Experimental Biology and Medicine

Two types of cortical cataract have been produced in salamander larvae by synthetic diets differing in their protein constituents. The first type, rarely detectable in the early stages and with a marked final shrinkage, was caused by a diet of purified casein and by diets of the separate muscle proteins. The second type, with an easily visible early zoning, large fluid content at maturity, and halted later changes, was caused by diets with high hemoglobin content. Prevention of the first type of cataract was attained by dietary supplementation with cystine, while prevention of the second type required all 3 components of glutathione and apparently some additional factor.

1. An aqueous liver extract effectively prevented the oxidation of added ferrous iron. 2. Due to the reducing action of a commercial liver extract, from 50 to 95% of ferric iron added to it was later present in the ferrous state. 3. The efficacy of liver and iron combinations in the treatment of the iron deficiency anemias may be partly due to this action of the liver extract in supplying or maintaining iron in the more easily absorbable ferrous state.

One of the most interesting aspects of the entire phage problem has been the question of how phage is formed. In earlier studies it appeared that phage was produced only in the presence of an actively growing bacterial substrate. This relationship found experimental verification in quantitative studies on the phage-bacterium reaction and led to the adoption of an equation in which phage production was expressed in terms of bacterial reproduction. 1 Further work, however, proved this relationship to be more apparent than real for it was found possible to impose conditions such that cell division was inhibited while phage continued to be developed at a very considerable rate. 2 , 3 , 4 The fact that phage can be produced at all in the absence of cellular reproduction indicated that the latter is not an essential conditioning factor for phage formation, even though the two phenomena ordinarily proceed concurrently and require careful adjustment of pH, temperature, electrolyte concentration, 5 , 6 etc., to be dissociated.

Attention was then devoted to the possibility that phage production might involve the autocatalytic transformation of an inactive precursor into phage. 7 Formal proof of the existence of the precursor in cell-free solution has not yet been obtained, although traces can be detected irregularly. 8 However, there is satisfactory evidence that the substance from which phage is developed exists within susceptible cells. 9 To demonstrate it, staphylococci are subjected to active growth in an oxygenated medium, after which they are washed and stored in Locke's solution at 5°C. These “activated” cells when mixed with phage in appropriate proportions, bring about a tenfold increase in phage titre within 2 minutes. 10

The plasma iodine is divided between the albumin and the globulin fractions of the plasma in proportion to the relative amounts of albumin and globulin present. There is no chemical evidence for the presence of a preponderant amount of thyroglobulin in the circulating blood.

The daily urinary citrate content and concentration of patients with calcium urolithiasis is much lower than those of normal individuals. This finding may be of etiological importance.

The purpose of this experiment was to see if methylene blue had any effect upon the O₂ consumption of shark (Mustelus) red blood cells in the presence of carbon monoxide or air. Since these red blood cells are nucleated and have a considerable rate of respiration, it was thought of interest to test Barron's conclusions that the action of methylene blue in increasing oxygen consumption is less marked, or even nil, 1 in nucleated erythrocytes or leucocytes as contrasted with such non-nucleated erythrocytes as mammalian. In addition it was also thought of interest to find out whether methylene blue antagonizes the effects of CO on the respiration of these nucleated erythrocytes.

The blood was collected fresh for each experiment into tubes by cutting the dorsal posterior fin of the shark. In some experiments the blood was centrifuged and the erythrocytes were subsequently washed several times in 1.1% NaCl and in others whole blood was used. No difference in the results was noted. Aliquot portions of 2 cc of blood were placed in the bottom of cups of Warburg respiration apparatus, temp. 20°C. Either 100% CO‡ or air was used in the system. CO₂ was absorbed by KOH in the insets in the usual way. The concentration of methylene blue added was .004% made in 1.1% NaCl. Tests were made to see if complete darkness affected the results with CO. No such effects were noted. The material was found to be remarkably uniform in behavior, and remarkably resistant to hemolysis in a wide range of NaCl concentrations.

The results are shown in Table I.

This table shows (1) that Mustelus blood has a considerable rate of respiration: (2) that in air methylene blue causes an increased O₂ consumption of 151% over the controls in 2 to 4 hours under the conditions of the experiment—this does not confirm Barron's conclusions that methylene blue has little or not effect upon the respiration of nucleated red blood cells; (3) that CO produces a decrease to 61% (it was not determined in these experiments whether this change in the manometric readings was due to residual oxygen remaining after the atmosphere was saturated with CO, or actual CO consumption); (4) that methylene blue counteracts this decrease, increasing the rate to 86%. This increased rate in CO is about the same in percent as that produced by methylene blue in air.

Two hypotensive extracts, namely Padutin and tissue extract No. 568, advocated in the treatment of important circulatory disorders, produced no demonstrable changes in blood pressure and no symptoms after oral and intramuscular administrations in very high (beyond therapeutic) doses in animals. Placed directly into ligated intestinal loops, both extracts remained unchanged and unabsorbed for 2 hours. All the animals reacted typically to control intravenous injections of both extracts. A range of fatal doses was determined intravenously only in pigeons, because very much larger doses given either by the oral or intramuscular route were without demonstrable effects. An experimental basis for the clinical use of the “hypotensive” tissue extracts, either orally or intramuscularly, is completely lacking. However, accidental intravenous injections, resulting from careless intramuscular or hypodermic uses, could conceivably cause alarming symptoms and even death. These extracts are undesirable, if not dangerous, for intravenous use.

Recently developed methods for the cultivation of Rocky Mountain spotted fever and typhus rickettsiae in agar slant tissue cultures 1 and in the yolk sac 2 of the developing chick embryo have opened new possibilities for the preparation of vaccines against these infections. We have been making comparative studies (a) of the immunizing potencies of vaccines obtained from rickettsiae grown in these ways and (b) of the relative merits of these procedures for the large scale production of vaccines.

These comparative tests have been made using an “eastern” strain of spotted fever rickettsiae of relatively low virulence. It had been maintained in agar tissue cultures for at least one year before being used in the present experiments. It was uniformly infectious for guinea pigs but unlike more virulent strains of spotted fever the mortality rate among diseased pigs was not high. In spite of this difference in virulence vaccines made from it protected guinea pigs against many lethal doses of “western” spotted fever rickettsiae, and vice versa.

For the production of vaccines 3 this rickettsial strain was carried both on agar slant cultures and in developing eggs. When grown in culture large test tubes containing Tyrode-serum agar slants were inoculated with infected tissue according to the technic described by Zinsser, Wei and Fitzpatrick. 1 The tubes were incubated at 35°C for 5, 6, or 7 days. At the end of this period smears were made from bits of tissue detached from the agar slants, strained and examined for rickettsiae. Cultures showing good growth of rickettsiae were harvested by scraping off the tissue with a small spatula, care being taken not to disturb the agar surface itself.

In an attempt to widen the range of compounds amenable to experimentation on hypersensitiveness in animals, using substances which may differ in the mechanism of sensitization, we undertook to sensitize guinea pigs with quinine which is known to cause allergy in human beings. Some positive sensitization results of moderate degree were obtained after a number of applications to the skin of a 5% solution in a mixture of one part of butyl carbitol (diethylene glycol monobutyl ether) and 3 parts olive oil. In several experiments in which this treatment was made instead on inflamed areas the number and degree of positive effects were increased; inflammation was produced with cantharidin, similar to previous work, 1 along with other irritants (xylene, croton oil, etc.). Likewise, marked sensitivity was achieved when quinine was applied to areas inflamed by 2:4 dinitrochlorobenzene in animals sensitive to the latter substance.

In well sensitized animals the reactions following the application of one drop of the above quinine solution are well developed by the following day and consist of intense reddening and slight elevation of the treated area. In less sensitive animals pronounced reactions appeared after 2 or 3 consecutive daily applications to the same site.

In a preliminary experiment the method of cantharidin burns gave positive sensitization effects also with a nickel salt (sulfate).

(1) Extracts of muscle tissue prepared with physiological saline at room temperature were obtained from 65 varieties of fish and examined pharmacologically. (2) It was found that extracts of muscle tissue obtained from ten different sets of fishes were poisonous for seedlings of Lupinns albus and for mice while suspensions of the other fifty-five specimens exhibited little or no toxicity. (3) A comparative study of the chemical and pharmacological properties of the poisonous fish muscle extracts with those of certain snake venoms is now in progress.

Guinea pigs were found to show a tolerance to delvinal sodium [5-ethyl-5-(l-methyl-1-butenyl) barbituric acid] on repeated administration of large doses of the drug. The average length of sleep dropped from 127.3 to 48.2 minutes following 8 semi-weekly injections of the drug, while the average length of hypnosis dropped from 191.9 to 104.1 minutes. The average weight of the guinea pigs increased from 250 to 380 g, which was more than a 50% increase in weight.

The principle of the electro-magnetic flow meter 2 is based on the fact that an electromotive force is induced in the blood as it flows through a magnetic field (the direction of flow forming preferably a right angle with the magnetic lines of force).∗ The induced e.m.f. is linearly proportional to the velocity of flow. It is tapped by two electrodes placed at right angles to the magnetic lines of force and to the direction of flow. Owing to the electrical conductivity of the blood vessel wall the pick-up electrodes merely have to touch the outside of the vessel to establish a sufficient electrical contact with moving blood. Fig. 1 shows the perspective view of the arrangement and the dimensions of the magnet.

The modification presented here employs an electro-magnet fed by an alternating current. The use of an alternating magnetic field eliminates the disturbances due to polarization and variable galvanic potentials and makes it possible to use an a.c. amplifier. It introduces, however, the new difficulty of eliminating the a.c. voltage induced in the lead wires at. zero flow. This interference has been abolished by the use of a special sleeve S and a compensating potentiometer P in conjunction with a filter F in the amplifying circuit. (Fig. 2.) The bakelite sleeve, S, contains a slit, S1, which allows a blood vessel to slip into the round channel of the sleeve. This establishes the proper contact between the vessel wall and the electrodes, E₁ and E₂ (2/56 brass screws screwed into the bakelite flush with the channel wall). A coil, C, of 3 turns of copper wire is wound around the sleeve imbedded in the groove, G.

It has been demonstrated that progesterone 1 , 2 and desoxycorticosterone 3 will elicit the copulatory reflex in the spayed guinea pig after preliminary estrogen treatment but that certain androgens will not do so. 4

We thought it of interest to determine whether aqueous extracts of the adrenal cortex which contain the life-maintaining principle would produce this reaction.

The method used to test the cortical extracts was similar to that described by other workers. 4 Two groups of guinea pigs, A and B, consisting of 60 and 11 animals respectively, were used. The results obtained from administering graded doses of the extract on successive weeks are shown in Table I.

It will be seen that there is a relation between the dose administered and the number of animals showing the copulatory reflex. The data on the small group are included since the response to each dose agrees so closely with that of the larger group. Furthermore, group B was given each dose of the extract one week prior to the administration of the corresponding dose to group A. Since these figures were tabulated, 30 animals representing one-half of group A have given a 50% response to a dose of 1 cc. This result offers further evidence of the reliability of the method.

The extract used in obtaining the data shown in the table contained 2.9 rat units 5 per cc. Other preparations which contained more than 2.9 rat units proved to be more active in producing the copulatory reflex. These preliminary findings indicate that there is a correlation between the life-maintaining and the copulatory-reflex activity of adrenal cortical extracts. It seems probable, therefore, that this reaction in the guinea pig might be used as a method for assaying such preparations.

The regularity with which pneumococci become resistant to the action of sulfapyridine under experimental conditions in vitro and in vivo 1-4 and the occurrence of such strains in infections in man, have led to the development of a method for their recognition through the use of blood agar plates, which is described below. The results of its application to stock laboratory cultures, to specimens obtained from patients who responded poorly to treatment with sulfapyridine, and to specimens from pneumonia patients submitted for routine bacteriological examination indicate that this test is suitable for use in connection with the control of chemotherapy.

Method. Specimens from pneumonia patients, or subcultures from such specimens, are inoculated on 3 blood agar plates (citrated horse blood, 5% in nutrient agar) to 2 of which sulfapyridine is added sufficient to make concentrations of 5 mg % and 10 mg %. The third blood agar plate, without sulfapyridine, serves as a control. Equal sized loopfuls of material are used in order to make the inocula as nearly as possible the same on all 3 plates. The growth on the plates is compared after 24 hours in the incubator, and pneu-mococcus colonies on the control plate without sulfapyridine are identified as to type by capsule swelling reaction and as to bile solubility by spraying dried bile over the colony. 5 The susceptible pneumococcus on the plates containing sulfapyridine produces some partial (alpha type) hemolysis where the inoculum is heaviest, but separate colonies are rarely visible and of minute size. Stained preparations from hemolyzed areas show aberrant bacterial forms, and subcultures sometimes show viable pneumococci. Pneumococcus growth on the plates containing sulfapyridine is seldom obscured by overgrowth of other organisms as these are also markedly inhibited.

Renin, the so-called “renal pressor substance” is vaso-inactive unless permitted to interact with a pseudoglobulin present in blood plasma, which has tentatively been denominated “renin-activator.” 1 The interaction of renin and renin-activator yields a crystallizable, dialyzable, thermostable product, angiotonin. 2 Intravenous injections of this substance in normal animals cause sharp pressor responses which diminish slowly with repeated injections. Nephrectomized animals are highly sensitive to the pressor action of this substance and do not show tachyphylaxis with repeated injections. 3 Slow intravenous infusions of angiotonin in dogs increase arterial pressure and cause renal vasoconstriction, predominantly of the glomerular efferent arterioles, but do not alter cutaneous temperatures. 4 The effects of angiotonin infusion therefore simulate those of renin, 5 although they are not as long sustained.

The present report is concerned with observations of blood pressure and renal hemodynamics during single injections and intravenous infusions of angiotonin into human beings. Electrocardiographic records and observations of venous pressure and skin temperature were obtained in some instances. These, together with further observations of renal functional changes will form part of a subsequent report.

Arterial pressure was determined by auscultation. Effective renal blood flow was calculated from the hematocrit ratio and apparent plasma diodrast clearance and is equal to approximately 90% of total renal blood flow. 6 Diodrast was determined in blood and urine by a modification of the method of White and Rolf 7 and inulin by the method of Corcoran and Page. 8 A mixture of diodrast, phenol red and inulin in 0.9% NaCl was given intravenously at a slow rate during the observations of renal clearances. 9

This study was undertaken to determine whether the concentration of vitamin A in the blood has any relationship to the vitamin A intake and to the storage in the liver. Groups of albino rats, 3 to 4 weeks of age, were fed amounts of vitamin A ranging from 0 to 1000 international units daily. The vitamin A supplement used for rats receiving 100 units or less daily was a reference cod liver oil obtained from the United States Pharmacopea Committee, whereas rats receiving 1000 units were given a vitamin A concentrate. After the rats received these preparations for 6 weeks, the vitamin A content of their bloods and livers was determined 24 hours after the last dose of the vitamin.

The method employed for the vitamin A determination of the blood and liver was based on the Price-Carr reaction. As the diet was devoid of carotene, this substance was not found in the blood or livers of the rats. The vitamin A in the blood was extracted directly with petroleum ether and alcohol as recommended by Clausen and McCoord, 1 whereas the liver was treated by a preliminary saponification. The extracts were evaporated in nitrogen. taken up with chloroform and then treated with antimony trichloride. The intensity of the resulting blue color was measured at 620 μM with the use of an Evelyn Photoelectric Colorimeter. 2 The reading was translated into biological units (international) by referring to a calibration curve obtained with a sample of halibut liver oil of known biological potency.

The relationship of the vitamin A blood level to vitamin A intake and to liver storage may be noted in Table I. It will be observed that when the intake was less than 50 units daily the vitamin A concentration of the blood was directly related to the vitamin A intake.

In a former communication 1 I have identified a previously unrecognized, Gram-negative, anaerobic bacillus as the specific cause of ulcerative enteritis of quail (so-called “quail disease”). I also gave details of the technic by which the organism may be demonstrated in suitable material, directed attention to the chronic carrier as a factor in maintaining and spreading the infection and recorded observations indicating transmission of the infection to young quail chicks through eggs laid by carriers.

The purposes of the present communication are: (a) to correct and contradict a statement in the previous paper relative to spore formation, (b) to give technic for isolation of the organism in pure culture, (c) to call attention to coprophagy as an important source of transmission in captive quail, and (d) to record successful immunization of birds by vaccination with killed cultures of the specific organism.

(a) The statement previously made that “occasionally spores can be found on slide preparations made direct from lesions” has been found to be incorrect. Examination of hundreds of specimens made from pure cultures and from lesions has failed to show spores. Reexamination of some of the original specimens on which the first statement was based reveals that what were taken for spores were something else, therefore contradicting the previous statement relative to spores.

(b) Pending development of differential culture media and cultural technic, and possibly more favorable culture media than is now available, attention is called to a medium and technic which are entirely satisfactory for isolation and maintenance of pure cultures of the organism This medium is the thioglycollate medium of J. H. Brewer, 2 which I modify slightly by the addition of 0.1% agar for semisolid cultures or 1% for solid cultures.

Infusion from 37.5 g pork per 100 cc.

Several diets were tested for adequacy in the nutrition of the guinea pig, and the necessity of the grass juice factor for this animal was confirmed. A diet high in the grass juice factor and adequate for normal guinea pigs, failed to maintain adrenalectomized guinea pigs given amounts of desoxycorticosterone acetate which were adequate when a normal diet was fed. Adrenalectomized guinea pigs can be maintained for at least 6 weeks on a normal diet if salt drinking fluid is offered.

Desoxycorticosterone acetate subcutaneously administered in daily doses of 0.5 to 1.0 mg produces normal growth in adrenalectomized guinea pigs.

Even though adrenalectomized guinea pigs had been maintained for over 5 months by salt and hormone therapy, they died on an average of 4.6 days after discontinuing this therapy.

Ligation of the common bile duct of rats prior to or 20 hours after the injection of L₇ organisms delayed the onset of arthritis, and the subsequent degree of joint involvement up to 12 days' time was markedly diminished. However, when duct ligation was performed after the onset of arthritis there was no effect upon the course of the disease. Since rat arthritis induced by the injection of hemolytic streptococci was not influenced by bile duct ligation, it seems probable that the effect of obstructive jaundice on the development and severity of L₇ arthritis in rats was due to the bile solubility of the L₇ organisms.

The care of dogs with cannulated gastric or intestinal fistulas is often difficult because many of the animals will pull their cannulas out at the first opportunity. In the latter part of the year 1938 we undertook to design a cannula which could not be pulled out. To accomplish this it was necessary to use a larger inner flange than could conveniently be installed when the cannula was constructed in the usual way. The cannula was. therefore, designed with a removable flange which could be inserted separately into the stomach or intestine; the remainder of the cannula could then be attached to it. Once the flange is inserted, no great force can be applied to it through the visceral wall because of danger of injuring the mucosa; consequently, it was necessary to provide a wrench which could be applied through the lumen for tightening the flange on the cannula. The cannula and the wrench are illustrated in Fig. 1.

To install the cannula, the viscus to be cannulated (stomach or intestine) is secured through an abdominal incision and an opening made, barely large enough to admit the separated flange. After the flange has been inserted, the appropriate end of the inner tubular portion of the cannula is inserted through the opening and screwed into the flange, as far as possible, by hand. The wrench is then assembled inside the cannula in such a way that the hooks on the lower ends of the 1/8 inch rods grasp the flange in its narrow diameter. The tapered plug with a knurled head is slid down the 3/8 inch rod and rotated until the fins which project from its sides fall into the grooves on the inner aspect of the cannula; the screw joint is then firmly tightened.

Small pellets of estradiol benzoate implanted locally into the uterus produced fibromyomatous tumors in 9 out of 12 guinea pigs (75%) after intervals varying from 32 to 150 days. Estrone in the same concentration did not produce tumors in 2 animals. From the findings made in 2 additional animals, it appears that androgen may have a tendency to limit the extent to which the estrogen is able to produce these tumors.

We wish to thank Miss Jane Smelser and Miss Anne Hammet for preparing the histological material and Mr. George Smelser for his advice and assistance.

We are indebted to Prof. B. P. Watson and to Dr. Carl Bachman for their help and criticism in preparing this material, and to Mr. Fred Roseberry for his careful drawings. The hormones used in these experiments were furnished by the courtesy of Mr. Robert Mautner of the Ciba Pharmaceutical Products, Incorporated.

The daily administration of 200 γ of androsterone for 15 days into sexually mature spayed rats caused no significant change either in pituitary weight or pituitary lactogen content and produced no detectable growth of the mammary glands.

Hydrogen has been used for the determination of the pulmonary capacity by VanSlyke and Binger, 1 who pointed out the danger of explosion and of poisoning with arsine. This procedure was replaced by oxygen dilution methods, particularly the Christie method, 3 which has been in use in this laboratory for the last 10 years. A recent report 2 has resulted in new interest in hydrogen, because of the ease and rapidity of analysis for this gas by thermal conductivity. A preliminary report is made at this time of a new method employing helium, because it has all the advantages and none of the dangers of the hydrogen method.

A closed spirometer system with a blower is used. Carbon dioxide is absorbed by soda-lime. An important precaution is the addition of a water bottle to replace water vapor lost in the soda-lime. Dead space is measured by the oxygen dilution method. Part of the mixture circulating in the respiratory system is shunted through a thermal conductivity cell∗ with precautions to assure complete saturation of the gas mixture with water vapor. A meter, which is connected to the cell, is calibrated for helium in air fully saturated with water vapor over a range of 0-14% and also for variation in oxygen concentration of the mixture from 18-23%. Seven hundred ml of 98.2% pure “Medical” helium is added from a graduated cylinder with a leveling bottle to 4,800 ml of air in the system. When this is mixed by the blower, the concentration is 12.5%. The meter reading is compared with the calibration curve to assure proper function of the indicating unit. Two hundred ml of oxygen are then introduced to approximate the concentration of air.

Bloch, 1 as a result of his discovery of the “dopa reaction” and because it had been impossible to demonstrate tyrosinase in the skin of any of the slightly pigmented vertebrates, postulated the existence of an enzyme that accelerated the oxidation of dihydrox-phenylalanine. He named it “dopa oxidase”, and claimed that this enzyme was responsible for melanin formation in human skin. Such an enzyme has not been isolated and its existence has been questioned many times. The controversy in regard to this has been discussed in comprehensive reviews. 2 , 3

Recently it has been shown 4 that the activity of the enzyme tyrosinase is regulated by the oxidation-reduction potential of substances added to the substrate. It was evident from this that both the enzyme tyrosinase and its substrate tyrosine could be present in a cell without melanin formation taking place if a sufficiently strong-inhibitor such as reduced glutathione was also present. With this in mind, an investigation was started to determine whether the enzyme responsible for the “dopa reaction” was a separate and specific dopa oxidase or tyrosinase associated with an inhibitor. To investigate this possibility chemically the rates of melanin formation were determined in the following series of reaction mixtures:

1. Buffered dopa alone.

2. Buffered dopa plus various reversibly oxidizable substances.

3. Buffered dopa plus tyrosinase.

4. Buffered dopa plus tyrosinase and glutathione.

5. Buffered tyrosine plus tyrosinase.

6. Buffered tyrosine plus tyrosinase and glutathione.

Series 1 was included to study the rate of non-enzymatic oxidation in air. Series 2 was included to see if any of the reversibly oxidizable substances would accelerate or inhibit oxidation in air. The last 4 series were included to determine and compare the effect of glutathione on the rate of enzymatic oxidation of dopa and tyrosine.

(1) Typical polar granules appear as dense spherical masses, or possibly plates, in electron micrographs of unstained preparations of C. diphtheriae grown on blood infusion agar. (2) In addition to polar granules, needle-like crystals appear in electron micrographs of unstained preparations of C. diphtheriae cells grown on potassium tellurite chocolate agar. (3) The needle-like crystals, as well as the black color, of cell masses of C. diphtheriae grown on potassium tellurite chocolate agar disappear upon the addition of small amounts of bromine water. It is inferred, therefore, that the black color is due to the tellurium metal which occurs in the form of needles. (4) It is to be further inferred that the tellurite ion is able to diffuse through the cell wall and is reduced with the precipitation of tellurium metal within the cell boundaries. (5) With the aid of the electron microscope it is now possible to obtain pictorial records of the location of sites of certain chemical reactions incident to the metabolism of the bacterial cell.

The influence of the reticuloendothelial system on estrone inactivation in the organism has been investigated. It is concluded that the reticuloendothelial cells have no part in this process and that the liver cell in itself is responsible for this inactivation. i. e., contains the inactivating factor (enzyme).

The in vitro oxygen consumption of brain-cortex slices from thyrotoxic guinea pigs is not above normal.

Radioactive phosphorus is incorporated into sphingomyelin to the greatest extent in the liver, the lung, and the intestinal mucosa. With the exception of the lung the tissues distribute themselves in approximately the same order for sphingomyelin activity as for total phospholipid activity. The lung is relatively higher in the case of sphingomyelin. Radioactivity in sphingomyelin reaches a maximum at about two days in most tissues. In the case of liver the total phospholipid fraction appears to be many times more active than sphingomyelin, indicating that this phospholipid, though possessing a certain definite activity, plays only a small part in phospholipid phosphorus metabolism in this organ.

The author is indebted to Miss Sylvia Levy for one-half of the radioactivity measurements and to Dr. William F. Bale and Dr. S. N. Van Voorhis for preparation of radioactive phosphorus.

The transformation of the normal female anal fin into a male-like gonopod is induced by prolonged administration of testosterone propionate. Such treatments influence this secondary sex characteristic in all cases, from female to male, over an interval of approximately 5 months. These experimentally induced transformations of the normal female anal fin into that of a male-like gonopod were observed in all of 91 specimens so treated.

In the 2 cows studied, 1.5% and 2.5% of the radio-iron administered orally was secreted respectively in the milk during a 78-hour period.

I am deeply indebted to Dr. M. D. Kamen for preparing the radioactive iron solution used and for counting the samples; to Dr. O. W. Schalm, Department of Veterinary Science, for the use of the cows and for the procurement of the blood and milk used; and to Dr. L. H. Duschak, Department of Mining Engineering, for use of high temperature ovens.

Sodium chloride added to an intraperitoneally injected glucose solution has no influence upon the absorption of the glucose but does increase glucose excretion by way of the kidney. This increased glycosuria appears to be a factor involved in producing the normal glucose tolerance observed in hereditary diabetic and partially-pancreatectomized rats administered sodium chloride with the glucose.

In studies of the iodine requirements of poultry a simple type of goitrogenic ration has been used at this Station. This has consisted essentially of ground yellow corn, dried brewers' yeast residuals, soybean oil meal, steamed bone meal, salt and vitamin D oil. This has supported fairly good growth and feathering and has allowed the production of severe goiter when feedstuffs selected for low iodine content were used. The goitrogenic effect has been readily overcome by the addition of iodine to the ration. However, it was found that both production and reproduction on this ration were poor, even when sufficient iodine was present to prevent goiter.

In order to study the iodine requirements of laying birds, it was necessary to correct this difficulty. Experience had shown that the replacement of part of the soybean oil meal with casein in the type of ration just mentioned would permit excellent growth. Accordingly, a number of single-comb White Leghorn pullets, reared on the goitrogenic type of ration, were divided into 3 pens at time of sexual maturity and were fed rations containing different proportions of casein and soybean oil meal. The protein, vitamin, calcium and phosphorus content of these rations were approximately the same. Oat groats were also added at the 15% level in pens 2 and 3. Pertinent data are given in Table I.

It is obvious from the data in Table I that reproductive performance in period A improved as the soybean oil meal was replaced with casein and oat groats. Nevertheless the results were not satisfactory. Hence in period B the amount of soybean oil meal was reduced to 6% in pens 1 and 2. Pen 3 was retained as control on the same level of soybean oil meal as in period A.

Duval's chromogenic acid-fast culture recovered from the tissues of human leprosy produced lesions in the rabbit that microscopically are identical to those of the human disease. The infection is progressive over a period of 6 to 8 months. It is significant that the causal microorganism steadily increases in number throughout this period.

The chromogenic acid-fast bacillus of Duval was recoverable from the experimental lesions as late as 8 months after the last inoculation. In vitro cultivation was more difficult in direct ratio to its residence in the host animal.

In the control animals (B. phlei and B. smegmatis) no gross or microscopic lesions were observed one month after the last injection. No culture was obtained or acid-fast bacilli observed in the tissues of the control animals which were sacrificed 3 months after the last injection.

The results of the experiments herein reported show that Duval's chromogenic acid-fast bacillus induces a chronic form of infection in the rabbit with characteristic tissue changes deep in the parenchyme of the various organs.

A list of acid dyes reported to be secreted in gastric juice has been reinvestigated. None of them appeared in histamine-excited gastric juice from Pavlov or Heidenhain pouches in dogs. This work gives support to the earlier generalization that only dyes in which the chromogen may be in the cation, appear in highly acid gastric secretions.

The low pepsin content of achlorhydric gastric juices from carcinomatous stomachs is of the same order as the low pepsin content of achlorhydric gastric juices obtained from patients not presenting carcinoma of the stomach.

Neither the fundamental mechanism of the Frei reaction, which until recently was the only basis for the certain diagnosis of lymphogranuloma venereum, nor the nature of the reagent in the antigen which provokes a positive Frei test has yet been clearly defined. It has been observed that high-speed centrifugation of certain Frei antigens yields dermally active sediments containing virus elementary bodies 1 but nonreactive supernatant fluids. 1 , 2 Furthermore, the passage of Frei antigens through Berkefeld filters 1 , 2 or Seitz K discs 1 was found to reduce greatly or, more commonly, to abolish completely the dermal activity of the filtrate; it should be noted that Seitz K filter? are permeable even to bacteria. These observations have led Findlay to the conclusion that the Frei test demonstrates an allergic reaction due to the presence of the elementary bodies of the virus in the material injected.

It has previously been shown that by propagation of the agent of lymphogranuloma venereum in the yolk-sac of the developing chicken embryo 3 or in the lungs of mice 4 there are obtained quantities of virus far greater than those present in other tissues. Inactivated suspensions of elementary bodies derived from the yolk-sac, and separated from tissue constituents by differential centrifugation, have been successfully employed as antigens for the Frei test 5 and for the demonstration of complement-fixing properties in the serum of persons infected with the virus; 6 in the latter test suspensions of virus from mouse-lung have also been used.

Complement-fixation tests 6 have been performed in which falling dilutions of selected lymphogranulomatous human sera, known to fix strongly, were titrated against falling dilutions of antigen in the form of suspensions of elementary bodies sedimented from yolk-sac or mouse-lung by high-speed centrifugation.

Menkin 1 has shown that inflammatory exudates of various kinds have the property of increasing capillary permeability as demonstrated by the so-called trypan blue test in rabbits. This consists in the localization and concentration of intravenously injected trypan blue at the site of an intradermal injection of the exudate or preparations thereof. He ascribes this property to a specific substance in the exudates which he terms leukotaxine, and which, presumably, is liberated into the exudate from the injured tissues. Bier and Planet, 2 as well as Rigdon, 3 have reported that extracts of normal tissues exhibit this capillary permeability effect. Since Rocha e Silva and Bier 4 have shown that suitable concentrations of histamine give a positive trypan blue test, and since Bier and Planet found that extracts of rabbit's skin, which is comparatively rich in histamine, gave a positive test, while extracts of other rabbit tissues, which are poor in histamine, gave negative tests, it appeared probable that the capillary permeability factor in normal tissues might be histamine rather than some unknown specific tissue constituent. To test this possibility further, extracts from a variety of tissues have been examined with respect to their ability to give a trypan blue test and with respect to their histamine content.

Method. Simple extracts were prepared by grinding 2 to 4 grams of tissue with sand in saline. The amount of saline was varied in different experiments, so that extracts of varying activity could be studied. The extracts were filtered and assayed for their histamine-equivalent content, using the isolated atropinized intestinal strip of the guinea pig for the assay. A solution of histamine acid phosphate of the same activity as the extract was prepared and both extract and histamine solution were injected into the abdominal skin of previously shaved rabbits.

A single, subcutaneous injection of thyrotropic hormone will result in a significant increase in mean acinar cell height within 8 to 16 hours, which is comparable to that heretofore reported in 72 hours following a series of daily injections. This measured response persists at least 48 hours following injection.

Recently Beutner, Landay and Lieberman 1 reported that hydroxy mercuri-methoxy-propyl carbamyl phenoxy acetate (salyrgan) will act as an anticonvulsant drug. They found that salyrgan prevented the usual procaine convulsions if the two drugs are mixed prior to the injection. They also noted that if the salyrgan is injected separately before that of procaine, it afforded no protective action against the procaine. They concluded from these experiments that the “anticonvulsant” action of salyrgan is not through a renal diuretic action but rather by virtue of a “tissue diuretic” action, i. e., decreased cellular permeability.

A series of experiments was performed in our laboratory in which the following intramuscular injections were made into guinea pigs: (1) procaine alone, (2) procaine and salyrgan into separate areas of the body, (3) procaine and salyrgan mixed before the injection, (4) procaine and mercuric chloride mixed before the injection, and (5) lead nitrate and procaine mixed before the injection.

Results. Our experiments confirm Beutner, et al., that salyrgan will protect an animal against procaine convulsions if the 2 drugs are mixed before being injected, but not if the 2 drugs are injected separately in different regions of the body. In 17 experiments performed on as many guinea pigs, 150 mg per kilo of procaine (10% solution) were injected intramuscularly into the right rear leg. Sixteen of the animals showed violent convulsions, from which they recovered in 15 to 40 minutes. In 12 experiments 20 mg per kilo of a 10% solution of salyrgan were injected intramuscularly and immediately following 150 mg of procaine per kilo were injected intramuscularly into the opposite extremity. All of these animals had convulsions which were as severe and lasted as long as those in which procaine was injected alone.

On the fifth day following lumbar sympathectomy the blood supply to the tibia in the rat is decreased. This paradoxical effect may be the result of disproportionately greater dilatation of the vessels in soft tissues.

The assimilation of carbon dioxide by heterotrophic bacteria was first established by Wood and Werkman 1 with the propionic acid bacteria. Recently the stable isotope of carbon (C¹³) 2 and radioactive carbon (C¹¹) 3 have been used as tracers of the carbon dioxide fixed in the fermentation of glycerol. The fixed carbon has been shown to be predominantly in the products propionic and succinic acids. Wood, et al., 2 demonstrated, in addition, that the fixed carbon in succinic acid is exclusively in the carboxyl group. These authors. suggested that all the carbon dioxide is fixed by the following reaction

and that propionic acid containing fixed carbon is derived from the 4-carbon compounds. Accordingly, the fixed carbon of propionic acid would be in the carboxyl group. Therefore, the report by Carson, et al., 4 of fixed carbon occurring in the α- and β-carbons as well as in the carboxyl carbon of propionic acid is of particular interest. These results imply that, in addition to the mechanism shown by Wood, et al., 2 the propionic acid bacteria have another method of fixing carbon dioxide. The latter method might involve the synthesis of the entire chain from 1-carbon compounds, since the fixed carbon is suggested by Carson, et al., 4 to be distributed among the 3 carbons of propionic acid. This observation places in question the present schemes of fermentation, for the implication is that the substrate may first be broken clown to 1-carbon compounds and then synthesized to the final products of fermentation. While it is clear that autotrophic forms of life must perform such a synthesis, it does not seem necessary for heterotrophic forms of life to break down the substrate to a 1-carbon compound and then resynthesize it to final products. We have, therefore, reinvestigated the problem. Contrary to the report of Carson, et al., 4 we find the fixed carbon is exclusively in the carboxyl group of propionic acid.

Recent reports 1 , 2 , 3 have established the principle that illnesses which on the basis of their similar clinical symptoms would be classified together as influenza can be caused by antigenically different viruses. The same principle must be taken into account when repeated attacks of influenza occur in an individual; that is, in a person who previously has had influenza a subsequent attack may be due to infection with a virus that is antigenically different from the one responsible for the earlier illness. That examples of that sort do occur will be shown by the results of the following experiments in which samples of serum obtained over a period of 4 to 5 years from persons who had had more than one attack of influenza were tested against two antigenically different viruses, the TM virus 1 , 3 and the PR8 strain of the virus of epidemic influenza. 4

Patient A had 2, and Patient B had 3 attacks of influenza during the period of observation. The serums, which were 14 from each patient, included samples obtained at the time of onset of the symptoms of the illnesses, samples obtained 3 to 4 weeks later, and samples obtained at reasonably regular intervening intervals. In order to make the comparison valid all of the samples from each patient were tested at the same time. Each serum dilution was mixed with an equal volume of a suspension (mouse lung) which contained between 100 and 1000 lethal doses of the respective virus per 0.03 cc, and after incubation at 37°C for 1/2 hour 0.06 cc of the mixture was given intranasally to mice which were under ether anesthesia.

It has been shown that Flexner's M.V. poliomyelitis strain carried in monkeys can be acclimated to rats 1 and subsequently retransferred from rats to monkeys, in which latter typical disease was produced. 2 The first 2 experiments of this report were performed in order to determine whether the virus could be retransferred from the collateral monkey generation back to rats.

Experiment I. Monkey No. 2379 was 4 generations removed from the original rat transfer. Generation I monkey had received intracerebrally 1 cc of a 10% lineal generation VI cotton rat cord virus. Generations II, III, and IV monkeys were given 1 cc of a 10% cord virus obtained from the generations I, II, and III monkeys, respectively.

Generation I Rat Re transfer. On 11/11/40, 5 cotton rats were injected with 0.06 cc IN, 0.06 cc IC, 0.5 cc SQ, and 1 cc IP of a 10% cord virus suspension of generation IV, Monkey No. 2379. (The doses mentioned here are the same in all the subsequent experiments of this type.)

Two animals died—1 on 11/27, the sixth day, and 1 on 11/28, the seventh day; both were weak, but neither had paralysis.

Generation II Rat Retransfer. On 11/29/40, the cords of the 2 animals dying in rat generation I were injected into 3 cotton rats. One rat died on 12/1 with no evidence of paralysis; the other 2 animals had paralyses and died on 12/5.

Generation III Rat Retransfer. On 12/6/40, a cord suspension made from the 2 animals that died on 12/5 in rat generation II was injected into 3 animals, all of which developed paralysis.

Thus, one can transfer Flexner's M.V. strain from the monkey to the cotton rat, back to the monkey and then to the rat again.

Fasted adrenalectomized rats treated with NaCl excrete nitrogen in amounts comparable with that of hormone-treated controls or that of normal animals, for at least 5 days of fasting. The survival time of such animals is also somewhat prolonged.

Analyses of the subcutaneous tissues in a 3 1/2-month-old, breast-fed female infant with sclerema neonatorum revealed the usual histologic and chemical alterations: chronic inflammatory reaction and presence of excess saturated fat. When the condition was improved repeat analyses revealed normal adipose tissue. During the course of the disease studies of the blood lipids were made. Early in the disease an increase in all the fractions of the blood lipids as well as a decrease in the iodine number, especially of the neutral fat, was found. When all signs of the disease had disappeared the blood lipids were normal. The lipemia is briefly discussed in the light of the alterations of the fat tissues, and it is suggested that a disturbance in fat deposition or in fat transport from the pathologic areas may be present in this disease. The clinical observation gives support to the idea that this tissue was meta-bolically altered.

Colchicine, by topical application in vaginal pessaries, permits the detection of the growth stimulating effects of estrogens, and progesterone in combination with estrogens, on the vaginal epithelium of menopausal castrates. In menstruating women, this technic demonstrates clearly the proliferative effect of these hormones of endogenous origin during both the follicular and premenstrual phases of the cycle.

Desoxycorticosterone is not androgenic.

The author gratefully acknowledges the technical assistance of Miss Margaret R. Kelleher.

A color reaction characteristic of many α-amino acids has been described.

1. At least 0.17 g of potassium per 100 g of diet was found necessary to secure approximately maximal growth of chicks under the conditions of these experiments. 2. More than 0.13 g of potassium per 100 g of diet was necessary to prevent heavy mortality. 3. Rubidium had a slight and only temporary alleviating effect on potassium deficiency.

The decarboxylating enzymes contained in the kidney are specific for certain amino acids and vary with the species.

We express our sincere appreciation to the Department of Urology of the Presbyterian Hospital, for help in providing us with the human material.

(1) A rapid and reliable method for quantitatively analyzing “tracheal” fluid by emission spectroscopy has been described in detail. (2) Evidence has been presented showing that a correction should be made for electrode impurities. (3) Typical analyses of potato tracheal fluid for B. P. Mn, Mg, Cu, and Ca are presented.

Continuing the study of the metabolic fate of testosterone in the human male, we have now been successful in demonstrating that etioallocholanol-3(β)-17-one (isoandrosterone) is a possible metabolite of testosterone. Previous communications have established androsterone and etiocholanol-3(α)-17-one as metabolic products of testosterone in the human male. 1 , 2 , 3 When testosterone was administered to the adult male guinea pig, etioallocholanol-3(β)-17-one was recovered in the urine. 4

The presence of etioallocholanol-3(β)-17-one has been established in the urine of a patient suffering from adrenal hyperplasia. 5 , 7 Recently, this substance has been isolated from the urine of normal women and also from the urines of both men and women suffering from cancer. 6

The urine was collected from a 30-year-old hypogonadal male during the time the individual was being injected intramuscularly with 30 mg of testosterone propionate† per day. During this period the daily excretion of androgenic substances exceeded 75 International Units (I.U.) per day compared to the value of 14 I.U. per day in the absence of any treatment. A total of 12 days of urine during this treatment was collected.

The urine was extracted in the usual manner and the neutral compounds separated into the ketonic and nonketonic fractions by means of the Girard-Sandulesco reagent. 2

The neutral ketonic compounds were fractionated by means of chromatographic analysis using aluminum oxide (Brockmann) as the absorbent and carbon tetrachloride as the solvent. That fraction which was eluted with carbon tetrachloride containing 0.1% ethanol yielded a crystalline residue on evaporation of the solvent. This crystalline residue was dissolved in 4 cc of hot 90% ethanol, and 170 mg of digitonin in 8 cc of hot 90% ethanol was added.

Egg white injury or acrodynia in the albino rat, which occurs when commercial dried egg white is included in the diet of this animal in a substantial amount develops much more slowly if 10% of the fat in the diet is in the form of corn oil instead of Crisco. If vitamin B₆ is injected into corn oil fed rats the signs and symptoms of the egg white acrodynia almost entirely disappear.

1. With Butler's phosphate buffer as a salting solution, prothrombin precipitates between 1.7 and 2.0 molar strength, which places it in the upper globulin fraction. 2. The bulk of proteins of solubility similar to prothrombin migrates electrophoretically as alpha globulin. 3. On the electrophoresis of whole plasma, prothrombin migrates with the albumin fraction.