Experimental Biology and Medicine

The nutrition of animal cells in tissue culture has been studied with particular reference to amino acids, vitamins, nucleic acid constituents and various accessory growth factors. The optimal concentrations of these substances for the maintenance of cell life in vitro have been established and a completely synthetic feeding solution has been devised. Although this mixture will not support cell life indefinitely, it has been found adequate to maintain chick embryo cells for an average period of 4 to 5 weeks. The mixture is being used as a basal synthetic medium for further studies on the unidentified growth-promoting substances known to occur in natural media.

1. The aureomycin semitivity range of 274 recently isolated group A, B, and C beta hemolytic streptococci was 0.02-0.5 μg/ml.

2. Aureomycin resistance was induced in 23 strains of group A, B, and C beta hemolyticstreptococci by 40 serial subcultivations on aureomycin medlium. The minimum increase in resistance was 2-fold in a group A organism, and maximum increase was 60-fold in a group C organism.

3. Serial transfer on control medium or passage through normal mice partially restored aurmmycin sensitivity to resistant variants.

4. Mouse virulence was decreased in only the 2 group C organisms which had shown the highest degree of aureomycin resistance.

5. In only 1 group B resistant variant was the group specificity lost after the develop ment of aureomycin resistance.

6. Growth on maximum concentrations of aureomycin stimulated transient conversion from clear to green hemdysis in organisms normally producing only a dear beta zone. Conversely, group C organisms with normal green hemolytic characteristics lost their green hemolytic zones when gmwn on maximum aureumycin concentrations.

8. Reduction in streptokinase production occurred in 2 of 5 group A and C resistant strains itnd diminished ribnuclease activity in 2 of 7 group A resistant variants. No change in stmptolysin S or pmteinase activity was observed.

9. The C203 MV strain of group A streptococcus failed to acquire aureomycin resistance after repeated exposures to sublethal amounts of the antibiotic either in mice or embryonated hens' eggs.

It has not yet been reported that beta hemolytic streptococci acquire resistance to aureomycin in vitro. Paine, Collins, and Finland( 1 ) reported that one strain of Streptococcus mitis and one strain oif Str. pyogenes acquired no increase in resistance to aureomycin even after 60 subcultivations on antibiotic-containing medium, Harvey, Mirick, and Schaub( 2 ) demonstrated an 8-fold increase in resistance to aureomycin by a microaerophilic streptococcus and a 16-fold increase in resistance by a Str. faecalis. These organisms were isolated from 2 patients during a relapse after several weeks of treatment with aureomycin for subacute bacterial endocarditis. The resistant Str. faecalis grew more slowly and formed smaller colonies on blood agar. The resistance of this strain was not permanent since streptococci of the original sensitivity reappeared after several transfers on blood agar.

The present study was undertaken to compare the in vitro and in vivo development of aureomycin resistance by group A, B, and C beta hemolytic streptococci, and to determine whether or not aureomycin altered the biology of the streptococcus in a manner similar to that produced by penicillin, ( 3 4 5 6 ) streptomycin,( 7 ) and bacitracin.( 8 , 9 )

Materials and methods. Resistance to aureomycin was induced in 23 strains of beta hemolytic streptococci: 8 of group A, 8 of group C, and 7 of group B. Parent strains and their designations are the same as those used in preceding studies. ( 3 4 5 6 7 ) Aureomycin sensitivity was determined by the whole plate method and was taken as the maximum concentration of antibiotic that permitted'good growth. Bacto heart infusion agar (Difco) containing 5% defibrinated sheep's blood and varying concentrations of aureomycin HOI (Lederle A377, Lot No. 7-8411) were used in 100 mm Petri dishes. Fresh dilutions of the yellow crystalline salt were prepared daily in physiological saline at a pH of 3.1.

Human gastric juice contains small amounts of vitamin B₁₂ or its equivalent as assayed by L. lactis. Crystalline vitamin B₁₂ was added to gastric juice and the solutions were incubated at pH 2, 3 and 7; there were no observed changes in the absorption spectra or in the microbiological activity over a 22-hour period. Again, crystalline vitamin B₁₂ was added to gastric juice, and after incubation at pH 2 and 7 at 37°, much of the vitamin B₁₂ was recovered. No evidence was found for a chemical reaction between vitamin B₁₂ and the intrinsic factor which results in a gross structural change in vitamin B₁₂. A possible reaction between vitamin B₁₂ and the intrinsic factor in which there was a very small structural change of the large vitamin B₁₂ molecule, and which could produce as little as 5 μg of orally active product is being investigated. A concentrate prepared from beef muscle having vitamin B₁₂-like microbiological activity was found to be hematopoietically active when administered by the parenteral route to pernicious anemia patients; thus, the extrinsic factor acts like vitamin B₁₂. These results support the previously derived hypothesis that the effect of the intrinsic factor is due to its ability to promote the absorption of the small amounts of vitamin B₁₂ or its equivalent which occur in natural foods. The mechanism of this enhancement is being studied further.

Cysteine greatly reduces the sensitivity of rats and mice to lethal amounts of x-rays delivered at either high or low dose rates provided that the amino acid is given before the exposure. Rather comparable protection is obtained when cysteine is injected intravenously in rats immediately or 1 hour before irradiation with 800 r (90% reduction in the 28-day mortality). Injection immediately after exposure or 6 or 24 hours before is without influence. Cysteine also improves survival significantly when it is given orally 30 to 60 minutes prior to the irradiation. Glutathione (I.V. but not oral) can also diminish radiation toxicity. Cystine, methionine, ascorbic acid, and sodium sulfide do not alter survival of the irradiated animal. Results with colloidal sulfur are equivocal. The possible significance of these observations is discussed.

Vit. B-12 has been shown to reduce blood levels of non-protein nitrogen and of 7 individual amino acids from the levels found in vit. B-12 deficient chicks.

Chicks given vit. B-12 grew more rapidly and utilized feed more efficiently than B-12 deficient controls, although the latter had higher blood levels of amino acids.

Vit. B-12 appears to function in metabolism by enhancing utilization of circulating amino acids for building fixed tissues.

Aureomycin. injected intraperitoneally starting 24 hours after intranasal infection of cotton rats with the virus of primary atypical pneumonia almost completely inhibits the development of pulmonary consolidation when given in daily doses of 1 mg, and is partly active at a dosage of 0.5 mg per day. Therapeutic activity with doses of 2.0 to 5.0 mg daily was also demonstrable when the beginning of treatment was delayed for 5 to 6 days.

Chloromycetin in doses of 5 to 20 mg per day had slight and irregular effects on the virus in cotton rats under the same experimental conditions.

Aureomycin inhibits the growth of the atypical pneumonia virus in chick embryos when injected into the yolk sac in two doses of 1 mg each 2 and 48 hours after amniotic inoculation of the virus.

The iodoacetate index was determined on 700 apparently healthy men and women of various age groups and found to be greater than 9.3 in all instances except in six cases in which normal values were obtained on repetition of the test. Seventy-five percent of the persons tested were less than 24 years of age.

The changes in endochondral ossification of the tibia accompanying acute pantothenic acid: deficiency in young rats have been investigated. Twenty deficient animals, 13 male and 7 female rats, were autopsied together with their controls at ages ranging from 3 to 16 weeks. The histologic changes observed were decreased growth of the tibia with marked impairment in chondrogenesis, osteogenesis and hematopoiesis.

Four stages of morphologic changes were observed. Stage I, in which the ages of the rats ranged from 21 to 33 days, was characterized by unusual trabecular resorption, decreased osteoblast proliferation and edema of the bone marrow. In Stage II the ages ranged from 33 to 63 days and marked retardation of osteogenesis with some calcification in the epiphysis and blunt diaphyseal trabeculae was observed. The dominant feature of Stage III, from 45 to 72 days, was the beginning of epiphyseal cartilage calcification and the cessation of osteogenesis. Stage IV, from 78 to 109 days, was characterized by complete absence of trabeculae and the formation of a heavy layer of sealing-off bone below the epiphyseal cartilage.

The role of pantothenic .atid and off additional factors such as pteroylglutamic acid, unknown vitamins and caloric restriction in these histologic changes is discussed.

Over a 3-year period the lipotropic agent choline was effective in significantly reducing the mortality rate due to recurrent coronary thrombosis with myocardial infarction in a series of 115 patients with proven coronary atherosclerosis.

1. Portal origin of blood in abdominal collateral veins of patients with portal hypertension may be demonstrated simply and rapidly by feeding fluorescein or thiocyanate and finding higher concentrations in the abdominal veins than in the antecubital veins.

2. Seven out of 12 patients with cirrhosis gave positive tests for the portal origin of abdominal collateral vein blood. Patients with acites are more likely to give positive results than those without ascites.

3. Evidence is presented for the acetylation of PABA by the human intestinal tract.

Excised fragments of gills of Mytilus edulis L., a pelecypod mollusk, were irrigated with artificial sea water containing P³². The rate of uptake of phosphate in the presence of 0.001 M inhibitors was compared with the control rate. Inhibition of phosphate uptake in the presence of cyanide, sulfide, and iodoacetate, and the lack of inhibition by azide and fluoride, are considered from the point of view of available knowledge of the penetration of such substances into cells. Cyanide and sulfide, being salts of weak acids, penetrate the cells and probably affect the turnover rate of intracellular phosphate. The lack of effect with fluoride is consistent with its existence in the completely dissociated state. Iodoacetate, also completely ionized, may be effective at the cell surface, affecting the permeability of the cell to phosphate. The apparent rise in the rate of phosphate uptake in the presence of cysteine remains unexplained.

The sulfhydryl content of serum proteins has been quantitatively determined through an adaptation of an amperometric titration. Among normal adults, the values noted, expressed as milligrams of cysteine, were uniform, reproducible and averaged 6.5±0.2 mg per 100 ml serum or 5.5±0.3 mg per gram serum nitrogen. The albumin component of serum contained 80% of the total sulfhydryl. The addition of desoxyribonucleic acid, creatine or formaldehyde did not change the sulfhydryl content of the serum. The addition of an SH reagent such as para-chloro-mercuri-benzoate abolished all reactant groups measured with this method.

Sera obtained from patients with various diseases showed a significant reduction in the sulfhydryl available for this titration. This quantitative reduction in sulfhydryl was still evident when corrected for differences in the albumin and globulin components and thus represented a qualitative change in the serum proteins. At the present time, the alteration in the sulfhydryl content of serum or its components is not characteristic enough to permit specific differentiation among the neoplastic, metabolic, or infectious diseases. The possible application of serial serum sulfhydryl determinations in animals and patients may be of value as an objective indicator of response and for study of the mechanism of action of chemotherapeutic and other agents.

A new method of demonstrating the union of antigen and antibody is described. The test is performed'on the surface of filter paper and submitted to the washing effect of a stream of isotonic NaC1 solution. While normal serum is washed together with the antigen, the immune serum produces the fixation of the antigen to the paper at the place where it was deposited.

Regarding its specificity, it has been observed that this test, studied only in brucellosis, produces responses which so far have agreed with the usual agglutination reactions.

Following the intravenous administration of testicular hyaluronidase to albino rats, the following changes were noted:

1. The maximum concentration of the enzyme was attained within 5 minutes and diminished rapidly, so that at one hour it was no longer detected by turbidimetric methods.

2. Approximately 2% of the hyaluronidase appeared in the urine in an active form.

3. The plasma hyaluronidase inhibitor level was depressed for 8 hours and returned to normal withini 24-72 hours.

4. No significant proteinuria resulted.

The results from this preliminary study demonstrate the capacity of astatine to induce an extreme degree of radiation injury of the thyroid gland in the rat without apparent involvement of the adjoining parathyroid gland.

Presidon and pentobarbital administered intraperitoneally, prevent the convulsions produced by procaine in dogs, rabbits, guinea pigs, and mice.

Presidon confers protection against the convulsive effects of procaine at one-fourth to one-third the hypnotic dose, whereas the protective dose of pentobarbital is from one-half to the full hypnotic dose.

It has been observed that the degree of inhibition of tubercle bacilli by subtilin in vitro parallels the degree of growth dispersion of the organisms. In a medium permitting diffuse growth, subtilin was highly active. Conversely, in a medium in which growth was clumped, whether because of omission of Tween or from antagonism of Tween by serum, no significant inhibition of growth by subtilin was demonstrable. When serum from rabbits injected with subtilin was tested against tubercle bacilli in a medium containing sufficient Tween, the observed antituberculous activity paralleled the activity against streptococci and staphylococci. If the amount of Tween added to such tests was insufficient to produce diffuse growth of tubercle bacilli, subtilin activity was greatly diminished or absent.

So long as tadpoles are maintained at temperatures from 0° to 5.5°C the destructive effects of x-ray exposure are very slight in the hematopoietic cells. The temperature of tadpoles at the time of x-ray treatment makes little if any difference in the destructive processes that develop later.

These destructive eifects depend primarily upon the temperature to which the tadpoles are subjected after irradiation.

Thyroid gland of guinea pig was subjected to low temperatures (−70°, or −190°C) and subsequently autotransplanted. In some instances pituitary thyrotropin was administered before or after transplantation in order to increase the growth potential of the transplant and overcome the injurious effects incident to transferring the tissue. There was one successful transplantation of thyroid gland in 12 attempts after exposure at −70°C and one parathyroid transplant. Eight viable thyroid grafts in 12 attempts were obtained after immersion at −190°C. The significance of these findings with respect to rapidity of freezing is discussed in their relation to the mechanism of survival of mammalian cells and tissues exposed to extremely low temperature. The relation of these results to evidence purported to indicate a viral transmission of transplanted frozen-dehydrated tumor material is also pointed out.

Simultaneous tests for agglutinins and complement-fixing antibodies for Brucella were carried out on the serum and cerebrospinal fluid of 13 patients having brucellosis. Complement-fixing antibodies and agglutinins were demonstrated in the cerebrospinal fluid of one patient with chronic encephalomeningitis and in one patient with a paravertebral abscess associated with Brucella spondylitis. A third patient having chronic Brucella encephalomeningitis had agglutinins in the cerebrospinal fluid. In 13 cases these two serologic tests when performed with cerebrospinal fluid have not aided in resolving the problem of the incidence of organic brain damage in patients with brucellosis.

The liver of rats maintained on a 50% casein diet retains its ability to inactivate estradiol in spite of avitaminosis B or inanition. However, animals on a 5 or 15% diet under the same conditions cannot inactivate estradiol.

Aluminum hydroxide gel (amphojel) reduced the level of aureomycin in the serum of 10 individuals within 24 hours after the gel was added to each dose of aureomycin. These findings indicate that the two drugs should not be used together.

Young white rats were fed diets containing 2% DL-lysine HCl and different levels of DL-hexahomoserine for 28 days. When the diet contained 0.08-0.33% DL-hexahomoserine, the growth rate was reduced without simultaneous development of anemia. When the diet contained 1% DL-hexahomoserine, growth was almost completely inhibited and the majority of the animals showed definite symptoms of anemia.

Mengo encephalomyelitis (ME) virus was readily propagated in the embryonated hen egg. Infected embryos showed extensive haemorrhages and were usually dead within 48 to 96 hours after inoculation. Sections showed widespread degenerative changes and haemorrhages throughout the entire embryo. The embryo was susceptible to inoculation of ME virus by the amniotic, yolk sac, and allantoic routes and onto the “dropped” chorio-allantoic membrane. After amniotic inoculation, there was a slight lag in virus multiplication, after which rapid multiplication occurred and titers of virus of 10^-7 were obtained in suspensions of whole embryos by 48 hours after inoculation. Similar titers were obtained in suspensions of whole embryos and yolk sacs after inoculation by the amniotic or yolk sac routes. The embryo was less susceptible to inoculations by the allantoic route. Irrespective of the route of inoculation, the titer of virus in the allantoic fluids was always less than 10^-2 up to 48 hours after inoculation. The behavior of ME virus in the embryonated hen egg was similar to what has been described after inoculation of eggs with Columbia SK, MM and encephalomyocarditis viruses.

The effect of DOCA on the insulin sensitivity of 6 insulin insensitive patients was studied. Five showed a marked increase, and one an equivocal increase in insulin sensitivity.

(1) In 3 experiments, systolic blood pressure measurements were made on 27 control rats and on 31 animals watered with hypertonic sodium chloride solutions for a period of 6 weeks.

(2)The animals watered with hypertonic saline solutions developed an arterial hypertension after a latent period of one to 4 weeks. At autopsy this was found! to be associated with an hypertrophy of the heart and kidneys relative to body weight.

(3)Some possible mechanisms for the development of this hypertension are discussed.

(4)The substitution of hypertonic sodium chloride solutions for the drinking water affords a simple, inexpensive, and dependable method for the production of arterial hypertension in the rat.

It has been shown that the adult female rat, whose growth curve has “pla-teaued”, can be made to develop an acute pantothenic acid deficiency when stimulated to grow rapidly by the continued injection of anterior pituitary growth hormone. The deficiency, so produced, develops more rapidly and is more severe if the deficient diet is high in fat. It is concluded that pantothenic acid is required for the synthesis of protein in the body.

Using erythrocytes tagged with radioactive phosphorus, the percentage replacement of blood in replacement transfusion can be determined. The values obtained agree well with mathematically derived values.

The complement fixation test using filtered muscle antigen can be employed for differentiation of the Coxsackie virus into serologically distinct types and for determining the presence of antibodies in human sera.†

The short column technic described is an adjunct to the chromatographic methods previously published(2) and makes avalibale a complete procedure for the preparation of certain subcellular particulates from the crude tissue to the purified fractions without recourse to the centrifuge at any stage.

Some of the advantages of the short column technic for the preliminary preparation of cytoplasmic fractions are speed, simplicity of equipment and operation, an adaptability to a controlled gas phase during manipulation, and a sharp separation of cytoplasmic elements from nuclei and viable cells.

Commarized insulin added to fresh pancreatic juice can be extracted from the juice and its effect noted on the glucose uptake by the diaphragm in quantities as small as 0.0045 unit per milliliter juice.

In extracting pancreatic juice alone, no clear-cut demonstration of insulin effect (i.e., an increase in glucose uptake of 50-100%) could be obtained, even though the extracts of as much as 7 milliliters of fresh juice per milliliter of substrate were tested. (The 20 to 25% averaged increase of glucose uptake by pancreatic juice is not of statistical significance.)

Thus if insulin is present in pancreatic juice, the amount extractable and detectable by our methouds must be less than .005/7 or .0007 unit per milliliter of juice.

When strychnine is applied topically to the cat's cerebellum, injected intravenously or into the dentate nucleus in doses of .018 to .022 mg per kilo, or injected into the cerebellar cortex in doses of 0.3 mgr the electrocerebellogram develops rhythmical waves with a frequency of 10-30 per second and a voltage of 100 to 400 microvolts occurring in periodic discharges of 30 seconds to 3 minutes duration and recurring at intervals of 1 to 3 minutes for 30 minutes to an hour.

Rabbit serum, contrary to previous observations, contains only one enzyme which hydrolyzes acetylcholine, and this one has the characteristics of a true cholinesterase. In addition, there is present in rabbit serum an enzyme which hydrolyzes only benzoylcholine, and which, since it does not hydrolyze acetylcholine significantly, should not be labeled a cholinesterase. This enzyme may be similar to the “benzQylcholin-esterase” described by Sawyer 10 as being present in guinea pig liver and kidney.

Pyrogen is the only substance which has been reported to increase significantly the renal blood flow in man. However, according to Goldring and Chasis, 1 it is not suitable for therapeutic purposes. Aminophylline seems to be effective only in patients with cardiac failure. 2 No treatment which will relieve renal ischemia has yet been found.

According to Meier et al., 3 Compound 5968 (hydrazino-phthalazine hydrochloride), supplied by Ciba, produces in animals a sustained fall in blood pressure. In man, we found that hypotension did not occur regularly following the subcutaneous injection of 10 mg. However, when measurements of renal plasma flow were performed, using the clearance technic, 1 it became evident that renal hyperemia could be elicited in nearly every subject. A similar effect has also been demonstrated in cats by thermostromuhr measurements. 3

Our data, presented in Table I, demonstrate that this drug can cause increase in renal plasma flow in man varying from 16 to 68%, averaging 38.6%. The glomerular filtration rate, as reflected by the clearance of thiosulphate, is not consistently affected. The filtration fraction is always decreased. In some cases, urine volumes are markedly increased. Blood pressure shows a definite reduction only in some hypertensive patients. Therefore, the influence of the drug upon the kidney is somewhat similar to that of pyrogenic substances. The responses obtained are moderately prolonged, lasting 1 to 2 hours or more. Toxic manifestations are relatively unimportant. Moderate tachycardia may be observed in some cases. Effects of the drug upon cardiac output are being investigated.

From these results, it can be concluded that the drug 5968 (Ciba) is a renal vasodilator in man.

A study has been made of the effect of 7 dyes of the phenazine, oxazine and thiazine series on the in vitro coagulation times of normal and heparinized rabbit plasma. Toluidine Blue, Azure A, Neutral Violet and Neutral Red effectively counteract the anticoagulant action of heparin but the amount of each dye required for this antiheparin effect is critical. At concentrations below the critical level the dyes had little or no effect upon the coagulation system studied. At concentrations above the critical level all the dyes produced a progressively increased incoagulability of the system. This anticoagulant effect of the dyes is related to concentration and the action is on some substance other than heparin.

Five 4-amino-pteridine compounds were studied for toxicity in rats. The 2-thiol-substituted pteridines were not found to be toxic in the doses used. The 2,4-diami-no- 6,7 -dihydroxy-pteridine and 2,4-diamino-pteridine interfered with the growth rate. The latter compound caused crystalline deposits in the renal tubules, and the extent of the deposits correlated well with the body weight effect. No histological effects were noted in the liver and bone marrow,‡ and none of the compounds altered the blood picture.

Cultures of Lactobacillus arab-inous have been stored at room temperature for 32 months lyophilized and for 38 months in Bacto-cooked meat medium (Difco product) . The assay performance of the cultures was not impaired by this treatment, although acid production in the cooked meat medium culture was reduced. It was concluded that these methods of storage may be substituted successfully for frequent subculture with Lactobacillus arabinosus and perhaps other lactic acid bacteria.

A form of progressive, chronic, “right-sided” cardiac failure has been produced in dogs by tricuspid valve avulsion and pulmonary artery stenosis. Three dogs so treated developed congestive failure with elevated auricular pressure and distended veins, decreased work tolerance, hepatomegaly, ascites, tachycardia at rest, and a relatively fixed heart rate during exercise. Studies of 2 of the hearts in the heart-lung preparation indicate clearly that cardiac insufficiency was present.

Rats under the influence of an alarming stimulus (formalin injection) showed decrease in thyroid function, the latter evidenced by diminished uptake of 1-131 per milligram of thyroid tissue. Desoxycorticosterone acetate and adrenal cortical extract failed to influence thyroid uptake of 1-131 significantly.

Electron micrographs of sections cut through tobacco leaves infected with tobacco mosaic indicate that the virus is often present in fibrous masses associated with chloroplasts which are severely affected. Virus can clearly be seen in the cytoplasm of the diseased cells sometimes as single filaments. The nuclei seem unaltered by the infective process and have appeared free of virus. The great length of these filaments, much in excess of 2800A, indicates an end-to-end arrangement of the rods in situ.

Guinea pigs which have been sensitized to tuberculin by the injection of heat-killed tubercle bacilli in paraffin oil show and increased in vitro tuberculin leucocyte cytolysis within 48 hours after a positive intracutaneous tuberculin reaction.

1. The total lymph volume is not measurable, so that the exact amount of activated protein that is present in the body lymph, following intravenous injection of iodinated protein, cannot be calculated. However, it appears from this experiment that a negligible amount is present in the thoracic duct lymph 10 minutes after injection.

2. During the first hour there is a rapid increase in the lymph content of radioactive protein followed by a more gradual rise.

In the mouse the growth potentials of the skin and its appendages are not appreciably affected by biotin deficiency, but normal differentiation and function are impaired. There is a slight increase in mitotic activity in the epidermis and in the sebaceous glands. The pilosebaceous units are blocked by keratinized debris and little or no sebum is released upon the surface of the skin. In the sebaceous cells, the stored lipid droplets are much larger than in those of normal animals. Unlike the sebaceous lipids of normal glands, those of biotin deficient mice are isotropic and negative to the Liebermann-Burchard test. There is abundant cellular fragmentation at the periphery of sebaceous glands and the lipids are apparently phagocytized by histiocytes in the dermis. The melanin-bearing cells around the sebaceous cells, when present, also contain lipid droplets. As a rule, neither histiocytes nor melanin-bearing cells contain demonstrable lipids in the skin of normal mice. There is an increase in the number of mesenchymatous cells, mast cells and leukocytic cells in the dermis of biotin deficient mice.

1. 21-Acetoxy-pregnenolone was selected for a limited study in 7 ambulatory patients with rheumatoid arthritis because in the rabbit synovial membrane screen it was at least as good as cortisone in decreasing permeability and it showed powerful anti-DOCA action on synovial membrane.

2. The effect on the end organ seen in the screen in rabbits manifested itself in each of the seven patients by reduction in swelling, lessening of the pain, and increased motion in the joints.

d-1 Nor-epinephrine (Arterenol) and the N-isopropylnorepinephrine (Isuprel) exert a powerful inotropic action on mammalian heart muscle.

The arginine requirement of the chick was estimated in relation to the protein level in the diet, from 15 to 30%. The requirement was found to be approximately 6% of the total protein in the diet, at each level of protein.

The development of experimental exophthalmos in the guinea pig is dependent on the accumulation of large quantities of intercellular ground substance and water in the connective tissue of the orbital contents. This is evidenced by the presence of considerable amounts of metachromatic material, of which hyaluronic acid is an important component, and by an increase in the hexosamine and water content of these tissues.

This is similar to the changes observed in localized myxedema in man, and suggests a common mechanism for the two conditions.

(1) The concentration-action relationships of DC and DCG upon aerobic respiration of rat brain cell suspensions indicate:

(a) Increasing amounts of DC and DCG produce increasing amounts of inhibition This relationship is reasonably linear plotting the logarithm of the amount of DC against per cent of inhibition produced.

(b) With increasing amounts of DC a longer period of time is required to attain maximal inhibition while a comparable effect is more rapidly attained with equimolar amounts of the water-soluble conjugate, DCG. The effect, once attained, remains constant without further increase or release of the inhibition.

(c) By this bio-assay the water-solubility of DC at 37.2°C is approximately 3.6×10^-3 Molar.

(2) These data suggest that, under the conditions used, the relatively large amounts of steroid necessary to produce inhibition of aerobic respiration in vitro entirely penetrate or are adsorbed by the affected system. These amounts exceed the concentrations of steroid which will affect other systems in vitro or that occur in living organisms. This does not necessarily exclude the possibility that steroidal inhibition of aerobic respiration may be of significance in the intact organism.

By labelling anti-rat-lung serum with radioiodine and injecting it into rats, it was found that the anti-rat-lung serum contained antibody which localized specifically in the lung. It also contained antibody which cross-reacted with and localized in the kidney. Anti-rat-brain, -liver, and -heart sera were also investigated and showed no specific localization, but these experiments were considered inconclusive.

The data presented show that kidney tissue is not unique in being able to localize antibody from tissue homologous antiserum.

The carcinogen, 2-acetylaminofluorene, produced a riboflavin deficiency in rats, reducing the liver riboflavin below control values. Increasing the riboflavin content of the diet prevented the appearance of the deficiency, keeping the liver riboflavin at normal levels. In the absence of protein in the diet, however, the increased riboflavin did not prevent the development of the deficiency.

The LD50 for pure eugenol, administered to rats by stomach tube, and without subsequent reaspiration, was estimated to be 1.8 ml (1.93 g/kg). Any use of this datum as a guide to safe dosage in man, however, may take cognizance of the fact that the conditions of the rodent studies are considerably more severe than any which might be encountered in clinical studies. In the first place, the rat experiments employed eugenol per se, whereas work with dog and man is done with aqueous emulsions of the irritant. Furthermore, the rat retains all of the eugenol administered, because of its inability to vomit, whereas dog and man can reject part of the irritant in this manner if the dose is excessive, and this affords an additional factor of safety.

The salient features of the toxic manifestations of eugenol in the rat were as follows: Paralysis occurred initially in the hind legs and the lower jaw. The forelimbs were unaffected unless general prostration or coma ensued. In those animals in which the acute symptoms subsided, the animals remained lethargic, showed signs of urinary incontinence and frequently hematuria, and gave evidence of impaired function of the hind legs for several days. Gross and microscopic observations of the tissues suggested that profound changes in fluid distribution had occurred in response to acute irritation of the gastrointestinal tract. The net impression of the effect of the eugenol, from the microscopic evidence, was essentially that of circulatory collapse with resultant congestion.