Experimental Biology and Medicine

Stable dry powders and cell-free solutions that possess hydrogenase activity have been prepared from cultures of B. coli communior.

Crude and purified gramicidin after initial stimulation inhibits the oxygen consumption of bovine spermatozoa completely in Ringer phosphate of acid pH and renders the cells immobile. By the use of sufficiently small amounts only stimulation is observed during the period of the experiment. In alkaline phosphate buffer only the increase of oxygen consumption is noted. However, in Ringer bicarbonate medium of the same pH, the respiration again is greatly inhibited; aerobic as well as anaerobic glycolysis are depressed on the average 40% and the motility of the spermatozoa is markedly impaired. Tyrocidine, in the concentrations studied, caused a small reduction in the oxygen uptake and the glycolysis.

The author has shown 1 , 2 that the methyl esters of linoleic and linolenic acids interfere with the utilization of carotene and vitamin A by vitamin-A deficient rats. It was found that this antagonism can be counteracted by the addition of soybean oil and is not apparent if sufficiently large amounts of carotene are fed.

Subsequent investigations have revealed that the antagonism between the unsaturated fatty acid esters and carotene can be prevented by the unsaponifiable fraction of soybean oil or by α-tocopherol; but not by choline, ethanolamine, or soybean lecithin or cephalin.

The growth obtained with carotene and α-tocopherol was only slightly better than that with carotene alone, but the addition of α-tocopherol to carotene and methyl linolate or linolenate gave a pronounced growth response. The antagonism between methyl linolenate and carotene was more pronounced than that between methyl linolate and carotene. The addition of α-tocopherol gave better growth with methyl linolate than with methyl linolenate.

It is improbable that α-tocopherol acts as a simple chemical antioxidant since in vitro experiments 2 have shown that the unsaturated fatty acid esters cause no direct destruction of carotene with the method of feeding employed. It appears that in the absence of α-tocopherol there is a physiological antagonism between unsaturated fatty acids and carotene which results in the inefficient utilization of carotene.

A β-anthryl-carbamido derivative of antipneumococcus III rabbit antibody retains the original immunological properties while rendering Type III pneumococci specifically fluorescent in ultraviolet light.

It is a pleasure to acknowledge the constant advice and help so generously given by Dr. John F. Enders and Dr. Allan L. Grafflin, and the kind interest shown by Dr. Louis F. Fieser.

The efficacy of the sulfonamides in the systemic treatment of certain types of infection has been definitely established. During the past few years several communications 1 , 2 , 3 , 4 , 5 , 6 have described and recommended the application of these drugs directly into raw wounds as a prophylactic or therapeutic measure against infection. Most of these reports based their contentions upon qualitative impressions gathered from the observation of a small series of patients rather than upon controlled studies. Before indiscriminately depositing these drugs into a raw wound it is essential to determine first of all whether they have any noxious effect upon the tissues involved in the healing of the wound. Bricker and Graham 7 reported that the systemic administration of sulfanilamide had an inhibitory effect upon the healing of stomach wounds in dogs during the first 7 postoperative days. Taffel and Harvey 8 found that sulfanilamide did not affect the healing of stomach wounds in rats during all of the normal phases of healing. The drug was given orally in doses sufficient to maintain an adequate and sustained “therapeutic” blood level.

This series of experiments was carried out to determine the effect of the local application of sulfanilamide upon the healing of a soft tissue wound. Adult white rats weighing about 250 g were used. The tensile strength of the wound served as an index of healing. In addition, histological studies were made of the wounds.

I. Experimental Group. 57 animals. This group was maintained throughout the duration of the experiment on a diet of Purina Dog Chow. On the 3rd day of the diet the stomach was delivered through an incision in the anterior abdominal wall and 0.2 cc of a 3% suspension of sulfanilamide in normal saline were injected through a fine needle into the wall of the cardia.

Ablation studies in the dog, cat and monkey reveal a functional type of localization within the anterior cerebellar lobe. This localization is so precise that functional units in single extremities, the neck and the labyrinths are discretely represented in isolated anterior cerebellar subdivisions.

This forms the second report on developmental rate in hybrid frogs. The purpose of these experiments, in hybridizing species that differ in rate of development, is to determine the earliest effect of the sperm on this particular character. Previously such a study has been made on hybrids between Rana pipiens and Rana palustris. 1 Both hybrids, pipiens ♀ × palustris ♂ and palustris ♀ × pipiens ♂ developed at the maternal rate during early stages. The first unmistakable deviation from the maternal rate appeared in the neural plate stage in both hybrids. Additional evidence on these points is derived from the following crosses.

The material used in these experiments is from the following sources: Rana pipiens Schreber from dealers in the Lake Champlain area of Vermont and Rana sphenocephala (Cope) from dealers in southern Florida. Although listed as separate species by many taxonomists the two are closely related and are thought best included under one name by Kellogg. 2 Regardless of their true taxonomic status the two frogs form viable hybrids and differ in rate of growth to such an extent that it is possible to analyze the developmental rate of their hybrids. At 15°C the rate of both species is identical. At higher temperatures sphenocephala develops more rapidly and below 15° less rapidly than pipiens. The egg diameter in the sphenocephala used in these experiments averaged 1.3 mm and in the pipiens 1.8 mm. Whereas the upper limit for normal development in pipiens is 28° or 29°, the Florida species can tolerate temperatures as high as 34.5°.

The first experiment was begun Jan. 20, 1939. Gametes of both male and female pipiens and sphenocephala were used, the eggs being secured by pituitary injections and sperm by cutting the testes in 0.1 amphibian Ringer's.

Serum protection tests in mice have been done and the results are significant as tested by a simple statistical method. The experiments are in favor of the view that the Lansing strain is immunologically related to human poliomyelitis.

We acknowledge, with thanks, assistance from the following: Mr. Louis Mihelich and Miss Frances Love (technical assistance), and Doctor Martin Bronfenbrenner (advice on statistical method).

The serum to be described was studied because the patient from whom it was derived had had hemolytic reactions following transfusions of apparently compatible blood. In this report we shall present the serologic findings; the clinical data will be described elsewhere. 1

The patient belonged to group 0, type N. The patient's serum agglutinated every one of 28 consecutive group 0 bloods. That we were not dealing with an autoagglutinin was proved by the absence of agglutination in mixtures of the patient's serum with her own cells. Evidently the serum contained isoagglutinins in addition to anti-A and anti-B.

Since the patient was Rh negative, 2 , 3 it seemed possible that anti-Rh isoagglutinins might be present in her serum. Accordingly the serum was absorbed with pooled, washed cells A₁MNRh- and BMNRh-. The absorbed serum was found to give reactions coinciding with those of standard Rh antisera, proving that the anti-Rh isoagglutinin was present. Since tests on Rh negative bloods with the unabsorbed serum showed that most of the bloods were agglutinated, it was evident that another irregular isoagglutinin was present. Difficulty was encountered in separating the unknown isoagglutinin from the anti-Rh isoagglutinin by absorption experiments. A separation between them was readily effected, however, when it was found that the anti-Rh isoagglutinin acted strongly at body temperature, and not at all at refrigerator temperature, while the unidentified isoagglutinin acted only in the cold.

In order to ascertain the nature of the fourth isoagglutinin, it was necessary to test a larger series of bloods. On account of the presence in the patient's serum of anti-A and anti-B isoagglutinins, bloods of groups A, B, and AB could not be tested directly. It was found that by the simple addition to the patient's serum of a solution of the group substances A and B (Table I), the action of the interfering α and β isoagglutinins could be completely neutralized without affecting the activity of the anti-Rh isoagglutinin or the other irregular isoagglutinin.

Although cyclopropane initially sensitizes the dog's heart so that the injection of adrenalin causes ventricular tachycardia, subsequently it may exert an adrenolytic effect on the cardiovascular system.

The view of the identity of the interstitial cell-stimulating and thyrotropic pituitary factors could not be substantiated by the results obtained on further chemical fractionation of such preparations.

The diencephalic areas have been invoked as important subcortical centers having to do with the emotions. 1 , 2 , 3 The emotional reactions of animals with hypothalamic lesions have not been differentiated as spontaneous and reflex phenomena. The former appear without provocation and vary somewhat between different subjects. The reflex emotions occur only after stimulation of one or another sensory modality. The monkey is an excellent animal for study of spontaneous emotionality. He threatens, grimaces, wiggles his ears, undergoes pilo-erection, and vocalizes either in anger or in fear. When approached he may attempt to run away or permits his anger to carry him into definite aggressions. Occasionally, in blind rage, he dashes himself against the mesh of the cage as if he meant to rend it apart. Cats show a good deal less of spontaneous emotional display than do monkeys.

Material and Method. Monkeys and cats were used in this study. In all instances, the diencephalic lesions were placed under more or less direct vision by following the stump of one optic nerve to the chiasmal region.

Observations. 1. Total destruction of the hypothalamus.

Such a lesion invariably results in stupor in which there are no spontaneous emotional demonstrations. The stupor is frequently associated with cataleptic phenomena. When the animal is painfully stimulated, there is a prompt reflex display of emotions. The threshold to such reactions is elevated but the responses are certain. One cat responded to pressure over bony prominences, with baring of claws, opening of the jaws, an awesome rumbling in his throat, and a battery of sneezes. A similar preparation in a monkey could be aroused by stimulation to adequate shows of emotions including grimacing and defense movements of all extremities.

1. It is shown that most strains of the more important saccharolytic, toxin-forming gas gangrene species, Cl. welchii, Cl. novyi, Cl. septicum and Cl. sordellii, produce an active fibrinolytic substance. Certain other saccharolytic species, Cl. tertium, Cl. fallax, Cl. aerofoetidum fail to produce this substance. 2. The more common proteolytic gas gangrene species, Cl. histolyticum, Cl. sporogenes and Cl. tyrosinogenes produce an equally or more rapid destruction of coagulated plasma. 3. Most cultures which are active against plasma from one species will break coagulated human, guinea pig and rabbit plasma. Sheep plasma is less readily attacked and not attacked by any strains of Cl. welchii tested. 4. A few strains in both groups of gas gangrene anaerobes produce anticoagulation factors which prevent CaCl₂ coagulation of plasma. It is shown that this is not a pH effect.

Three groups of female and 2 groups of male guinea pigs were injected with 7 to 20 mg of desoxycorticosterone acetate to determine its effect on pituitary weight and on the lactogen content of the AP. A significant increase in pituitary weight was obtained, but there was no increase in the lactogen content of the AP.

A slight increase in serum inorganic phosphorus without a significant increase in serum calcium after multiple injections of anterior pituitary extract in rats is regarded as insufficient proof of the presence of a parathyreotropic factor in the extract examined.

Various authors (Borrel, 1 Roskin, 2 Lipschütz, 3 Fischer, 4 Zweibaum 5 ) have indicated that the structure of Rous sarcoma cells shows certain peculiarities, which differentiate them from normal mesenchyme cells.

These indications are very fragmentary and there is little agreement between the various descriptions. The morphological features of the Rous sarcoma cell are still not sufficiently known.

The structural peculiarities of the Rous sarcoma cells appear with great clarity and they are easily detectable in pure culture of sarcoma cells. Our studies of cell strains, cultured without addition of normal tissue revealed that the sarcoma cells in vitro constantly show profound morphological alterations of their cytoplasma and nuclei, often associated with marked, indeed extraordinary hypertrophy of the entire cells.

In this note we report on the changes taking place in the nuclei of the sarcoma cells. The studies were made on cultures (preparations in toto and serial sections) fixed in Carnoy and stained with Giemsa.

The changes in the nucleus are very profound and may lead to definite disturbances of the entire nuclear structure. The nuclear ground substances, which is finely reticulated, or finely granular in normal spindle cells, and almost structureless in normal lymphoid cells, in sarcoma cells often appears as if precipitated. In numerous nuclei particulate masses become visible. The individual particles are more or less uniform in size and stand out distinctly on the almost colorless background. At first the particles are equally distributed throughout the nucleus; but later the granular material gathers in the central parts of the nuclei, leaving a wide or narrow zone, a clear halo, between the particulate aggregates and the nuclear membrane (Fig. 1). This zone appears empty, but in some preparations we can see that it is crossed by very fine threads running radially.

We wish to report on experiments undertaken in order to investigate the cellular composition of pure Rous sarcoma cell colonies in vitro. They were carried out on cultures, 20-50 passages old, of our Rous sarcoma strains, which were cultivated without the addition of normal tissue. They include observations on living cultures and on preparations in toto and serially sectioned ones, fixed in Carnoy or Zenker Formol, and stained with Giemsa.

The results of these investigations can be summarized as follows:

The pure Rous sarcoma culture consists of two cell types: spindle cells and round cells. The structure of the spindle cell corresponds exactly to that of the normal fibrocyte. The round cell is characterized by a strongly basophilic, completely homogeneous or finely granular cytoplasm and a relatively large, round, somewhat excentric nucleus. The nucleus has a thick, unfolded membrane, 1-2 heavy nucleoli, and more or less finely divided chromatin. The size of the round cells varies between 5 and 15 μ. They are almost round, with regular or somewhat angular contours. Their pseudopodia are slightly developed, their movements are sluggish and they phagocytize but little.

The fibroblast-like cells and the basophilic round cells are not distinct cell types but different aspects of the same cell. In every culture numerous transitional stages enable us to follow, step by step, the change of one cell form into the other. The transformation of the spindle cell into the basophilic round cell takes place by means of contraction of the former. The fibroblast-like cell draws in its processes; its body becomes round, the cytoplasm more and more basophilic, the nucleus becomes more compact, its membrane easily visible and the nucleoli very prominent. The basophilic round cell, arising from the contraction of the spindle-cells, recalls at times lymphocytes and even more haemocytoblasts as seen in cultures of blood and hemopoietic tissues.

Young albino rats are more susceptible to diphtheria toxin than are adult animals. No difference seems to exist between young and old rats regarding their resistance to bacillary infection.

Propazone (5,5-dipropyl-2,4-oxazolidinedione) has been given intravenously and by mouth to a group of patients. All stages of hypnosis and anesthesia have been produced without ill effects. The absence of side reactions, the striking degree of relaxation which comes on in even the early stages of hypnosis and its long duration of action indicate that this substance might be of therapeutic value. Its use in epilepsy is being investigated.

Evidence has been presented that adrenaline and sympathin are liberated reflexly in unanesthetized dogs as a result of the brief hypotension produced by intravenous injection of acetylcholine. 1 The diphasic action of acetylcholine on intestinal motility was interpreted as resulting from a direct stimulatory effect of the compound on the intestinal smooth muscle followed by inhibition of the intestine by sympathomimetic substances. Adrenal demedullation prolonged the direct excitatory phase and reduced the inhibitory phase. It was considered that the inhibition that still resulted after adrenal demedullation was caused by sympathin liberated at the endings of adrenergic nerves activated reflexly by the fall in blood pressure.

Wiggers and Green have suggested that the evidence that a part of the acceleration of the denervated heart after acetylcholine injection is caused by adrenalin would have been more convincing had it been shown that the acceleration is reduced after excision of the adrenal medullae. 2 Such an experiment should also determine the cardiac effects of the sympathin produced during acetylcholine hypotension. The effect of a given dose of acetylcholine on the rate of the denervated heart can not be readily determined for the same dog before and after adrenal-demedullation, because the nutritional state of the animals with denervated hearts does not permit carrying them through additional operations. However, records have been obtained from 2 series of dogs with denervated hearts, one series having intact adrenal glands and the other series having the adrenals demedullated prior to the cardiac denervation.

Data obtained by the methods previously described 1 show that adrenal demedullation greatly reduces, but does not entirely eliminate, the acceleration of the denervated heart following acetylcholine injections. These facts provide additional evidence that acetylcholine causes the liberation of sympathomimetic substances from both adrenal and extra-adrenal sources.

Bromsulfalein, injected intravenously in a dosage of 2 mg per kilo of body weight, is rapidly removed from the blood stream (85-95% in 5 minutes; 100% in 30 minutes), but its elimination in the bile continues over a period of several hours. This suggests that two separate or related mechanisms are involved and that simultaneous investigation of the rapidity of removal of the dye from the blood and the curve of its elimination in the bile might yield information of interest.

The concentration of bromsulfalein in the bile was determined as follows: 0.1 cc of bile was added to 15 cc of distilled water and 0.1 cc of 10% NaOH, and mixed by inversion. A blank was prepared for each specimen, containing 0.1 cc of bile, 15 cc of water and 0.1 cc of 10% NaCl. If turbidity developed the mixtures were filtered. After 10 minutes, readings were made in the Evelyn photoelectric colorimeter, using filter 580, and the concentration of bromsulfalein determined on the basis of a calibration curve obtained by adding known amounts of the dye to bile. In the case of very high concentrations, 30-45 cc water may be used instead of 15 cc.

Studies were made upon 6 cholecystectomized, bile-fistula dogs (45 determinations), 10 patients with T-tubes in the common bile duct (32 determinations), and 24 subjects in whom bile was obtained by duodenal intubation (60 determinations). Bromsulfalein was injected intravenously (2 mg per kilo), blood was withdrawn at the end of 30 minutes for estimation of the degree of retention of dye and determination of the serum bilirubin concentration, and bile was collected by continuous drainage in 15-minute fractions over a period of at least 2 hours.

In experiments on dogs, after the intravenous injection of acacia, it was found that (1) the rate of glomerular filtration is essentially unchanged; (2) water excretion shows a diphasic response; (3) chloride excretion is markedly increased; (4) plasma proteins and hematocrit values diminish in comparable extent.

The frog is a satisfactory animal for the assay of gallbladder contracting materials and has the advantage over others of time and expense. Assays on normal summer frogs give lower figures than at other seasons. There is no significant variation in the assays on spring, early summer, male and female frogs, or winter frogs. Starved frogs have gallbladders greatly distended with thick bile. Such distended bladders do not respond well to S.I. The response of the frog's gallbladder to S.I. is not due to the presence of histamine or choline-like substances in the extracts. A modification of Ivy's method for the preparation of S.I. has resulted in the isolation of a more potent preparation and the recovery of much active material lost by the original procedure.

Actinomycin is found to be a powerful bacteriostatic and bactericidal agent in vitro. The presence of serum does not diminish the efficacy of this substance. However, no protection is afforded to mice inoculated with cultures of Streptococcus hemolyticus or Pneumococcus Type I, or to guinea pigs inoculated with Brucella abortus. The lack of in vivo activity may be due, among other things, to the rapid disappearance of actinomycin from the blood. Actinomycin is extremely toxic to all animal species, death apparently resulting from respiratory failure. Most deaths do not occur until 15-20 hours after actinomycin inoculation.

Although Bacteroides vulgatus grows in the absence of oxygen and is killed by air, its growth at low oxidation-reduction potential in the presence of 3 to 8% oxygen is characterized by oxygen consumption.

Pellets of estrone were implanted in the subcutaneous tissues, the spleen, and the transplanted ligated spleen, respectively, of groups of normal adult male rats. At the time of implantation of the pellet the right testis was removed in half the number of animals of each group. Marked atrophy occurred in the testes and related genital organs when the pellet was present in the subcutaneous tissues or in the transplanted ligated spleen. No changes occurred in these organs when the pellet was present in the normally situated spleen, indicating that when the absorbed estrone passes through the liver of normal adult male rats before reaching the organs it specifically affects, it is inactivated.

The need of vitamin E for the successful completion of pregnancy in rats is well known. 1 More recently it has been shown to be essential in mice also. 2 , 3 We have found no reference in the literature describing resorption of the embryo due to vitamin E deficiency in guinea pigs. The failure to demonstrate this heretofore has been due undoubtedly to the fact that guinea pigs on vitamin E-low diets usually die of muscular dystrophy before the onset of sexual maturity.

It seems worthwhile, therefore, to place on record certain observations which indicate that the guinea pig, like the rat and mouse, requires an abundant supply of vitamin E, and that an inadequate intake results in death of the embryo in utero.

Experimental. The basal diet V consisted of:

This was supplemented by 5 cc of tomato juice and 25 g of fresh lettuce daily. Lettuce was added to supply a necessary water soluble growth factor, the need for which in guinea pig nutrition has been indicated by the work of Kohler, Elvehjem and Hart, 4 and of Cannon and Emerson. 5 The amount of vitamin E contained in this amount of lettuce has been found by us in previous experiments inadequate to protect against muscle dystrophy.

Of 3 controls on this basal diet, 2 died after 101 and 312 days with severe muscular dystrophy. The third died at 286 days without muscle lesions.

Confirming our previous experiences, the addition of 25 g of lettuce daily did not provide sufficient vitamin E to protect 2 of the 3 animals against muscular dystrophy, nor to ensure a successful pregnancy.

Fifteen guinea pigs received a weekly supplement of 5 or 10 mg of alpha-tocopherol acetate (Hoffman-LaRoche).

From the above data it appears that on an 18% casein diet, the supplementation of 75 mg of cystine in addition to 200 γ of pantothenic acid per rat per day markedly decreases the time required for the replacement of the grey hair of nutritional achromotrichia. Since better growth results when this amount of cystine is administered, the findings of Mulford and Griffith 1 that 18% casein diets do not furnish enough cystine for maximum growth of the rat, are substantiated.

The authors wish to acknowledge the technical assistance of Mr. Alois E. Rempe in this work.

Experiments reported in this paper suggest that female mice are more resistant than males to an intradermal implantation of a small dose of a mouse sarcoma, as evidenced by the smaller incidence of takes and greater incidence of regression in the female animals.

Although in muscular atrophy, either due to final or upper neurone lesions, or due to tenotomy, there is a distinct weight loss after 9 to 10 days, the muscles have at that time still the same contractile power per weight unit and the same birefringence as normal muscles. But 3 to 5 weeks after onset of the atrophy, only muscles atrophying due to upper neurone lesions or to tenotomy have still the contractile power per weight unit and the birefringence of normal muscle. However, for denervated muscle, contractile power and birefringence are distinctly diminished at that time. Only in the latter type of atrophy does the submicroscopic crystalline structure, so essential for the contraction process, become seriously impaired.

Butler and Marrian 1 in reporting the isolation of a steroid compound pregnane-3-17-20-triol, from the urine of 2 women with virilism caused by “enlarged adrenals,” also noted the presence of “appreciable amounts of pregnandiol.” Venning, Weil and Browne 2 later reported the finding of considerable amounts of sodium pregnandiol glucuronidate (12 mg to 30 mg per day) in 2 cases of virilism, one of which was subsequently found to have an adrenal cortex carcinoma and, the other, adrenal cortex hyperplasia.

During the past 2 years, we have been performing pregnandiol excretion studies in women with the arrhenomimetic (virilism) syndrome. In the present communication, we wish to report the results of these studies.

Methods and Materials. A total of 7 cases was studied. These consisted of 2 cases proven by operation to be adrenal cortex carcinomas and 5 cases of amenorrhea and hypertrichosis of undetermined etiology. Sodium pregnandiol glucuronidate determinations 3 were performed on single 24-, 48-, or 72-hour specimens, for periods varying from 6 to 32 days.

Results. Pregnandiol Excretion in Patients with Adrenal Cortex Carcinoma. In the 2 patients in whom adrenal cortex carcinomas were found, the pregnandiol excretion varied in one from 9 to 20 mg per day and, in the other, from 12 to 18 mg per day. In one patient (I.E.), in whom it was possible to obtain post-operative urines, these were found to contain no detectable amount of pregnandiol after removal of the tumor.

Pregnandiol Excretion in Patients with Arrhenomimetic Syndrome (without adrenal cortex carcinoma). In the 5 cases in this group, X-rays of the adrenals after intravenous pyelogram and peri-renal insufflation failed to reveal any evidence of adrenal tumor or enlargement. Two of these (E.W. and T.S.) were explored surgically without revealing any evidence of adrenal neoplasm.

In one of 2 hypoproteinemic individuals exhibiting the “nephrotic stage” of chronic nephritis, intravenous administration of amino acids was, on two occasions, followed by a well marked rise in the level of the serum proteins. The rise occurred in the albumin fraction. The other subject failed to respond.

Cl. welchii was grown in a medium free from peptone or other substances of large molecular weight. Toxins were consistently produced which were equal to or more potent than those produced in glucose peptone beef infusion broth. The pH of the culture media did not fall below 6.0. This may in part be responsible for the higher potency of toxin obtained in this medium. The basal medium alone did not support growth; when it was supplemented with pantothenic acid, pimelic acid, nicotinic acid, riboflavin, or liver-extract, there was a marked increase of growth and toxin-production.

The fact that the highest bacterial-nitrogen value was obtained in glucose-peptone beef-infusion broth, appears to indicate that the casein hydrolysate still lacks one or more chemical ingredients required for maximal growth.

The work is being continued in an effort to identify a chemically defined medium for maximal growth and consistent toxin production by Cl. welchii.

This note presents the radioactive phosphorus (P³²) and the non-radioactive phosphorus (P³¹) content of tissues obtained from a patient dead of chronic lymphoid leukemia, who had received orally 19 days before death a single administration of a sodium phosphate solution, containing radio-phosphorus, which emitted 20 millicuries of beta radiation on the day of administration. 1

Materials and Methods. The radio-phosphorus was produced by the Berkeley cyclotron. 2 The patient was a 65-year-old carpenter who on August 16, 1940, complained of fatigue and headaches of 2 weeks' duration, and who on physical examination had an enlarged spleen and enlarged inguinal glands. The blood findings were: Hemoglobin, 37%; r.b.c., 2,000,000; w.b.c., 125,600. 99% of the latter were small leukemic lymphocytes. On October 20, 1940, the patient took orally 120 cc of a solution containing 3.6 g of sodium phosphate and emitting 20 millicuries of beta radiation. Between 10/28/40 and 11/7/40 the patient received 2350 cc of blood without marked alterations in the blood findings. The patient died 11/8/40. Fortunately it was possible to obtain at postmortem all of the organs and one entire lower extremity. Typical gross pathological findings of leukemia were observed. Thirty-eight organs and tissues were weighed as were small aliquots (5 g approx.) of each. Then the whole organs and tissues and their aliquots were placed in separate crucibles and all ashed at 400°C. The ashes (all of which weighed less than 200 mg each) of the aliquots were immediately measured for radioactivity by use of an electrometer. The ashes of the whole organs and tissues were thoroughly mixed and 100 mg aliquots were assayed for radioactivity. Quantitative phosphorus analyses were made by the method of Pregl. 3

Spermatogenesis was maintained by subcutaneous injection of 3 mg daily of testosterone propionate in 4 guinea pigs hypophysectomized for 60 to 155 days. Tubular atrophy in the pig may not be complete as late as 84 days after hypophyseal ablation. Adrenal atrophy occurs after removal of the pituitary, but increase in body weight does not necessarily cease.

We reported the conversion of S-benzyl-l-cysteine to the corresponding acetyl derivative in the rat, rabbit, dog 1 and man 2 and suggested the possibility of the inversion of S-benzyl-d-cysteine to the acetylated l-derivative via Knoop's acetylation mechanism 3 which postulates the formation of a keto acid from the d-amino acid followed by the interaction of the keto acid with ammonia and pyruvic acid to give the l-acetyl amino acid. duVigneaud, et al., 4 reported results which apparently indicated such an inversion of S-benzyl-d-cysteine in the rat. On feeding the d-isomer to rats, some l-acetyl derivative together with dl-acetyl benzylcysteine were isolated from the urine. When d-benzylcysteine was fed to rats and humans, 2 significantly smaller yields of the acetylated products could be isolated from the urine as compared to those obtained after the administration of comparable amounts of l-benzylcysteine. We drew the conclusion at the time 2 that these findings did not conform to the postulates of Knoop's acetylation theory which presupposes that if the acetylation of an amino acid takes place via the keto acid, then the amounts of the acetylated product excreted in the urine of animals which were fed d- or l- amino acid should be the same. Indeed, du Vigneaud and Irish, 5 who revived Knoop's theory, stated that “this would be expected if the theory were correct, since both enantiomorphs should be deaminated to the keto acid.” The lower yields of the acetyl derivatives in the urine after the feeding of d-benzylcysteine to rats and man 2 we later attributed 6 to a possible destruction of the intermediate keto acid which was presumably formed from d-benzyl-cysteine prior to its conversion to the acetyl derivative.

No significant changes in cell volume, total erythrocyte count and hemoglobin values were observed in trichina-infected dogs. All animals exhibited increases in eosinophiles which reached a maximum 10 to 15 days after infection. Significant increases in serum calcium, which reached a maximum (16-18 mg%) 11 to 18 days after infection, were observed in only 4 dogs. It would appear that hypercalcemia can only be elicited by a massive infection, whereas an eosinophilia will always result from ingestion of fewer organisms.

(1) The absorption of many drugs introduced into the bone marrow was studied in various animals. (2) Aqueous solutions of all such agents were rapidly absorbed as indicated by their characteristic systemic effects. (3) Intramedullar injections offer an especially useful means for pharmacological study of oils and oily solutions. (4) Volatile oils are rapidly absorbed through bone marrow but the fixed oils are absorbed more slowly, thus retarding absorption of active principles dissolved therein. (5) Epinephrine in oil, introduced into medullary canals, produces a marked and long-sustained rise in blood pressure, an effect which cannot be achieved by intramuscular injection of such an oil solution.

The therapeutic effectiveness of sulfanilamide may be markedly enhanced in the presence of other antibacterial agents, notably immune serums. The question presents itself as to whether or not a similar synergistic action may be obtained by the combined use of two different chemotherapeutic substances. In vitro studies on the combined bacteriostatic activity upon pneumococci of sulfapyridine or sulfathiazole and optochin hydrochloride (ethylhydrocupreine hydrochloride) or beta hydroxyethylapocupreine dihydrochloride (Parke, Davis & Co.) failed to reveal such a synergistic effect. 1 On the other hand, it could be shown that with E. coli as test organism, the combined use of pyridium (phenylazo-alpha-alpha-diamino-pyridine mono-hydrochloride) and sulfamido compounds resulted in bacteriostatic effects beyond that exerted by either compound alone. 2 The activity of sulfamido compounds and azochloramid used in combination against hemolytic streptococcus Group A and enterococcus is reported in the following communication. Azochloramid (N, N'x-Dichloroazodicarboxamidine) (Wallace and Tiernan products) was chosen as a representative of chlorine compounds. It is a bacteriostatic and bactericidal agent which is relatively stable and whose effectiveness in the presence of organic matter is inhibited to a slight degree only.

As test microörganisms a strain of beta hemolytic streptococcus (Group A Lancefield) which was isolated from the chest fluid of a patient with empyema and a strain of hemolytic enterococcus that was isolated from the urine of a patient with infection of the urinary tract, were used. Brain heart infusion and 1/4% maltose phenol red broth (Difco) were employed as culture media. Sulfanilamide [p-aminobenzenesulfonamide, prontylin, repurified for injection (Winthrop)] and sulfathiazole [2-sulfanilamidothiazole (Squibb)] were dissolved in appropriate amounts in broth by heating in a water bath. These solutions as well as the broth media were sterilized by autoclaving at 15 lb pressure for 12 minutes.

Accelerative effects of biologically conditioned casein-peptone medium, when added to fresh medium in proportions of 1-5 parts in 10, have been reported previously for Colpidium campylum. 1 Subsequently, it has been found that an increase in the proportion of conditioned medium induces inhibitory effects on the growth of Colpidium campylum and Glaucoma piriformis. ∗ Observations on the latter are presented below.

Series I and II. A culture filtrate was prepared from 96-day flask-cultures of G. piriformis in casein-peptone (Difco “tryptone”) medium containing KH₂PO₄ (1.0 g per 1). The cultures were filtered through paper (Whatman No. 12), as previously described by Hall and Loefer, 1 and the filtrate was divided into two portions. One portion (Series I) served as the control medium. To the other (Series II), the following growth-factors were added: Merck's thiamine hydrochloride (1 × 10^-6 g per cc), Merck's riboflavin (1 × 10^-7 g per cc), and S. M. A. Corporation's nicotinic acid amide (1 × 10^-7 g per cc). Each medium was tubed in 10 cc amounts and sterilized in the autoclave at 122° C for 20 minutes. All tubes received 0.5 cc inocula from a 3-day culture of G. piriformis in the basic medium. In both series, the initial pH was 6.9; initial count, 4,000 ciliates per cc. All cultures were incubated in darkness at 24°C, and counts were made at the indicated intervals (Fig, 1, A).

Series III and IV. The procedure was the same as above, except that 109-day flask-cultures were used as the source of the filtrate, and a 21-day stock culture was used for inoculation. Series III served as the control; in series IV, the medium received the 3 growth-factors. In both series, the initial count was 6,400 ciliates per cc; initial pH, 6.9.

It has been shown that on a purified basal ration 0.5% sulfaguanidine greatly reduces the growth rate of young rats. When liver extract is fed with 0.5% sulfaguanidine, optimum growth is obtained. p-amino benzoic acid fed with 0.5% sulfaguanidine from the beginning of an experiment gives a definite growth response, but gives no response during the first week when fed to rats which have received sulfaguanidine alone for several weeks. The bearing of these results upon the possible synthesis of unidentified rat growth factors by intestinal bacteria is discussed.

The blood coagulation time in methyl methacrylate (boilable “lucite”) tubes was found to be twice as long as the coagulation time in glass tubes. The blood coagulation inhibiting effect of this material follows Lampert's rule of surface adhesion.

When histidine and related compounds are treated with bromine at pH 1 to 1.5, the excess bromine removed, and the solution alkalinized and aerated, significant amounts of ammonia are recovered from the imidazole ring. Copper carnosine, copper acetyl histidine, and imidazole lactic acid yield amounts of ammonia which correspond to a complete cleavage of the ring nitrogen. Histidine yields sufficient ammonia to account for all of the ring nitrogen plus almost one-half of the alpha-amino nitrogen. Other compounds tested gave 13-55% of their imidazole nitrogen as ammonia.

In connection with work on hypertension it was of importance to have a simple method for testing a large number of extracts for their renin content. Rabbits were used for the test as their blood pressure is conveniently measured in the artery of the ear by a membrane manometer. 1

After several preliminary trials a procedure was standardized, which, despite its simplicity gives reliable and reproducible results, as accurate as one would expect from a bioassay.

The test was carried out in a warm room. The unanesthetized animals were placed in a box with open front and top; after some training they would sit quietly. The left ear was placed between the capsule and light of the manometer without forcing it away from its natural position. Measurements of the blood pressure were taken at about 1/2-minute intervals; after a few minutes the readings were constant, fluctuating not more than one millimeter from the average level. After a constant level had been maintained for at least 3 minutes, the extract was injected intravenously in the right ear. The introduction of the needle had no effect on the blood pressure, as the right ear had previously been denervated. Injection of 5 cc of physiological saline solution also had no effect on the blood pressure.

The injection time chosen was 60 seconds, but practically the same results were obtained if the injection was made in 30 seconds. When the injection was started, measurements of the blood pressure were done in rapid succession, about 3 per minute. The maximum response to renin was usually obtained about 3 minutes after the start of the injection. For each extract 4 animals were used and amounts of renin were injected which would cause a rise in blood pressure of 20 to 40 mm mercury.

Ten adult, male, New Zealand albino rabbits were used to determine the percentages of body weight of blood, skin and fur, viscera, bone, brain and muscle. The brain weight is a constant percentage of body weight, the bone weight fairly constant and the other tissues vary within 6%.

1. The disappearance of pyruvate added to blood in vitro is caused by the blood cells and not the serum. 2. Hemolysis of the cells by saponin or freezing increases the speed and extent of this reaction. 3. The extent and rapidity of the reaction is not altered by Vitamin B₁ deficiency of the cell donor, nor by the addition of cocarboxylase to the cells in vitro. 4. Anaerobiosis has no effect on the reaction. 5. The reported effect of cyanide upon the reaction is due to an artefact. 6. The pyruvate which disappears is neither decarboxylated nor changed to other ketonic acids or lactic acid.

A modified electro-magnetic flow meter has been described which may be applied to investigation of a wide variety of cardiovascular problems involving flow and pressure measurements in intact animals.

Liquid, semi-solid and solid media, tissue cultures, and developing chick embryos have been utilized for the growth of Bartonella bacilliformis. 1 , 2 , 3 , 4 , 5 Of these media, the semi-solid leptospira medium of Noguchi and nutrient blood agar have aided (1) in proving B. bacilliformis as the etiological agent of Oroya fever and verruga peruana, the two entities of Carrion's disease in Peru, (2) animal experimentation, (3) studies on transmission, and (4) limited immunological studies with the organism. Further progress, particularly with the biochemical characteristics of the organism and the immunology of the disease, has been handicapped by the slowness and relatively sparse growth of bartonella in leptospira medium, and the difficulty of making suspensions with organisms when grown on blood agar. Thus, it was believed that the development of methods for cultivating greater quantities of the organisms would stimulate attempts to solve some of the current problems of bartonellosis.

A fruitful approach to the problem of improving known media and devising new ones, appeared to be linked with the need in leptospira medium for serum and particularly a solution of hemoglobin from laked red blood corpuscles. The necessity for these ingredients indicated that certain vitamins and growth-promoting factors found in the cellular elements of the blood might be the key substances needed for growth.∗ Furthermore, B. bacilliformis in Oroya fever invades and multiplies within cells of the reticulo-endothelial system, 4 a fact which suggested the possible advantageous use of ingredients in monocyte media 6 , 7 for enhancing the growth of bartonella.

Extensive experiments with protein digests; blood, yeast, and liver extracts; peptones; vitamins and developing chick embryos have yielded numerous new media that surpass the results obtainable with leptospira medium and blood agar.

In 3 cases of healed massive necrosis of the liver Vitamin A was more abundant in the parts of the liver from which hepatic cells had disappeared than in those where these cells had not been destroyed. Presumably it was stored in the Kupffer cells, which had been spared by the agent which produced the necrosis.

Various sulfonamide derivatives prepared in this laboratory have been tested against Trypanosome equiperdum with negative results. 4-Nitrobenzoic acid which has a slight antibacterial action 1 , 2 was found to have a trace of trypanocidal activity. Investigation of the isomers revealed that the meta derivative showed increased activity against trypanosomes, although without effect upon bacteria; 2-nitrobenzoic was inactive against both types of infection. A review of the literature revealed that Morgenroth found 3-hydroxybenzoic acid to possess some trypanocidal action. 3

A more detailed study of 3-nitrobenzoic acid and some derivatives was undertaken. Mice were inoculated intraperitoneally with a quantity of trypanosomes that would cause death in 3 to 5 days. Therapy was usually begun on the second day after inoculation. The dosage used was in most cases one-half of the maximum tolerated single dose; this was repeated daily for 3 to 6 days to surviving animals. A few of the compounds were synthesized by one of us (H.B.). The others were obtained from Eastman Kodak Company.

The average survival time was calculated from the day of inoculation, using a maximum of 30 days.

Compounds with some activity are shown in Table I. The following derivatives also showed traces of activity; dosages in grams per kilo, administered subcutaneously, are shown in parenthesis. The soluble sodium salts of the acids were employed. Insoluble compounds were injected in olive oil. Dosages represent approximately one-half the single M.T.D.

3-Nitrobenzyl chloride (0.5), 3-nitrohippuric acid (2.0), 5-nitrosalicylic acid (0.5), and 3,5-dinitrosalicylic acid (0.1).

The following compounds were inactive:

2-Nitrobenzoic acid (0.5), 3-nitrophenol (0.25), 3-nitrochlorobenzene (0.25), 3-nitrobenzene sulfonamide (0.25), 3-nitrobenzene sulfonic acid (1.0), 3-nitrobenzoyl-2-amino-pyridine (0.4), 3-nitroanisole (0.5), 3-nitrophenetole (0.5), 3-nitroacetophenone (0.5), 3-aminobenzoic acid (2.0), 3-aminobenzene sulfonamide (1.5), nicotinamide (1.0), isophthalic acid (1.5), 3-nitrophthalic acid (2.0), 4-nitrophthalic acid (2.0), 3-nitrophthalimide (1.0), 3-nitrosalicylic acid (0.5), 3,5-dinitro-benzoic acid (0.25), 2,4-dinitro-benzoic acid (0.5), 3-nitro-4-hydroxy-toluene (0.5), 3-nitro-2-aminotoluene (0.25), 3-nitro-4-aminotoluene (0.5), 3-nitro-2-iodotoluene (0.4), 3-nitro-4-amino-anisole (0.5), 3-nitro-4-amino-phenetole (0.75), 3-nitro-4-acetylaminophenyl acetate (0.5), 3-nitrodiphenylene oxide (0.5).

During the past 8 years sporadic outbreaks of an unidentified, highly fatal malady of newly-born pigs have come to the attention of the Illinois Agricultural Experiment Station. This, or an indistinguishable malady, has been arbitrarily referred to as baby pig disease.∗ In connection with different outbreaks on widely separated farms an increasing number of naturally affected pigs has been delivered to the laboratory for examination, while a limited number of affected herds has been inspected to observe methods of managing the pregnant sows. So far as could be determined the rations fed the pregnant sows appeared adequate. However, further information regarding the relation of feeding of the pregnant sow to the disease in newly-born litters is desirable.

Pigs that suffer from the disease referred to present a characteristic syndrome. In typical outbreaks newly-born pigs, though apparently normal at birth, suddenly develop symptoms. Pigs that immediately display similar or indistinguishable symptoms at birth are purposely excluded from consideration in this report. In the typical syndrome, apparently normal litters at approximately 24 to 48 hours of age show symptoms of shivering, dullness and inappetence. Affected pigs often emit a weak crying squeal. Coincident with the loss of appetite and weakness, the hair coat becomes rough and the affected pigs leave the nest and lapse into coma. Death of several or all pigs in the affected litters often occurs within 24 to 36 hours after the first symptoms are manifested. The extent of the loss has ranged from one to 22 litters, representing approximately 5 to 95% of the pigs farrowed in some herds.

At autopsy no gross pathologic lesions have been observed. Supplementing gross autopsy examination of typically affected pigs repeated efforts have been made to demonstrate in the tissues the presence of pathogenic agents such as bacteria, filtrable viruses, protozoa and toxins.

When grown in sucrose broth some strains of group H streptococci produced large amounts of material reactive with types 2 and 20 antipneumococcus and with antileuconostoc serums; little or none was produced by the same streptococci when grown in dextrose broth. This reactive material was different from the streptococcus antigen involved in the usual Lancefield grouping test; as much of the latter was produced in dextrose as in sucrose broth culture.

In addition to indicating an interrelationship of the different Gram-positive cocci the data furnished an example of the influence of a particular carbohydrate upon the capacity of some microörganisms to elaborate a serologically reactive polysaccharide.

A medium has been developed on which a specific oral streptococcus can be selectively isolated from mixed bacterial populations. The value of this selective medium as a detector of oral contamination of restaurant eating utensils is indicated by the distribution studies, which show that this bacterium is found normally in the human mouth and on articles which have come in direct contact with human lips.

(1) The ability of normal systemic blood plasma to neutralize angiotonin is markedly decreased after circulation through the partially ischemic kidney.

Dehydration is antiketogenic when ketosis is measured by the level of ketone bodies in the blood of fasting albino rats. Protein catabolism is increased during dehydration and it is probable that the larger amount of carbohydrate provided by the protein break-down accounts for the antiketogenesis.

By means of the vibrocardiograph 1 it is possible to record all the cardiac vibrations (audible and inaudible) that are transmitted through the chest wall. A study of the vibrocardiograms of 100 normal young adults, 2 taken from the usual auscultation areas, established the presence of dominant vibration groups at the onset of systole and at the second sound. These curves were dissimilar to the usual stethogram, as the vibration groups were modified by low-frequency (inaudible) components with which the sound elements are mixed. In 5% of the tracings there appeared low-frequency waves (approximately 1-5 dv. per sec.), the largest mound occurring in mid-systole and two or more in diastole. These waves were of very low amplitude.

The vibrocardiographic curves of 102 individuals over 55 years of age, with and without evidence of coronary disease, were examined. The cases fell into three groups: those with clinical and electrocardiographic evidence of coronary disease (34 cases); those with objective evidence but with normal electrocardiograms (33 cases); and those with no evidence of heart disease. Cases having definite evidence of coronary disease, despite the presence or absence of electrocardiographic change, all showed a prolongation of the total vibration complex at the onset of systole. Normally this complex averages 0.22 second in duration; in these cases the average length was 0.33 second. In some instances of severe myocardial damage, deflections of low frequency with occasional steep slopes occupied nearly all of systole, and on auscultation the sounds were “impure” and muffled. In most of these tracings large, low-frequency waves appeared in systole and diastole similar to those seen in some normals, except that they were definitely augmented in amplitude, occasionally becoming as tall as the most dominant sound deflections; they were in phase with each other and with the onset of ventricular systole.

The glycogen content of biopsy samples of normal livers was highest in patients that were fed glucose prior to operation. When extensive liver damage was present, liver glycogen was low despite glucose feeding.

Tissues from 16 leiomyomas were tested for contractility using normal uterine muscle for control. The stimulating substances used were pituitrin, histamine, and ergot.

In 11 cases the tumor tissue showed definite response. Uterine muscle usually showed increasing tone in the dying phase while the tumor tissue became unresponsive without change in tone.

When an intracutaneous injection of bacterial filtrates or heat-killed organisms into sensitized rabbits was followed by intravenous injections of sufficient amounts of killed or living organisms, a hemorrhagic reaction frequently developed at the site of the intracutaneous injection.

The most intense hemorrhagic reactions were observed in animals with the highest degree of cutaneous sensitization. We did not observe hemorrhagic phenomena in non-sensitized animals treated in a similar manner.

Blood pantothenic acid values from six normal subjects varied in the present study over the usually obtained normal range of from 19.7 to 33.6 μg%. These values are ordinarily decreased in cases of pantothenic acid deficiency. Six patients with advanced multiple sclerosis failed to show any such decrease in the concentration of pantothenic acid in the blood indicating that this vitamin is probably not an etiologic factor in this disease.

In addition to thiamin, riboflavin, nicotinic acid, pyridoxine, and pantothenic acid, young puppies also require choline and probably other factors of the vitamin B complex for normal growth under the conditions of these studies. Possible factors altering the requirement of choline for dogs on synthetic rations are discussed.

Details are given of a method for primary anaerobic cultivation in small plates of spirochetes from human and experimental fuso-spirochetal exudates, and for their pure subculture in plates and tubes. Successful primary plates were obtained in 23 instances out of 29 cases of human infection, and from all of 21 experimentally infected guinea pigs. Eleven strains of spirochetes have been isolated and subcultured in a short period with little difficulty.

The total sulfur, total nitrogen and the amino acid content of total serum proteins from normal and arthritic subjects were compared. No essential difference in values between the two groups could be found, invalidating any suggestion that an altered composition of the total serum protein of arthritic patients reflects a disturbed sulfur metabolism.

Eastwood 1 showed inositol to be one of the bios factors required by certain yeasts. Recently Woolley 2 , 3 has demonstrated that the mouse requires inositol for normal growth and the prevention of alopecia. Pavcek and Baum 4 have also found inositol to be effective in preventing lack of growth and “spectacled eye” in rats maintained on certain purified diets. These results together with the isolation of phytic acid from chicken blood by Rapoport 5 suggested that the chick might also require inositol.

Growth responses varying from 18 to 52 g in 4 weeks have been obtained upon the addition of inositol† to several different simplified chick rations (Table I). It is clear that inositol has a definite growth-promoting action although the response varies with the amount of inositol in the ration and the adequacy of the supplement used to supply unidentified growth factors.

Ration 477 is the same as ration 470 described previously 6 with the following additions: 3% of extracted kidney residue, .15% choline, 15 mg of pantothenic acid, 100 mg of nicotinic acid, and 4 mg of pyridoxine per kilo. The thiamine and riboflavin content have been raised to 3 and 4 mg per kilo respectively and additional MnSO₄ has been added to raise the level of manganese to approximately 60 p.p.m. Ten percent of blackstrap molasses replaced kidney residue in groups 2 and 3 as a source of the antidermatitis factor previously studied. 7 SLE eluate designates a norite eluate prepared from solubilized liver extract‡ by adsorption at pH 3 and elution with 5% ammonia.

These results extend our knowledge of the rôle of inositol in the nutrition of the mouse, rat, and yeast to that of the chick as well.

The preliminary administration to immature rats of high dosages of stilboestrol or alpha-estradiol dipropionate produces a significant increase in the effect of chorionic hormone on the resultant ovarian weight.

The estrogenic hormone content of the blood of women was determined at 4-hour intervals during the course of one day in 5 instances. A significant diurnal rise was noted once, a minor increase in 3 cases, and a constant level was found in one case.

The blood serum and blood cells of a group of 20 non-pregnant women were examined for their estrogenic hormone content by the intravaginal application of pellets made from desiccated material. The blood cells were found to contain two or more times as many rat units per unit volume as the blood serum.

Beckwith and Chanutin 1 state that “in the hypertensive partially nephrectomized rat, the plasma volume was increased, the red cell volume decreased and the total blood volume remained unchanged.” In referring to a paper by Griffith and Ingle, 2 who found an elevated blood volume in hypertensive partially nephrectomized rats, they say, “On inspection of their procedure for estimating blood volume, it appears that they neglected to take the red cell volume into consideration and the values reported by them probably represent plasma volumes… the increased plasma volumes (found by Beckwith and Chanutin) … agree with the reinterpreted data of Griffith and Ingle.”

The blood volume method used by Griffith and Ingle was that previously reported by Griffith and Campbell. 3 In this method 0.3 cc of 5% vital red is injected intravenously and 4 1/2 minutes later 50 cmm of whole blood are removed and placed in 2.95 cc of physiologic saline, the resulting volume being 3 cc and the blood dilution 1:60. After centrifuging, 2 cc of the supernatant fluid are removed and compared in a colorimeter with suitable standards. The red cells remain in the 1.0 cc left in the test tube, and a slight error is thus introduced in that the volume which the cells occupy is included in the 3 cc but does not contain an aliquot portion of the dye. The dye is actually contained in 3 cc less the volume of the cells. If the cells comprised 50% of the total blood, the figure would be 3.0 cc less .025 cc or 2.975 cc. This represents an error of 0.8%, and would make the calculated figure too low by that amount.

It has been described in a previous note that in cultures of certain bacterial strains belonging to various species tiny colonies, similar to young colonies of the pleuropneumonia group of organisms develop. 1 During the last year these colonies were isolated in pure culture from a strain of flavobacterium and one of Bacteroides funduliformis respectively. The colonies isolated from the flavobacterium always died out after 2 or 3 transplants; those isolated from the funduliformis apparently can be indefinitely maintained in cultivation. Colonies resembling those of the organism of pleuropneumonia bovis were first isolated by Klieneberger from cultures of Streptobacillus moniliformis. 2 She designates them with the letter L. Recently she described the isolation of similar cultures from a Gram negative streptobacillus causing suppurative lesions in guinea pigs. 3 The characteristics which differentiate these L type colonies from the usual bacterial colonies are the following:

The colonies are very tiny. The growth extends not only on but beneath the surface of the medium. The growing elements of the colonies are tiny pleomorphic granules and fine filaments which swell up and may form large round bodies of 5 to 10 micra. The pleomorphism of the cultures is accentuated by the softness of the elements. They are disfigured by the slightest tearing and for this reason are not recognizable in dry smears. The colonies have a marked tendency to autolyze.

A strain of funduliformis was isolated from a case of generalized peritonitis. Immediately after isolation the bacterium was similar in many respects to Streptobacillus moniliformis. It grew in the form of Gram negative segmented filaments which in fully developed colonies became pleomorphic and many of the bacteria swelled up into large round bodies.

Klarmann, Shternov and von Wowern 1 showed that the substitution of one, 2, and 3 atoms of chlorine into the phenol molecule led to a progressive increase in the germicidal action of the resulting compounds. Unfortunately, at the same time the solubility of the phenolic compounds decreased markedly with substitution. The sodium salts of these halogenated phenols possess a considerably greater solubility than the phenols themselves, but there is no explicit information available in the literature as to the actual germicidal power of the phenolates.

Tilley and Schaeffer 2 showed that the germicidal efficiency of phenol decreased with an increase in percentage of sodium hydroxide added until the percentage of sodium hydroxide equalled or slightly exceeded that required to neutralize the phenol. Phenol is a weak acid with the acid dissociation constant K'_a = 1.06 × 10^-10 at 25°C. 3 Consequently a completely neutralized solution of phenol will be strongly alkaline, and an attempt to evaluate the actual germicidal action of the phenolate is complicated by the alkalinity of the solution. The substitution of chlorine atoms into the phenol molecule increases the strength of the molecule as an acid and makes it possible to evaluate the relative germicidal action of some undissociated phenols and their phenolates without undue interference by excess acidity or alkalinity.

In this investigation 3 substituted phenols: o-chlorophenol, 2, 4 dichlorophenol and 2, 4, 6 trichlorophenol were tested in moderately acid and alkaline solutions. The acid series of solutions were made by diluting weighed quantities of the several phenols with M/60 phosphate buffer of approximately pH 6.1. The alkaline solutions were made by adding standard sodium hydroxide to weighed samples of the phenols to adjust them to the neighborhood of pH 9.8 and diluting with M/60 carbonate buffer at pH 9.8.

Since the first description of uroporphyrin by H. Fischer 1 this substance has been believed to be excreted only in the urine. Numerous publications concerning the excretion of porphyrins under normal or pathological conditions have failed to mention the occurrence of uroporphyrin in the feces. We have found it in the form of the zinc complex in the feces of four out of five cases of idiopathic porphyria.† Its presence was questionable in the fifth. Although uroporphyrin was readily identified in the feces of cases 1 and 3, the method of purification available at the time these were studied was inadequate to permit crystallization. By means of an improved method entailing chromatographic analysis, crystalline uroporphyrin has been isolated from cases 4 and 5. The method employed was as follows: The untreated feces was ground in a mortar with methyl alcohol saturated in the cold with hydrochloric acid gas. The methyl alcohol extract was filtered from the fecal residue on a Buchner funnel, and the residue was then extracted twice more by grinding in a mortar with an additional amount of methyl alcohol HCl. The combined methyl alcohol extract was allowed to stand overnight. It was then mixed with chloroform and several volumes of water and shaken in a separatory funnel. The chloroform fraction was washed twice with water and once with 10% NH₄OH. Shaking with an equal volume of 7% NaCl quickly breaks any emulsion. The chloroform was washed twice more with NaCl solution. It was next filtered through chloroform moistened paper and mixed with 10 volumes of petroleum ether. The filtrate was then passed through a column of Brockmann's Al₂O₃ (Merck) 1.5 × 12 cm in dimensions.

The urines of patients suffering from idiopathic porphyria have often been noted to exhibit strong Ehrlich reactions, in many instances at least, not due to urobilinogen. Waldenström's studies 1 , 2 clearly demonstrated that the chromogen responsible for the Ehrlich reaction in these urines is quite distinct from urobilinogen, which is of course, most often implicated in other pathological states. During the past 3 years we have had opportunity to investigate urine samples from 5 cases of so-called “acute” idiopathic porphyria.† In each instance the urine contained the zinc complex of uroporphyrin in considerable amount. The subject of zinc uroporphyria as a disease entity will be considered in a separate communication. These urines also exhibited Ehrlich reactions in varying degree, at times very intense. In the first 4 cases, the freshly voided urine was already red-brown in color, becoming darker on exposure to light. In the fifth case, the fresh urine was for the most part normal in color, exhibiting a strong Ehrlich reaction. On standing in the light for several days the urine from this case likewise became a deep reddish-brown, containing now both the zinc complex of uroporphyrin and the reddish-brown porphobilin. 2 Waldenström pointed out that porphobilinogen is much less soluble in organic solvents than is urobilinogen. 1 , 2 In addition to confirming this, we have found that the porphobilinogen aldehyde compound as formed in the Ehrlich reaction is wholly insoluble in chloroform, while that of urobilinogen is readily and easily extracted with this solvent. The reaction as we have carried it out is as follows: Equal parts of urine and Ehrlich's reagent‡ are mixed in a test tube.

A synthetic, chemically-defined medium has proved satisfactory for the study of the bacteriostatic effect of the sulfonamide compounds upon staphylococci. Sulfathiazole and sulfadiazine have a definitely greater inhibitory effect upon growth compared to sulfapyridine and sulfanilamide, with sulfathiazole superior to sulfadiazine. While p-aminobenzoic acid inhibits this bacteriostatic action of all the compounds studied, the greater the effect of a compound upon growth of Staphylococci, the less inhibitory is p-aminobenzoic acid against this action of the compound.

Recently the first case of sectioning the nerves to the choledochoduodenal junction in a patient was reported. 1 The reasons for believing that this procedure would not prove effective in relaxing the human sphincter of Oddi have already been set forth. 2 To test this more thoroughly, selection has been made of a laboratory animal in which inhibitory reflexes from the gut tract to the gall bladder have already been demonstrated 3 and in which the detailed distribution of nerves to the choledocho-duodenal junction has been worked out 4 —namely the cat.

At laparotomy, the following procedures were carried out upon approximately 30 cats: (1) various autonomic nerves in the abdomen were severed (or doubly ligated); (2) insulated electrodes were sewed to the caecum by a flap of the rubber tube through which the enamelled wires were brought to the laparotomy wound; (3) the gall bladder was filled with lipiodol (Whitaker method). Some 15 hours later, by which time the animals had fully recovered, the latter were fed a mixture of egg yolk and milk by tube and then x-rayed at appropriate intervals with the object of ascertaining the rate of emptying of the gall bladder and the presence of inhibitory reflexes emanating from faradically induced contraction of the caecum.

Severance of the gastroduodenal nerve and plexus—specific nerves in the lesser omentum leading to the choledocho-duodenal junction—resulted, in 8 animals, in retarding the evacuation of lipiodol. (The studies of DuBois and Hunt 5 were used as controls); and interruption of these nerves did not abolish the inhibitory reflex from the caecum to the gall bladder. These experiments, therefore, fail to support the view that such operations in man would relieve a spasm of the sphincter.

Paranitrobenzoic acid and the salt compare favorably with sulfathiazol in their bacteriostatic power against Type II pneumococci in vitro.

The principle of the method to be described for testing pituitary chromatophorotropic substances is based on the fact that the dermal melanophores of excised skin of Anolis carolinensis respond to pituitary chromatophorotropin 1 by dispersion of the melanosomes.

The use of hypophysectomized frogs and lizards and light adapted frogs involves several undesirable conditions. They are all time-consuming, and in the case of operated reptiles, endocrine imbalance, especially thyroid effects, are involved. The use of excised skin of Anolis as a test object for measuring intensity of effect, is very rapid, entails no difficult operative procedure, requires only small amounts of material, and eliminates the influences of other possible endocrine or neural disturbances. The test has been found to be reliable. Uniform responses are produced on skin of wellfed animals 100% of the time without difficulty. The complete dispersion of melanosomes is easily detected since the color change from green to brown is very striking.

Acetone-dried pituitaries of several vertebrates (man, beef and dog) have been tested and complete assay of pituitaries of Rana catesbeiana has been made by this method. It has been found that frog material is best employed in dilutions of 1 mg in 20 cc cold-blood Ringer's solution. Stronger concentrations react upon the skin too rapidly for accurate observations to be made. The solution is refrigerated 24 hours and then filtered. The filtrate is saved for the test. It has been found that the unfiltered solution shows partial inactivation of the hormone after 24 hours at room temperature. Complete inactivation is accomplished after 48 hours.

Lizards used for such tests should be well-fed and watered daily, since variations in reactions or complete unresponsiveness results when the animals are not fed and watered regularly.

Studies were conducted at two large institutions during an outbreak of influenza A which was proven by complement-fixation tests. Persons at both institutions received a living virus and complex formalinized vaccine previous to the outbreak. An epidemiological survey revealed some protection at one institution, but none at the other. There was no obvious difference in the prophylactic efficacy of the two vaccines in the relatively small group of 829 individuals studied.

The rat omentum, following the injection of particulate matter of various types, undergoes hypertrophy due to accumulation of histiocytes which are transformed into macrophages. The macrophages collect just beneath the surface and phagocytose the material which enters through and between the mesothelial cells. Secondary collections of macrophages appear about the smaller blood vessels and take up the particles which have traversed the vascular walls. The taches laiteuses increase in size and produce numerous macrophages.

With the advent of phagocytic activity the Golgi net increases in size and undergoes fragmentation. The fragments become associated with the ingested granules and apparently aid in bringing about their coalescence into masses which are stored by the cells. The changes found in the Golgi material never occur in cells which do not collect and store particulate matter.

The development of a simple procedure 1 to determine both thiamin and the pyrimidines in human urine permits the investigation of the possible relationship between the two.

Schultz, Atkins and Frey showed that the rate of glucose fermentation by a yeast is directly proportional to the concentration of thiamin present. In addition to thiamin, the pyrimidines give mol for mol stimulation effect on the rate of fermentation. Such pyrimidines are found normally in human urines. Since the pyrimidine nucleus constitutes an integral part of the thiamin molecule, it is of interest to determine the relationship between thiamin and urinary pyrimidine.

The total fermentation is a measure of thiamin and pyrimidine in the urine. The fermentation after oxidation of the free thiamin is a measure of the pyrimidines; the difference represents the free thiamin. This technic combines the gas method with the initial steps of the thiochrome procedure.

The urinary excretion of the thiamin and pyrimidine of a group of patients was studied under various conditions. Three patients were given a diet completely deficient in B₁ for 10 days. Two of these were normal and one had ileo-jejunitis. Fig. 1 and 2 and Table I illustrate that complete deprivation of dietary thiamin for a period of 10 days changed the thiamin-pyrimidine ratio from approximately 9:1 to 1:9. During this 10-day deprivation period, the absolute amount of pyrimidine excreted remained at approximately the same level, while the free thiamin disappeared almost completely. It becomes apparent, then, that the determination of free thiamin in the urine is an index of the dietary intake immediately preceding the measurement, since the urinary excretion of thiamin decreases so rapidly on deprivation. Therefore, low values for free thiamin need not necessarily indicate a state of chronic insufficiency with respect to this vitamin since the same values are obtained with temporary, acute deprivation.

Reports concerning the action of amphetamine sulfate (A.S.) on gastro-intestinal motility are somewhat variable. Myerson and Ritvo 1 summarizing the results of their studies on man state that A.S. is a sympathomimetic drug which is of great value in diminishing or abolishing spasm of the G. I. tract. Ivy and Krasno, 2 summarizing the clinical literature state that 10-30 mg orally delays the rate of evacuation of the stomach, increases the tone of the pylorus, shows no marked constant effect on the small intestine and variable effects on the colon. Ersner, 3 after treating 500 cases of obesity with the drug, reports it to cause mild constipation, in 10 mg doses.

Experimental. Our study of A.S. consisted in measurement of the action of various doses on effective peristalsis and egestion time in intact animals and its action on isolated segments of colon.

The animals were fasted for 4 hours before each determination in order that they would quickly eat their test meal which consisted of a 3 g pellet made up of the following parts by wt: bread 1, milk 4, hamburger 5, and Fe₂O₃ 1. The A.S. dissolved in water was injected intraperitoneally, immediately after the food pellet was eaten.

The action on effective peristalsis 4 was measured by comparing the rate of progress of a test meal dyed with Fe₂O₃ in treated and non-treated litter mates of the same sex, paired data being secured in all determinations; 30 min. after eating the animals were killed by decapitation, the gastro-intestinal tract quickly excised, straightened out on a warm moist plate and slit open with fine scissors; thus the distance which the dyed mass had been propelled from the stomach could be measured accurately.

The intravenous injection of trypsin into rabbits results in a prompt reduction in the total blood histamine with an associated leukopenia. The addition of trypsin to heparinized rabbit's blood in vitro results in a shift of histamine from cells to plasma. In both respects the effects of trypsin are similar to those produced by antigen in sensitized animals.

The osmotic activity of pouch gastric juice samples relative to that of the plasma is presented as a function of the sodium content of the gastric juice.

The general features of the resulting distribution are in qualitative agreement with the predictions based on the two-component theory of Hollander and the buffering process which it implies.

Observations on the development of the melanophores in normal and hypophysectomized Xenopus show contraction of the melanophores in the latter at a late tail bud stage, about 48 hours after fertilization. The pituitary at this stage is undifferentiated. It is inferred that the secretion of melanophore hormone begins at or possibly before this time.

Sulfathiazole and sulfadiazine are inhibited to varying degrees in different media. In blood broth sulfadiazine is selectively inhibited to a greater degree than sulfathiazole. In human serum sulfathiazole is inhibited to a greater degree than it is in blood broth, in human plasma, in defibrinated blood or in horse serum, while sulfadiazine is inhibited to about the same extent in these media. In liver infusion medium sulfadiazine and sulfathiazole are equally effective in a concentration of 5 mg % but sulfathiazole is more effective in lower concentrations. The possible error of attempting to compare therapeutic efficacy of different sulfonamides on the basis of the results of in vitro tests is discussed and the importance of the proper selection of media for the performance of in vitro tests of sulfonamide drugs is emphasized.

When small inocula are planted in a semi-synthetic medium, sulfadiazine and sulfathiazole are bactericidal in concentrations as low as 1 or 2 mg % on E. coli, Shigella paradysenteriae (Flexner) and S. choleraesuis (suipestifer); and in 5 mg % on S. enteritides, S. schottmülleri and S. typhimurium (aertrycke). In the latter concentration they are bacteriostatic for S. paratyphi and in 10 mg % they are bactericidal for Klebsiella pneumoniae, type A.

In a previous paper 1 it has been reported that temperatures of 0-5°C during roentgen irradiation (1,300 r) decreased the amount of injury produced in the skin of new-born rats. In a second series of experiments 2 this increased resistance of animals irradiated at 0-5°C was studied quantitatively for dosages ranging from 300 to 3000 roentgens. The purpose of this investigation was to determine whether the resistance would change quantitatively with increasing temperature.

The animals were of the same strain used in the previous studies.† The temperature was controlled during the irradiation by placing the animals in a double walled box, constructed of transparent sheet plastic, containing water at the desired temperature. The temperature of the skin before and after the irradiation was determined by means of a thermocouple. Experiments indicated that the rectal temperature changed very rapidly to that of the skin at low and at high temperatures, but at room temperature the interior of the body was slightly warmer than the skin.

The radiation used was unfiltered except for 1 mm of cardboard and 2 sheets of 0.5 mm plastic. The radiation was delivered at 130 K.V. peak and 5 ma. The half-value layer was ca. 1.5 mm aluminum. The intensity in the box at 26°C was 137 roentgens per minute. The target distance was 30 cm.

The exposed skin was a 6 × 9 mm area to one side of the mid-ventral region of the abdomen. Both ventral and dorsal skins were examined microscopically 2 weeks later, and the extent of injury determined in units as described in a previous report. 2

Fig. 1 shows some typical results of irradiation at different temperatures. These animals were litter mates and each was given the same dosage.

Vitamin K (potassium 2-methyl-1,4-naphthohydroquinone disulfate), injected into eggs prior to their incubation, produces vitamin K stores in newly-hatched chicks. The injected vitamin protects against the rapid and extreme fall of the pro-thrombin level which occurs when chicks are placed on a vitamin K-free diet.

The vitamin K requirement of the newborn infant is shown to be extremely low (approximately 1 μg of synthetic vitamin a day). Milk contains enough preformed vitamin K to meet this minimal requirement.

In an attempt to disclose the possibility of a “sparing action” of fatty acids on pyridoxin deficiency, varying levels of ethyl linoleate were fed in conjunction with a subnormal and an optimal level of pyridoxin; the “sparing action” was not observed.

Ansbacher 1 has reported that p-amino benzoic acid would cure the characteristic graying of areas of the pelage of rats maintained on a vitamin B deficient diet supplemented with thiamin hydrochloride, riboflavin, pyridoxin hydrochloride, calcium pantothenate, nicotinic acid, inositol and choline chloride. On the appearance of this paper a considerable number of characteristically grayed rats were available to us for experimentation. They had been raised on a somewhat different ration than that employed by Ansbacher, for they received a vitamin B deficient diet supplemented with only three substances, to wit: thiamin hydrochloride, riboflavin and pyridoxin hydrochloride. They were divided into four groups as follows:

(1) Controls: Animals continued on the same diet and supplements which had produced the graying.

(2) Animals receiving daily, in addition to the 3 supplements mentioned, 100 μg pantothenic acid.

(3) Animals receiving daily, in addition to the 3 supplements mentioned, 3 mg of p-amino benzoic acid.

(4) Animals receiving daily, in addition to the 3 supplements mentioned, both 100 μg of pantothenic acid and 3 mg of p-amino benzoic acid.

After 25-30 days on the diet, a marked darkening of the fur amounting practically to a cure (only “stippling” remaining) was noted in the group supplemented with calcium pantothenate alone and with calcium pantothenate plus p-amino benzoic acid. The animals receiving p-amino benzoic acid alone were not altered in appearance and were indistinguishable from the controls. There was an evident stimulus to growth as well as cure of the graying in the animals supplemented with the calcium pantothenate alone or the pantothenate and p-amino benzoic acid. The p-amino benzoic acid alone, which had no influence on the graying, also evoked no growth response.

In spite of the negative curative efforts just enumerated, there remained the possibility that graying could be produced with the exact Ansbacher diet regardless of the fact that it was supplemented with 500 μg of calcium pantothenate daily and that such graying could be prevented or cured with p-amino benzoic acid.

In tests made on 41 normal young White Carneau pigeons daily injections of 1-60 units of insulin during 10 hours to 10 days enlarged the adrenals, produced mitosis and cellular activity. These cytological effects were more pronounced in cortical cells than in medullary cells. Wholly comparable effects were produced in the adrenals of 16 additional birds previously deprived of their anterior pituitary glands. The cytological changes in the cortex following insulin are identical with those induced by adrenotrophic pituitary extracts. Food consumption of the test animals and of 73 controls was regulated at different and definite levels. The relation of these results to the alarm reaction and to the rôle of the adrenals in carbohydrate regulation is discussed.

Pregneninolone alone and injected subcutaneously with estrone into castrate virgin female mice caused the development of the lobule-alveolar system of their mammary glands, having a property similar to progesterone in this respect. The injection of estrone with the pregneninolone enhanced this activity of the pregneninolone by five times. Pregneninolone has one-half the activity of progesterone in stimulating mammary lobule-alveolar growth when both are injected with estrone.

An inflammatory exudate readily induces an increased capillary permeability as evidenced by the rapid accumulation of trypan blue from the circulation into cutaneous areas treated with the exudative material. Dialysis of the exudate allows the outward diffusion of leukotaxine. The indiffusible residual material remaining after dialysis of the exudate is essentially incapable of increasing capillary permeability with the characteristic homogeneous pattern. The exact original effect of the exudate, however, can be readily reconstituted by merely adding leukotaxine to the indiffusible fraction of the dialyzed exudate. This cannot be obtained by adding histamine in the concentration in which the latter is recovered from exudates. These facts therefore add further support to previous observations that leukotaxine and not histamine is the primary factor concerned in the mechanism of increased capillary permeability in inflammation.

Evidence is presented that abnormally great hemo-concentration occurs in the erect posture in a subject with orthostatic hypotension and tachycardia. The magnitude of the effect is great enough to permit the conclusion that abnormal filtration rates aggravate the circulatory embarrassment in this condition.

The production of renal ischemia by molded gauzecollodion capsules, as demonstrated to us by Dr. John Williams, appears to be the most satisfactory method of producing cardiac hypertrophy in rats.

p-Nitrobenzenesulfonamide is rapidly reduced by rat liver suspensions. After oral administration p-nitrobenzenesulfonamide is rapidly reduced by rats; a high percentage of the dose is excreted in the reduced form, and the extent of conjugation of the reduced form is large. A small amount of the drug is excreted in a state of oxidation above that of the amine. Azobenzene-4,4′-disulfonamide administered subcutaneously to rats is excreted slowly, reduced to only a small extent and conjugated to only a low degree.

p-Nitrobenzoic and m-nitrobenzoic acids are readily reduced to amino-compounds by tissue suspensions. o-Nitrobenzoic acid, in contrast, is reduced to only a small extent by tissue suspensions.

When p-nitrobenzoic acid or m-nitrobenzoic acid is administered to rats only a small fraction of the dose is excreted in the urine in the reduced form, the remainder is present in a state of oxidation above the amine. The reduced fraction is conjugated to a high degree. o-Nitrobenzoic acid is also reduced by rats; an appreciable amount of free amine is recoverable from the urine.

The existence of hydrogenase, the enzyme activating molecular H₂, has been demonstrated in cultures of Azotobacter by a methylene blue reduction method as well as by measurements of H₂ uptake in the Warburg microrespirometer. The enzyme is found in cells grown in the presence or absence of H₂ and using either free or combined nitrogen. Cells of the pea organism taken directly from the nodule possess the enzyme but those grown on the usual laboratory media do not.

Epinephrine, by slow intravenous injection, increases the prothrombin activity of human and dog plasma. An identity of type seems to exist between the forces capable of exciting hyperglycemia and increases in plasma prothrombin activity.

1. Gramicidin and tyrocidine depress the surface tension of aqueous solutions. Tyrocidine is more active in this regard than is gramicidin. 2. The ability of gramicidin to depress surface tension is improved by the addition of organic solvents such as glycerin which increases the solubility of gramicidin. 3. Serum decreases the activity of tyrocidine less than it does the activity of gramicidin, sodium oleate and aerosol OT. 4. The bactericidal and hemolytic effects of gramicidin are destroyed by heat but its property of altering surface tension is heat stable.

The unpreserved, refrigerated crude latex was consistently the most efficient parasiticide tested, the preserved latex somewhat less effective. A 10% solution of the freshly opened crystoid compared favorably with the refrigerated latex but exposure to air rapidly diminished its potency. The amorphous ficin, while relatively stable, proved to be only about half as efficient as the fresh crystoid. The most specific action of these products occurred on whipworms, provided the large bowel was previously free of feces. The effect on hookworms was almost uniformly less satisfactory. Considerable amebostatic and, at times, amebicidal action was demonstrated.

A method for the determination of ascorbic acid in whole blood is proposed. The oxidation of ascorbic acid by oxyhemoglobin is prevented by reduction of the oxyhemoglobin by alternate evacuation and treatment with CO₂ under pressure before deproteinization with HPO_3.

There was no significant change in whole blood, plasma, or urinary excretion of ascorbic acid following administration of 180 mg of phenobarbital daily to human subjects. Twenty-five mg of crystalline ascorbic acid daily is insufficient to maintain whole blood or plasma values when the subjects take an ascorbic acid-free diet.

It is quite apparent that colloidal Ca₃(PO₄)₂ diminishes the pressor effect of submucosally injected epinephrine.

Various observers (Reiss and Haurowitz, 1 Avery and Johlin, 2 Kabat and Dennis, 3 Selle and Witten, 4 and Himwich, Alexander and Fazekas 5 ) have reported that young animals are much less susceptible to anoxia and asphyxia than adults. The present studies were undertaken to determine whether the primitive respiratory mechanism (gasping) itself survives longer in the young than in the old.

Movements of the mandible of various species (dogs, cats, rabbits, and rats) were recorded mechanically following complete and rapidly induced anoxia produced by ligation of the cerebral vessels or by decapitation. In preliminary experiments it was found that the character and duration of the gasps induced by either of these methods were essentially the same and that the gasping movements of animals in which the cerebral vessels were ligated corresponded to and were simultaneous with labored respiratory movements of the trunk.

It is evident that the respiratory center of the young is much more tenacious and viable than is that of the adult, and that the survival of gasping itself is inversely proportional to age for animals up to or slightly past the weaning period. Thus, the isolated head of a rat 7 weeks of age or older gasps 5 to 8 times over a period of 10 to 20 seconds and remains motionless thereafter; whereas, the head of a younger animal may continue to gasp for many minutes.

Unlike adults, rats under 6 weeks of age display 2 periods of gasping movements: an initial series which consists of 6 to 12 gasps and lasts for a period of 20 to 80 seconds; and a second series which begins after an interval of 30 to 50 seconds following cessation of the initial or first series.

The inactivating effect of ultraviolet light on viruses as well as bacteria has been observed repeatedly. That viruses so inactivated may be effective as immunizing antigens has already been shown for at least 2 viruses capable of causing disease in man. With a vaccine prepared by irradiating mouse-brain infected with rabic virus, Webster and associates 1 successfully immunized mice and dogs against a subsequent injection of active virus. Salk, Lavin, and Francis 2 compared the antigenic potency of epidemic-influenza virus following irradiation with that of active virus. In high concentrations, irradiated virus was nearly as effective an immunizing antigen as active virus; when lower concentrations were tested, a hundredfold loss in immunizing capacity was found to have occurred during irradiation. Ultraviolet light has been applied to the virus of equine encephalomyelitis, Eastern strain (E.E.E.), by Sharp and associates; 3 they studied the molecular stability of ultraviolet-treated virus. The preparation of an immunizing antigen produced by irradiation of E.E.E. virus with ultraviolet light is reported here.

Chick embryos 7-days-old were inoculated with 0.1 cc 10^-3 suspension of E.E.E. virus-infected embryo in 0.85% saline solution. Embryos removed from the egg 18-20 hours after inoculation were rinsed in saline solution, ground in a mortar and made to a 10% suspension in saline or Tyrode's solution. This suspension was centrifuged at 2000 rpm for 10 minutes; the centrifugation was repeated with the supernate; the supernate then obtained was spun in a Swedish angle-centrifuge at 4000 rpm for 45 minutes. About 30 cc of the final supernate were transferred to a quartz test-tube, with an internal diameter of 2.1 cm, which was placed in the center of a quartz-mercury resonance lamp in the form of a spiral∗ with an internal diameter of 9 cm.

This report deals with a marked reaction in the eye of the rabbit following the intravenous injection of Shwartzman toxins.∗ It consists of miosis, photophobia, lacrimation, congestion of the iris and conjunctiva with a marked pericorneal ring of dilated capillaries and in some instances with gross conjunctival hemorrhages. Ophthalmoscopical examination is rendered difficult by the turbidity of the dioptic media although a marked congestion of the fundus may be revealed. The ciliary bodies show enhanced permeability to fluorescein injected intravenously. The aqueous humor of the anterior chamber is usually under high pressure and coagulates immediately upon removal. The fluid is clear in appearance although numerous fibrin-threads, crystals, and epithelial cells may be seen microscopically. The reaction reaches its maximal intensity within 2 hours following the intravenous injection of potent toxins but it completely disappears 24 hours later. The toxins may be given intravenously and intraäbdominally, the cutaneous route is only successful when a vascularized area of the skin is used (the upper third of the ear). Microscopically, severe reactions show massive conjunctival hemorrhage with dilatation and engorgement of blood vessels of the recti muscles. No lesions have been seen in the iris, retina, cornea, and ciliary bodies.

Incidentally, other primary toxic effects following the intravenous injection of the Shwartzman filtrates may be noted, i. e., severe diarrhea, enhanced fragility of the capillaries of the skin, serous fibrinous exudation in the joints, some enhanced permeability of the choroid plexus to fluorescein and a high mortality-rate. These effects may be considerably diminished by a previous local preparation of rabbits to the Shwartzman phenomenon (observed on about 50 animals). No similar protection is afforded by local preparation with chemical irritants and inflammatory agents devoid of Shwartzman principles (cantharidin plaster, turpentine, diphtheric toxin, etc.).

The neutralizing effect of homologous immune serum on the rabbit papilloma-virus protein is quantitatively reversible 1 by simple dilution over the whole range 2 of serum-virus relations accessible to study by infectivity-measurement. 3 Under proper conditions of serum-virus proportions, precipitates occur in the mixtures, consisting presumably of virus and specific antibody. In the present study the distribution of virus between the soluble and insoluble phases in one region of serum-virus quantities has been determined.

The results reported here were derived from 8 correlated experiments with papilloma-virus protein and an antipapilloma rabbit-serum designated as D. R. 496. The findings of 7 experiments with this serum have been tabulated in detail elsewhere. 1 , 2 An additional study made in the desired region of serum-virus relations is described here.

Papilloma-virus protein procured under standard conditions 3 by ultracentrifugation was mixed with undiluted immune serum D. R. 496 as previously reported 2 so that the final concentration of total virus was 24.4 γ per 0.1 cc (virus pI 4.6, serum pD 0). 1 , 2 After 11/2 hours the precipitate forming at room-temperature was sedimented in an ordinary horizontal International Centrifuge No. 2 at full speed for 30 minutes. The clear supernatant fluid was carefully pipetted from the pellet which was then suspended in 0.9% NaCl solution to the volume of the supernatant fluid (4.5 cc). The supernatant fluid and the pellet-suspension were inoculated 3 in successive twofold dilutions in a group of 32 rabbits, a number statistically adequate 3 for the problem at hand. Twofold dilutions of untreated viral protein were inoculated in the same animals for standardization.

With the serum-virus relations described, the free virus of the whole mixture without separation of the precipitate is 3 γ per 0.1 cc as previously reported, 2 indicating neutralization of 21.4 γ of the 24.4 γ initially present.

Nicotinic acid and nicotinamide do not always produce equivalent growth-promoting effects upon microörganisms. A series of microörganisms is presented showing a ratio of activity of amide to acid varying from one-tenth to infinity. The last case is represented by certain organisms of the Pasteurella group whose growth and respiration is promoted by nicotinamide, but not by nicotinic acid. A further summary is presented of synthetic abilities of different microörganisms with respect to the entire enzyme-coenzyme complex.

2-(Sulfanilamido)-5-ethyl-4-thiazolone (sulfaethyl-thiazolone) has less antistreptococcic activity and approximately the same antipneumococcic activity as sulfapyridine. Its antistaphylococcic activity is of the same order as that of sulfathiazole and sulfadiazine.

Sulfadiazine produces urolithiasis medicamentosa capable of causing death by acute suppression of urine in mice and rats, whereas sulfaethylthiazolone is free of this defect, but may cause fatal anemia.

Erythrocytes contain an apparently specific phosphatase 1 , 2 , 3 classified by Folley and Kay 4 as phosphomonoesterase A_4. This enzyme in laked red cell systems readily dephosphorylates monophenylphosphate, 2 the pH range of activity for the reaction showing an optimum at 5.8-6.0 but extending to well below pH 5.0. 2

In estimating the “acid” phosphatase activity of blood serum 5 , 6 by a recently described adaptation 7 of the King and Armstrong “alkaline” phosphatase method, 8 monophenylphosphate substrate is used with citrate buffer at pH 4.9. These conditions also permit of hydrolysis by such erythrocyte phosphatase as may be present if sufficiently hemolysed samples of blood serum are employed. The determination of serum “acid” phosphatases (which has certain clinical applications 9 , 10 , 11 ) therefore gives misleading results in markedly hemolysed blood samples. If the use of such samples is unavoidable, a correction can be made after hydrolysis in the presence of NaF, which in proper concentration differentially inhibits serum “acid” phosphatases.

Experimental. Effect of hemolysis on phosphatase activity at pH 5.0. Fresh, non-hemolysed, normal sera exhibit slight “acid” phosphatase activity: 1.4 and 1.6 units/100 cc respectively in the 2 subjects cited in Table I. Defibrinated whole blood samples from these normal subjects showed more than 100 times greater phosphatase activity when determined at the same pH (5.0) by the same method, because large amounts of erythrocyte phosphatase are present in the hemolysates. Fresh, non-hemolysed sera of patients with metastasizing prostatic carcinoma contain prostate “acid” phosphatase and show elevated “acid” phosphatase levels (Table I). Defibrinated whole blood samples from these patients exhibit further increases in phosphatase activity at pH 5.0, due to the added effect of erythrocyte phosphatase.

1. There is a rise above the normal of blood pyruvic acid in heart failure. 2. This elevation approximates the degree of failure.

Croxatto, Huidobro, Croxatto, and Salvestrini 1 published a paper showing an increase of almost 100% in the choline-esterase activity of the renal blood of the cat, during neural stimulation of the leg muscles. In an attempt to verify these findings, similar experiments were performed on the frog as well as on the cat with modified technics.

For the frog experiments, a perfusion technic was used. The cannulae were introduced into the dorsal aorta and the ventral abdominal vein as described by Fenn, et al. 2 The perfusion fluid was a mixture of gum acacia, Ringer's solution, and beef red blood cells. 3 Five experiments were done, analyzing only the supernatant fluid after the red cells had been removed by centrifugation, and one experiment in which the whole perfusion fluid was analyzed. In all cases, analyses were made of arterial samples and of venous samples taken, (a) in the resting state, (b) during about 10 minutes of electrical stimulation of the leg muscles through the sciatic nerve, and, (c) after stimulation had ceased. The stimulation was accomplished by means of a condenser discharge type stimulator. The frequency and intensity of the shocks were adjusted to produce maximal contraction and were not constant. The samples were analyzed for choline-esterase with the differential volumeter apparatus of Fenn 4 according to the principle used by Stedman and Stedman. 5 One cc of bicarbonate buffer (.65% NaCl and .4% NaHCO₃), and one-half cc of 1% acetylcholine solution in saline were placed in the bottom of the flask, and 1/32 to 1/4 cc of the test solution, depending on the activity, was placed in the side arm. In cases where there was appreciable hemolysis of the red blood cells, the degree of hemolysis was determined by measuring the hemoglobin content of the supernatant fluid by dilution and color comparison.

Nicotinic acid has no coagulant effect when tested in vitro with heparinized recalcified plasma. Its coagulant action is comparable to that of distilled water when tested in vitro with heparinized whole blood. Nicotinic acid is an active hemolytic agent and its coagulant effect is apparently due to the release of thromboplastin from the disrupted elements of the blood. Compounds of nicotinic acid which do not produce significant hemolysis have no appreciable coagulative potency.

The preventive and curative effect of cholic acid on dietary gizzard erosion of chicks has been reported. 1 , 2 The effect is similar in the case of gizzard erosions produced by the use of either a basal diet or a practical rearing diet to which cincophen has been added. 2 We desire to report at this time evidence for the existence in cow's milk of a labile substance which acts like cholic acid.

Dried milk products were fed by mixing in the diets; liquid milk products were given to the chicks in place of the drinking water. The diets and the details of the experimental procedure have been described. 1 , 2

The results given in the table show no effect on the characteristics measured in the case of dried whole milk. This has also been found true in the case of commercial dried skim milk, dried buttermilk and dried whey. On the other hand, liquid milk products distinctly reduce the severity of gizzard erosion, increase the gall bladder bile volume per chick and increase the quantity of cholic acid per chick. The data show that this effect of liquid milk products is stable to heating up to the boiling point. Other experiments have indicated that the activity is not removed by steam distillation. The effects of liquid milk products are very distinct with both types of erosion producing diets.

The evidence presented is consistent with earlier reports 1 linking gizzard erosions with a deficient formation of bile, especially of cholic acid, within the chick. It is further indicated that a substance with an activity like that of cholic acid exists in cow's milk. Attempts to detect cholic acid in skim milk yielded only negative results.

The apparatus and methods for the precise measurements of small differences in the high frequency conductivity and dielectric effect of the intact egg and samples of the contents of the egg have been described.

The relative conductivity and dielectric effect of the intact egg at frequencies of 27.3 and 54.6 megacycles gave results indicating that fresh infertile eggs have a higher conductivity and a tendency towards lower dielectric constant than fertile eggs.

In dissected eggs the difference between the fertile and infertile egg in relation to conductivity and dielectric effect was observed to occur principally in the albumen. The conductivity of the albumen of fertile eggs was 7.4% lower than that of infertile eggs.

The conductivity values for various parts of the egg were maximum at about 15 megacycles. There was no frequency dependence for the percentage separation between yolk and albumen over the range from 2 to 60 megacycles.

Instruments for the measurement of tissue and cell respiration have steadily moved, since the introduction of Warburg's convenient manometer, in the direction of smaller volume and greater sensitivity. Gerard and Hartline 1 took advantage of the greater stability afforded by reducing the tissue chamber of a volumeter to capillary dimensions (0.5 to 1.2 mm diameter) and having this inside the relatively large “differential” chamber. Index drop movements, followed with an ocular micrometer, were consistent over 5-minute intervals, even when corresponding to volume changes of about 0.01 cmm. The diver technique, introduced by Linderström-Lang, 2 , 3 is of the same order of sensitivity and consistency; and the electrical method 4 gives promise of superior performance.

We have further developed the capillary method so that it is convenient to follow the respiration of 10 tissue samples at once, and it is possible to measure absolute gas volume changes of 0.001 cmm, minute by minute, with an error of some 8% minute by minute, under 1% for longer intervals. The respiration even of bits of frog sciatic nerve weighing a fraction of a milligram can thus be followed at half-minute intervals.

The tissue chamber, also containing filter paper bits soaked with acid or alkali and separated from each other and the tissue by dry paper guards, is a short length of capillary of 1.2-1.5 mm internal diameter. The “open” end of this, after the insertion of materials, is plugged with plasticine. Into the other end has been cemented a fine drawn capillary of about 0.2 mm diameter. At the close of the experiment, this capillary is broken off and its end diameters accurately measured by end on examination with an ocular micrometer.

Each of 7 local anaesthetics derived from p-aminobenzoic acid partially or completely blocked the in vitro bacteriostatic effect of sulfapyridine upon a strain of B. coli grown in a simple medium. None of 9 local anaesthetics not derived from p-aminobenzoic acid displayed any antagonism to the sulfapyridine. Each of 3 local anaesthetics derived from p-aminobenzoic acid blocked the bacteriostatic effect of sulfathiazole on B. coli and Staphylococcus aureus. None of 3 local anaesthetics not derived from p-aminobenzoic acid displayed any antagonism to the sulfathiazole.

In the promulgation of current methods of prothrombin assay 1 , 2 there has been some investigation of plasma dilutions but no direct experiments to show the ability to recover prothrombin added to plasma. This is important in connection with the possibility that clot-inhibitors 3 , 4 , 5 , 6 may complicate the interpretation of the assay. It is recognized, for instance, that heparin interferes with prothrombin determinations 2 and heparin can act both as an antiprothrombin 6 and as an immediate antithrombin, 5 in the presence of the respective plasma co-factors. The natural antithrombins also include a progressive thrombinolytic factor. 4 As an example of the kind of interference which may be expected, it has recently been found that a series of prothrombin dilutions, subsequently activated with Ca⁺⁺ and brain thromboplastin, gives more than the theoretical thrombin yield, as determined from the clotting-times for purified fibrinogen, using a similar dilution series of the same (full-strength) thrombin as the standard of reference. 6 In the absence of equivalence it is, of course, impossible to assay prothrombin both in terms of an arbitrarily fixed clotting-time and a definite thrombin dilution value. Inherent in the clinical tests is a false assumption that inhibitors do not influence the results obtained. Actually, there must be a rôle for the natural inhibitors of the types mentioned. The Iowa workers 2 recognized only the progressive antithrombins and succeeded in controlling these by dilution. It is an unproved assumption that immediate antithrombins and antiprothrombins do not interfere with the prothrombin assay. In the following experiment, in which a prepared prothrombin is assayed in the presence of plasma, these problems are readily brought to test.