Experimental Biology and Medicine

Colorimetric methods for the determination of inulin in blood serum or plasma and urine based on the reaction of inulin with diphenylamine have been described in recent years. The most widely used procedure is that of Alving, Rubin and Miller, 1 in which the inulin-containing solutions are heated in closed tubes with diphenylamine and hydrochloric acid dissolved in ethyl alcohol. Tightly capped tubes are necessary because of the volatile solvent, and because the solution must be heated for 60 minutes at 100°C to obtain the maximum color. In this laboratory as well as in others, this method has been troublesome and the results not consistently accurate.

For this reason a modification has been devised in which a less volatile solvent is used, thus permitting the use of open tubes. With this reagent a shorter period of heating is necessary for maximum color development and accurate results have been obtained in the determination of inulin in blood serum or heparinized plasma in concentrations as low as 4 mg per 100 cc. The determination can be carried out on 0.2 cc.

Method. Reagent. Three grams of diphenylamine, reagent grade, are dissolved in 100 cc of glacial acetic acid and to this solution are added 60 cc of concentrated hydrochloric acid.

This reagent is stable if kept in an amber bottle in a refrigerator at 6°C, and the color production with inulin is found to remain constant for at least one month. A few crystals of diphenylamine may precipitate out of solution but this does not affect the reagent. If kept at room temperature the color in the blank determination becomes progressively darker, but if correction be made for the blank as is automatically done in a photoelectric colorimeter of the Evelyn type the calibration curve is not changed.

While evidence has been accumulating concerning the importance of a nervous factor in the etiology of traumatic shock,1-3 similar studies of the problem of burns have not been carried out.

A comparison was made of the effects of thermal trauma to one hind leg in control cats and in spinal animals. Transection of the spinal cord at L₁ or T₁₁ effectively eliminated the influence of afferent nerve impulses from the burned leg but left the major vasoconstrictor outflow from the spinal cord intact.

The experiments were performed on 83 adult cats, anesthetized throughout the procedure with pentobarbital sodium. One hind leg was burned with a Bunsen flame for 10 or 15 minutes. The spinal cord was transected about 30 niiiiutes preceding application of thermal trauma. Observations were made of blood pressure (carotid cannula), hemoglobin and specific gravity of whole venous blood, (falling drop technique), respiration, body temperature, survival, local fluid loss in the burned limb and pathology.

In control cats, burning resulted in an abrupt rise in arterial pressure from 120 to 160 mm Hg. The blood pressure remained high and only reached the original level after 10 hours. On the other hand, arterial pressure fell sharply in spinal cats during thermal trauma from an initial 90 mm Hg to 60 mm Hg; it was restored in the course of 1.5 hours and then maintained between 100 and 80 mm Hg. While arterial pressure fell quickly to 90 mm Hg as a result of spinal transection alone, without burning it remained at about this level for many hours. The rise in blood pressure from burns in control cats is thus apparently reflex and masks an initial sharp fall due to non-nervous factors.

In the spring of 1941, during the occurrence of an epidemic of infectious mononucleosis, we attempted to isolate a transmissible agent from sterile Berkefeld filtrates of nasal washings and whole blood by inoculation into the chorio-allantoic membrane of the chick embryo. Material was obtained from patients who gave a characteristic history, blood picture, and whose heterophile antibody titers ranged from complete agglutination of 160 to a high of 2560. We also attempted passage from patients whose clinical history and leucocyte picture were typical and who later gave heterophile agglutinations in the range mentioned. The nasal washings and blood from some patients were frozen for 12 hours before they were used. Two additional cases made their appearance this fall. In all, a total of 8 attempts was made to transmit the agent from 8 cases; positive results were obtained from 4. The sterile filtrate, when inoculated onto the membrane, showed small pearl-gray reactions with a spreading cloudiness about them. This reaction occurred after the third to the fifth day of inoculation and continued to grow until the membranes were harvested for study. Of 66 membranes which were inoculated with sterile nasal washings, 19 showed positive reactions. Of 30 membranes inoculated with whole blood, 13 showed positive reactions. Cultures from all infective membranes were bacteriologically sterile. Nasal washings and blood from 2 normal individuals were negative. Microscopically, the positive membrane showed an ectodermal proliferation and heavy monocytic cell infiltration. Although special stain was applied, (Mallory phloxine-methylene blue) no inclusion bodies were found. We are able to continue the reaction through 4 to 14 passages obtaining positive results with about 50% of the membranes. The agent finally died out, showing a dramatic drop of positive takes in the last 2 generations of each passage.

Growth of rats was obtained when the sole source of nitrogen in the diet was provided by parenterally injected casein digest.

Tolerance to both gold sodium thiosulphate and to gold sodium thiomalate can be developed in rats, as well as a cross tolerance between the 2 compounds. A minimum dose is necessary for tolerance formation, but with a sufficient dose, tolerance can be developed with only 2 doses. The tolerance lasts for considerable time.

1. The volume of sulfocyanide available water of cats is definitely increased after hemorrhage. 2. The chloride concentration of serum water was found to be decreased in all experiments save one. 3. The total water and sulfocyanide available water content of cardiac muscle, pancreas, pylorus, duodenum, colon, skin and liver (chloride available water) was increased after hemorrhage. No significant differences were found in the case of skeletal muscle (gastrocnemius and diaphragm). 4. The significance of the above findings is discussed.

In the treatment of psychoses, insulin coma 1 (or the period of unconsciousness) is generally not allowed to persist beyond an hour or an hour and a half. In cases inadvertently allowed to linger in coma beyond that period, an irreversible insulin coma 2 frequently follows that can no longer be relieved by intravenous glucose. This dangerous and sometimes fatal complication has, however, directed the attention of several investigators to the important fact that patients who survive several hours or days of this relatively irreversible coma often show dramatic psychiatric improvement on awakening. Kraulis 3 attempted to utilize this phenomenon for therapeutic ends by prolonging insulin coma with periodic small glucose feedings sufficient to sustain the patient but not sufficient to rouse him. The difficulties in the clinical management of these sporadic feedings, however, and the frequent presence of gastric retention 2 (which Kraulis did not suspect) made this mode of treatment too dangerous for clinical application.

It can be assumed that the cerebral injury which ensues in accidentally protracted coma is due to the diminution of brain metabolism (by withdrawal of substrate) below levels adequate for cell maintenance over long periods of time. In our cases (30 experiments on 10 subjects), we have succeeded in prolonging hypoglycemia as long as 21 hours and coma as long as 18 hours, without mishap, by sustaining brain metabolism above this dangerous level, but still below levels which support consciousness and other associated cerebral functions. This has been achieved by administering infusions of 5 % glucose in physiological saline (Sterisol) intravenously at controlled rates. Inasmuch as large doses of insulin exert their hypoglycemic effect for many hours after administration, 4 one need only maintain the blood glucose at a moderately high level (e.g., 35 mg per 100 ml) to prolong coma for many hours. When the insulin effect begins to subside, a fresh intraiiiuscular iiljection of insulin can be administered.

Although a number of studies have been made on male urine to determine the presence or absence of estrogens, comparatively few have been made upon testes. This is rather surprising in the light of the work of Zondek 1 who reported enormous quantities of estrogenic substance (more recently identified as alpha-estradiol and estrone by Beall 2 ) in the testes of the stallion.

Zondek 1 while more often credited with the report on the stallion testes, examined also the bull testes. Perhaps because of the relatively minute amounts (less than 0.1 %) found in the latter as compared to the former it was not thought to be worth recording in the later literature. Zondek assumed that “the mass production and excretion of the estrogenic hormones in the testes is a peculiarity of the equines.”

There is considerable difference of opinion among those who have examined testes for estrogens. A number of workers have failed to secure any results from their extracts, but on the other hand sufficient data have been recorded to indicate that estrogens occur in the testes of several species. Fellner, 3 Brouha and Simonnet, 4 Dorfman, et al., 5 all reported the presence of estrogens in bull testes, while Laqueur and deJongh 6 report them in human testes and Dodds, et al., 7 in “mammalian” testes. With the exception of Zondek all of the workers who have found estrogens in the testes have reported only qualitative results.

The desirability of further quantitative study is evident, but any quantitative study based upon extraction and the assay is bound to be more or less inaccurate. About the most one can say is that there is at least as much hormone as the assay indicates.

1. The agent of Lymphogranuloma venereum in yolk sac material can be completely inactivated by the addition of 1 or 2% urea and also by extraction with ether. 2. Crude suspensions of yolk sac material treated with urea or ether constitute antigens (devoid of anticomplementary activity) of considerably higher potency than the high speed sediments obtained from the same suspensions.

The technical assistance of Miss Augusta Hollender is gratefully acknowledged.

Nicotinic acid, thiamin chloride, cocarboxylase, riboflavin, pyridoxine and para-amino benzoic acid have been shown to have no effect on the photometrically determined growth rate of S. aureus and E. coli in simple media. Calcium pantothenate definitely accelerated the rate of multiplication of S. aureus but not of E. coli when present in a concentration of 200 μg per milliliter. None of these substances except para-amino benzoic acid antagonized sulfathiazole bacteriostasis although present in amounts 40 times as great as the sulfonamide. Concentrations of sulfonamide 2,500 times that of the accompanying para-amino benzoic acid were completely ineffective in inhibiting bacterial growth.

These observations, therefore, support the view that the sulfonamides directly affect that part of the microbial metabolic system which specifically utilizes para-amino benzoic acid, since relatively large amounts of 6 other important growth factors completely failed to inhibit their bacteriostatic activity.

About 50% of the strains of meningococci which have been superficially “typed” as belonging to Group II have been found to form an independent and homogeneous lot. Those who have worked with individual strains agree that many of them give a definite Quellung reaction, they are good antigens, and produce sera that protect mice well against infections by meningococci of the same group. But there is no cross protection with sera representing other groups.

Because of the close agglutinogenic relation to other Group II meningococci, it is suggested that, for the present, they be designated as Group II alpha, following the precedent set by Dopter in his studies of his parameningococcus since designated as Group II.

The amount of thiamin required for growth of albino mice has been determined. Five micrograms per mouse per day was found to maintain body weight attained at the time of weaning. Ten micrograms per mouse per day is the minimal level for normal growth. Less than 5 μg per mouse per day results in loss of weight and eventual death.

In adrenalectomized rats the toxicity of phosphate injected intraperitoneally is increased, and approximates that of potassium.

Thyrotropic hormone produces a fall in the blood cholesterol of hypercholesterolemic rabbits regardless of the presence or absence of the thyroid glands.

At 37°C viable cells and vaccinia virus can be demonstrated in cultures composed of Tyrode's solution and chick embryonic tissue during about 4 weeks following inoculation. These results are contrasted with those previously obtained in roller tube cultures where survival of both cells and virus persists much longer and the multiplication of the virus is greatly increased.

During the course of experiments previously reported 1 a male cat was prepared with an extensive bilateral lesion probably involving the left side of the eighth cervical segment and the right side of the first thoracic segment of the spinal cord. The lesion was produced on March 26, 1940. At the time of writing, 21 months after the operation, the animal appears to be in excellent condition. Its weight remains constant at about 3.04 kg, which is slightly above the preoperative value. There have been no pressure ulcers. The appetite is good, and although neither micturition nor defecation occurs spontaneously, the output of urine and feces is normal in quantity. Active spermatozoa are produced and are ejaculated in response to appropriate stimulation. Sensory anesthesia and volitional paralysis are apparent below the level of the lesion. Breathing is entirely diaphragmatic. The functional signs are those of complete interruption of the spinal pathways.

In addition to the usual spinal reflexes, which appear in this animal with regularity and vigor, there is occasionally exhibited a mass reflex of exceptional character which I have not seen described for laboratory animals. It is often elicited by rubbing the shoulder region in such a way as to evoke the typical scratch reflex. Upon continuation or intensification of the stimulus, the scratch response gives way to a violent flexor spasm of the muscles of the abdomen, which brings the hind feet into a position forward of the head, with the legs slightly abducted. As the flexor spasm begins, the rubbing of the shoulder region is discontinued, and the subsequent events occur without further external stimulation.

The globulin fraction of the serum of rabbits, immunized with living Hemophilus pertussis, was shown to protect mice against experimental infection with this organism. 1 The intramuscular injection of doses of from 4 to 7 ml of the refined serum into infants, in the early stage of whooping cough, produced an immediate increase in both the mouse-protective and agglutinative titers of the infants' sera. 2 In certain instances, there seemed to be a beneficial effect upon the clinical course of the disease, and frequently a definite decrease was noted in the total number of mononuclear cells of the blood.

The present report concerns the prophylactic use of the antiserum† in 20 infants exposed to pertussis by family contact.

When an older sibling was found to have whooping cough, the exposed infant was given an intramuscular injection of from 2 to 10 ml of the concentrated antiserum (after preliminary testing for sensitivity). The exposed infant then remained at home in contact with one or more infected siblings, and was observed carefully by clinical examination. In 13 cases the presence of pertussis in the family was confirmed by nasopharyngeal culture.

In the group of 20 infants 13 (65%) were completely protected. Of the 7 infants who developed pertussis after receiving the serum, however, 5 experienced mild cases, whereas the disease was unmitigated in the other two. Furthermore, in 3 of these 7 instances, the infants began to cough 5, 5, and 4 1/2 weeks respectively after the serum was injected (the time of onset was not ascertained in 3 cases).

Aminoacetic acid affords some protection against convulsions produced by the intravenous injection of metrazol. It is insignificant as compared to other substances, as pyridine, nicotinamide and phenylcinchoninic acid. Aminoacetic acid affords a great deal of protection against convulsions produced by the subcutaneous injection of metrazol.

(1) Tyrothricin and actinomycin A inhibit fibrinolysis by beta hemolytic streptococcus cultures or supernatants thereof. (2) These substances also interfere with coagulation of plasma by pathogenic staphylococcus.

For more than ten years the automatic microtome knife sharpener described by Fanz 1 has been standard equipment in most large pathology laboratories, saving many hours of tedious labor while producing a far better cutting edge on the knives than hand work could possibly do.

In the apparatus presented here, the glass plate described by Minot and Fanz has been incorporated but the mechanics have been simplified. The time needed for sharpening has been reduced by swinging the knife through an ellipse rather than an arc alone.

Description of Machine. The whole apparatus (Fig. 1) is mounted on a pedestal which has a heavy base fitted with 2-inch casters. P is a 1 20 H.P. geared motor with a 3/4-inch shaft, 4 inches in length, presenting vertically. This shaft carries the small gear I and the 8-inch disc C. The disc C in turn mounts the three 3-inch vacuum cups on which rests the 24-inch glass plate B. This type of mounting holds the plate securely in place but absorbs the torque of starting and stopping. The larger 10-inch gear J is mounted on the supporting plate and attached to it is one of the flat bearings which, with its mate, holds the carriage K for the knife F and turning mechanism G-H. In the center of each bearing is a socket for a locking pin which, when placed in the bearing on J rotates the knife through an ellipse, and when placed in the second bearing rotates the knife through an arc only. The knife may be held fixed in any position desired on the plate by disengaging the small gear I with the lever O.

Mounted on the knife carriage K is the fixture bearing the small motor and turning mechanism H as well as the knife arm and counter weight M.

The erythrocytes contain an abundance of phosphoric esters hydrolyzable by phosphatase, 1 but the plasma contains only 0.2-0.5 mg phosphorus per 100 cc 2 in the form of phosphatase hydrolyzable esters. The phosphatase activity of a fracture callus increases rapidly beginning 3-4 days after the injury. 3 , 4 , 5 Although the site of an old fibrous non-united fracture may exhibit low phosphatase activity 3 and infected fractures contain somewhat reduced amounts of this enzyme 6 there is no evidence that defective calcification of bone or delay in fracture healing is primarily a result of a quantitative deficiency of this enzyme. It appears possible that one of the factors limiting the speed of the calcification process is the low content of plasma with respect to the substrate of bone phosphatase and this consideration suggested that some cases of delayed or nonunion of fractures might be so explained. It seemed desirable, therefore, to investigate the effect of the intravenous injection of sodium betaglycerophosphate on the healing of fresh fractures.

The right radius of rabbits, weighing between 4.25 and 4.75 lb, was fractured while the animals were anesthetized. The fractures were produced by grasping the middle of the shaft of the bone between two adjacent and parallel Kocher forceps and exerting steady rotation of the forceps in opposite direction until the bone was heard to break. A few animals, in which breaks of both the radius and ulna were produced, or which displayed marked bruising of the skin, were discarded. Roentgenological examination showed that the fractures usually were transverse and that the fragments were in good alignment with little comminution of the bone. The undamaged ulna served as an effective splint and the animals were able to use the limb upon recovery from the anesthesia.

Two closely related isomers of thyroxin have been tested biologically. Meta thyroxin had little if any effect upon the B.M.R. of the rat in doses up to 500 mg per kg of body weight, while ortho thyroxin was found to have an activity of 1/25 to 1/50 of that of thyroxin.

The 1-(+)-lysine requirement for optimum growth of the young chick is approximately 0.9% of the diet.

The conventional method of manometric recording of blood pressure in animal experiments with a mercury manometer and a float, which carries a writing point, has been essentially the same since Ludwig described it in the last century. A vertical kymograph and specially treated paper (lamp black, etc.) are necessary, as any substantial friction between the paper surface and the writing point causes the float to submerge. This happens occasionally in spite of all precautions. A comparatively wide manometer tube has to be used in order to support the float, which has to carry the writing point and to overcome the friction on the paper; a wide tube draws several cc of blood from the vessels of the animal, when a rise in blood pressure occurs, and therefore a manometer cannot be used in small animals.

To eliminate these shortcomings of manometric recording an apparatus was designed where the float is substituted by a light beam (Fig. 1)

A slide carries a lamp and a phototube. It has a female thread, which engages a threaded brass rod. This rod is rotated by a reversible electric motor. When the motor runs clockwise, the slide—and with it the lamp and the phototube—are raised, at a speed of 2 cm per second, and it is lowered at the same speed when the motor runs counterclockwise. After amplification† the phototube current operates a relay. The relay contacts are connected with the motor in such a way that the motor runs clockwise when the relay is open, and counterclockwise when it is closed.

When the light beam from the lamp strikes the phototube (the phototube housing has a hole 2 mm in diameter), the relay closes, the motor runs counterclockwise and the slide goes down, until the light beam is cut off by the mercury in the manometer tube.

The occurrence of an Rh antibody in the breast milk of a woman with an erythroblastotic child is described. The patient's serum, too, contained a very potent antibody against Rh blood cells.

1. In mice, rats, and dogs, the acute toxicity of p-aminobenzoic acid by mouth has been determined. The drug is more toxic to mice and dogs than to rats. By intravenous injection the sodium salt of p-aminobenzoic acid is more toxic to rats than to mice. 2. Oral doses of p-aminobenzoic acid greater than 1.0 g per kg to dogs may cause death accompanied by acute gastro-enteritis and hemorrhages into the small intestine. Acute necrosis of the liver may result from doses of 2.0 g per kg or larger. 3. Rats tolerate daily doses of 1.4 g per kg by mouth for about a month without inhibition of growth or pathological changes.

Further studies on the rôle of the Lactobacillus casei ∊ eluate factor in chick nutrition are reported. Purified preparations of the factor promote growth, hemoglobin formation, and normal feathering.

Three groups of male rabbits were injected with 3 different levels of estrone in order to determine whether estrogen could effect a simultaneous rise in the lactogenic hormone of the pituitary and blood stream. Assays of the pituitaries revealed that a considerable increase in lactogen was obtained, although the highest dosage of estrone (5000 I.U.) gave a smaller increase in lactogen than was secured with the two lower levels (500 and 1000 I.U.). Assays of the whole, untreated rabbit blood also showed that a definite increase in the release of lactogen into the blood stream was secured following the injection of estrone. These findings are believed to account for the initiation of lactation following suitable estrogen treatment.

It has been suggested that copious lactation can be initiated only when (a) there is a well developed mammary gland present and (b) when there is a high lactogen content in the anterior pituitary. 1 After the middle of pregnancy one of these two factors is present, namely, a well developed mammary gland, but the production of lactogenic hormone by the pituitary remains at too low a level to initiate abundant lactation. Thus in the rat, 2 3 guinea pig, 4 and rabbit, 1 4 the lactogen content of the pituitary remains as low during pregnancy as in the non-pregnant state. Almost immediately following parturition, however, there is a great increase in the lactogenic hormone of the pituitary (from 200 to over 400%), and full lactation is initiated. 2 3 4 An increased lactogen content in the urine has also been demonstrated in postpartum women. 5

Inasmuch as the mammary glands of the rabbit become well developed during pseudopregnancy, it was of interest to determine whether the content of lactogenic hormone in the pituitary at the end of pseudopregnancy would account for the absence of any marked degree of milk secretion in such animals. Seven normal, mature New Zealand white rabbits served as control animals, while 11 similar rabbits were each injected intravenously with 100 I.U. chorionic gonadotropin (Korotrin)‡ in order to induce pseudopregnancy. Twenty days later both the control and pseudopregnant rabbits were killed, and their pituitaries were removed for assay. In 6 of the 11 pseudopregnant rabbits it was found possible to express either a slight amount of milk or serous secretion from the teats. The pituitaries of each group were assayed quantitatively for lactogen by the Reece-Turner pigeon method. 2

Ovulation in Amphiuma can be induced prior to the normal egg-laying season by injections of cattle and frog pituitary suspension and extract.

Meningococci are more resistant to the lethal action of certain chemicals than the gram-negative organisms usually employed in germicidal tests.

Rats on a choline-poor diet containing liberal supplements of carotene develop fatty livers poor in vitamin A. The kidneys of these animals are extremely rich in vitamin A.

The lid-closure (wink) reflex of amphibians—retraction of the eye bulb upon tactile stimulation of the cornea or surrounding skin—appears comparatively late in development. Urodeles display it first in advanced larval stages, anurans at or about the time of metamorphic climax. In the latter group its dependence upon the metamorphosing agents was demonstrated by experiments in which metamorphosis was either suppressed (hypophysectomy) or advanced (hyperthyroidism); 1 in the former case, the reflex failed to appear unless thyroid was administered, while in the latter case its onset was advanced almost, though not fully, as much as was the transformation of the body. Both the sensory (trigeminal) and motor (abducens) components of the reflex develop full functional capacity in very early larval stages, but their central linkage into a workable reflex mechanism is not effected until at the time of metamorphosis.

The close association with metamorphosis suggested that maturation of the central mechanism of the reflex might be under direct control of the thyroid hormone. In order to test this possibility, an attempt was made to precipitate the development of the reflex relative to the metamorphic changes in the rest of the body by exposing the hind brain locally to supernormal concentrations of the hormone. As Hartwig 2 has shown, implantation of thyroxin-soaked agar under the fin skin of salamander larvae causes local metamorphosis of the surrounding tissues. A similar procedure was, therefore, adopted for the present experiments.

Agar pellets were soaked in thyroxin (1:1000) and then implanted into the fourth ventricle of tadpoles of Rana pipiens.

A new type of antagonistic substance, designated as streptothricin, has been obtained from a soil Actinomyces. The substance appears to be formed from certain amino acids in the medium. Streptothricin is insoluble in ether, petrol ether or chloroform, and is carried down by precipitating the medium with ethyl alcohol. It is best separated from the medium by adsorption on norit-A, followed by elution with dilute mineral acid. Further concentration is accomplished by neutralization of the acid and concentration in vacuo. The active substance is low in nitrogen, is not affected by proteolytic enzymes and can withstand 100°C, for 15 minutes. Its properties are those of an organic base.

The bacteriostatic action of streptothricin is unique. The addition of 0.1 mg of crude material to 10 cc of nutrient agar inhibits the growth of E. coli and various other gram-negative bacteria; among the gram-positive bacteria, some, like Bac. subtilis and micrococci, are more sensitive, whereas others, like Bac. mycoides, are far more resistant. Streptothricin possesses also marked bactericidal properties, especially upon certain gram-negative bacteria.

Recent work in this laboratory 1 has demonstrated the requirement by a strain of Cl. tetani for a component of liver extract originally described in another connection by Snell and Peterson 2 and subsequently studied by Stokstad. 3 This material, together with thiamine and riboflavine, fully accounted for the activity of a fraction of liver extract which had been found essential to the growth of our tetanus organism. The active component of Snell and Peterson's fraction has been further investigated by Mitchell, Snell and Williams. 4 who have carried the purification considerably further, and have named the agent “folic acid” since they find green leaves to be a convenient source. Its chemical constitution has not yet been determined.

Through the courtesy of Professor Williams it has been possible to test his purified material as to its growth-promoting effect on the tetanus bacillus. Three specimens, representing varying degrees of concentration have been made available. The amounts required to produce half maximum growth of L. helveticus were given as follows:

These preparations were used in varying quantities to replace the Snell and Peterson or Stokstad factor in an otherwise complete medium. 1 After seeding with our strain of Cl. tetani and anaerobic incubation for 24 hours, relative growths were determined in a photoelectric nephelometer. The results, expressing degree of growth approximately as percentage of maximum are shown in Table I.

It is evident that “folic acid” is essential to the growth of this strain of tetanus, and that its effect is exerted by extremely minute quantities.

Evidence has been presented to show that Hydrosulphosol is an available and convenient source of -SH radicals. The compound rapidly decolorizes methylene blue and supports the growth of the anaerobic organisms Clostridium sporogenes and Clostridium perfringens under aerobic conditions. The compound is relatively nontoxic to embryonic chick heart tissue fragments cultivated in vitro.

The possibility of antagonism in systemic toxicity between the sulfonamides and other compounds has begun to receive attention during the past year. McCarty 1 found that para-aminobenzoic acid has no effect on the acute fatal toxicity of sulfapyridine in mice. David, et al., 2 reported that pentobarbital exerted a protective action against the toxicity of intravenously administered heparin-sodium sulfapyridine mixtures in dogs. Richards 3 found that small doses of urethane or pentobarbital decrease the fatal toxicity of intravenously administered sodium sulfapyridine in rabbits and dogs. In the present paper it is shown that the acute toxicity of 2-sulfanilamidopyrimidine (sulfadiazine) in the mouse is reduced by phenylazo-alpha-alpha-diaminopyridine hydrochloride (pyridium). The toxicity of sulfanilamide is not affected by this compound.

The percent mortality in a group of mice injected subcutaneously with a given dose of sulfonamide was compared, under the same experimental conditions, with the mortality in groups receiving this dose and, in addition, doses of 10, 30 or 100 mg of pyridium per kg. For each experiment, 3 groups consisting of 8 to 15 mice each were employed. A total of 269 mice was used in the experiments here reported. The sodium salts of sulfadiazine and sulfanilamide∗ were injected subcutaneously in concentrations of 5 to 10%. Pyridium was injected in a 0.2% solution or a 0.5% suspension. When both sulfonamide and pyridium were employed, they were injected within a few seconds of each other into widely separated sites. The mice were given water and food ad lib. and the number of deaths occurring in 4 days noted.

The ovaries of 24- to 26-day-old rats and the testes of day-old White Leghorn chicks showed an increase in weight following the injection of a suspension of a single male rat pituitary gland in 8 divided doses over a 4-day period. The average increase in ovarian weight was 309% and that of the testes was 62%.

1. Stimulation of nerve by electrical fields associated with the charging and discharging of condensers has been obtained, although the efficacy of increasing and decreasing fields is not identical; 2. a strength-duration curve of excitation with diminishing electrical fields associated with the discharge of condensers has been determined wherein E is at first a decreasing and then an increasing negatively accelerated function of the time. The region of inversion is between .0001 and .00038 mscs; 3. the curve is in marked contrast with those obtained by discharging condensers through nerve tissue, in that these (a) are not characterized by an inversion, (b) show E as a diminishing negatively accelerated function of the time; 2 4. there are great differences in the stimulability of preparations.

As a result of the development of radio apparatus and of new types of vacuum tubes, it became possible to devise stimulators having desirable features impossible to inductorium or neon tube stimulators, i. e., greater range of frequency of stimulation, greater range of intensity of each shock, and methods for recording the frequency of the stimulation. Many instruments, incorporating some or all of these features, have been devised, outstanding examples being those of Scheminzky, 1 Schmitt, 2 Nicolai, 3 and Bouman. 4 Although these instruments can be considered as great improvements over earlier types, they all have one or more of the following undesirable features: (1) large, complicated and expensive, (2) operate on batteries, (3) unwanted and irregular leakage currents, and (4) the wave form of the stimulating current is not independent of frequency.

A recent instrument which is especially noteworthy for its portability is that devised by Cullen. 5 The attached signal magnet, however, always vibrates at a frequency of 60 times per second and so provides no means of indicating the frequency of stimulation. Furthermore, after having used this particular instrument the authors have found that the high amperage delivered to the signal magnet considerably shortens its life when the ordinary classroom type is used. Another undesirable feature is the presence of “stray charges in the instrument” which renders it unfit for precision work.

The authors have designed a portable (operating on 110 volts, A.C.) and inexpensive stimulator which does not have the disadvantages discussed and enumerated above.

gExperiments on post-pubertal albino rats indicate that androstenediol and pregnenolone prevent the testis atrophy otherwise produced by small doses of testosterone. Since androstenediol possesses only a weak “male hormone” or testoid action and pregnenolone is completely devoid of it, it is evident that the gonad-protecting power of a steroid compound is independent of its testoid action. The experiments suggest, furthermore, that by combining moderate doses of testosterone with a non-testoid compound such as pregnenolone, therapeutic doses of testosterone may be given in cases of eunuchoidism without the danger of producing testis atrophy.

The separated colloid fraction of either arsphenamine or neoarsphenamine is retained long in the body in comparison with the crystalloid fraction which is rapidly eliminated.

Perosis was reported 1 to be a symptom accompanying the egg white syndrome in chicks. In the present investigation, single-comb White Leghorn chicks were placed on the following diet at hatching: Yellow corn meal, 55 g; wheat middlings, 20; dried skim milk, 10; commercial casein, 10; ground limestone, 2; steamed bonemeal, 2; alfalfa meal, 1; choline chloride, 0.1; NaCl, 0.5; MnSO₄, 0.05; fresh raw egg white, 30 cc. The wet mixture was spread in thin layers and dried at 45°C, after which 0.3 g of fish oil blend (3000-A, 400-D) was added. In addition to dermatitis, 2 symptoms of perosis developed in from 50 to 70% of the chicks at the age of 3 to 5 weeks. If egg white was omitted from the diet, or if cooked egg white replaced raw egg white, perosis did not develop. It was found in a preliminary experiment that all the symptoms were prevented by injecting a biotin concentrate† at a level which supplied approximately 3 μg of biotin daily. In the next experiment, crystalline biotin methyl ester‡ was injected into the breast muscle. Each chick received a total of 13 μg divided into 7 injections during a period which extended from the 12th to the 32nd day. Eight chicks were used in the injected group and 11 uninjected chicks served as controls. The results shown in Table I were observed on the 38th day.

Feathering was superior in the injected group. The level of biotin administered, which averaged only 0.34 μg per bird per day for the duration of the experiment, was not sufficient to prevent dermatitis completely but was sufficient to prevent perosis.

In recent publications 1 , 2 reviews have been given which point out that the best evidence of lens regeneration from the dorsal iris is limited to a small group of salamanders (Triturus). It is of interest to know whether or not all of the Triturus group possess this power of lens regeneration. Therefore the effect of lensectomy in Triturus torosus was investigated.

Stone and Sapir 2 reported that reimplanted lens tissue of the adult Triturus viridescens did not survive. Since the reimplant did not persist its inhibitory effect on lens regeneration could not be tested satisfactorily in their experiments. Therefore, in Triturus torosus the excised lens was reimplanted to test its capacity for survival. In case it did survive, any inhibitory effect on lens regeneration could be observed.

Lenses were completely removed in 100 eyes of Triturus torosus larvae ranging in length from 15 to 25 mm. For histological studies the animals were killed at close stages (2 to 50 days) after operation. The initial stages of lens regeneration were the thickening of the edge of the iris with subsequent depigmentation and separation of the cell layers. These three stages are distinct and progress at about the same rate of development. In all eyes fixed 5 days after operation the thickening of the dorsal iris was observed. At 7 days all specimens showed the first indication of slight depigmentation of the iris and from 9 to 14 days all eyes showed further depigmentation and separation of the cell layers of the dorsal iris. In cases fixed from 14 to 24 days after operation the regenerating lenses varied in developmental rate, especially in the formation of the fiber-forming pole.

The mode of action of sulfonamides upon susceptible bacterial species has been the subject of much study. It appeared to us that one of the most fundamental aspects of this problem was to determine whether active compounds differ from closely related inactive substances in their respective abilities to reach or penetrate the bacterial cell. It was conceivable that p-aminobenzene sulfonamide (sulfanilamide) diffuses into the streptococcal cell (and is concerned in its metabolism) whereas its chemotherapeutically inert isomers, the ortho and meta compounds, do not diffuse into the organism. Since this does not appear to be true, the inactivity of certain compounds cannot be explained by this one fundamental property. In the light of our results diffusion into the organism may or may not be a requisite for a chemotherapeutic agent.

Twenty-liter batches of broth cultures of Streptococcus hemolyticus, strain C-203, were grown for 48 hours. The organisms were then collected by means of a continuous flow Sharples centrifuge and washed with 250 cc normal saline 6 to 8 times. The cells were collected after each washing by means of horizontal centrifugation. After final washing the bacteria were dried over P₂O₅ in a vacuum desiccator. The same procedure was repeated with cultures in broth containing 20 mg % sulfanilamide, orthanilamide and metanilamide, respectively. The drug broth cultured bacteria were washed 6 to 8 times until the wash fluid contained no demonstrable sulfonamide compound as determined by the Marshall test. Accurately weighed samples of dried organisms from each of these test cultures were quantitatively extracted in 3% trichloracetic acid for 2 hours at 37°C and the amount of each substance so eluted was also determined by the Marshall method.

The infectivity of Bartonella muris can be preserved for at least 11 weeks when infected defibrinated rat blood is rapidly frozen in a mixture of dry ice and alcohol and then preserved in dry ice.

Through the adaptation of a strain of poliomyelitis virus to the mouse by Armstrong, 1 a mouse neutralization test has been made possible. Haas and Armstrong 2 have already applied this test to post-epidemic surveys, but with results which are difficult to interpret. Thus, we felt it wise to investigate its significance somewhat further by testing a series of acute and convalescent sera from poliomyelitis patients against this same virus. Twenty-three pairs of such sera have now been tested. They were collected from 3 different outbreaks over a period of 2 years. Sera from 1940 and 1941 from the Los Angeles County Hospital were supplied by Dr. John F. Kessel. Another group, collected during the Tacoma, Washington, epidemic in 1940, were obtained from Miss Beatrice Howitt. None of the patients were seen by us, but the clinical diagnoses were made and checked by clinicians well trained in infectious diseases. The diagnosis of the non-paralytic case, in particular, is liable to error, but spinal fluid examination in all instances, together with serum neutralization tests to eliminate the virus encephalitides in several instances, reduced the possibility of error. From one non-paralytic case poliomyelitis virus was isolated from the stool by Miss Howitt.

Method. Both the admission (acute) and the discharge (convalescent) serum from each patient were tested simultaneously to insure comparable results. Positive and negative controls were always tested at the same time. A minimum of 8 mice was used for each serum, and tests on many pairs were repeated to check results. The first series was run by the method described by Armstrong, 2 employing 3 twofold dilutions of virus, but later a technic was adopted which is more comparable to that used for the yellow fever virus, using a large number of mice and only one dilution.

1. The maximal survival in vitro of an individual Eustrongylides larva at 37.5°C was 346 days. The medium was Bacto Yeast extract 0.5% + NaCl 0.5% + Glucose 0.5%. 2. The maximal average survival of a series of worms was 157 days in a medium containing Bacto-broth 0.8% + NaCl 0.5% + Glucose 0.5%. 3. The molecular concentration of the media could be varied in rather wide limits without material change in the length of survival. 4. The presence of 0.5% glucose was decidedly beneficial. 5. The worms produced acids. 6. No moulting or other sign of development was observed.

A three-year accumulation of data obtained from the analysis of more than 1,000 blood samples indicated that the average plasma ascorbic acid level of the patients in this hospital was less than .40 mg %. The lowest results, averaging .20 mg %, were obtained during the months of April, May and June and the highest during October and November when an average of .59 mg % was achieved. These data on ascorbic acid and others on various nutritional essentials which will be described in subsequent reports indicated that a large mental institution is an exceptional laboratory for conducting studies of human nutrition and a project has been started to make use of this opportunity. The regulated routine of the majority of the patients, the supervision of the dietary intake, and the choice of age and sex from the large numbers available make it possible to have better controls than is ordinarily so easily available.

A modification of the Mindlin and Butler 1 method was used to determine the plasma ascorbic acid. No cyanide was added to the blood but the plasma was separated from the cells and precipitated with metaphosphoric acid within 30 minutes after the blood was drawn in order to minimize ascorbic acid oxidation. All recent estimations have been made with the aid of a Cenco Spectrophotometer. Prior to January, 1941, a Cenco Photelometer was used with a green filter. All blood samples were taken in the morning before breakfast.

Table I presents the average ascorbic acid levels of different groups who were in residence at this hospital during the months March through June. The differences demonstrated are the direct results of differences in the diets.

Exposure of citrated or oxalated plasma to an air current is followed by a rapid diminution in its prothrombin activity; this is corrected by the addition of CO₂ and not affected by oxygen.

Ovulations were induced in hens by the intravenous injection of a luteinizing preparation into hens pretreated with pregnant mare's serum for 6 to 11 days. Ovulation was induced in some hens following each of 3 successive LH injections. As many as 7 multiple ovulations were effected in individual PMS pretreated hens following a single LH injection.

1. Evidence is presented that there is a relationship in the reactions of the thymus, spleen, and lymph nodes to the effects of castration, thyroidectomy, and treatment with thyroxin or testosterone propionate, or combinations of these in the male rat. 2. Castration and thyroxin cause comparable increases in the weights of thymus, lymph nodes and spleen. Thyroidectomy and treatment with testosterone propionate tend to decrease the weights of these tissues. Thyroidectomy and testosterone propionate prevent the increase in the weights of thymus, spleen and lymph nodes which follow castration. The order of decreasing sensitivity to the stimuli employed is as follows: thymus. cervical lymph nodes, mesenteric lymph nodes and spleen. 3. A positive coefficient of correlation of 0.591 ± 0.080 was calculated from the data derived from the above experiments for the thymus and the cervical lymph nodes. This is further evidence of a similarity in reaction of these tissues.

The electrophoretic pattern of the cerebrospinal fluid proteins resembles that of the plasma. Alterations in the composition of the serum proteins produce similar changes in the spinal fluid patterns. Colloidal gold activity in pathological fluids is associated with the gamma globulin.

These data indicate that Azochloramid potentiates the bactericidal effect of sulfonamides. This effect may be due to the inactivation of sulfonamide inhibitors by the chlorine compound.

The rotameter has been used to measure cardiac input and mean blood flow in the arteries and veins of the anesthetized dog. Typical records are shown. Tests indicate that in routine use the instrument will give reliable blood flow values with an error of less than 10%. Its use enables the experimenter to determine at a glance the moment to moment flow during the time that flow is actually being measured, an advantage not possessed by any other known method. The rotameter is so simple in operation that it should also serve a very useful purpose for the measurement of blood flows in student experiments in the classroom for which as yet no simple and reliable method has been available.

Cantharides blisters were produced on the skin of allergic human subjects. After removing the stratum corneum over the blisters, the denuded areas were covered with saline, and the release of histamine and protein into the saline was determined before and after production of local allergic reactions by injection of antigen. Both the histamine and the protein content of the saline rose sharply within a few minutes after injection of the allergen and fell to the original level within 60 minutes. An increase in exudation of fluid and the appearance of a substance similar to the “slow reacting substance” were also observed.

Propionin injected intraperitoneally in mice has an L.D. 50 of 1.7 ml per kg. Propionin administered by stomach tube to rats has an L.D. 50 of 15.3 ml per kg.

A number of materials have been used as substitutes for blood in the treatment of shock and hemorrhage. Acacia solutions were used rather extensively for this purpose during World War I, mainly as a result of the work of Bayliss. 1 Later investigators have shown that there are many dangers attendant upon the intravenous use of acacia. Andersch and Gibson 2 reported that about one-half of the injected acacia accumulated in the liver. Yuile and Knutti 3 found that after repeated injections of acacia in dogs the liver weight increased 5 or 6 times and that sometimes as much as 8 to 10% of acacia (by weight) was found in the liver.

Pectin sols, having physical properties similar to those of acacia, should show the same advantages for transfusion purposes and yet, due to their different chemical constitution, might not cause damage by storage in the liver. The characteristic constituent of acacia, a gum exudate from certain tropical trees, is aldobionic acid which is unusually resistant to hydrolysis. Pectin, however, is a normal constituent of fruits and vegetables used for human food and is rather easily hydrolyzed in mildly acidic or alkaline systems and by many enzymes. Its molecule, with a weight of 150,000 to 300,000 is composed of chains of partially esterified galacturonic anhydride units. 4

A general study of the effect of pectin in the circulatory system of rabbits has been in progress in this Department for the past 3 years. Various types of pectin in solutions of different concentrations have been injected intravenously into rabbits, particular attention being given to the effects upon organs involved with the circulatory mechanism. No ill effects of any kind have been observed.

Regeneration of plasma proteins was found to occur in dogs with depleted proteins stores and lowered plasma proteins on a nitrogen-free diet receiving intravenously an enzymatic hydrolysate of casein as the sole source of nitrogen. Parenterally administered casein digest constitutes an effective method of parenteral nutrition.

It has been generally accepted as a fact that iron in transport in the blood plasma following its ingestion occurs in combination with globulin. The use of radioactive isotopes enables us to distinguish between iron† and copper‡ normally found and that recently added to the body economy. In earlier experiments we reported that, whereas about 80% of the radio iron in plasma shortly after feeding was in the filtrate following precipitation of the proteins by trichlor-acetic acid, nearly all of the iron in the red cell was in a form which was precipitated with this acid. 1 Below we tabulate further data relating to this subject.

It can be seen that in a longer series of observations the fraction of iron in the plasma which is not precipitated by trichloracetic acid constitutes about 90% of the total iron (Table I) under the conditions of these experiments. Furthermore, the time after ingestion does not seem to be related to the amount of iron found in this fraction.

In Table II are listed the results obtained in an extension of these observations. By various procedures, the amount of iron in combination with protein was determined and found to be about 95% of the total. However, only about 15% of the iron was found to be actually bound to globulin as determined by half-saturation with ammonium sulphate.

The adductor muscle of Pecten consists of two parts, the larger of which is responsible for the rapid movements during swimming, and the smaller, which contracts slowly, is responsible for keeping the valves closed. The peculiarities of this muscle have been studied repeatedly. 1 , 2 , 3 A satisfactory answer has not been given to the question of how the slow muscle can withstand rather large tensions for indefinite periods without appreciable fatigue and at the same time be able to relax very quickly when necessary.

Reflex Relaxation. In Pecten stimulation of the mouth in a normal animal leads to rapid relaxation of the slow muscle. In our experience the sensitive area for this reflex is much wider than hitherto reported, and includes all parts in the vicinity of, and innervated by the cerebral ganglion. Stimulation of the branchial nerves also leads to relaxation, although they do not connect directly with the cerebral ganglion, but with the visceral ganglion. That they nevertheless find their reflex center in the cerebral ganglion is shown by the fact that after cutting both cerebro-visceral commissures their stimulation elicits no relaxation of the slow muscle. As long as either commissure is intact, the reflex relaxation takes place. We were not able to find in this species the reported difference in action of the two commissures. 1 , 2 As long as the nerve bands between the visceral ganglion and the slow muscle are intact, mechanical stimulation of these bands results in relaxation. This, too, is most likely reflex as this kind of stimulation becomes totally ineffective after cutting the bands. 2

Peripheral Inhibition. The relaxation following a contraction produced by direct stimulation of isolated slow muscle is much slower than the relaxation observed when preganglionic mechanisms are stimulated.

Since pancreatectomy has been shown to abolish the hypoglycemic response of the adrenalectomized animals to anoxic anoxia, it may be concluded with Gellhorn that anoxia normally stimulates the parasympathetic (insulin response) as well as the sympathetic (adrenal response) system.

1. Whole egg contains an excess of avidin, as the amount of biotin in the whole yolk is unable to neutralize the amount of avidin in the white of the same egg. 2. Biotin in egg yolk is present in a high molecular, undialyzable form which is physiologically active in yeast growth tests or in tests made on rats with egg-white injury.

The results presented indicate the necessity for determination of the purity of the P.G. isolated from non-pregnant urine. It has been clearly shown that the modifications reported by Venning 1 do not necessarily remove all impurities from the final product. The implications of these findings, for much of the data in the literature, are clear. In the light of our results, the finding (first reported by Hamblen, et al., 2 that P.G. may be present in the absence of a secretory endometrium, particularly warrants critical reëxamination.

The artificial induction of aseptic pleuritis by the injection of talc into the pleural space has been reported by a number of different workers. 1 This procedure has been recommended for the purpose of producing adhesions which will prevent recurrent spontaneous pneumothorax from collapsing the lung, or may allow a selective collapse, with only an affected portion of the lung collapsed. It has also been advised as a preliminary to operations on the lung, such as drainage of abscesses, cysts or cavities, or before partial or complete amputation of a lung, to diminish the extent and severity of subsequent infection. 2 In view of the frequency of tuberculous involvement of the pleura, it seemed desirable to study the effect of such talc pleuritis on intrapleural tuberculosis before endorsing its use in such conditions.

Three rabbits were each given 2 g of talc (U.S.P., Merck), suspended in 5 cc of normal saline solution, injected into the right pleural cavity. Four months later these rabbits, together with 3 other previously untreated animals were each inoculated, in the right pleural cavity, with 0.001 mg of a virulent culture of bovine tubercle bacilli. One month after this infection, X-ray examination showed extensive densities throughout both lungs in the 3 animals which had received the talc, with but little change detectable among the 3 controls. At autopsy, 2 months after infection, the talc-treated rabbits showed extensive nodular caseous tuberculous lesions in both lungs, scattered small caseous nodules in both kidneys, caseous mediastinal lymph nodes and extensive pleural adhesions in the right side. The control animals showed much less extensive lesions, limited mainly to the intercostal and mediastinal lymph nodes, with little or no pulmonary or renal involvement, and no pleural adhesions.

The effect of carbon dioxide gas upon the motility of the gastro-intestinal canal has been studied chiefly on excised sections of the gut1-4 or by the ingestion of water saturated with CO₂ or by the intragastric injection of the gas itself. 5 As far as we know the effect of CO₂ + O₂ inhalations upon gastric and cecal motility induced by the intragastric injection of air or by the subcutaneous injection of pilocarpine or physostigmine has not been reported. Furthermore, it may be emphasized that the results to be reported were obtained without opening the peritoneal cavity or the use of balloons but simply by inspection of the intact abdomen.

The method is a slight modification of one described sometime ago: 6 preliminary narcotization to reduce or abolish the inhibitory effects of external stimuli; to accomplish this 150 mg sodium barbital per kilo were injected subcutaneously about 30 minutes before the test; after cutting the abdominal hair, which is readily done with a blunt-pointed scissors curved on the flat, both the cecum and most of the stomach including the preantrum may be easily observed through the intact abdominal wall; the antrum itself is not visible, but its contraction is readily indicated by a bulging of the preantrum. If the stomach was not well-filled, 60-120 cc air were injected into this viscus through a hypodermic needle plunged through the abdominal wall. The gas mixtures were 50% each CO₂ and O₂ delivered from tanks at a rate of 150-300 cc per minute each; this gas mixture was administered to the rabbit through a funnel held at such a distance over the nose or the tracheal tube that the respirations were definitely deepened. All observations were 5 minutes long: before, during and after the gas administration.

The influence of the sympathetic and parasympathetic nerves on the intrahepatic vascular system has been deduced mainly from indirect evidence based on data obtained from plethysmographic changes in volume of the liver and from changes in hepatic inflow or outflow, as well as from variations in portal pressure during stimulation of the nerves. The object of this study was to observe directly, by transillumination of the intact liver in the anesthetized animal the effect of stimulation of the sympathetic and parasympathetic nerves on the intrahepatic vessels and to determine whether or not the intrahepatic vascular system possesses an active vasoconstrictor or an active vasodilator mechanism or both.

The literature on the subject discloses a number of different views held by investigators who employed various methods for determining the effect of stimulation of the autonomic nerves on the vascular ramifications within the liver. Bayliss and Starling 1 obtained a rise in portal pressure by electric stimulation of the splanchnic nerves. Cavazzani and Manca 2 produced constriction of the portal radicles by asphyxia after sectioning the vagus nerves, but not after sectioning the splanchnic nerves. They stated that the vagus nerves cause direct and not reflex dilation of the portal radicles within the liver. In another publication they reported that the intrahepatic branches of the hepatic artery constrict during vagal stimulation and dilate during stimulation of the celiac plexus. François-Franck and Hallion 3 obtained a decrease of hepatic volume and a rise of portal pressure in the dog when the splanchnic nerves were stimulated. The same results were obtained after ligation of the hepatic artery.