Douglas bags.

Very weak currents (coil distance 11-12 cm. or at an angle) were used for 1-3 seconds and the pupillary reaction was measured with a telescope.

The results were uniform and showed that under the influence of 643% O₂ the threshold for pupillary dilatation was raised.

Through numerous investigations it is known that the increased respiratory response to CO₂ is due less to its effect on peripheral receptors than on the respiratory center itself, whereas a similar response to oxygen deficiency occurs only when the carotid sinus nerves are intact. The removal of the afferent fibers by elimination of all known ”buffer nerves” results in a failure of respiration during O₂ deficiency (Selladurai and Wright, 1 Schmidt, 2 v. Euler and Liljestrand, 3 Smyth, 4 Gemmill and Reeves, 5 and Wright, 6 but cf. Dautrebande and Wegria 7 ). Under such circumstances the blood pressure reaction to O₂ deficiency may also be converted from the rise obtained in the intact animal to a fall (Selladurai and Wright, 1 and Brewer 8 . However, the question whether the organization of the vasomotor center is similar to that of the respiratory center can only be investigated when the respiration is kept constant and thereby secondary effects of a reduced respiratory volume eliminated.

Such studies were carried out on narcotized dogs (Na-barbital, Na-amytal, chloralosane) with pneumothorax and artificial respiration. O₂-N₂ gas mixtures (1.0 to 9.8% o₂) were prepared by means of flowmeters, analyzed and inhaled from Douglas bags for periods of from 1 to 3 minutes and the blood pressure effects were recorded. After the control reaction consisting of a rise in blood pressure was obtained both vagi were cut in the neck and both carotid sinus regions were denervated. Hereafter the same O₂-N₂ mixture was inhaled and invariably an immediate fall of blood pressure was observed which occured regularly no matter whether the blood pressure level was unchanged, higher or lower than under the control conditions.

1. The anterior and posterior presumptive epidermis of the early gastrula of Hyla regilla possess essentially the same degree of competence for neural plate formation. 2. In the middle and late gastrula stages the posterior presumptive epidermis is less competent than the anterior. 3. Thinning of the ectoderm by removal of at least a large part of its inner layers of cells (the so-called nervous layer) in the advanced blastula stage does not impair the competence of the ectoderm.

Vitamin D causes a rise in the concentration of lead in the blood stream and in the bones of rats suffering from lead poisoning.

In the preceding paper 1 a marked effect of vitamin D on the blood and bone lead concentrations of lead-fed animals was demonstrated. In view of the great importance attached to the dietary calcium and phosphorus content in lead poisoning 2 , 3 , 4 investigations were undertaken to determine the part these factors play upon the previously noted action of the antirachitic vitamin.

The basal diet used in the previous experiment contained traces of calcium (0.03%) and small amounts of phosphorus (0.246%) to which basic lead carbonate was added. To this basal diet 2.5% of calcium carbonate was added to produce a high calcium low phosphorus diet; and 2.75% of anhydrous Na₂HPO₄ was added to produce the high phosphorus low calcium diet. The diets are described below:

Albino rats raised in our laboratory from an original Wistar strain were used. The mothers were kept on the stock diet of Bills, et a. 5 The young were weaned at 21 days, at which time they were placed on the stock diet. At the age of 23 to 25 days this was replaced by the experimental diets described above. One half of each group was given 33 Steenbock units of vitamin D. (Mead Johnson's 250 D-viosterol in halibut liver oil, diluted in maize oil.) After 23 to 2.5 days the animals were sacrificed. The lead content of the blood was determined by the method of Willoughby, et al. 6 The lead content of the femora was determined by a modification of the above method (to be described).

The results of the experiments are presented in Table I. The results of the experiments are presented in Table I.