Experimental Biology and Medicine

This method enables one to analyze rapidly for salicylates in biological fluids in which there are no suspended solids to occlude the apparatus. The time required is short-one hour for the hydrolysis, the time necessary for the actual analysis depending upon the skill developed by the operator of the Van Slyke apparatus. The accuracy of this method was found to exceed that of other existing methods and does not depend upon one's ability to match colors in a colorimeter.

Kurloff 1 first described granular inclusions within the cytoplasm of what he called lymphocytes in guinea pigs' blood. It was shown subsequently that the cells containing these bodies were monocytes. 2 Wright's stained blood smears reveal the Kurloff bodies as dark red, homogeneous masses in the cytoplasm. Although many workers have observed and studied these inclusions, little is known concerning their origin and function. They appear to be found only in the blood of guinea pigs.

Bender and DeWitt, 3 while studying the blood picture of tuberculous guinea pigs, observed that their normal control pigs showed an unexpected eosinophilia. At the same time, Kurloff bodies appeared in the blood. They decided that there was a relationship between eosinophilia and Kurloff bodies. They were able to reduce the eosinophiles with mercurial medication, but not the number of Kurloff bodies.

During an investigation 4 of eosinophilia in trichinous guinea pigs about 3 months old obtained from one source, I observed the number of Kurloff bodies in several hundred Wright's stained blood smears. The cells were counted 3 times a week for 6 to 8 weeks. All the animals had Kurloff bodies in their blood when first examined. Six normal control pigs had varying levels of eosinophiles. There was a definite, direct relationship both in the healthy and infected pigs between the number of eosinophile cells and the number of Kurloff bodies in the monocytes so that the high levels of eosinophile cells were accompanied by the greatest number of Kurloff bodies. It was possible to reduce markedly the number of eosinophile cells in trichinous animals by a staphylococcus infection, but this did not essentially affect the number of Kurloff bodies.

We have already reported the blood iron content of the adult to be an average of 50 mg. per 100 cc. for men and 45 mg. per 100 cc. for women, and the blood copper 0.132 mg. or 132 micrograms per 100 cc. for men and women. 1 , 2 In determining the blood iron and copper in various pathological conditions accompanied by anemia, we observed an inverse relationship between the copper and the iron in the blood. As the blood iron fell, the blood copper rose. Hypercupremia was the usual response to hypoferronemia. 1 , 2

We have already reported the blood findings in a white woman, age 60, suffering from polycythemia vera. 2 We now report another case which verifies our first findings. The aim of the treatment in vogue in this condition, which is manifested by an increase in the red cell count beyond the normal, is to cut down the number of red cells to the physiological level. In this type of therapy it is often advisable to diminish the red cell count even below the normal in order to postpone the rapid return of the polycythemic state. The drug used, phenylhydrazine hydrochloride, is a cumulative poison, so that its destructive effect upon the erythrocytes goes on after its discontinuance. A polycythemic individual therefore in the course of the treatment now prevailing may be converted to an anemic individual with a low red cell count. In periods of non-treatment the red cells have a tendency to increase to the polycythemic stage. The periods of treatment alternated by periods of non-treatment offer a chance to study the reciprocal relationship between blood iron and blood copper.

The gradual descent of poliomyelitic infection, after intranasal inoculation, through the olfactory tract, the basal ganglia, midbrain, pons and medulla to the spinal cord was demonstrated in experimental animals (monkeys) by Faber and Gebhardt, 1 using the method of recovering virus from bits of nervous tissue excised on successive days of the preparalytic period. Evidence was presented by Faber 2 that the infection in man may follow a similar pathway. Since this method of recovering virus permits only a rough localization, other means were sought to obtain a more precise definition. Evans and MacCurdy 3 reported successful vital staining of poliomyelitic lesions in monkeys, but failed to state the dye used or the period of infection when the animals were examined. McClellan and Goodpasture 4 were able, by means of intravital staining with trypan blue and subsequent clearing of the tissues, to visualize the lesions of another axon-conducted viral disease, herpetic encephalitis. Their method has been used in the present study of the sites and character of the lesions in the preparalytic stage of experimental poliomyelitis after intranasal inoculation carried out by the technic of Schultz and Gebhardt. 5 †

Results in 3 rhesus monkeys are here reported: 1. a control; 2. an animal killed 3 days after inoculation; 3. an animal killed 6 days after inoculation, before paralysis or weakness occurred.

In the control, which was given no inoculation with virus but received trypan blue in the same manner as the inoculated animals, there is no visible staining of the nervous tissues proper. The dura, velum interpositum and choroid plexuses are deeply stained, while the leptomeninges are faintly stained. The smaller arteries and veins are inconspicuous and there is no evidence of capillary dilatation.

The data here reported show clearly that fluorine additions of 8 to 14 ppm. to a basal diet containing 3 ppm. results in growing rats in increases in the fluorine content of the bones and teeth, which are marked and roughly proportional to the level fed. They show sodium fluoride and bone meal to be equally effective in causing these changes. They suggest that analysis for fluorine is a more sensitive measure than the enamel pigment changes which are also noted at these low levels. The data indicate that the fluorine content of the bones and teeth increases with age during growth.

The methods used in the purification of tetanic toxin were based on the general principles outlined in a previous paper on the purification of diphtherial toxin. 1 The crude toxins were prepared in meat-infusion containing 2% of Bacto-peptone, and 1% dextrose. Cultures of Cl. tetani were grown 14 days under vaseline and filtered. Lots 4 and 5 were about one year old, and lots 6 and 8 were fresh when the purification was started.

Purification. The toxic preparations should be kept in a refrigerator except when reagents are added or when being centrifuged. The optimal proportions of reagents vary somewhat with different lots of toxin. These variations have been indicated in the directions for the process given below. Amounts are for the purification of one liter of crude toxin.

1. To one liter of crude toxin 100 to 125 cc. of 10% solution of ferric ammonium sulphate are added gradually. The pH is kept between 5.5 and 6.0 by the addition of normal sodium hydroxide. The resulting precipitate will dissolve in an excess of the ferric salt below pH 5.5. After settling for a few hours the precipitate is collected and washed with distilled water. The toxin is then eluted with 400 cc. of 2.5% sodium citrate at a pH of 7.5 to 8.0 for 3 days or until most of the precipitate has dissolved. The insoluble residue is centrifuged down and the pH of the supernatant adjusted to 7.0.

2. Four hundred cc. of the eluted toxin are diluted to one liter and the calculated concentration of sodium citrate adjusted to 1.5 to 2.5%.

During a recent study of the vitamin C needs of children which included determinations of the daily urinary excretions of ascorbic acid, one child developed a slight upper respiratory infection which interfered with night rest. He was given on 2 successive nights 2.5 grains of aspirin; and water ingestion was forced during 2 days. Since his food intake remained normal and his temperature was only slightly elevated, the experiment was not discontinued as is usual when a child who is under observation develops a cold. Analysis of the urine following aspirin administration showed an increased output of ascorbic acid. This was quite out of line with our general findings to the effect that following physiologic adjustment, at a given level of ingestion the daily excretions of ascorbic acid of the children under observation were surprisingly constant during the 10-day period of study (Everson and Daniels 1 ). Was the increased excretion of ascorbic acid due to conditions producing the elevated temperature, the effect of the acetylsalicylic acid, or to a washing-out process following the larger water intake? That infections may deplete the stores of vitamin C has been suggested by Dry, 2 by Harde, Rothstein and Ratish, 3 and by Harde and Benjamin. 4 On the other hand, Giardina 5 has shown that sodium salicylate will precipitate scurvy and death in guinea pigs receiving diets containing an insufficient amount of the antiscorbutic vitamin in a shorter time than in pigs on similar diets with no sodium salicylate.

To obtain further data regarding the effect of aspirin on vitamin C metabolism in the human organism, the 3 children between 4 and 6 years of age who were under observation were given constant weighed diets during which period the ascorbic acid intakes and excretions were determined daily.

No anterior-pituitary-like thyrotropic activity could be demonstrated in beef, sheep or hog ovaries.

Small, but demonstrable amounts of estrin were found in the urine of pregnant rats; no estrin could be demonstrated in rat placentae.

In view of the fact that there is a vigorous opsonic response to the parenteral injection of antigenic fractions of Brucella cells, an unusual opportunity is offered for measuring the phagocytic activity of the various types of bone marrow cells collected directly from the marrow, or as they are found in the peripheral blood in myelogenous leukemia.

Studies have been made on the phagocytic activity of marrow cells aspirated from the cavity of the femur of normal guinea pigs, and those immunized to Brucella, and on marrow cells in the peripheral blood of one human case of chronic myelogenous leukemia.

The cells in the blood of the pigs were measured for their Brucella phagocytic activity in vitro prior to the collection and examination of marrow cells. The cells in the whole blood of normal pigs showed no phagocytic activity. The segmented and stab neutrophile forms in the whole blood of the immunized pigs were found to ingest at least SO or more bacteria; thus indicating the presence of immune opsonins in the serum for Brucella.

The phagocytic activity of marrow cells from normal and immunized guinea pigs was measured in vitro in the presence of both normal guinea pig serum and serum containing immune opsonins.

The phagocytic activity: of the marrow cells in the peripheral blood of the case of myelogenous leukemia was examined in the presence of the patient's serum and in the presence of a serum obtained from another person possessing a high immune opsonin titer for Brucella. The mixtures of cells, serum and bacteria, were incubated in a water-bath at 37°C. for 30 minutes before making spreads and staining.

The corpus luteum hormone corporin as obtained from the corpus luteum will produce premenstrual development of the endometria of castrated monkeys and traumatization of such uteri stimulates the formation of epithelial proliferations characteristic of normal implantation sites. 1 , 2 It is also known that oestrin will promote growth of the uterus, cornification of the vagina, development of the sex-skin, and metaplasia of the cervical glands. 3 , 4 When effective doses of oestrin and corporin are administered simultaneously oestrin does not prevent the action of corporin on the endometrium nor does it prevent epithelial proliferation following trauma, Corporin, however, minimizes the effects of oestrin on the sex-skin and vagina, and prevents metaplasia of the cervical glands. 5 , 6

Experiments have been conducted to determine whether or not the synthetic corpus luteum hormone, progesterone, possesses the above physiological properties known for the natural substance. The material used was prepared synthetically from Stigmasterol by the method described by Butenandt and furnished by the Schering Corporation, Bloomfield, N. J.

The type of experiment employed is illustrated by the following protocol: Monkey Al. 54, adolescent, body weight 4075 gm., was given pituitary extracts resulting in marked ovarian development without luteinization. The animal was castrated and given 100 RU of oestrin daily for 15 days, followed by a combination treatment of 100 RU of oestrin plus 4 international units of progesterone daily for 16 days. The uterus was traumatized on the eighth day of oestrin-progesterone treatment and the animal was killed at the termination of the experiment.

Histological examination of the genital tract of this animal showed a premenstrual endometrium approaching secretory exhaustion, marked epithelial proliferations surrounding the traumatic areas, an absence of squamous metaplasia of the cervical glands, and a reduced oestrin effect on the vaginal mucosa as indicated by relatively few1 mitoses, subepithelial leucocytic infiltrations, and the absence of Dierk's layer over extensive areas. These results agree with those previously reported for experiments in which combinations of oestrin and corporin were used, 5 , 6 and indicate that synthetic progesterone has the same physiological prop erties as the progestational hormone extracted from corpus luteum tissue.

We are reporting experiments elsewhere on the effects of cysteine and cystine on the metabolism of rats (Goldfarb, Fazekas, Himwich 1 ). The present communication is a report of further experiments on isolated brain tissue. The observations were limited to the effects of cysteine, since cystine was soluble in solutions too alkaline or acid to support respiration. In all experiments the tissue was suspended in a phosphate medium buffered at pH 7.4 with lactate as a substrate. The tissues were inserted in the Warburg respirometer and one-half hour elapsed, the time required for equilibration, before observations were begun.

The data disclose that the O₂ consumption of the brain tissue treated with cysteine exhibits a diphasic response. During the first 30 to 45 minutes there is a marked stimulation which is followed by a profound depression in the later periods. Cysteine, moreover, retains an early stimulatory effect on O₂ consumption despite the presence of cyanide.

In order to obtain further information as to the character of the increased O₂ consumption, a group of observations was made on the R.Q. after the addition of cysteine hydrochloride in a concentration of 0.01 M. In most instances the R.Q. of unwashed, minced brain tissue is approximately 0.9, (Himwich, Fazekas, Barker, Hurlburt 2 ) and in these studies the addition of cysteine resulted in a depression of the quotient. In 8 experiments they were found to be 0.50, 0.65, 0.65, 0.54, 0.59, 0.52, 0.30, and 0.83. The presence of the sulphydryl group had obviously caused a consumption of O₂ without the simultaneous production of CO₂.

Removal of the posterior lobe of the hypophysis with only slight injury to the anterior lobe does not increase the sensitivity of rats to the convulsive action of insulin. Removal of the anterior lobe only, leaving the posterior lobe intact, results in a marked increase in insulin sensitivity equal to that observed after complete hypophysectomy.

Observations regarding the effect of ultra violet light upon the fermenting power of yeast have been made by various authors. Owen and Morley 1 report an increase in activity of yeast after administration of appropriate dosages, while Tanner and Byerly 2 demonstrated an adverse effect. The first noted authors seem convinced that irradiation of the medium by means of ultra violet energy before introduction of yeast results later in enhanced growth of the microorganism, but Tanner and Byerly were unable to confirm this finding.

The object of our series of experiments was to determine whether exposure of yeast to ultra violet light is attended by subsequent increase in formation of ethyl alcohol.

For our purposes, we have utilized equipment set up as follows: The source of ultra violet was a quartz tube of approximately 24 inches length and one centimeter external diameter, of the type known ordinarily as “Cold Quartz” which operates with emission of very little heat and with much energy in the region of 2500-2800 A°. Placed outside of this quartz electrode, was an ordinary glass tube. Materials to be treated with the ultra light thus could pass between the outer wall of the quartz tube and the inner surface of that made of glass. The system was set up in a vertical position and fluids to be treated entered at the bottom and were withdrawn at the top. A variety of glass tubes of different diameters enclosing the inner quartz electrode permitted alteration of the thickness of layer of the fluid to be treated. Proper mixing during the period of treatment was brought about by intermittent surges of air introduced likewise at the lower end of the vertically placed apparatus.

A rise in blood pressure may be produced in dogs either by partial obstruction of the renal arteries 1 or by ligation of the ureters. 2 Since the latter procedure often causes a marked decline in the renal blood flow, 3 it seems likely that the hypertension is in both instances related in some way to ischemia of the kidneys. It was shown by Tigerstedt and Bergman 4 that saline extracts of the kidney of rabbits produced a sustained rise in blood pressure when injected into other rabbits. The object of our experiments was to determine whether extracts prepared from ischemic kidneys, removed from dogs with hypertension, had a greater pressor effect than similar extracts of normal kidneys.

The freshly removed kidneys were chopped up with scissors and ground with carborundum and 0.9% salt solution. The suspension so obtained was centrifuged and the supernatant fluid was kept in the ice box until used. Amounts corresponding to 5 gm. of kidney tissue were administered intravenously to normal unanesthetized dogs. Changes in blood pressure were measured by the cuff described by Ferris and Hynes, 5 the passage of the pulse wave being determined by palpation of the dorsal artery of the foot. 6

The results which have been obtained are summarized in Table I. Extracts of the normal kidneys frequently caused a marked preliminary decline in blood pressure, followed in a few minutes by a gradual rise which persisted for 30 minutes or longer. The degree of rise varied from zero to 60 mm. of mercury above the control values. Extracts of the kidneys of dogs with renal hypertension usually caused less preliminary decline and a more marked secondary rise in blood pressure.

Recent studies by Martin 1 Mooney and Winslow, 2 and Clifton 3 , 4 , 5 on the metabolic activities of bacteria indicate that the rate of metabolic activity per cell varies widely at various phases of the growth-cycle. The maximal oxygen-consumption, carbon dioxide-production or ferricyanide-reduction per cell per unit-time was noted near the end of the lag-period of growth and could be only in part explained by increased cellular size during the same period of growth. The metabolic activities per unit-volume of the cultures, as measured by the above indices, reached maximal values near the end of the logarithmic period of growth and then decreased rapidly as the age of the cultures increased. Peptone or other complex media were employed in these studies.

The present paper reports the influence of the concentration of lactic acid (sodium salt) on the oxygen-consumption, carbon dioxide-production and growth of Escherichia coli (K-12) in an inorganic medium of the following composition: NH₄Cl, 5.0 gm.; Na₂SO₄, 2.0 gm.; MgSO₄, 0.1 gm.; K₂HPO₄, 3.0 gm.; KH₂PO₄, 1.0 gm.; FeCla (0.5% solution), 0.5 ml.; CaCl₂ (0.5% solution), 0.5 ml.; distilled water to make 1000 ml.

Oxygen-consumption was measured by the usual Warburg technic and carbon dioxide-production by the technic described by Walker. 6 All tests were carried out at 37.5°C.

The influence of the concentration of lactic acid on the growth of Esch. coli, as measured with the aid of a photoelectric turbidimeter (Clifton 7 ), is illustrated in Fig. 1, A. Concentrations of sodium lactate greater than 0.1 N markedly inhibited the growth of this organism.

The influence of 4 different initial concentrations of lactic acid on the oxygen-consumption of Esch. coli is illustrated in Fig. 1, B, in which cubic millimeters of oxygen consumed per minute per millimeter of the cultures is plotted against time in hours.

Differentiation of Salmonella pullorum from Salmonella gallinarum cannot be made by agglutination or agglutinin-absorption, because of a similar antigenic structure. This similarity and the fact that the 2 diseases produced in fowls are alike in some respects have caused certain European investigators 1 , 2 , 3 to consider them as variants of the same species.

Mailman's 4 work on the use of sodium-mucate agar, Jordan and Harmon's on tartrate-agar, 5 and Naidu's 6 and Nobreg's 7 studies on differentiation by bacteriophage, are examples of investigations which supply further evidence that these organisms are separate entities. This report deals mainly with the use of cysteine-gelatin as another differential medium.

The cysteine-gelatin was prepared according to a method used by-Valley 8 for the study of anaerobic growth. Enough Difco granular gelatin is added to the 0.15% cysteine-broth described by Valley 9 to make a 12% solution. After dissolving the gelatin in a water-bath, the medium is tubed and sterilized at 15 pounds extra pressure for 15 minutes. Tubes of 12 mm. and of 16 mm. diameter were found to be equally satisfactory, when at least 5 cc. of medium were used. The cysteine-gelatin retained its potency for at least 2 months, when stored in the refrigerator and dehydration was prevented.

The action of 61 strains labeled S. gallinarum, 120 S. pullorum, and 11 S. typhi, was studied in this medium. Tartrate-agar 5 was inoculated with the same strains, for comparative purposes. A summary of these results is given in Table I. The tartrate-reactions support Mailman's findings. The results obtained with the 11 strains of Salmonella typhi are included also for record. Kauffmann and were designated “Duisburg.”

Our previous findings with respect to the distribution of the 4 blood groups and the M factor among the Blackfeet Indians have been confirmed and a surprisingly high incidence of the A 1 sub-group was observed.

The pH of guinea pig's bile has been determined and an analysis of the principal constituents has been made.

Mature mice showing regular oestrous cycles were injected with 0.1 mg. of testosterone in 0.1 cc. oil twice daily. (I am indebted to Messrs. Ciba, Limited, for the supply of testosterone.) During the period of injections extending over 2 weeks or more oestrus was abolished. Following cessation of injections oestrous cycles were reestablished. Injected mice were placed with males for a week; no mating was observed and no vaginal plugs were found. Normal cycles were continued in control mice injected with oil alone.

Mature ovariectomized mice were injected with oestrone in doses sufficient to produce cornification, together with testosterone. The simultaneous administration of testosterone inhibited the oestrous reaction of the vagina. The oestrogenic effect of 0.0001 mg. of oestrone (a dose which produces cornification in 50% of our mice) was inhibited by 0.4 mg. of testosterone.

These findings show that there may be a direct antagonism between the female and the male hormones in their actions on the vagina of the female mouse, and are the more remarkable in that testosterone in large doses has been shown to produce oestrogenic effects in the infantile rat (Butenandt and Kudzus 1 ).

Further experiments are now in progress to investigate quantitatively the relation between the actions of oestrone and testosterone on the vagina, and to determine whether other compounds with male hormone properties have effects similar to testosterone.

Of the various existing tests for the male sex hormone, that of the capon comb is generally recognized as being the most satisfactory. The cost of capons and their upkeep is a hindrance to wide use of this test. If baby chicks can be utilized to test for male hormone properties, the cost of such tests will be materially reduced.

The purpose of this investigation was to determine whether the comb of the baby chick is sufficiently sensitive to the action of male sex hormone to warrant the more extensive experiments necessary to establish that the baby chick may be used as a test object. †

As a preliminary experiment 3 groups of day-old, Single-comb White Leghorn chicks (7 chicks to the group) were injected subcutaneously with the male hormone product testosterone. A fourth group of 7 chicks was carried as controls. Group 1 received 25 gamma daily, and group 3 received 100 gamma. After 4 injections, 2 chicks of Group 1, 5 chicks of Group 2 and all those in Group 3 showed a definite increase in comb growth. Injections were continued on 3 chicks selected from Group 3. After 14 daily (except Sunday) injections these 3 chicks were photographed with their controls. Fig. 1 shows one of these chicks with its control.

In a second series of experiments, 117 other chicks were injected, some with testosterone and some with androsterone. Single-comb White Leghorns and Rhode Island Reds of both sexes were used. Their ages varied from 1 to 10 days of age, and injections of various amounts of material were made either in the base of the comb or in the breast muscle. Table I shows the results obtained.

Tooth pulps were collected by cracking teeth. Fibrous, calcified and putrescent pulps were discarded. For human teeth, the sample of pulps weighed 0.43 gm. Micro-lipid analyses were carried out using the Bloor oxidative procedures. The total lipids were 0.91% (moist weight), phospholipids 0.70%, and cholesterol 0.11%. For cow teeth, 214 incisor pulps weighing 33.72 gm. were collected. After lipid extraction, saponification of the residue gave 0.16% lipids unextracted. The total ether-soluble lipids (0.85% of moist weight) contained 50% unsaponifiable matter; of the saponifiable fraction, the fatty acids composed 76.6%, iodine number—72.3. Using the Twitchell procedure, 79.9% liquid fatty acids and 3.0% solid fatty acids were found.

The Rein “Thermostromuhr”, 1 the best method available for measuring flow in unopened vessels, is limited to mean flow measurements. In this laboratory a new method was developed which can follow rapid flow changes and which, besides, possesses the further advantage that the deflection bears a linear relation to the flow.

This flowmeter is based on the principle that an electromotive force is induced in a conductor moving so as to cut the lines of force in a magnetic field. If a wire is moving through the field in a direction perpendicular to its own axis and to the lines of force, then the potential difference at the ends of the wire is given by the expression :

where E denotes the induced voltage, H is the strength of the magnetic field, 1, the length of the wire and v, its velocity.

A steady potential difference can be obtained by moving an endless metal belt, mercury or an electrolytic solution between the magnet poles. Here 1 would indicate the width of the conductor used. The induced electromotive force generated in the case of the solution can be tapped by introducing 2 non-polarizable electrodes through the wall of the tube through which the liquid is flowing. Since the induced voltage is proportional to the velocity of fluid flow, the current flowing through the galvanometer connected to these electrodes will be a linear function of the rate of flow. This was verified by actual trial with solutions of various concentrations of copper sulphate and sodium chloride using copper electrodes for the former and silver-silver chloride electrodes for the latter.

Development and secretion of mammary parenchyma is normally demonstrable in male rats beginning at puberty. Castration prevents the proliferation of the mammary gland in the immature male, just as it does in the female. In adult males, in which the mammary gland is already developed, castration is followed by involution of the gland. Testosterone in doses sufficient to produce mammary gland development does not cause vaginal cornification either in the normal or the ovariectomized immature female rat.∗

In a single case of a hypophysectomized ring dove, Riddle, Bates and Dykshorn 1 reported an increased weight of the crop gland to the extent of about 300%, with secretion of “crop-milk” following 4 daily injections of prolactin. Observations in our laboratory have shown that the injection of purified lactogenic hormone, galactin, immediately after hypophysectomy in cats, rats and guinea pigs, failed to initiate or prevent the rapid cessation of lactation. It is of interest, therefore, to determine whether galactin would cause the proliferation and the secretion of “crop-milk” of the crop glands of hypophysectomized pigeons. Common pigeons were used. The operation, which was carried on by the oral or parapharyngeal route, was well tolerated and the high post-operative mortality was minimized by the routine injection of glucose solution. In all instances, the injections of lactogenic hormone were started in 2 to 3 days after hypophysectomy, by the intramuscular or intradermal route for 4 consecutive days, the birds being sacrificed 24 hours after the last injection.

Of a total of 28 operated pigeons, 16 were found to be completely and 12 incompletely hypophysectomized at autopsy. The crop glands of the completely hypophysectomized birds which received galactin in amounts ranging from 4 to 10 mg. intramuscularly or intradermally over the crop sac all showed no evidence of proliferation. Three of the incompletely hypophysectomized birds which received 5 mg. and 2 which received 10 mg. of galactin intramuscularly, showed proliferation of both crop glands with an average rating of plus 2.6 and 1.25, respectively, while 7 which received 4 mg. intradermally over the crop sac showed proliferation only in the gland immediately above the site of injection.

We reported 1 that the normal male rat pituitary contains an appreciable amount of the lactogenic hormone and that the injection of the estrogenic hormone definitely increases the galactin content of the pituitary gland.

Employing the same technique as that which we described in the paper referred to above we have extended the study of the galactin content of rat pituitary glands. The results are summarized in Table I.

From the table one can see that as the male rat continues to grow the pituitary gland likewise increases in weight. The bird units of galactin per pituitary gland increase as the animal matures, but this is due largely to the increase in the weight of the gland as one notices that the galactin concentration remains quite constant. The daily injection of 500 international units of Oestroform B into male rats for 7 days increases the weight of the pituitary gland and increases the galactin content of the gland while the content in bird units of galactin per mg. of fresh pituitary tissue remains nearly constant. The injection of thyroxine in increasing dosages decreases the weight of the pituitary glands, decreases the total potency of the glands, and decreases the galactin concentration per mg. of fresh pituitary tissue. The injection of thyroxine at the same level each day, 0.01 mg., produces the same results but to a less extent. Male rats castrated for 60 days possess much larger pituitary glands; the bird units of galactin per pituitary gland, however, remain constant.

The pituitary gland from the immature female rat contains about 3 times the amount of galactin of the immature male rat, this increased galactin content being accompanied by an increased glandular weight and an increased concentration of the galactin. As the female rat matures there is an increase in weight of the pituitary gland as well as an increase in concentration of galactin. At 12 days of pregnancy there is a decrease in the number of bird units of galactin per pituitary gland as compared with glands from normal estrous cycle females, and at 21 days pregnancy there is an increase in potency of the glands as compared with those from rats pregnant for 12 days. Forty-eight hours postpartum there is a doubling of the galactin content of the pituitary gland over that of the glands from either the normal estrous cycle female or the female pregnant for 21 days. At 10 days postpartum the galactin content of the glands decreases somewhat, being about midway between the glands from 48 postpartum rats and glands from normal estrous cycle females.

The site of action of phlorhizin in phlorhizin diabetes is now recognized as being in the kidneys, 1 the disturbance caused by the drug being apparently due to inability of the tubule to reabsorb glucose. This, according to Lundsgaard, 2 arises from the fact that the poisoned tubule is unable to carry out phosphorylation of glucose. Moreover, several glucosides, including phlorhizin, are now known to inhibit phosphorylation in vitro, and glucose resorption in the isolated perfused frog kidney is also inhibited. 3 It occurred to us that the glycosuric effect of phlorhizin might be a property common to all these glucosides. The action of arbutin, amygdalin, and salicin on healthy, fasting dogs was studied according to the procedure and methods of urine and blood analyses customary with phlorhizin.

Arbutin is, like phlorhizin, a phenol glucoside. When injected subcutaneously in sterile distilled water twice daily in fasting dogs, it caused a diabetes similar to that produced by phlorhizin with lowered blood sugar and acetone bodies in the urine, the ratio of dextrose to nitrogen in the urine being 1.45, 1.56, 1.50, and 1.92 in 4 different dogs. The urinary reducing substance was dextrose, for after yeast fermentation the urine no longer reduced Benedict's reagent. In contrast to the diuresis of phlorhizin diabetes, the urine of the animals receiving arbutin, likewise of those receiving the amygdalin and salicin, was very scanty unless water was administered by stomach tube, but the voluntary intake of water also was very small.

Three fasting dogs injected subcutaneously with salicin, an alcohol glucoside, in sterile distilled water exhibited no glycosuria nor ketonuria; nor did 3 dogs similarly injected with amygdalin, an aldehyde glucoside.

Lundsgaard 4 found that phlorhizin inhibits glycolysis of excised tissues.

In previous papers the isolation and description of numerous variant forms of Micrococcus tetragenus was reported. 1 It was at first believed that these interchangeable variants composed a complex system of bacterial variation different from the orderly one associated with other bacteria. A clue to the solution of the problem was provided many months later by the detection of typical rough colonies among the pink colonies already studied. An orderly arrangement of most of the variants and a basis for comparison with other bacteria was then made possible. 2

With the rough-pink form at hand, this form and the mucoid-pink and pink were grouped together as the usual M, S, and R culture-phases of a given bacterial type. On this basis it was predicted 2 that the yellow, white, pink, pink-yellow, and brown forms represented distinct types of M. tetragenus and that each possessed the respective culture-phases. In subsequent investigation involving the aging of 100 cc. broth cultures of the types at hand for months at room-temperature, most of the predicted forms missing from the scheme were detected, isolated, and studied, namely, the mucoid-pink-yellow, mueoid-brown, rough-yellow, rough-pink-yellow, and rough-brown forms. The rough form of the white type has not yet been obtained although several methods known to favor dissociation were employed. The white type under these manipulations changed either into mucoid or translucent form and yellow colonies occasionally appeared. The studies will be published in detail elsewhere. The variant forms of M. tetragenus isolated were arranged as follows:

It appears that M. tetragenus provides a unique opportunity for the study of type and culture-phase transformation.

In thyroidectomized young guinea pigs anterior pituitary extract of cattle exerts its growth-promoting effect on the cartilage of the joint as well as in non-thyroidectomized animals, and the same arthropathic changes characteristic of acromegaly develop in both. Lack of the thyroid gland does not only not prevent the growth-promoting effect of anterior pituitary extracts, but in thyroidectomized guinea pigs the phenomena are even more pronounced than in normal animals which were treated with the extract. These experiments provide, therefore, further evidence for the conclusion that the extract exerts its influence on bone, epiphyseal line and joint cartilage without the intermediation of the thyroid gland.

Cardiac glycogen is found to be raised in depancreatized cats, and to be well maintained in fasting phloridzinized rats, both with and without an accompanying injection of epinephrine. Attention is drawn to the importance of these findings to current views of carbohydrate change in the body.

In my study of the development of the behavior of Amblystoma it became imperative to know whether or not eggs or embryos that had been subjected to a low temperature, in order to prolong the experimental season by retarding growth, followed the normal order of development of movements. To meet this requirement I conducted experiments in 1930. Regarding behavior the results were conclusively in the negative, but it seemed desirable to vary the conditions in further experiments before publishing the results, especially in consideration of the numerous and extreme structural defects that appeared in the experimental animals. But inasmuch as I have not been able, and probably shall not be, to repeat the experiments the most obvious results are here presented on account of their bearing on experimental morphology. The work was done in the biological laboratory of the Effingham B. Morris Biological Farm of the Wistar Institute of Anatomy and Biology. The special advantages of this laboratory are gratefully acknowledged.

Embryos from 2 clutches of eggs, designated 30M and 30E, were used for the experiments. They were collected from Gould's mill-pond in the Pocono Mts., Pa, April 13 and removed from the egg-masses, but not from the individual envelopes, on April 14. On April 20 they were placed in a mechanical refrigerator, the clutches in separate finger-bowls, where they remained till May 27. On this date the refrigerator was found not to be operating and defrosted, although it was operating on the previous day. The period of chilling was, therefore, 36 days. The temperature of the interior of the refrigerator fluctuated considerably with that of the room, which was also subject to rather marked changes according to the weather.

In an attempt to bring the suspended particles of an aqueous spinal cord suspension containing rabies or poliomyelitis virus into more intimate contact with inactivating agents for vaccine production trials a “solution” of the cord was made with the aid of urea. The effect of urea in strong concentration on these viruses proved interesting. As first recorded by Spiro 1 and in more detail by Ramsden 2 urea in aqueous solution has a remarkable ability to “dissolve” proteins. When spinal cord of the rabbit or monkey, moist with saline, is placed in a mortar and urea crystals added the cord on trituration quickly passes into an opaque syrupy “solution” containing innumerable lipid droplets in suspension. In this manner a 50% cord “solution” containing from 30% to saturation with urea is easily obtained.

Rabies Virus. Typical results are presented in Table I. The fixed rabies virus usually killed within 6–11 days after intracerebral inoculation. The non-urea treated virus suspension was a 20% concentration of cord and brain in saline. The urea treated preparation contained 50% of cord-brain and about 40% of urea. Rabbits 3 and 4 were injected intracerebrally within an hour after the preparation of the cord “solution”. They were given a second injection of 0.2 cc. each of a urea-treated preparation 10 days after the first ones with no ill effects. Rabbits 5 and 6 were given injections of 0.1 cc. each of the non-urea-treated preparation 3 weeks after the first injections. Both of them died within a week. Rabbit No. 7 was given a weekly “vaccination” by a 1.0 cc. subcutaneous injection of the 50% urea-treated rabies cord preparation for 6 weeks and then inoculated intracerebrally

In a previous communication 1 some of the effects of thorium, zirconium, titanium and cerium on enzyme action in vitro were reported. Due to the peculiar physico-chemical properties of these metals, especially in respect to their precipitability in protein solutions it was deemed advisable to determine whether these effects could be observed in vivo. Roussy, Oberling and Guerin 2 have reported that thorium dioxide is carcinogenic when administered intraperitoneally to rats. In this investigation an attempt is being made to determine the effects produced by the intraperitoneal injection of varying amounts of soluble thorium nitrate, Th(NO₃)₄, on a variety of the blood and liver enzymes of the adult white rat.

The effects upon the following enzymes were investigated: blood amylase, blood lipase, blood esterase and blood phosphatase. The following liver enzymes were investigated : liver amylase, liver lipase, liver esterase, liver phosphatase, liver xanthine dehydrogenase, liver glucose dehydrogenase, and liver lactic dehydrogenase. Amylase was determined by the Willstatter-Schudel method, 3 lipase and esterase by the method suggested by Sure et al., 4 phosphatase by the method described by Bodansky 5 and the inorganic phosphorus determined by the method of Fiske and Subbarrow. 6 The dehydrogenase activities were estimated by the Thunberg technique as adapted by Collett. 7

Twenty-one adult white rats were used for the study. Two sets of controls were run. One set of controls was inoculated with physiological saline and the other received an injection of dilute hydrochloric acid equivalent to the acid strength of the thorium injected into the experimental animals.

Supersonic radiation appears to be the most suitable method yet studied for preparing fat emulsions, suitable for intravenous use in man.

An emulsion of fat that could safely be given in quantity by vein was first developed by Yamakawa and his collaborators 1 in Japan. Recent observations in Latin America and in this country 2 have confirmed and extended their work and have furnished very suggestive clinical and experimental evidence of the value of such preparations. The technique used by Japanese and American workers is essentially similar: the fat or oil is mixed with purified egg lecithin (Merck's or Kahlbaum's) and water and passed through a 2-stage dairy homogenizer operating at 3000 to 4000 lb. pressure, after which the product is sterilized by heat in sealed containers. When suitable quantities are used, it is possible to obtain an emulsion in which practically all the lecithin-coated fat globules are less than 2μ in diameter with only an occasional one as large as 3 or 3.5μ. Such an emulsion passes through the lung capillaries with ease, 3 avoiding the danger of fat embolism, which may occur when the particles exceed 4μ in diameter. 4

A difficulty that has been experienced—both with Japanese and American preparations—is the limited time during which these emulsions are stable. In the course of a few weeks, more or less, the larger fat particles which are in less active Brownian movement tend to rise to the surface, forming a visible cream layer. Although the formation of the cream does not necessarily indicate an increased particle size, nevertheless the large fat particles are in close apposition, and there is danger of their coalescing to form larger aggregates.

Our methods of producing typhus vaccines with the murine strains of Rickettsiae has depended upon the inoculation of rats in which resistance had been reduced by a variety of procedures, the most consistently successful of which has been preliminary X-ray radiation. These methods have persistently failed to give adequate results with the European virus. This fact in itself constitutes further strong evidence that the two types of Rickettsiae are distinct and biologically fixed varieties. After much effort to apply the “rat” methods to the organism of the classical European disease we were finally persuaded that other methods of approach must be sought for obtaining accumulations of the European Rickettsiae adequate for practical purposes of immunization.

In a paper, now in press, the writers have reported the results of experiments in which the active immunization of guinea pigs against the European typhus virus was accomplished, both with the use of formalinized tissue culture vaccines and by methods of sero-vaccination. While this work was going on, Kligler and Aschner 1 published observations on a method of successful animal immunization with formalinized tissue culture vaccine, similar in all important principles to the method which we are using.

Since the development of a tissue culture technique for producing vaccines in amounts adequate for practical purposes appeared the most hopeful direction of effort, we have tried for a long time to improve the technique of making such cultures, particularly in regard to increasing the volumes of the tissue cultures themselves.

In studying the general problem of tissue cultures, one of the writers (Zinsser) in collaboration with Schoenbach, turned his attention to observations on what may be called the physiology of tissue cultures in general, particularly as regards changes in pH, oxygen consumption and oxydation-reduction potentials.†

The observation by Roberts of an increase in serum phosphatase activity in patients with jaundice has been confirmed by a number of investigators. Similar results have been obtained in dogs with experimentally produced obstructive and nonobstructive hepatic jaundice 1 , 2 , 3 and elevated values have been reported in portal cirrhosis. 4 , 5 In view of the uniformity of the previously reported data, particularly in obstructive jaundice, and the importance of this uniformity in connection with any attempt to explain the pathogenesis of this phenomenon and its significance, the results of our observations upon cats with total bile stasis are of interest.

Determinations of serum bilirubin concentration and serum phosphatase activity were made in 8 normal cats and 30 adult cats at various stages of total bile stasis produced by ligation of the common bile duct. No case is included in which, at autopsy, there was evidence of the presence of any complication or any indication that stasis was not complete. The animals were maintained upon a diet of fresh raw scrap meat and milk. Serum bilirubin was determined by the method of van den Bergh and serum phosphatase by the method of Bodansky. 6

The findings are presented in Table I. The control values ranged from 0.95 to 3.84 units of phosphatase. Of 45 determinations made upon 30 cats with total stasis, 41 during stasis of 2–21 days' duration were within the limits of the control values. Higher values were obtained in only 4 instances, as follows: 11.03, 5.92, 5.8 and 4.6 units, with stasis of 16, 29, 21 and 8 days' duration, respectively. There was no consistent relationship between serum phosphatase activity and either the duration of stasis or the degree of bilirubinemia.

Studies with the Hamilton and Highman test 1 of parathyroid function have been performed in 6 patients with hyperthyroidism. The results compared with those obtained in normal subjects indicate increased circulating parathyroid hormone in the blood of 5 of the 6 thyrotoxic patients. This investigation is being continued to evaluate the significance of the findings.

Strophanthidin can induce emesis in cats after very extensive denervation of the liver, and also after dehepatization, thus confirming the results Hanzlik and Wood obtained with pigeons. How the vomiting is brought about in animals deprived of the liver, or its nerve supply, is a problem for further study.

In the majority of specimens of drainage bile obtained from biliary fistulae in the human patient, cholesterol is present only in the free form unless the bile is contaminated with blood. One case is reported of the occurrence of combined cholesterol in bile draining from a cholecystostomy.

A direct relationship exists between the molecular weight and the toxicity of the alcohols up to that with 4 carbon atoms: the relationship is inverse with their anhydrides.

The first 2 anhydrides are more toxic than their corresponding alcohols, while the anhydrides of the higher homologues are less toxic, or possess about the same degree of toxicity as their precursors.

Streptococcus hemolyticus whole-culture is capable of inhibiting the inflammatory fixation which is normally elicited by aleuronat in the skin of the rabbit.

It has been clearly demonstrated that the dissemination of streptococci, staphylococci, and Pseudomonas aeruginosus from an inflammatory focus in the skin to the regional lymphnodes and thence to the blood stream is largely governed by the efficiency of the fibrinous barrier which is responsible for what Menkin has termed “inflammatory fixation”. 1 , 2 , 3 It appears to be quite clear that the necrotizing property of Staphylococcus aureus is responsible for the characteristic localized nature of staphylococcal infections, by eliciting a prompt and intense inflammatory response. 3 Menkin 4 , 5 has suggested and maintained that Streptococcus hemolyticus paradoxically owes its characteristic invasiveness to a lack of irritating properties and consequent delay in eliciting an inflammatory response. The experimental data given by Dennis and Berberian 1 and the clearly defined results given above indicate that one need not resort to a paradoxical interpretation of the observed phenomena. If Streptococcus hemolyticus is capable of inhibiting the formation of an inflammatory barrier in the presence of an irritating-substance such as aleuronat, it seems not unreasonable to suppose that this ability to interfere with the normal inflammatory response may play an important rôle in the dissemination of streptococci from a portal of entry under natural conditions.

Inflammatory fixation is promptly induced in acute local streptococcus inflammation in the presence of streptococcus antiserum, in contrast with the marked delay in fixation in the presence of normal serum.

The experiment presented above has been repeated on several occasions. Five different strains of Streptococcus hemolyticus, and 4 different commercial brands of polyvalent so-called antitoxic-antibacterial streptococcus antisera have been used with essentially identical results.

The data given above suggest that the factor produced by Streptococcus hemolyticus which is responsible for the active suppression of the formation of the local inflammatory barrier is antigenic and is readily neutralized by streptococcus antiserum. A possible alternative explanation is that the inflammatory fixation is part of an Arthus phenomenon due to the introduction of a mixture of antigen and antibody into the skin. However, in view of the ability of the “inhibitory factor” to suppress fixation in other types of inflammatory response, this explanation would seem to be improbable, and attempts to test the hypothesis experimentally have given negative results.

Ward and Lyons 4 , 5 have shown that the virulence of hemolytic streptococci is ultimately dependent upon the resistance of the organism to phagocytosis. However, invasiveness (i. e., the ability of the organism to disseminate from the portal of entry) is a vitally important corollary of virulence. We have found that avirulent strains of streptococci may rapidly reach the blood stream following injection into the skin, but are quickly disposed of by the cellular defenses of the body; on the other hand the speed with which organisms which are resistant to phagocytosis kill the host may be determined by the ease with which they reach the blood stream. Menkin 6 has recently demonstrated that the early establishment of a nonspecific inflammatory barrier at the site of injection into the skin may protect rabbits against otherwise lethal doses of pneumococci known to be highly resistant to phagocytosis.

The hexahydric alcohol, sorbitol, has recently become available in quantities at a low price. It is non-toxic, has enormous water binding capacity, is rapidly excreted by the kidneys after intravenous injection and has 1.88 times the osmotic pressure of the same percentage sucrose solution. Its solutions are less viscous and more easily injected than those of sucrose and are entirely stable to heat sterilization. It apparently possesses all of the properties desirable in a physical diuretic.

A study of the diuretic action of a 50% sorbitol solution (prepared from the syrup supplied by Atlas Powder Company) as compared with that of a 50% sucrose solution has been made in 3 dogs. The dogs were placed under nembutal anesthesia and kymographic records made of arterial pressure, respiration, and urine flow from a catheter placed in the bladder. Urine volumes were measured. Fig. 1 shows the rates of urine excretion (volume per 15-minute period) obtained after intravenous injection of 50 ml. of 50% solutions of sucrose and sorbitol. The curves are numbered in the order of injection. When injected at the rate of 50 ml. in 2 minutes (38°C.) neither sucrose nor sorbitol caused appreciable changes in blood pressure or respiration. The curves indicate sorbitol to be a much more efficient diuretic in dogs than sucrose.

In the case of Dog 1, sorbitol was injected first (Curve 1), followed by sucrose (Curve 2), and then a second injection of sorbitol was given. No replacement of body fluids during the experiment excepting the water given with the solutions injected. Curve 3 shows that sorbitol had a much more powerful diuretic action than sucrose even after the dehydration produced by the previous injections.

The mechanism by which choline chloride influences the deposition of fat in the liver is by no means clear. Best, Channon and others have shown that this nitrogenous component of lecithin is capable of preventing or curing the fatty infiltration of the liver which is produced in the rat on a high fat diet or in the dog on a lean meat-sucrose diet. The finding that diets essentially free of choline cause an infiltration of fat into the livers of rats, a condition curable by the addition of choline chloride to the diets, led Best, et al., to suggest that choline may be an essential dietary factor.

The most obvious hypothesis as regards the manner in which choline produces its “lipotropic” effect would involve perhaps the formation of lecithins and other choline-containing phospholipids from fat, phosphate and ingested choline, thus favoring the transport of lipid materials. There are obstacles to the immediate acceptance of such a view, of possible significance among which is the lipotropic inactivity of aminoethanol, the nitrogenous constituent of the phospholipid cephalin. Of greater import is the finding (Best, et al.) that betaine, the naturally occurring acid corresponding to choline, is lipotropically active. This substance is incapable of entering into the formation of phospholipids unless it is first reduced to choline by the organism, a conversion which would be of considerable biochemical interest.

Pharmacological investigations of the phosphorus and arsenic analogues of choline and acetylcholine, 1 , 2 molecules in which arsenic replaces nitrogen as the nuclear element, suggested that the arsenic analogue of choline chloride (“arsenocholine” chloride) might prove useful in furnishing essentially a “tagged molecule” the assimilation of which by the organism might be followed.

The skin of the monkey (Macacus rhesus) is suitable for local passive sensitization to ragweed pollen extract by the intradermal injection of either serum from a sensitized guinea pig or serum from a human case of ragweed hay fever.

Hypertension produced in dogs by means of compression of the renal artery has not been shown by the method employed to be caused by liberation of pressor substances into the blood stream which act directly on the peripheral blood vessels to cause vaso-constriction. Nor is the known pressor secretion of the pituitary gland present in amounts large enough to be detected by the method employed, in the blood of animals with hypertension. These conclusions are based on the following evidence: 1. Alcoholic extracts of plasma of dogs with hypertension when injected into anesthetized cats raises the cat's arterial pressure no more effectively than extracts from plasma of normal animals. 2. Removal of the hypophysis in animals with experimental hypertension does not alter the character of the pressor response to extracts of plasma.

Although the use of an electric field to force drugs into the skin has been known for many decades, surprisingly few experiments have been performed with local anesthetics. That the unbroken skin can be anesthetized by the electrophoresis of drugs into the skin has been known for many years, but this method has not been developed in the light of the modern theory of electrolytes.

It is of interest both theoretically in connection with electrokinetic properties of the skin, and practically in hay fever, to determine if the anesthetization of a small area is without essential influence on the development of moderately or markedly positive skin reactions.

By the use of an electrode especially designed to anesthetize a large number of small areas of the skin simultaneously, it has been possible to make intradermal tests with ragweed, timothy, dust, and other inhalants without any discomfort to the patient. Anesthesia lasting sufficiently long to perform skin tests can be obtained after 10 to 15 minutes electrophoresis of (1) procaine base in alcoholic or acetone solution, (2) butyn base, (3) cocaine base, (4) procaine hydrochloride, of various concentrations. Some effect was obtained with procaine borate. Agar jellies of the foregoing anesthetics were also effective. Control tests of allergens without the anesthetic have shown with few exceptions only the variability in skin reactions usually found on comparing 2 different skin sites. In fact, because of the anesthesia, the cooperation of the patient is more than usual and the fine needle may be inserted with greater care, Of the anesthetics employed, procaine base containing a trace of alkali has given the most consistent results with current densities of approximately 0.5 milliampere.

Three cases of yellow atrophy of the liver have occurred in each of 3 widely separated plants and under different management within a year or two. In each instance where the illness appeared, the men were exposed to a chlorinated naphthalene heated above the melting point and giving off fumes. Yellow atrophy of the liver is a rare disease and there have been no cases reported where chlorinated naphthalene was the proven etiological agent.

The following experiments were conducted to determine if some of the chlorinated naphthalenes might produce such lesions. Three naphthalenes obtained from one of the above factories were used in this work. They are designated then as A, B, and C.

Compound A is a mixture of tri- and tetra-chlor-naphthalene. Compound B is a mixture of tetra- and penta-chlor-naphthalene and may contain some tri-chlor-naphthalene. Compound C is a mixture of penta- and hexa-chlor-naphthalene which is plasticized with a relatively small percentage of asphalt.

Groups of 5 rabbits weighing approximately 2 kilos were used in testing each substance. Thirty milligrams of the compound dissolved in 2 cc. of paraffin oil were injected subcutaneously each day until the rabbit died.

None of the animals receiving injections of “A” died. The first death in the group receiving injections of “B” died on the 12th day and all were dead by the end of the 15th day. In the group receiving “C” the first rabbit died at the end of the 12th day and the last of the group on the 26th day. None of the rabbits receiving injections of sublimate from “B” died.

Previous studies with the strictly autotrophic sulfur bacterium, Thiobacillus thioöxidans, growing on elemental sulfur have shown that the sulfur is rapidly oxidized to sulfate without the accumulation of intermediate products. 1 The strictly autotrophic sulfur bacterium Th. thioparus transforms thiosulfate to the 2 products, sulfate and elemental sulfur. 2 No question has arisen concerning the initial stages of transformation of thiosulfate, but, by reason of the relatively large size of the particles of elemental sulfur compared to the tiny bacterial cells (about 0.5 × 0.8μ) it has been suggested that some initial transformation to a reduced or oxidized substance may precede passage of the sulfur material through the cell membrane, after which the reactions leading to the release of energy for growth takes place. 3 However, McCallan and Wilcoxon present evidence which shows that the vapor pressure of elemental sulfur is sufficiently high to permit sulfur vapor to enter cells which are not even in contact with the solid sulfur. 4

Inorganic media containing sulfur or thiosulfate and supporting growth of these 2 bacteria have been examined for the presence of sulfide. No sulfide or other reducing substance was detected in cultures of Th. thioöxidans by titration with 0.01N iodine. No substance formed from thiosulfate by Th. thioparus was found by titration with iodine. Tests for sulfide with nitroprusside were negative in both cases. The results do not support the contention of von Deines that the sulfur material precipitated by Th. thioparus during growth on thiosulfate is a highly sulfured polysulfide. 5

Although sulfide does not appear in detectable amounts in the media, sulfide is produced by both organisms during growth.

A number of observers have found that extracts from the kidneys of various animals may have pressor effects. 1 – 7 The object of the present investigation was to determine whether extracts of kidneys from human beings and dogs with hypertension had greater pressor effects than those of control kidneys.

Kidneys were obtained at autopsy from 15 patients with hypertension and from 17 control subjects. The hypertensive group consisted of 9 patients with benign hypertension, 2 with malignant hypertension, 2 with chronic glomerular nephritis, and 2 with pyelonephritis and secondary contracted kidneys. The material was put through a meat grinder, crushed in a mortar, shaken for 30 minutes with saline (one cc. per gm.), filtered and kept on ice. The tests were most satisfactorily performed on an unanesthetized dog with a Van Leersum (carotid) loop. 8 In several instances records were obtained by direct cannulation of the carotid artery of anesthetized dogs and cats. Injections were made intravenously, using the equivlent of 7 gm. of tissue for the unanesthetized dog and smaller doses for the anesthetized animals.

A single injection usually resulted in a transient depressor effect followed by a gradual and prolonged rise which lasted from 10 minutes to over 3 hours. The average rise in blood pressure in the hypertensive group was 28 mm. of mercury, the range being 9 to 60 mm. of mercury. The average pressor effect in the non-hypertensive series was 12 mm. of mercury, the range being 0 to 48 mm. of mercury.

The pressor action in general was more prolonged in the hypertensive than in the non-hypertensive group. No striking differences in the preliminary depressor effects were observed.

It has been shown recently 1 , 2 that many cases of lymphogranuloma inguinale develop hyperproteinemia, comparable in degree, incidence and fractional distribution to that long known to occur in kala-azar. This observation, it has been pointed out, affords an explanation for certain peculiarities of the serum in lymphogranuloma inguinale—peculiarities which appear to be common to most sera with definite hyperglobulinemia, irrespective of etiology: 1. increased erythrocytic sedimentation rate 1 ; 2. anticomplementary properties of the serum in a significant proportion of cases 3 ; 3. apparent discrepancy in acid-base equivalence of the blood in that the sum of the determined acid-equivalents appears to exceed the total base. 2 A further property of the serum in lymphogranuloma inguinale, likewise ascribable to hyperglobulinemia, is described in the present study. It was found that the formol-gel test, used extensively for the diagnosis of kala-azar, is positive in cases of lymphogranuloma inguinale presenting hyperproteinemia.

The total protein of the serum was determined by difference on 1 cc. samples, the macro-Kjeldahl technic being employed for total N and Folin's method with Nesslerization for non-protein N. Partitions were carried out in duplicate on 0.5. cc. samples by Howe's method. The formol-gel test was performed as follows: to 1 cc. of serum in a test tube (8 mm. bore is a convenient size) add 2 drops of 30–40% formaldehyde solution, mix, and allow to stand at room temperature. Normal control sera showed no change in viscosity or transparency in 24 hours.

Results. The formol-gel test was carried out on the sera of 13 cases of lymphogranuloma inguinale, all Negresses with positive Frei reactions, of whom 12 were under treatment for rectal stricture.

Experimental production of exophthalmos has been accomplished repeatedly by stimulating the sympathetic innervation of the eye. MacCallum and Cornell, 1 Code 2 produced, by electrical stimulation of the cervical sympathetic ganglion of dogs and cats, a contraction of the smooth muscle elements in the orbit which caused exophthalmos. Schockaert, 3 Loeb, 4 Friedgood, 5 and Marine 6 , 7 have reported exophthalmos resulting from the injection of thyrotropic anterior pituitary extracts into ducks, normal and thyroidectomized guinea pigs, and by the injection of methylcyanide into normal and thyroidectomized rabbits. Loeb and Schockaert noted that the exophthalmos produced was functional, i. e., did not persist during anesthesia or after death. Marine showed that the protrusion resulting from injection of either pituitary or cyanide was accomplished through a nervous mechanism, presumably upon the unstriated orbital muscle of Müller, and that it could be prevented by removing the cervical sympathetic ganglion.

In the following experiment exophthalmos has been produced which does not depend upon a nervous mechanism but is due to an increase in orbital structures. It is readily obtained by the injection of beef anterior lobe extracts in thyroidectomized, but not normal, guinea pigs of both sexes. Twenty-six guinea pigs were injected with a thyreotropic anterior pituitary extract for 3 to 9 weeks with a daily dose equivalent of 250–2000 mg. of acetone dried beef anterior lobe powder. Loss in body weight and intense stimulation of the thyroid, as indicated by high epithelium, almost complete loss of colloid, and gross enlargement of 6–7 times, occurred. Of these only 3 showed slight indications of exophthalmos. Besides these questionable cases the eyes remained normal or became slightly enophthalmic.

Depression of the blood inorganic phosphates after the administration of dextrose has been demonstrated by Perlzweig, Latham, and Keefer. 1 This observation has been confirmed by Sokhey and Allen, 2 Harrop and Benedict, 3 , 4 and Bolliger and Hartman 5 with both humans and animals receiving dextrose orally and parenterally. Although this fall in blood inorganic phosphates after the administration of dextrose appears to be the rule, it does not occur invariably. Blatherwick, Bell, and Hill 6 in a series of experiments in which dextrose was given orally to human subjects, found no consistent change in the phosphate level and state that there is “sometimes an increase, or a decrease or no change at all.”

Insulin, on the other hand, has been found by Wigglesworth, Woodrow, Smith and Winter 7 to produce a marked drop in the plasma inorganic phosphate level of the rabbit. Cori and Cori, 8 Perlzweig, Latham and Keefer, 1 and Vollmer 9 have reported similar results, and further, have demonstrated that injections of epinephrin produce the same effect. Cori 10 and Cori and Cori 11 in seeking an explanation for these changes found a marked increase in the hexose-phosphate content of the skeletal musculature of the normal animal after the injection of either insulin or epinephrin. No change in blood or muscle phosphates could be demonstrated in the adrenal-ectomized animal, and they concluded that the depression of the blood inorganic phosphate level following: the injection of insulin was the result of an increase in the secretion of epinephrin called forth by hypoglycemia. Ellsworth and Weinstein 12 found varying degrees of change in the blood inorganic phosphates in adrenalectomized dogs after the injection of insulin.

Experimental data is presented confirming the work of Farmer and Abt that the reduced ascorbic acid content of the blood plasma in the “normal” individual parallels the vitamin C intake. Confirmation of this is presented in a controlled dietary experiment using rhesus monkeys. For satisfactory comparative data, determinations should be made on fasting blood specimens. On the basis of the studies recorded, we believe that fasting plasma ascorbic acid levels below 0.7 mg. % are probably sub-optimal. Levels langing between 0.7 and 0.9 mg. % would appear adequate. Optimal levels probably lie above this range. Reduced ascorbic acid plasma levels below 0.5 mg. % must be considered low.†

In biometric studies of rats, it is desirable at times to compare simultaneously and graphically (1) Various organs by weight with (2) age and (3) body weight of the animal. By ordinary two dimension graphs, relationships between any 2 of these data can be well illustrated, but only by “three dimension” graphs is it possible to illustrate simultaneously the interrelationship of all 3–age, body-weight and weight of any given organ.

The construction of such graphs is simple (Chart 1) : Ordinate AB forms with abscissae BC and BD an angle of 120°. AB represents age, the only constant uniform with all rats; BC body-weight and BD gland-weight. The “age-weight” is easily plotted, the line B₁C₁ paralleling BC and forming an angle of 120° (AB₁C₁). Gland-weight is plotted from B₁ (corresponding to age) on line AB to D₁ (representing gland-weight), which point must be on a line perpendicular to AB projected from C₁ (corresponding to body-weight).

For normal rats all lines representing “age-body-weight” (B₁C₁) will parallel abscissa BC and form angles of 120° with ordinate AB, differing only in length. The lines representing “age-gland-weight” (B₁D₁) will vary in length and will parallel BD only when the increase in weight of the gland, with respect to age is directly proportional to the increase in body weight. If the gland is small for the age, angle D₂B₁A will be greater than 120° and line D₂B₁ short. If the gland is large, then line D₃B₁ will be longer and angle D₃B₁A more acute.

By ordinary two-dimension graphs, relationship between age and body weight in rats is well illustrated as well as relationship between various organs and age or body weight. Only by three dimension graphs however is it possible to illustrate the interrelation of age and body weight and also the weight of any given organ. 1 Such graphs have been particularly helpful in evaluating the influence of thymus extract on the various endocrine organs of the rat, whose general body growth is so much more rapid than normal. Thus it affords comparison with age-weight controls, in contrast to controls of comparable age but lower body weight or comparable weight and greater age. Eight thymus-injected rats exhibiting average precocity are used to illustrate the usefulness of these graphs.

Thymus-treated rats appear grossly normal, differing mainly in their size with respect to age. Therefore the “age-weight” graph of the injected rats is not parallel to the normal (Chart 1), nor is the “age-gland-weight,” since the thymus gland in thymus-treated rats is consistently heavier than normal. This increase in size is apparently due, on histologic examination, to the cortex being slightly wider than normal and less sharply demarcated from the medulla, both being crowded with lymphocytes. Hassall's corpuscles are less easily distinguished, seem fewer and are composed of fewer reticular cells than normal controls.

We gain the impression, through sacrificing rats and weighing the adrenals, that they tend to be below normal; plotted three dimensionally however (Chart 2) this does not seem to hold, nor was any deviation from normal observed histologically in the ordinary stains. (The graph for normal adrenals is the average between males and females for the plotted age-weight).

Warburg has devised the methods which make it possible to examine the respiration of isolated cells at a constantly maintained equilibrium between the cell and the gas medium and to measure quantitatively the influence of various oxygen tensions upon respiration in such a way as to eliminate diffusion as a limiting factor. 1 By these methods Warburg has determined the effect of low oxygen tensions upon the respiration of bacteria and isolated animal cells. He found the rate of oxidation of the nucleated red blood cells of geese, which he examined at 0°C., to be unchanged at oxygen pressures varying between 5 and 75 mm. Hg. (=0.7 and 10 vol. % O₂). 2 In micrococcus candicans, at 1°C. the rate of respiration even at an oxygen tension of 10^-5 atm. (=0.0076 mm. Hg., =0.001 vol. % O₂) was the same as in air. 3 Also the respiration of non-nucleated red blood cells of rabbits poisoned by phenylhydrazin, examined at 38°C. showed no dependence upon oxygen tension. 4 Since these investigations it has been a generally accepted axiom of physiology that cell respiration is independent of variations of oxygen tension. 5

The only exceptions seemed to be the nitrogen-fixing bacteria and pneumococcus which were both examined with the Warburg methods under conditions of optimal gas diffusion. The relative rate of respiration of azotobacter at 2 vol. % O₂ and in air was 1 :2 (Meyerhof 6 ) and that of pneumococcus at 2 vol. % O₂ and in air was even 1:4.5 (Schlayer 7 ).

Daily injection of crystalline vitamin C increased the tolerance of tuberculous guinea pigs to repeated large doses of tuberculin.

Ether anesthesia causes profound constriction of the spleen and usually produces a marked increase in the erythrocyte count, the value for the hemoglobin and the hematocrit value while sodium amytal causes the spleen to dilate markedly, which removes a considerable percentage of erythrocytes from the circulation. Our results are in full agreement with those of Searles and Essex.

Crude or crystalline trypsin in proper concentration coagulates human, dog, rabbit, guinea pig and horse plasma or whole blood. It does not clot fibrinogen directly, but reacts with prothrombin to form thrombin. This activation is independent of the presence of either calcium or platelets. It follows that neither of these is necessarily an intrinsic part of thrombin. Trypsin also coagulates blood in vivo, which suggests that circulating blood contains free and reactive prothrombin, rather than an inactive hypothetical complex from which prothrombin is liberated only after blood is shed.

An excess of trypsin digests fibrinogen, prothrombin and thrombin; and too little trypsin has no effect. The activation of prothrombin therefore takes place within a relatively narrow zone of enzyme concentration, which varies directly with the amount of protein present. At this optimum zone, there is a dynamic equilibrium between the activation of prothrombin to thrombin and the destruction of both reagents by the enzyme.

Since trypsin, in activating prothrombin, has qualitatively the same effect as the physiological system calcium plus platelets (or calcium plus tissue extracts), it is tentatively suggested that these systems contain a proteolytic enzyme similar to trypsin which reacts with prothrombin to form thrombin.

It has been further found that the proteolytic enzyme papain, freed of calcium, nevertheless coagulates whole blood, plasma, or fibrinogen. In this case, the enzyme does not activate prothrombin, but acts directly on fibrinogen to form a soft fibrillar gel resembling fibrin, with a marked tendency to resolution. If one admits this clot to be fibrin, this constitutes strong evidence that the physiological coagulant thrombin is also a proteolytic enzyme with a specific reactivity for fibrinogen.

During a study of peptic ulcers produced by the administration of cinchophen we had the opportunity of studying the effects of large amounts of cinchophen on the liver and other organs of the dog. In view of the divergence of opinions regarding the effects of cinchophen on the livers of experimental animals, it seems advisable to record our observations on this subject.

Specimens of liver were obtained from 131 dogs which were normal in every case before the experiment was begun. They were given the routine kennel care and were fed a balanced diet of a calculated weighed amount sufficient for their caloric requirements. Cinchophen was for the most part administered orally; in a few cases it was given rectally, parenterally, or through intestinal fistulas.

Thirty-one dogs, whose average weight was 17 kg., were daily given 2 gm. of cinchophen well mixed with their food for a period varying from 3 to 60 days. The average length of time over which administration of the drug to this group was continued was 27 days; the average total dose of cinchophen was 50 gm.

Fifty-four dogs were given 2 gm. of cinchophen daily, with an occasional rest day, for an average of 30 days but in a few cases over a period of time as long as 114 days. To this group the amount of cinchophen administered varied from 36 to 228 gm. during the course of treatment.

Thirteen dogs, whose average weight was 13 kg., were given varying doses of cinchophen by routes other than by mouth, the usual dose exceeding 1 gm. daily. To members of this group an average of 21 gm. of the drug was given in an average of 13 days.

In the present investigation we have attempted to determine changes in the blood electrolytes following the production of hepatic parenchymal damage with arsphenamine. The studies included determinations of the blood non-protein-nitrogen, urea, sugar, CO₂ content, sodium, chloride, potassium calcium, phosphorus, magnesium, total proteins, and lactic acid, as well as complete hematologic studies.

Liver damage was produced in 6 dogs by injection of arsphenamine intravenously. Animals were kept on a diet of raw meat to which was added 2 gm. of salt daily for 2 weeks before the experiment was started. Arterial Wood for determination of various electrolytes was collected anaerobically before injection of arsphenamine. Forty to 80 mg. per kilo of body weight of freshly prepared arsphenamine was then injected intravenously. About 30 minutes later this was followed by vomiting which lasted for a few minutes before subsiding entirely. In 2 animals, a single injection of the drug was adequate to produce icterus. Four of the 6 animals required subsequent injections of arsphenamine to produce jaundice. Death followed in all animals about 20 to 24 hours after the last injection of the drug. Arterial blood samples for electrolytes were again collected when the animal appeared definitely icteric. Six to 8 hours after appearance of jaundice the dogs became comatose and died. At no time were there convulsive seizures.

Upon death a necropsy was performed and sections of liver, kidneys, and intestines were removed for microscopic study. In 2 instances considerable congestion of the small intestine and rectum was found. The kidneys appeared grossly normal in all instances. The livers felt firm, but had a distinctly mottled appearance. The lobules were well outlined and were surrounded by punctate hemorrhages.

When voluntary muscles are put under tension against resistance through conscious effort, and then allowed to relax with the resistance removed, there follows an involuntary contraction in the original direction. Thus, as has long been known, if one stands against a wall, pushing the hand strongly against it, with the arm held stiff, on stepping away from the wall, the arm slowly rises toward a horizontal position. This phenomenon, which may be called after-contraction, has been studied by Kohnstamm, Csiky and others, who have given various explanations of its mechanism, none of which, however, seems adequate. In view of the more recent advances in the physiology of the nervous system, it seemed to us that further study of the after-contraction would be of value. The present report deals with some aspects of the reaction.

The after-contraction may be obtained from any voluntary muscle group, and in these experiments the movement studied was flexion at the hip joint, the leg being extended. The subject stood on a platform, supporting his weight on one foot. To the other foot, hanging free, was attached a cord, passing over a pulley and carrying a fixed weight. Also attached to this foot was a string connected with a system of levers arranged to record the leg movement on a kymograph. At a given signal, the subject lifted the weight off its support by raising the extremity, and held it in a fixed position for a definite period. Then, at a second signal, the leg was relaxed and the weight disconnected. After a variable latent period the leg slowly rises, without conscious effort on the part of the subject and, indeed, somewhat to his astonishment.

1. Twenty-four-hour cultures of B. typhi, B. coli, Staph. albus, B. subtilis on agar slants and in beef tea were immersed in liquid air. In addition, strips of sterile filter paper that had been impregnated with the above cultures were also placed in liquid air. At the end of one week, the cultures and the filter paper were removed from the liquid air. They were then transferred to new media. After incubation all tubes showed growth.

2. A suspension of a 24-hour culture of B. typhi was made in physiologic saline solution and standardized to one million bacteria per cc. by cytometric count. Five cc. of the suspension were placed in sterile test tubes 150×13 mm. The tubes were sealed in a blast-lamp and then placed in liquid air. Daily, for a period of 10 days, a tube was removed and melted and the number of survivors determined by the McCrady most-probable-number method, using 3 tubes of beef tea for each dilution. There was a drop from one million to 10,000 bacteria per cc. at the end of one day. The succeeding days gave the same count of approximately 10,000 per cc.

3. The technic was the same as in 2. At monthly intervals a tube was removed and the number of survivors was determined by the most-probable-number method, and by the plate-count. This was continued for a period of 13 months and further counts were made after 16 and 19 months.

The plate-counts and the most-probable-number sometimes agreed and at other times disagreed widely. There was shown no consistent decrease in bacterial numbers over the period of 19 months.

Though Monilia albicans and Monilia Candida have so far been shown to give the same serological reactions, 1 nevertheless in disagreement with Stone and Garrod's conclusion, 2 we consider 3 , 4 , 5 , 6 that there are sufficient cultural, biochemical, and pathogenic differences to justify placing the organisms in 2 different species. A prominent difference is the coagulation of milk by M. albicans in striking contrast to M. candida's inability. The purpose of this study was to determine the conditions under which coagulation occurred and to see if in all cases a constant, definite difference in the species was observable. In addition, another objective was to demonstrate that the rennet-like coagulation of the albicans type is probably distinct from the adherent mat or mycelial pad formation of M. candida which produces a pseudo-coagulative effect.

In the work with coagulation the procedure for the preparation of the calcium-lactate milk medium is that formerly described. 3

While it was found that coagulation could readily be accomplished through the use of any of the M. albicans cultures themselves yet the utilization of an extract from that species would permit a better comparison of clotting results with commercial rennin. Now in order to perform experiments with extracting agents it was necessary to obtain a heavy cell yield, and for this purpose large Blake bottles containing Trommer's malt-extract agar were used. After several extracting agents had been employed without marked success, sterile distilled water in 20 to 25 cc. portions per Blake bottle was used to wash down the growth. The suspension, kept at room temperature, was shaken at intervals and centrifuged at the end of a week.

It has been shown in previous papers from, this laboratory that the respiratory rate of yeast suspended in non-nutrient phosphate buffer solutions or in glucose phosphate can be markedly increased on treatment with proper concentrations of 2–4 dinitrophenol (DNP), 4–6 dinitro-o-cresol (DOC) and some related compounds. 1 – 5 We report herewith the results of experiments in which it is shown that the effects of such metabolic stimulants upon the respiration of yeast is profoundly affected by the type of “exogenous” 6 carbohydrate fuel available.

A pure culture of Saccharomyces cerevisiae and pure culture methods were used throughout. The experimental procedures were the same as described previously 2 , 3 except that yeast was first grown 48 hours on Orla-Jensen agar medium, then taken up in 0.2M phosphate buffer, pH 6.8, and starved 24 hours under aerobic conditions in Fraser tubes 7 at 25° ± 0.02°C. before the beginning of each experiment. Respiration was measured in the conventional form of Warburg apparatus at 25° ± 0.02°C. Each set of quantitative comparisons was made on yeast from a single subculture. The observations reported represent typical findings in a series of 8 experiments involving 7 Warburg vessels each.

Fig. 1 illustrates integral curves showing the total amount of oxygen consumed in cmm. at N.P.T. as a function of time when previously starved yeast is suspended in 0.1M phosphate buffer, pH 6.8, or in the same buffer containing 1% glucose (for comparison), 1% d'-arabinose (levo-rotatory) or 1% l'-arabinose (dextro-rotatory).

In this experiment DOC was added from the sidearms of the Warburg vessels after a 60-minute control period. The strength of the DOC was such as to furnish a concentration in the Warburg vessel of 5.05 × 10⁻⁴M (giving 7.96 × 10⁻⁶M free acid at pH 6.8), which was found optimal in a series of preliminary experiments. After addition of DOC, the slope of the integral curves in the presence of glucose (not shown in Fig. 1, but can be inferred from Fig. 2) and of 1'-arabinose is greater, in the presence of d'-arabinose the same, as in the non-nutrient control. Since the integral curves for the two pentoses practically coincide before addition of DOC and draw apart afterward (Fig. l), this finding constitutes an interesting case of stereoisomeric preference on the part of the yeast cell.

The relation of the vitamin C intake and the urinary excretion of the vitamin has been recently studied by a number of observers. 1 – 4 But little could be found in the literature regarding the effect of the diet on the vitamin C content of human milk. It is generally agreed that human milk is richer in the antiscorbutic vitamin than cow's milk and that the clinical manifestation of scurvy is very rare in breast-fed infants. In the case of cow's milk its vitamin C content is said to remain quite constant in different seasons despite the changes in the diet of the cow. 1 One may ask whether the human milk constantly provides an optimal amount of vitamin C regardless of the dietary supply of the same.

The present report is based upon the result of simultaneous determinations of vitamin C in the milk and urine of 2 lactating mothers for 26 and 27 consecutive days respectively. Their diet was adequate in the caloric intake but very low in vitamin C and consisted of food articles which were usually taken in the Chinese families of lower class. The diet was kept constant throughout except for a short period beginning on the fifth day of the study in which large doses of additional vitamin C substance were given. In case 1, the additional vitamin C consisted of 1000 cc. of canned orange juice (“Absopure”) daily for 12 days; while in case 2, 600 mg. of ascorbic acid crystal (Schering-Kahlbaum) were given daily for 8 days. A total amount of 3500 cc. of fluids was allowed daily in each case.

It was found that a sudden increase in the urinary excretion of vitamin C did not happen until the large dose of vitamin C substance had been given for 6 days in case 1 and for 3 days in case 2, showing that the storage of vitamin C in the tissue of these mothers was very low at the beginning of the experiment. Once the peak was reached the daily urinary output of the vitamin C tended to be fairly constant which amounted to about 60% of the intake. The excretion dropped abruptly with the discontinuance of the added vitamin in the diet.

Daily dosage of white rats with acetylsalicylic acid for periods as long as 29 weeks, at high levels, produces no visible ill effects, and does not change the blood picture, except that a pronounced rise of white cell counts occurs on the second day after withdrawal of the drug from animals accustomed to it. It is obvious that the white rat is not suitable for testing the question of the possibility of ill effects in human beings from prolonged dosage with this drug.

In the urine and stool of normal individuals there is a small amount of coproporphyrin I present. In many pathological conditions, however, the amount excreted is considerably increased. 1 , 4 and in some conditions, notably lead poisoning, coproporphyrin III is excreted rather than the normal coproporphyrin I. Although it is probable that both types of porphyrins are produced in various diseases, the predominance of one type has made the isolation of both types difficult. In the case described below both coproporphyrin I and coproporphyrin III could be isolated from the feces of a woman suffering from chronic porphyria. Thus, the simultaneous formation of these 2 isomers is established.

The patient in whom this condition occurred was found in the psychiatric ward of the Strong Memorial Hospital. Since exogenous causes, such as lead and drugs, could be excluded, the condition is one of chronic porphyria. The patient was not anemic, did not suffer from abdominal colic and was not photosensitive; there was no evidence of abdominal bleeding. The patient was placed on a meat-free diet and the stool and urine collected. Because of the mental condition of the patient, collections were difficult to make.

The stool contained relatively large amounts of coproporphyrin, small amounts of a coproporphyrin ester and deuteroporphyrin and somewhat larger amounts of protoporphyrin. Only in the case of the coproporphyrin fraction was sufficient material present for identification. The porphyrin was methylated to the methyl ester and the product recrystallized from chloroform-methyl alcohol. After 3 recrystallizations crystals nearly uniform in appearance separated rapidly from the solvent. These melted indefinitely at 192°C. Further recrystallization raised the melting point to 194°C. and finally to 210–212°C.

An active adrenal cortex extract has been found to increase the ketosis of fasting female rats when administered in large doses. The ketosis of fasting male rats is only slightly increased. Adrenal cortex extract abolishes the partial oxidation of B-hydroxybutyric acid when fed to fasting rats.

Allantoin stimulates slightly but not to a significant degree the growth of fibroblasts from cardiac explants in tissue culture.

1. Cevitamic acid, properly buffered, may be administered intramuscularly without discomfort or damage to tissue. 2. Studies of the urinary excretion and the blood content of vitamin C following the administration of cevitamic acid intramuscularly demonstrated that it is used by the body when administered by this route. 3. The height of increase in the blood level following the intramuscular injection of cevitamic acid is reached more slowly and is maintained for a longer time than after intravenous injections. 4. In cases of vitamin C deficiency, where cevitamic acid is improperly absorbed through the gastro-intestinal tract, or where the intravenous mode of administration is not feasible, the intramuscular route may be used.

As the metabolic balance studies on the “so-called normal” adults 1 , 2 and 22 children 3 , 4 as well as the growth observations on 530 infants 5 have progressed in this laboratory over several years we have grown more and more impressed with the wide physiological variations found. These fluctuations are evident not only among individuals of the same age, body size and living under the same environmental conditions but of the same individual when kept under constant conditions and observed continuously over, not days, but weeks. Obviously, it becomes necessary to build up some knowledge as to how much variation one may expect to find in a healthy individual living under highly standardized conditions before it is possible to interpret the degree of differences of results found under specific experimental conditions or in disease. Moreover, in the case of children, it is exceedingly important to build up some concept of the changes that occur incident to growth and development alone, before one is able to interpret satisfactorily the significant effect of specific food or other amenable factors on the metabolism of the child.

To illustrate not only the degree of variability that may occur among healthy individuals of like age and body size but also the increments and decrements that occur in metabolism from time to time as the observations progress the present report records the calcium balances for only 25 to 65 consecutive days of 6 of the 22∗ typically healthy children who were maintained on simple diets of 70 to 100 calories per kilo of body weight per day appropriate for the age and size of the child and approximately uniform in mineral and nitrogen content. The dietary furnished one gram of calcium per day for each child.

This work was originally begun with the idea of determining, if possible, the nature of the “inhibiting substance” which Collip et al., 1 – 5 have described and designated as “antihormones.” The controversy which has arisen involves the question as to whether the action of the “antihormone” may not in reality be due to an antibody developed against the protein, which is always found in hormone preparations derived from human or animal source. It is our belief that the gonadotropic inhibiting substance is separate and distinct from the antiprotem and both are present in the antiserums obtained from rabbits which have been persistently injected with large doses of pituitary extract or P.U.∗ hormone.

One male and 3 female adult rabbits were injected over a period of 3 weeks, receiving 5 cc. of pituitary extract (E. R. Squibb and Sons) daily, totaling 900 growth units each. The antiserums, inactivated at 56° for 1/2 hour, gave a precipitin reaction with pituitary extract. On testing the pituitary extract for gonadotropic hormone, we found that 1/6 cc. injected intravenously into a female† rabbit gave corpora lutea. With the addition of 1 cc. of the antiserum to 1/3 cc. of pituitary extract, a heavy precipitate formed. The supernatant along with the precipitate was injected intravenously into an immature female rabbit. Examination of the ovaries 48 hours later showed no stimulation. Similar results were obtained with the other 3 antiserums.

We attempted to remove the anti-bovine protein present in our pituitary antiserums by treatment with normal bovine serum. Two cc. of bovine serum (1–100) incubated 2 hours with 2 cc. of pituitary antiserum gave a heavy precipitate which was centrifuged out.

The author wishes to present a brief report concerning a peculiar group of symptoms, in many ways suggestive of catalepsy, which has been observed in 8 cats following a one-stage operation in which both frontal lobes of the cerebral cortex were removed. These operations were done with every precaution to secure good hemostasis and prevent hemorrhage from complicating the interpretation of the subsequent observations. Careful autopsies revealed that in no instance was there any sign of hemorrhage at the base of the brain or of blood in the ventricles. The observations on animals (cats) were made on relatively acute preparations. Cats 5, 6, 7, 8, 9, 10, 11, and 12 survived for 28, 3, 19, 2, 8, 13, 6 and 15 days, respectively. Animal 9 died after a laboratory accident; animal 6 of pneumonia, and animal 8 of an infection contracted post-operatively. All others were sacrificed.

These animals all showed a remarkable plasticity of the muscles of the limbs and back. Such plastic tonus was especially well observed if the animals were placed in a supine position in a wooden trough-like structure. The limbs of these animals were usually rigidly extended when in this position. If they were passively flexed by the observer, there was considerable resistance, and the limbs remained flexed for exceptionally long periods of time, hours in many instances. When passively extended, there was likewise considerable resistance, and the limbs remained extended for very long periods. Furthermore, these animals made little or no effort to escape when in the supine position, but would lie quietly, breathing regularly. They were neither moribund or comatose. Moving objects placed before the eyes elicited a following movement of the eyes.

It was demonstrated recently 1 that ovarian ablation in the rat during the last third of pregnancy need not interrupt this condition, providing the litter being carried has been surgically reduced to one fetus and that the placentae of the fetuses removed have been allowed to remain intact. Under these circumstances, however, the animals fail to deliver and the young, to be obtained alive, must be recovered by operative procedure; retention in utero much beyond the normal time of parturition serves progressively to compromise their viability. It seems improbable that failure of birth is due to the manipulations involved since control animals, receiving similar treatment save for retention of one ovary, will litter at the usual time. In failure of the birth mechanism the rat appears to differ from other species reported to carry to term following upon oophorectomy; the guinea pig, for example, spayed during the latter half of pregnancy, frequently continues to term and to normal delivery; 2 substantially the same situation prevails for the mare 3 and, as is well known, for the human being. 4

The marked rigidity and contraction of the uterus in cases of uninterrupted pregnancy suggest either that it has become insensitive to, or that it lacks, the stimuli involved in the processes of labor. Since oestrin serves as a powerful, and in certain respects a specific, sensitizer of uterine tissue 5 it seemed probable, in the absence of ovaries, that deficiency in this principle might be an underlying factor in failure of birth. The present communication is concerned with the results of attempts at testing this possibility.

Lead was suggested as an etiological factor in multiple sclerosis by Putnam. 1 Cone, et al., 2 examined a series of 40 spinal fluids for lead, using the qualitative hexanitrite test of Fairhall. 3 In their series were 8 patients with multiple sclerosis. In 6 of these the tests for lead were positive. Rabinowitch, et al., 4 about the same time reported the results of tests of 50 spinal fluids, including those from 27 cases of multiple sclerosis. These authors used the Fairhall hexanitrite method and checked their results by spectographic determinations. These authors found positive tests for lead in only 2 of their 27 cases of multiple sclerosis; in 2 additional cases they obtained positive tests after administering ammonium chloride to their patients. Boshes 5 tested 28 specimens of spinal fluid for lead, by the same method. Sixteen of his patients had multiple sclerosis; in only one of these did the spinal fluid yield a positive test for lead. All the above authors found occasional positive tests for lead among their control cases.

Because the above findings depended on a qualitative test that is difficult to control, we believed it would be worth while to investigate the lead content of a series of spinal fluids, using a precise quantitative micro-chemical method. Such a method has been devised by Wilkins, et al. 6 This method involves the titrimetric extraction of lead with a standard solution of diphenylthiocarbazone in the presence of ammoniacal cyanide. The original method was designed for use with blood; we have adapted it to spinal fluids in the manner described below.

Lumbar puncture was done with a dry, sterilized needle, and the fluid allowed to drop directly into a lead-free Pyrex flask. A measured volume of fluid, varying from 25–50 cc. was transferred to a Pyrex evaporating dish, and evaporated to dryness on a steam bath. The Pyrex dish was then transferred to an electric muffle furnace and ashed for 16 hours at 450°C. The ashed sample was dissolved in warm 5% nitric acid and transferred to a Pyrex separatory funnel, in which the titrimetric extraction was carried out. Simultaneous blanks were run with each set of determinations.

It has been shown 1 that vitamin G as needed by the rat contains at least 2 major components: lactoflavin and vitamin B₆, the lack of the latter being associated with specific skin lesions (“rat pellagra” or, rather, “rat acrodynia” 2 ).

By feeding to rats a vitamin B-free diet complemented by vitamin B proper, the antineuritic factor (B₁), and lactoflavin, or by vitamin B₁ and a vitamin B₆ concentrate, it was possible to obtain quantitative data^{2, 3} concerning the distribution of lactoflavin and vitamin B₆ in different foodstuffs. For the estimation of the lactoflavin values, the basal vitamin B-free diet was supplemented by crystalline (or highly purified) vitamin B₁ (3 international units daily) plus vitamin B₆ concentrate. For the estimation of vitamin B₆ values, vitamin B₁ was similarly provided but supplemented by pure lactoflavin (10 γ daily). The vitamin B₆ concentrate consisted of a yeast preparation such as may be obtained according to the method of Peters, et al., 4 by adsorption of a yeast extract on charcoal and subsequent elution with alcohol containing hydrochloric acid. It has been found that 1 cc. of this so-called Peters' eluate is equivalent to about 1 “rat-day dose” of vitamin B₆.

The values for lactoflavin were determined by means of the growth test, skin symptoms being too irregular and frequently insufficiently obvious. The “rat-day dose,” taken as provisional unit, was defined as the minimum quantity of the substance which would give rise to a gain in weight of about 10 (9 to 11) gm. per week for at least 4 weeks.

The values for vitamin B₆ were based directly on the curative effect against dermatitis.

The growth-promoting effect of lactoflavin or of lactoflavin-5-phosphoric acid is independent of the way in which it is administered, that is, whether orally or parenterally. There are no biological differences, as shown by growth tests, between lactoflavin and lactoflavin-5-phosphoric acid, the rat-day dose for both being about 7 to 10 γ. It can therefore safely be assumed that the phosphorylation of lactoflavin is not only an intestinal but also a general cellular reaction.

Experimental rickets was produced according to the technique of Steenbock and Black, 1 with the modification of our paired feeding technique, so as to eliminate the influence of the plane of nutrition. 2 The animals were 29 to 55 days of age, and weighed 50 to 58 gm. at the beginning of the experiments. Since the ricketic type of diet employed allows only very small increases of weight, the size of the animals at the time of sacrificing yielded insufficient amount of blood for all the blood serum enzyme determinations; hence, some groups were taken for blood serum amylase and esterase, and others for blood serum phosphatase. Of the total numbers of groups studied, 24 showed by the line tests severe experimental rickets; 4, advanced; 8, moderate; and 2, mild.

There was a total of 658 titrations carried out in this investigation in duplicate, the results of which are summarized in Table I.

It will be noted that no noteworthy changes are apparent in concentration of blood and tissue enzymes in rickets developed in the albino rat compared with enzyme concentrations on the same diet supplemented with vitamin D supplied by irradiation of the ricketic ration.

The significant point in this study is the failure to obtain in experimental rickets large increases in concentration of blood serum phosphatase observed in human rickets. 3 ' 4 As a matter of fact, Bodansky and Jaffe 4 claim that the concentration of this blood enzyme is of even greater diagnostic value than blood phosphorus and calcium. It would appear then that experimental rickets is not the analogue of human rickets as it is generally assumed.

We have reported 1 that in vitamin A deficiency there is a marked decrease in the concentration of blood serum esterase, an appreciable decrease in hepatic esterase, and a marked increase in hepatic lipase. In vitamin B₁ deficiency there is a marked decrease in the digestive efficiency of pancreatic esterase and a moderate decrease in the concentration of pancreatic and hepatic lipase, suggesting a disturbance in the digestion of fats in this avitaminosis. An appreciable increase in the concentration of blood serum phosphatase was found in a deficiency of the vitamin B complex.

In this communication we are submitting summarized data on the influence of multiple avitaminosis (deficiency of vitamins A and the B complex) on the concentration of blood and tissue enzymes of the albino rat. The animals were 6 to 9 weeks old and weighed 106 to 118 gm. at the beginning of the experiments. A total of 304 titrations were carried out in duplicate. The results are given in Table I.

We conclude that the multiple avitaminosis of vitamins A and the B complex did not show increased disturbance in enzyme concentrations. The pronounced disturbance in blood serum esterase is due to vitamin A deficiency. The marked decrease in pancreatic esterase and the moderate decrease in hepatic lipase are to be credited to vitamin B₁ deficiency.