Experimental Biology and Medicine

Keys 1 recently dismissed our findings of heightened thiamine and choline requirements in tropical heat with the brief statement that they were unconvincing. The only basis given for his conclusion was his own study of human subjects exposed to high temperatures for only one week or less. In this brief interval of heat exposure, he failed to find any improvement in work performance with accessory vitamin administration (with men on regular army rations).

Reference to the published literature would have shown that no deficiency effects should have been expected in so short a period. Rat growth and food consumption rates begin to show the influence of vitamin deficiency only during the second (or some subsequent) week. This was plainly stated and made clear in the data presented in our paper on heightened thiamine need in the heat. 2 Others 3 have shown that the metabolic adaptation to change in environmental temperature begins in the second week of exposure and is largely accomplished by the end of the third week. Human tests covering one week or less therefore have no valid bearing upon the problem and provide no basis for a conviction one way or another.

Since any real heightening of vitamin requirement in tropical heat is a matter of great importance in human and animal nutrition, it is well to eliminate every possible uncertainty. With all vitamins added to our diet mixture and new food given only once or twice a week, it was possible that the destruction of thiamine and choline was more rapid in the feed jars at 90°F than at 68 °F and that this may have been the basis of the apparent heightening in requirement.

Six injectable liver extracts were found to contain choline or a choline-like substance, since their acetylation produced vasodepressor activity which was antagonized by atropine.

Evidence has accumulated which indicates that phosphatases occur in the erythrocytes of dog, beef, horse, rabbit, rat, and man. To provide further information about phosphomonoesterase occurring in the normal human red blood cells the enzymatic activity of red cell hemolysates was studied as to (1) the presence of alkaline and acid pliosphatase, (2) the effect of fluoride and magnesium upon the enzymatic activity, (3) the relation of erythrocyte phosphatase to plasma phosphatase.

Procedure. The erythrocytes were washed 3 times with normal saline. The washed cells obtained from x cc of oxalated or heparinized blood were hemolyzed by adding distilled water to bring the total volume of the hemolysate up to 1.5 times x cc. If the hematocrit value is known the phosphatase activity of the erythrocytes can be calculated from the activity of the hemolysates. The phosphatase activity of the hemolysate as well as of the plasm was tested by detertnining the amount of phosphorus (according to Fiske and Subbarowl) liberated from a sodiuni glycerophosphate substrate, or by measuring the amount of phenol freed from a discd' Jium phenylphosphate substrate, according to King and Armstrong. 2 Michaelis' 3 veronal-acetate buffer was used preferably, since certain buffer salts interfere with the colorimetric determination of phenol. In a few of the experiments with glycerophosphate as substrate, citrate and veronal buffers were used. The results are expressed in mg of phosphate or phenol liberated per 100 cc of hemolysate or plasma during one hour of incubation at 37°C.

Results. (1) Phosphatase activity of red cell hemolysates was found to be constantly present (Fig. 1). Strong activity was observed between pH 4.8 and pH 6.1. Below and above this zone of acidity hydrolysis drops abruptly. The optimum pH of the phosphatase of human erythrocytes was found around pH 5.3. Its action upon P-glycerophosphate is slow compared to the strong action upon disodium monophenylphosphate. A second zone of optimal activity at alkaline reaction as reported by Roche and Bullinger 4 for horse blood cells, was missing. If confirmed, this result would preclude the occurrence of an “alkaline phosphatase” in human erythrocytes.

The doses of X-rays which produce immediate hemolysis in vitro have been determined for different conditions. The X-ray dose producing complete hemolysis is dependent on the concentration of the irradiated erythrocyte suspension and on the medium employed. Erythrocytes suitably irradiated at high dilutions (0.1%) in saline become stable to X-ray hemolysis.

Acquired, non-specific cellular resistance to equine encephalomyelitis virus has been described and differentiated from specific immunity induced by vaccination or passive immunization.

Results obtained with sutureless nerve reunion by means of cuffs of live or frozen-dried artery in 21 rabbit, 30 cat, and 81 monkey nerve splices have confirmed for the larger mammals the conclusion previously reached in the rat, that optimal nerve regeneration may be secured by this method.

Full functional recovery may be obtained in cats and monkeys after the bridging of a nerve gap of several centimeters by grafts of frozen-dried nerve, stored for several months and rehydrated before use.

Virus studies were carried out on 75 patients suffering from various sporadic central nervous system infections of unknown etiology. Nine cases of lymphocytic chorio-meningitis proven by virus isolation or detection of specific antibodies are presented. The cases were clinically divisible as follows: (1) encephalomyelitic type, (2) meningeal type, and (3) an “influenzal” type without neurologic signs.,. Negative results were obtained in tests for St. Louis encephalitis and western equine encephalomyelitis.

The method of unstained cell counts provides a simple quantitative means of determining the toxicity of antiseptics to cells. The antiseptics tested differed markedly in their cytocidal activity, sodium sulfathiazole being the least and mercuric chloride the most toxic. Tinctures of merthiolate and zephiran were more toxic than the corresponding solutions. The polymorphonuclear leukocyte was more resistant to most of the reagents tested than the lymphocyte.

Acetylcholine (ACh.), when applied in minute amounts to the eserinized cerebral cortex, induces marked excitatory effects; 1 it was inferred that the ACh. stimulates or facilitates various cortical synapses. The experiments here reported indicate that ACh., on direct application in extreme dilutions, stimulates a motor nucleus, namely the hypoglossal nucleus; this nucleus is readily accessible, as the trigonum hypoglossi, immediately beneath the floor of the fourth ventricle. 2

Methods. Decerebration under ether was by the Sherrington decerebrator. Both common carotids were tied or, in other experiments, both internal carotids were tied and also the branches of external carotids, but leaving lingual arteries intact; essentially similar results were obtained in both preparations. The skull was held in a special clamp, so that the entire tongue could be observed. The floor of fourth ventricle was exposed by removing median parts of cerebellum. Acetylcholine chloride (ACh. Cl) (Merck) was dissolved in buffered Ringer-Locke solution and was applied in small rectangles 2 mm x 6 mm of spot test paper to the XII nuclei of both sides.

Results. Preliminary localization of effects from the XII nucleus was by unipolar faradization with a fine silver wire, ending in a minute bulb; the diffuse electrode was a saline-soaked pad on animal's back. Secondary distances varied from 35 to 50 cm, using a large Palmer inductorium (Fig. 1).

The following effects of ACh. were obtained in a decerebrate cat (3100 g), having lingual arteries intact. Preliminary eserinization was by 0.25 mg eserine sulphate (Merck) intravenously. A rectangle of spot test paper soaked in ACh. Cl. 1:50 millions was applied to both XII nuclei: in 4 sec. a slight median furrowing appeared at base of tongue between circumvallate papillae (Fig. 2); the furrowing, which coincided with inspiration, increased and was accompanied by simultaneous retraction of tongue base, elevation of soft palate and dilatation of remainder of mouth cavity.

A single intraperitoneal injection of 200 mg/kg of alloxan into white rats causes typical diabetes mellitus. The histologic changes are necrosis and complete disappearance of the beta cells from the pancreatic islets.

A method for studying the periodicity and survival of the primitive respiratory mechanism of young animals is described. Certain chemical and phvsical factors capable of influencing frequency, duration, and pattern of these periodic movements are indicated.

Soon after the introduction of insulin into clinical practice, it was observed that the amount of the new hormone required to control the glycosuria in patients with diabetes mellitus depended in part on the amount of available carbohydrate in the diet. Elaborate attempts were made to establish a quantitative relation between the insulin requirement of the patient and the amount of carbohydrate obtained as such in the food together with that which might arise through the conversion of protein and fat. Some success attended these efforts. It was soon found however that wide variations in the insulin requirement of certain patients occurred from time to time in spite of a constant diet and that enormous amounts of insulin were needed for certain patients. 1 The concept of insulin sensitivity and of insulin resistance thus arose. The precise nature of these states seems to be little understood and the present report is presented in the hope that it may contribute to the solution of this problem.

In a recent monograph, Jensen 2 states that about one unit of insulin per kilogram of body weight is required per day to maintain normal carbohydrate metabolism in the depancreatized dog. In our experience this figure is too low. Most of these animals require from 2 to 3 units per kg if good control of the glycosuria is desired. In comparison with these figures the amounts of insulin required to establish a comparable control of glycosuria in dogs with certain types of partial pancreatectomy seem surprising.

Nine male dogs, varying in weight from 8.2 to 13.4 kg, were operated upon and all of the pancreas removed save for a small portion in the region of the lesser duct.

The vitamin A-enhancing property of crude soybean phosphatides is due principally to the tocopherols present in these phosphatides.

A resurgence of interest in the basal ganglia has accompanied the recent excellent studies on these nuclei. 1 It is perhaps surprising, therefore, that the comparative morphology of the striatum and the pallidum has received so little attention. The present report presents data on the absolute volumes of these areas in a closely graded series of primates and in one sub-primate brain. Because of the linking of the basal ganglia with the cortex and the general problem of the relationship of brain size to development, the volumes of the cortex and the brain have been included in this study.

The method followed is that suggested by Dornfeld, Slater and Scheffe, 2 wherein the total volume is estimated by constructing a profile from the areas of sample cross sections (measured by planimeter) throughout the extent of the structure studied. In the case of the basal nuclei approximately every 20th or 40th section was measured, and in the case of the cortex and the brain approximately every 100th section was taken. All measurements were made on one side of the brain only (usually the right), hence the figures given in Table I are half-values for the total brain.

The measurements in this series were made on preparations stained for fibers and do not take into account variations of cellular density which may be quite marked, particularly in the case of the pallidum.

The brains selected consist of one sub-primate, the sheep (Ovis); 2 primitive primates, Galago and Perodicticus (potto); 2 monkeys, Cebus and Cercocebus, comparable in development to the often studied rhesus monkey, Macaca mulatta; and the chimpanzee∗ (Pan).

Relative size is indicated in Table II where the volume of each gray mass is expressed in percentage of the total brain volume.

A method is described for collection of air-borne bacteria, by means of dispersing air at a rate of 30 liters per minute from a Folin aeration tube into 20 cc of broth contained in a test tube. The apparatus is about 90% efficient, is readily constructed from simple equipment, and may be adapted to estimation of the bacterial content of textiles.

Albino mice kept on a diet devoid of fat developed the Burr and Burr syndrome. The deficiency could be cured or prevented by the administration of lard.

Quantities of acetoxypregnenolone 10 to 20 times greater than the anti-fibromatogenic threshold of progesterone were unable to prevent the fibromatogenic and other toxic actions of estradiol. This gives new evidence in favor of the following 2 assumptions: first, that the ketonic oxygen at C₃ is essential, or highly important for the antiestrogenic and antifibromatogenic activity of steroid hormones; and second, that the antifibromatogenic activity is often concomitant with the progestational one.

These experiments demonstrate an unusual type of hemolysis dependent jointly upon a high concentration of erythrocytes, extreme potency of cold hemagglutination and, presumably, damage as the result of shaking.

Of the samples of human thermal sweat investigated by us only two showed any ascorbic acid present and in these cases the amounts found could be considered practically negligible. Dehydroascorbic acid determinations indicated the presence of this acid in most samples. The amounts found were not sufficient to suggest the induction of vitamin C deficiency through excessive sweating. The use of rubber bags in the collection of sweat may lead to high titration values and appears responsible for some of the high values for ascorbic acid in sweat that have been reported.

Sexual abilities have been described in castrate men (Rowe and Lawrence, 1 Lange, 2 McCullagh and Renshaw, 3 Kolle 4 ), but the possibility of some degree of stimulation by androgens from extragonadal sources has remained largely an undetermined factor. After removal of both testes men continue to excrete appreciable quantities of urinary androgens (Gallagher and associates, 5 Callow and co-workers, 6 Hamilton and colleagues; 7 see summary of literature by last authors), and in a rare case the amount excreted is within the range observed in normal men (Callow and co-workers 6 ). There is evidence that an androgenic substance found in the urine of normal and castrate men, dehydroisandro-sterone, may be derived from secretions of the adrenal glands (Callow, 8 Fraser and associates 9 ).

Feinier and Rothman 10 attempted to define the quantity of urinary androgens in a 53-year-old castrate who claimed erectile ability, but the description of their findings seems confusing. They reported an entire absence of androgens in the urine, although, as indicated above, some quantity of these substances is found almost invariably witn the use of adequate methods of extraction and assay. Description of their technic was omitted. They commented that neither testosterone nor androsterone was present in extracts obtained apparently from 3,000 cc of urine, an odd statement since testosterone has never been found in urine. It would have been difficult to identify specific compounds present in this small quantity of urine, and since the extract appears to have been used for assay in capons, it is doubtful that the authors intended to imply that the isolation of compounds was attempted.

The present communication pertains to 2 men castrated for 13 and 18 years, respectively, who demonstrated during repeated examinations a pronounced degree of erectile ability despite the absence of any considerable stimulation by androgens from extragonadal sources, as indicated by low titers of urinary androgens and supranormal values for urinary gonadotropins.

Propenyl ethyl ether produces prompt surgical anesthesia in many species of animals upon inhalation. The anesthetic index is higher than ethyl ether and the compound appears to be without deleterious action upon the myocardium, the normal histology of the liver or kidney, or upon the red blood cell in anesthetic concentrations. However, this ether in common with its isomer, cypreth ether, produces irritation of the upper respiratory tract in animals and especially in man. For this reason we are of the opinion that the compound is unsuited as a volatile anesthetic in man.

Two cases of sprue are reported in which the values of the serum potassium were found to be at low levels, 1.1 meq/L to 1.6 meq/L, before the institution of treatment.

Three varieties of anti-Rh agglutinins have been encountered, designated as anti-Rh (standard), anti-Rh₁, and anti-Rh₂, and agglutinating, respectively, approximately 85%, 70%, and 35% of bloods from white individuals in New York City. Human sera containing agglutinins anti-Rh and anti-Rh₁ together are designated anti-Rh' sera containing the two agglutinins anti-Rh and anti-Rh₂ are designated anti-Rh”. By means of the 3 sorts of Rh agglutinins, 5 varieties of Rh agglutinogens are demonstrable, designated as Rh₁, Rh₂, Rh, Rh′, and Rh”. These in combination give rise to 8 Rh blood types (including the Rh-negative type), of which all but one, the rarest, have actually been encountered. A theory to explain the heredity of the Rh blood types is proposed in which the existence of 6 allelic genes is postulated. Evidence in support of the theory is presented and some of the implications of the theory are mentioned.

A method of making nucleated erythrocyte counts per 10,000 red blood cells is presented. Routine counts within 24 hours of delivery are recommended on newly-born infants and more than 2 nucleated red blood cells per 10,000 are to be considered abnormal. Regardless of Rh compatibility the mortality among children with 5 or more nucleated cells per 10,000 was 67% in our small series, although among the Rh incompatible infants there were more nucleated red blood cells than among the Rh compatible group. The possibility of ill effects when the mother is Rh-positive and the infant Rh-negative is suggested by the data.

Evidence was obtained which indicates that L. arabinosus 17-5 may be used for the assay of 1-tryptophane. Of other related substances tested only anthranilic acid and indole produced a growth response and these are removed by ether extraction.

Saliva, bile, and succus en-tericus obtained from man do not inactivate penicillin. Gastric juice destroys this antibacterial substance rapidly especially at body temperature. Hydrochloric acid, and not pepsin, is apparently responsible for this action of gastric juice.

In 2 subjects with pernicious anemia the absorption of penicillin, when administered by mouth, was greater than that observed in normal subjects.

The authors wish to thank Marjorie Jewell, Thelma Maxon, and Grace Helm for their assistance.

1. Sudden reduction of atmospheric pressure to 70 mm Hg caused pulmonary hemorrhage and death, in mice. 2. Fasting had the effect of reducing the degree of hemorrhage. 3. Bile salts, given tc the fasted animals increased the degree of hemorrhage.

Endotoxin-containing extracts of Shigella paradysenterie Flexner, Salmonella typhimurium, and Rhodospirillum rubrum when injected intraperitoneally into mice pregnant from 12 to 18 days induce decidua-placental hemorrhage which results either in abortion or resorption of the embryos.

It is pointed out that such decidua-placental hemorrhage induced by the endo-toxins of gram-negative bacteria may fall into the same class of physiological action as hemorrhage induced in implanted tumors following administration of endotoxins of gram-negative bacteria.

A definite reduction in the incidence of thrombus formation was noted in dogs previously treated with Dicumarol in a dosage equivalent to a therapeutic and very safe quantity commonly administered to human beings.

We wish to acknowledge with gratitude the aid of Dr. C. H. Bunting who examined the microscopic sections and confirmed the observations here recorded.

In the rabbit the administration of adrenalin is followed by a markedly augmented excretion of organic sulfates. This appears to indicate that this drug is inactivated in vivo by conjugation with sulfuric acid. Conjugation with glucuronic acid does not occur. These observations confirm those reported by Richter in 1940.

Atabrine is a strong inhibitor of choline esterase. This finding is discussed in relation to toxic and antimalarial activity and chemical configuration of drugs.

In the search for a conveniently small animal in which morphine addiction might be developed preparatory to a study of its effect on tissue metabolism, the hamster was found to be so resistant to the drug that the extremely high lethal dose was thought to be worthy of note. Coincidentally, the toxic symptoms observed differed from those reported in the literature for other species. 1

The present communication describes the toxic effects of morphine in the hamster. The lethal dose 50 and the lethal dose 100 are presented.

Morphine sulfate was injected subcutaneously, intramuscularly, and intraperitoneally; the route of administration did not alter the response. In all cases where large quantities had to be given at a single time, a combination of the 3 routes was used. Dosage levels ranged from 1 mg to 2000 mg per kilogram body weight. Normal hamsters of both sexes were included. Body weights were from 26 to 130 g with age corresponding from 1 to 8 months (life span of the hamster usually 12 months). Neither sex, age, nor weight of the animal made any apparent difference in the response to morphine. The seasonal or temperature variations during the course of the investigation also caused no difference in response.

In doses up to and including 150 mg per kg of body weight of the hamster, morphine had no noticeable effect. An increase to 300 mg per kg resulted in stimulation of salivary secretion, some incoordination in the extremities, increased activity, and hyperirritability to handling or to any direct contact stimulus. At dosage levels of 500 to 600 mg, the animal also showed some discomfort as indicated by circling movements and by the rubbing of its ventral surface on the floor of the cage.

In the course of studies on the hereditary obesity of yellow mice first described by Dan-forth, 1 , 2 the following observation was made.

A group of S obese females was permitted unlimited amounts of water and Purina dog chow pellets throughout the experiment. Their food intake was measured at 2-6-day intervals, and the caloric intake per mouse per day calculated from a value (3.50 cal./g) obtained by chemical analysis. At varying intervals they were also allowed to drink for a few days one of the following at a time: 10% and 20% solutions of dextrose, 50% sucrose, olive oil, and human plasma with a protein concentration of 17.3%. The unrestricted daily caloric intake per mouse of each of these (excepting olive oil) was calculated. Simultaneously, identical steps were taken with a group of 3 normal albino female mice.

Fig. 1 shows the results. Briefly, none of these substances caused a gain in body weight of the normal mice; within the limits of error their caloric intake remained practically constant (see below). But the obese mice gained weight in three 4-day periods: 2.2 g with 20% dextrose, 3.0 g with 50% sucrose, and 4.4 g with olive oil. The caloric intake during 2 of those periods increased by about 12 calories and 24 calories (20% and 50% sugar, respectively), enough to explain most or all of the increased body weight as fat deposition. Moreover, when the sugar or oil was withheld, body weight fell to the original level during a few days in which there was an appropriately reduced caloric intake; surprisingly, under the same conditions, there was also a transient reduction of caloric intake cf the normal mice without loss of weight.

Tubercle bacilli die more or less rapidly in solutions in water of glycerol, glycerophosphate, phosphoglycerate, glycerate, lactate, glyceraldehyde, or dioxyacetone. Similar suspension in distilled water in solutions of inorganic salts and in pyruvate survive for a prolonged period.

Three newly isolated strains of bovine tubercle bacilli that were unable to grow on Long's medium grew luxuriously on this medium after the addition of 0.1% pyruvate. Of 3 laboratory strains of “dysgonic” bovine tubercle bacilli, 2 showed a similar behavior. Addition of pyruvate had no effect on the growth of 12 human strains of tubercle bacilli.