Experimental Biology and Medicine

With ad libitum feeding and with lactose as the sole carbohydrate, the growth and appearance of albino male rats fed butter fat was superior to that of animals fed corn oil. Removal of the flavoring agents from butter fat by chromatographing or the addition of one such agent, diacetyl, to corn oil had little effect on the comparative nutritive value of the two fats. Rats fed butter fat or corn oil on rations containing dextrose as the sole carbohydrate grew at equal rates and were of normal appearance. Flavor played no part in these dextrose experiments.

Two compounds, toluidine blue and forbisen, have heen found to possess therapeutic action against murine typhus infections in mice.

The test for spirocheticidal action of drugs in vitro (Eagle²) has been used with certain antibiotic substances. Penicillin, originally presumed to be inactive in vitro, has proved to be active under these conditions in the relatively high concentration of 800 to 1600 Oxford units per 0.8 ml. Inadequate therapy of syphilis in a rabbit resulted in the production of a more resistant strain of Treponema pallidunz and emphasizes the importance of adequate initial therapy.

Vitamin K produces lowering of blood pressure in rats rendered hypertensive (by silk perinephritis. The hydroquinone compound Synkayvite shows no such effect. The trial of vitamin K in human hypertension is being considered.

At the present time biological assay methods are the sole means of determining penicillin potency. A number of such methods are in use in various laboratories; 1 however, the Oxford cup method, 2 with several modifications, 3 has continued to be regarded as a procedure in which a commensurate accuracy and speed may be obtained with a minimum of labor. The following filter paper disc modification should reduce the labor and time involved in setting up this test, while the case with which extra replicates may be run should increase the test's accuracy.

The principal modification lies in the use of a thick filter paper disc saturated with the penicillin sample, substituted for the sample-containing small cylinder used in the Oxford cup method. These discs may be conveniently set up on the seeded plates at a rate of about 6 per minute, a rate considerably more rapid than that in the original method. Another advantage of the discs is that the test plates may be manipulated freely to facilitate reading. Experience with a large number of tests has indicated that the zones of inhibition obtained with the discs are more consistent and more sensitive to variations in the penicillin content of the sample than the zones obtained with the cup method. This improvement may be due to a more consistent contact of the penicillin solution with the agar and to a more even diffusion from the disc.

The test organism, Staphylococcus aureus H† (Oxford strain), tends to produce a somewhat granular growth in nutrient broth. With the use of peptone broth as the seeding medium, a more diffuse growth is obtained and this in turn effects a more even seeding of the test plates.

A method of following the course of liver cirrhosis and splenic enlargement induced by feeding butter yellow to albino rats is described. The procedure consists of intracardiac injection of 0.5 cc of thorium dioxide sol (thorotrast) followed by roentgenograms of the liver and spleen.

The intravenous treatment of donors with purified A and B specific substances isolated from animal sources leads to a considerable increase in isoantibody titer. The substances used were highly purified and did not contain any demonstrable hog or horse protein. The administration of even small amounts is effective and results in the production of sera which are characterized by high titer, speed and specificity. The procedure described easily permits the production of large quantities of very potent typing serum.

Except for sulfadiazine, nomosulfanilamide and its N1-methyl derivative were the most effective drugs in local treatment of experimental gas gangrene of the compounds tested. The acute toxicity of homosulfanilamide was less than that of its derivative. The chronic toxicity of homosulfanilamide in mice appeared to be no greater than that of sulfanilamide. These two compounds were not effective in the treatment of infections in mice with Strep. hemolyticus C203, H. influenza: 62/B, or Staph. aureus Smith, but they were bacteriostatic in vitro for staphylococci. Although infection with Cl. perfringens in mice is not strictly comparable with infection in man, these results indicate that homosulfanilamide may be worthy of a clinical trial in the local treatment of gas gangrene.

It has been shown by Badger 1 that Fievre Boutonneuse, a disease of the Mediterranean basin transmitted by Rhipicephalus sanguineus ticks, and Rocky Mountain spotted fever are related, for one disease protects against the other in cross immunity tests in guinea pigs. On the other hand, Davis and Parker 2 have shown that a vaccine prepared from inactivated Rocky Mountain spotted fever rickettsiæ (tick vaccine) which protects against Rocky Mountain spotted fever affords no protection against Fievre Boutonneuse. While the reverse has not been demonstrated, namely, whether a vaccine prepared from inactivated Fievre Boutonneuse rickettsiæ confers an immunity to Rocky Mountain spotted fever, the work referred to indicates that while there is a group relationship between these diseases, there are some antigenic differences.

Because of the cross immunity conferred in guinea pigs by these two disease laboratory differentiation may be difficult. The following communication presents data to indicate that these two diseases can be differentiated by means of specific complement fixation

The antigen for the complement fixation test in Rocky Mountain spotted fever is best prepared from tissue cultures on agar. 3 This antigen is specific and with it Rocky Mountain spotted fever can be differentiated from either epidemic or murine typhus fever. This method is used routinely In the Division of Virus and Rickettsial Diseases at the Army Medical School for the Diagnosis of Rocky Mountain spotted fever.

The antigen for Fievre Boutonneuse is prepared in the identical manner as those described for epidemic and murine typhus. 4 The antigen is prepared from yolk sac cultures from fertile hens' eggs. The infected yolk sacs are harvested and inactivated with 0.5% formalin. The rickettsiæ are extracted and further purified by centrifugation in the cold in an angle centrifuge at 4,000 r.p.m. for one hour and washed 4 times in buffered saline, pH 7.0.

It has been shown by Golden and Sevring-haus 1 that rats in which the ovaries have been transplanted to the mesentery remain anestrous, owing to inactivation in the liver (previously reported by Zondek 2 ) of the estrogen thus secreted into the portal circulation. Thereafter for approximately 11 months

This phenomenon was more extensively studied by G. R. Biskind 3 , 4 , 5 , 6 using pellets of crystalline steroids implanted in another organ in the portal circulation, the spleen. Castrate rats on a complete diet with pellets of estrone or estradiol thus implanted likewise remained anestrous. During the course of an investigation on the effect of nutritional deficiency on the estrogen-inactivating function of the liver,^7-9 M. S. Biskind and Shelensyak 10 demonstrated that animals with one ovary removed and the other transplanted to the spleen showed no estrual reactions so long as the diet was adequate to maintain the functional integrity of the liver.

This observation seemed to provide an excellent means of ascertaining the effect on the ovary of protracted stimulation by the pituitary, under conditions in which the latter organ is freed from the normal inhibitory action of the secreted estrogen.

According, a series of adult female rats of the Sherman strain were castrated and one ovary was transplanted to the spleen† After an interval of approximately 2 weeks, all the animals had daily vaginal smears to rule out those with vascularized adhesions that permitted ovarian estrogen to by-pass the portal circulation. Those showing estrous reactions were discarded. The others were maintained on a complete diet§ thereafter for approximately 11 months. At the end of this time 9 animals remained; they were again smeared daily over a period of 3 weeks to determine whether any estrogen was reaching the systemic circulation.

A strain of Endomyces vernalis was obtained by growing the yeast in media containing pyrithiamine. This strain was not affected in growth by concentrations of pyrithiamine 25 times greater than that required to inhibit the growth of the parent strain halfmaximally. The new strain still required thiamine or its pyrimidine portion as a growth factor. In the absence of thiamine, pyrithiamine was 50% as active as the vitamin for growth of the variant. When grown in small concentrations of pyrithiamine and in the absence of thiamine, the new strain converted half of the pyrithiamine to the pyrimidine portion of thiamine.

It has been postulated by various investigators 1 that in traumatic shock, substances increasing capillary permeability are liberated in the injured tissues. It might be expected that such substances appear in the lymph draining the traumatized region in higher concentration than in blood, In an attempt to demonstrate such factors, canine lymph was collected during shock and tested in rabbits for its effects on capillary permeability.

Shock was produced by vigorous intestinal massage, peripheral circulatory collapse being verified by blood pressure, hematrocrit and autopsy observations. Lymph was obtained from a duct lying between the first and second intestinal nodes, prior to massage, and shortly before death during the period of permanently depressed blood pressure (below 70 mm Hg.). simultaneously femoral vein blood was drawn for plasma and serum samples. Permeability effects were determined in rabbits according to Menkin's method 2 (intradermal injection of 0.1 cc samples of test fluids, followed by intravenous injection of 2.0 cc per kg of a 1% saline solution of Trypan Blue).

In an initial series of 12 dogs and 15 rabbits, in which heparin was used as anticoagulant, the skin reactions did not show any significant difference between the responses to control and to “shock” fluids: a slight to a marked increase in permeability occurred with each type of fluid. Heparin controls were negative.

In a second series of 12 dogs and 6 rabbits, in which defibrinated fluids were tested without heparin, again no differences were observed between reactions to control and to shock samples; in these instances the responses were only slight or negative. Similarly, both control and shock fluids caused practically the same degree of extravasation of dye in a third series of experiments using 8 dogs and 8 rabbits, in which the non-protein fractions of lymph and of plasma were compared after the proteins had been removed by acetone precipitation.

When succinylsulfathiazo is incorporated into a diet containing all known vitamins plus ten essential amino acids, inadequate nutrition is found to result.

The feeding of isoriboflavin to rats receiving a sub-optimal intake of riboflavin inhibited the growth-promoting action of the vitamin. The antagonistic effect of the isomer was prevented or overcome by the feeding of an adequate level of riboflavin.

Hydrogen sulfide production by bacteria varied greatly with the indicator ion employed; bismuth was superior, as a detector, to iron and lead. Iron was the least toxic. Occasionally the tryptophane content but rarely the guanidine configuration serve as limiting factors in peptones used for differential bacterial tests.

By the methods used, there was no evidence that the agglutination of B-proteus OX19 antigen would be of value in determining the presence of cancer.

Hill 1 and Kuno and Ikeuchi 2 inhibited sweating in human subjects by cooling isolated portions of the body. They made no attempt to correlate the temperature of the bath cooling the part, the temperature and humidity of the environment, and the size of the area cooled, with the quantity of sweat and the comfort of the subjects. Nor were attempts made to evaluate the efficiency of local cooling on the adaptation of a man to a very uncomfortable, hot, and humid environment. The present studies were undertaken to throw light upon these problems.

Methods and Materials. The studies were conducted in an air-conditioned room with the temperature and relative humidity controlled at 75° ± 1°F and 50% ± 2%, respectively. The temperature and relative humidity of the room could be raised within 15 minutes to the desired levels as indicated below. The room in which the subjects rested contained very little of the recording apparatus in order to reduce the psychic disturbance to a minimum. Twenty-six (25 white and one negro) young adults, trained subjects who had had considerable experience around research laboratories, were used. They varied from 22 to 37 years of age. Three of the subjects were females.

The rate of water loss was measured from the skin of the right index fingertip, right forearm, and midepigastrium by a method described by Neumann, Cohn and Burch, 3 The oral and rectal temperatures were measured and recorded photographically by means of copper-constantan thermocouples. The variations in the volume of pulsations of the right index fingertip and right second toetip were measured by means of a modification of the sensitive pneumoplethysmographic method of Turner. 4

Lactic acid in concentrations of 0.05 to 0.3% injected into the exposed zone of innervation of the sternomastoid muscle in the white rat produced sudden destruction of most of the motor end plates within 30 seconds to 25 minutes dependent upon the concentration of the acid. This liquefaction of the hypolemmal axons of the end plates was preceded by a first phase of retraction and a second phase of expansion and fragmentation. Lactic acid caused a progressive depletion, in both the hypolemmal and the epilemma1 axons, of auriphilous substances. The blood vessels were enlarged, hyperemic, and contained clumps of agglutinated red blood cells in the rigorous muscle paralyzed by lactic acid. I t is suggested that the acid metabolites which accumulate during rigor mortis of muscle are responsible for the destruction of the end plates and that during life certain processes that result in the intramuscular accumulation of acid, such as vascular congestion and anoxia, likewise may result in the destruction of the motor end plates.

Following oral administration of sulfapyridine, a hydroxysulfapyridine and a water-soluble hydroxysulfapyridine glucuronide have been isolated from dog urine.

Pyridoxine has been separated from pseudopyridoxine by using the yeast Sacchatomyces cadsbergensis and the bacterium Lactobacillus casei. It has been shown that Sacch. carlsbergensis assays and utilizes only pyridoxine while L. casei assays and utilizes only pseudopyridoxine.

Estrogens injected into lactating rats have been shown to be transmitted to the suckling young inducing changes in urogenital organs of both sexes 1 The effects are of such a nature that it seemed advisable to make further observations on similar animals, at intervals after cessation of treatment, in order to determine the permanency of the condition and the course of any subsequent changes. It has been reported that female rats given prepuberal injections of androgen^2-4,6 or estrogen^5-7 have severe reproductive deficiencies as adults. Because of variations in Methods of administration, hormones used, and length of treatment, the present study brings out differences thought to be of significance.

Methods. We have observed that in untreated female littermates of immature animals given daily injections of diethylstilbestrol, the vaginas open at the same time as in injected animals. This has been confirmed repeatedly with large litters containing only one injected animal. Similar changes have been observed in litters confined to cages in which stilbestrol was sprayed upon shavings used as nesting material. Injection of cagemates or contamination of nesting material with stilbeestrol may therefore be used as alternative methods of administering hormone to immature animals.

Five groups of rats, totalling 154 animals, were treated with estrogen for various lengths of time, the initial treatment being given at various times between birth and the 28th day of life. Animals of Group I were suckled by mothers injected with .1 mg stilbestrol daily; in those of Group II the mothers received .5 mg estrone daily. Group III received direct injections of .01 mg stilbestrol, while those of Group IV were affected by stilbestrol injected into littermates given .01 mg daily. Group V consisted of animals exposed to small quantities of environmental stilbestrol on nesting material.

When the transmissible fowltumor (Olson) was frozen slowly and stored at −65°C to −76°C for 10 to 391 days, no significant reduction or alteration in its capacity to produce typical tumors in the pectoral muscle was detected by methods employed.

A “folic acid concentrate” and a crystalline L. casei factor were found to be strong inhibitors of tumor growth.

We failed to demonstrate alkaline phosphatase in the inclusion bodies of vaccinia, herpes simplex, fowlpox, and tracheolaryngitis by the use of a slightly modified Gomori technic.l0 While our evidence is not conclusive, it strongly favors the hypothesis of HoagIand, Ward, Smadel, and Rivers that the phoiphatase activity of the inclusion body is due to tissue contamination.

When 68 strains of pathogenic staphylococcus were subjected to the in vitro action of sodium penicillin, 8 of the strains, or approximately 12%, were found to be quite resistant, requiring from 0.4 units per cc to 0.8 units of penicillin before growth was completely inhibited. The growth of the remaining 60 strains necessitated the following amounts of penicillin before growth was inhibited: 22 strains, or about 3276, required 0.2 units; 25 or approximately 3776, required 0.1 unit; while 12 or about 1870, failed to grow in the presence of 0.05 unit. About 28% of the strains were found to be highly resistant to the antistaphylococcic action of sodium sulfathiazole. There was no relationship between resistance to penicillin and to the sulfonamide. The evidence at hand would indicate that the mechanism whereby staphylococci become resistant to the sulfonamides is different and unrelated to the development of resistance to penicillin. 10-14

Since some strains of staphylococcus isolated from patients appear to be naturally resistant to penicillin, and yet, sensitive to sodium sulfathiazole, it would not be unreasonable to assume that a combination of therapy with penicillin and sulfathiazole might be advantageous in the management of some types of staphylococcic sepsis. Further clinical observations are necessary to substantiate this, but preliminary investigations appear to bear out this assumption.

Strains of staphylococcus adapted to grow in the presence of the sulfonamides are just as invasive as other strains which are inhibited in growth by the sulfonamides. Whole, human blood has no greater bactericidal action on the sulfonamide-resistant strains than on the sulfonamide-sensitive strains. Strains of staphylococcus adapt themselves to grow in the presence of penicillin at a slower rate and to a lesser degree than is apparent for the sulfonamides. And probably of considerable clinical importance is that an increas d resistance to penicillin is accompanied by the development of strains which are more susceptible to the bactericidal action of whole blbod, and possibly to the other defense mecbanisms of the host.

The administration of alloxan in one suitable dose produces a pancreatic experimental diabetes. Satisfactory animals exhibit a prolonged high blood-sugar level that is maintained for weeks. KO renal lesions are observed in such animals and the fat and glycogen content of the liver is within normal limits. Adrenal and pancreatic islet cellular changes may explain the character of the acute phase.

Thymonucleic acid reacts with cysteine in H₂S0₄ at 40° in two different ways depending upon which of the two reactants is in excess. The reaction products are colored and can be used for the detection of cysteine in a dilution of 1:10⁶ and of thymonucleic acid in 1:10⁴. Glutathione also gives a positive reaction in a dilution of 1:300,000. The procedure for the two reactions is as follows:

I. To 1 cc of a solution of Na thymonucleate (0.01 to 0.1%) are added, with cooling, 0.04 cc of a 5% solution of cysteine hydrochloride and 5 cc of a mixture of 190 cc of H₂O and 450 cc of a 5% solution of cysteine hydrochloride and 5 cc of a mixture of 190 cc of H₂O and 450 cc of concentrated H₂SO₄. The mixture is put into a water bath at 40° for 5 minutes. A deep pink color appears, which is stable for hours. It shows a sharp absorption maximum at 500 μ. Cystine, homocystine, methionine, and dithiodiglycolic acid do not react even in high concentrations. Glutathione reacts like cysteine in equivalent concentrations. Thioglycolic acid reacts also, though a little less strongly than cysteine. Propyl mercaptan also gives the reactions, but pyruvic acid and acetone do not. When the desoxyribose is destroyed by heating the thymonucleic acid in 2% H₂SO₄ for 2 hours at 100°, the reaction with cysteine disappears completely. All these facts indicate that the colored substance is the product of a reaction between the desoxyribose of the thymonucleic acid and the sufhydryl group of the cysteine (glutathione, thioglycolic acid.)

The reaction can be used for the detection and probably for the colorimetric determination of small amounts of thymonucleic acid.

In all rats with 5% of pancreas remaining after partial pancreatectomy, cataracts appear after a period of time which in the cases we observed was from 50 days onward. This cataract is progressive and its degree is in direct relation to the level of fasting blood sugar. Microscopic examination shows well marked lesions in the lens.

In rats with 20% of pancreas, the lens lesions take over 200 days to appear and are less serious in direct relation to the less serious character of the diabetes.

We have not observed any cataracts like these in normal rats.

From these observations, it would appear that potassium-sulfon-succinate is not utilized as a substrate nor acts as a reversible carrier. It is capable, however, of inhibiting the catalytic activity of succinate when the latter is present in minute quantities.

This latter inhibition presents an analogy to the activity of para-aminobenzoic acid and the sulfa drugs. Owing to the widespread distribution of succinate in tissues and bacteria, the possible bacteriostatic action is being investigated.

Thiouracil is relatively nontoxic. Continued large doses cause cachexia, tremor, convulsions, bloody urine and feces. Most internal organs show hemorrhagic congestion; kidneys and liver seem to be damaged invariably; edema of the lungs is often, but not always, present.

It has been shown that growing trout require a diet containing fresh meat. Mixed diets devoid of fresh meat did not produce any increase in the growth of trout, Varying the nature of the dried liver fractions used did not stimulate growth. When these diets were enclosed in gelatin capsules and force fed to the fish, a marked gain in weight was obtained. Supplementing the meatless diets with gelatin resulted in satisfactory initial growth. The supplemented diets did not sustain continuing growth beyond 7 weeks suggesting that further deficiencies exist in these rations.

In a previous report, 1 the results of immunizing farm flocks with fowl pox vaccine prepared from entire chick embryo were described. The vaccine consisted of a composite of the chorio-allantoic membrane, yolk, yolk sac, albumen, embryo proper, and fluids from previously inoculated embryos that showed gross lesions on the chorio-allantoic membrane. While a high concentration of virus was noted in the chorio-allantoic membrane, the presence of virus in other parts of the embryo was also demonstrated. 2 The dehydrated vaccine was suspended 1-100 in tryptone broth and applied by the 3-stick method in 35 healthy farm flocks, comprising 25,164 chickens. Nine days after inoculation, more than 6000 birds, approximately one-fourth of the vaccinated birds, were observed with 95.85% showing takes. Forty-three days after inoculation, 71 culled chickens from 3 of the flocks were brought to the Laboratory and artificially exposed to fowl pox virus by the feather follicle method. Eighty-eight and seven-tenths per cent proved refractory.

The present note concerns an observation on the duration of the immunity produced by entire embryo vaccine. Seventy-one birds selected from 4 of the flocks were exposed, one year subsequent to vaccination, on the patagium by the 3-stick method to fowl pox virus in a 1-100 suspension in tryptone broth. The exposing virus was of the same strain as that use for vaccination, but consisted of the chorio-allantoic membrane which represents a greater concentration of virus than the entire embryo vaccine. The artificially exposed chickens were given three inspections at weekly intervals for lesions of fowl pox following exposure. Results of the exposure are noted in Table I.

Out of the 71, 3 susceptible birds were encountered. These were from the same flock.

Twenty-three senile dogs showing varying degrees of glomerular injury and at the same time a fairly diffuse change in the type of epithelium in the proximal segment of the renal nephron from a normal order of cuboidal cell to one of a flattened, less specialized type of cell when subjected to the anesthetic action of chloroform for 45 minutes fail to show, as the result of the use of this body, a reduction in the elimination of phenolsulphanephthalein, or a reduction in the reserve alkali of the blood from that value which these animals have established as their pathological normal. In a group of 29 control senile animals, with a normal urine, both chemically and microscopically, with the same order of change in the glomeruli but with, in general, a normal order of cytological structure in the proximal segment of the renal nephron when anesthetized for 45 minutes with chloroform show a marked decrease in the elimination of phenolsulphanephthalein and a reduction in the reserve alkali of the blood.

At least in certain cases, rutin appears to have the property of decreasing capillary fragility in subjects in whom this fragility is initially increased. In this effect it resembles hesperidin.

In a previous paper, 1 it was shown that a single dose of 0.5-5.0 mg of alpha-tocopherol administered on the 15th day of life to rats born of mothers on an E-deficient diet, and maintained after weaning upon vitamin E low diet, produced a significant delay in postpuberal degeneration of the testis. Similar doses given on the 5th day, or on the 28-30th day, were less effective than when given on the 15th day.

This paper is concerned with the effect of larger single doses, either given to the young directly, or through the mother's milk.

Experiment I. The mother was a third generation rat on E-deficient diet, 10 mg of alpha-tocopherol acetate having been given at the beginning of each pregnancy. Litter H₂ was the second litter; the first litter of 7 young did not survive 4 days. The young of H₂ were given 1 drop (25.6 mg each) of alpha-tocopherol acetate on the 15th day of lactation. Semi-castration was performed when they were 107 days old, and they were killed on the 182nd day. Repeated fertility tests were made by mating the rats with females which had been kept on a stock diet. During the mating period, male and female had access only to vitamin E-deficient diet. The birth of a litter was considered a positive test.

Experiment II. Each suckling rat of a second litter was given 51,2 mg of alphatocopherol acetate on the 15th day. The mother was again a third generation rat on E-deficient diet. The results are summarized in Table II.

From previous data, it has been found that the average weight of one testis in untreated animals under otherwise identical experimental conditions is as follows:

At 75 days, 2 of the 36 untreated control rats had motile sperm, but no positive mating was obtained.

It is apparent that the single dose received on the 15th day of life has exerted a prolonged delaying effect upon testicular degeneration.