Experimental Biology and Medicine

This report describes a stable marker chromosome for the transplantable Dunning rat leukemia R3149 in Fischer rats.

The karyotype of this acute leukemia was female and pseudoeuploid with a giant sub-metacentric marker chromosome.

Quantitative x-ray diffraction analyses of histological tissue zones representing progressive stages of endochondral and periosteal bone formation reveal that both amorphous and apatitic calcium phosphate salts appear throughout the entire mineralization process. The amorphous-crystalline mineral sequence exhibited by these tissue zones suggests that calcification in both cartilage and bone may entail an initial deposition of amorphous calcium phosphate followed by the conversion of this kinetically metastable, non-crystalline mineral phase to crystalline apatite.

To investigate a possible relationship between fat emboli and hypoxic decompression, rabbits were exposed to simulated altitudes of 30,000 feet and 60,000 feet in a decompression chamber. Normal rabbits, cholesterol-fed rabbits, and ethionine-treated rabbits were decompressed and autopsied, and a search was made for fat emboli in the lungs. None of the animals developed a significant number of emboli compared to control animals that were similarly treated but not decompressed. These data suggest that hypoxic decompression in the rabbit will not result in the formation of fat emboli, even in the presence of fatty liver or hyperlipemia. The factors responsible for fat emboli in fatal human decompression sickness remain unclear.

Pulmonary hyaline membranes developed in guinea pigs exposed for several days to 95% oxygen at atmospheric pressures. This process was accompanied by increased antiplasmin activity in plasma and decreased pulmonary plasminogen activator activity. The latter phenomena may promote the formation of alveolar hyaline membranes by interfering with enzymatic removal of fibrin derived from pulmonary exudation.

We wish to acknowledge the devoted assistance of G. Hauser, H. B. Lassman, I. B. Mink, and V. Zaniewski.

Isolated lungs of dogs and cats have been shown to retain their diffusion capacity for many hours(1,2), and excised dog lungs have also displayed a fair ability to function as human kidney(3). In view of the durability and adaptability of pulmonary tissue, an investigation was performed in which donor lungs were attached to the circulation of live animals, and the effects of this shunt upon blood oxygenation and upon survival of the recipient animals were studied.

Although time and circumstances did not permit additional experiments for resolution of the problems involved in this procedure, this report is presented in the belief that the findings may be of use to other investigators working in this field.

Experimental procedures. Mongrel laboratory dogs were used to supply the donor lungs. The animals were anesthetized (pento-barbital 30 mg/kg i.v.), heparinized (10 mg/100 ml blood), and exsanguinated; the chest was opened through a midsternum incision. The thoracic aorta was ligated in several places in order to block fluid outflow from the bronchial arteries; ligatures were also placed on the superior and inferior venae cavae, and on the azygos vein. The pulmonary artery was cannulated via the right ventricle, and a second cannula was placed in the left atrium. Polyethylene tubes were attached to the cannulae, the chest walls were sewn together with tubes emerging, and 4 to 6 liters of a 6% dextran-saline perfusion solution were pumped at low flow rate and pressure (less than 20 mm Hg) via the tubes through the lungs for several hours in order to wash out the residual blood and other antigenic substances. Then the pulmonary blood vessels were left filled with the heparin-ized perfusion fluid at a pressure intermediate between umbilical arterial (70 mm Hg) and umbilical venous (10 mm Hg)(4).

The concentrative transport of calcium by everted intestinal loops in vitro and the permeability of the small intestinal wall to this ion were determined in intestinal preparations from vitamin D deficient and vitamin D treated magnesium deficient and control rats. These experiments indicate that concentrative transport of calcium by rat small intestine in vitro is altered by the presence or absence of magnesium in the medium but not by magnesium depletion of the animals. The permeability to calcium of the intestinal preparations was not influenced by magnesium deficiency. The effects of magnesium depletion of the rat and of the presence of magnesium in the medium on concentrative transport of inorganic phosphate by the small intestine in vitro were also studied. Concentrative transport of phosphate by intestinal loops incubated in Mg++ containing buffer was less than that by those incubated in Mg++ free buffer. Intestinal loop preparations from magnesium deficient animals transferred phosphate to a greater extent than those obtained from control rats; this difference was observed both in the presence and absence of Mg++ in the buffer solution. Magnesium deficiency in the rat also reduced the concentration of serum phosphorus and increased the excretion of phosphate in the urine. These findings are consistent with the theory that magnesium influences the phosphate transport systems which control phosphate concentrations in extracellular fluids.

Sexually mature male rats in metabolism cages were adapted to the tube-feeding of a medium carbohydrate diet. Some rats were treated with cortisone acetate and with glucagon-free insulin, separately and in combination and in the presence and absence of implanted tumor. The severity of the glycosuria was proportional to the dose of cortisone. Glycosuria was markedly suppressed by insulin, but there was no suppression of the level of urinary nitrogen in the presence or absence of cortisone. Cortisone suppressed the growth of Walker Carcinoma 256 and of Jensen Sarcoma and caused atrophy of the adrenals, thymus and partial atrophy of spleen. None of these effects of cortisone overdosage were prevented by insulin.

Red cell content in liver was measured in normal and in shocked dogs using two independent methods of measurement. The mean concentration of red cells in the liver of a series of dogs under control conditions was 0.027 ± 0.002 (SE) ml/g liver tissue. After hypovolemic shock and retransfusion of the shed blood, the red cell content in liver tissue increased to an average of 0.052 ml/g. Under control conditions, the red cell label of peripheral blood was mixed adequately with the red cells in liver tissues. After shock, differentially labeled red cells mixed more slowly with the red cells in the liver and in some instances did not become equilibrated during the course of the experiments. This suggests a pool of noncirculating or slowly circulating red cells in the liver after shock. These changes may affect adversely the precision and interpretation of blood volume measurements made by conventional techniques and sampling schedules.

Treatment of FV infected mice with homologous antibody at different stages of viremia resulted in a significant reduction of infectivity titers and of splenomegaly with a concomitant increase in life span. When treatment was begun 4 hours prior to virus infection the animals survived longer than 120 days with no apparent symptoms of the disease. The therapeutic effects achieved in animals treated after virus infection are considered to be due to the ability of the injected antibody to neutralize extracellular virus as it is liberated which thus delays the induction of the Friend disease.

Sheep red blood cells have been freeze-cleaved and the exposed fracture faces have been replicated with carbon, shadowed at a 45° angle with carbon-platinum, and examined in the electron microscope. The cytoplasm appears to be filled with tightly packed particles, the replicas of which measure approximately 120A° in diameter. In large areas, these particles occasionally form extended linear configurations which may be straight or curved. Typically, large numbers of these lines are parallel to one another. It is suggested that the lines in these replicas may represent areas of molecular packing within the cytoplasm of sheep erythrocytes. The possibility that the individual particles may represent a contaminant adsorbed onto the cold freshly cleaved surface prior to replication or that the orderly pattern seen in replicas may be an artifact introduced during glycerination, freezing, or cleaving cannot be entirely eliminated by this data.

The authors thank Mrs. Marilyn Schaefer for typing the manuscript and Miss Joyce Jakobsen for preparing the illustrations.

Nematocyst toxin from Physalia physalis, when injected intravenously into dogs, activated ectopic cardiac pacemaker centers and induced “re-entry” extrasystoles; these effects could be reversed by infusion of KCl. After injection of a fatal dose, these arrhythmias progressed into ineffectual ventricular beats and cardiovascular collapse. In addition to cardiac arrhythmias, sub-lethal doses of toxin caused increased systemic blood pressure and cardiac output, elevation of serum K, decrease of serum Na, elevated breathing rate, and hemolysis.

Seven virus strains, all isolated from forest mosquitoes collected in eastern Trinidad between 1955 and 1959, have been shown to represent 3 unrelated new viruses, designated Bushbush, Ieri and Lukuni. Neutralizing antibody to Lukuni virus has been demonstrated in human serum. No relationship has been found between either Bushbush or Ieri virus and recognized arboviruses, but Lukuni virus has been found to be related to Anopheles A virus in complement-fixation and neutralization tests. Some of the biological properties of the 3 viruses are described.

The technical assistance of Fitzroy James, Shaima Ali, Harold Drysdale, Raymond Manuel, Desmond Seupersad and Enrique Preddie is gratefully acknowledged.

A reliable plaque assay system has been developed for several recently isolated frog viruses. Cell lines from reptiles, amphibians, and fish were tested for their ability to be maintained under various solid overlay materials. Starch gel was found to be the most useful overlay material. The most sensitive assay system was shown to be Terrapene heart (TH-1) cell culture.

Oral administration of 500 IU of vitamin D₂ or vitamin D₃ to rachitic rats resulted in the elaboration of a Ca-binding factor which could be detected in the supernatant fraction of duodenal mucosal homogenates at 72 hours after administration of the vitamin. This factor was found to be associated with the initial or protein containing portion of the supernatants after percolation through Sephadex G-25; this indicates that the factor is a macromolecule, presumably a protein. Further separation of the supernatants by Sephadex G-100 gel filtration showed that the increased binding activity seen after vitamin D administration was mainly associated with only one of the protein peaks present in the Sephadex G-100 fractions. This particular peak was much less prominent in the fractionated supernatant of the mucosal homogenates from rachitic rats. The results suggest that the vitamin D-dependent binding activity is due to the presence of a specific calcium binding protein in the supernatant fraction of duodenal mucosal homogenates from vitamin D-treated rachitic rats.

The authors gratefully acknowledge the technical assistance of Mr. Philip Shapiro and Mrs. Gabriele E. Collins.

The paradoxical insusceptibility of mouse cells in vitro to mouse-lethal coxsackievirus A6 was investigated further by infection of mouse fetal cells and newborn mice with RNA extracted from coxsackievirus A6 and mengovirus. Mengovirus RNA induced viral replication in vitro and lethality in vivo, while coxsackie A6 RNA failed to do either. Intact coxsackievirus A6 was also incapable of multiplication in non-trypsinized suspensions of mouse fetal tissue. These studies suggest that loss of viral receptor sites on mouse fetal cells, subsequent to dispersion by trypsin and cultivation, does not explain the insusceptibility of mouse cells in vitro to coxsackievirus A6. The insusceptibility of mouse cells in vitro may represent either disruption of the virus-synthesizing potential of cultivated cells, rather specific in nature, or the biologic integrity of the viral genome.

The authors would like to express their sincere gratitude to Dr. Leroy C. McLaren for advice concerning the initial scope of this work, to Dr. Ernest D. Gray and Mr. Joseph Ferretti for consultation regarding biochemical methods, and to Dr. David T. Imagawa for review of the manuscript. Miss Ann Barna provided stenographic assistance in the preparation of the manuscript.

Adenosine triphosphate and related compounds inhibit hemolysis in the acid and acid-thrombin tests of paroxysmal nocturnal hemoglobinuria. The mechanism of action is related to the capability of such agents to chelate a heavy metal (Mg) which is essential for these in vitro hemolytic tests.

The chelating property of these nucleotides rather than their high free energy, as usually presumed, may also explain their inhibitory action in other in vitro hemolytic systems.

We thank Dr. Daniel Turner for his technical advice, and Mrs. Alma Staffin and Miss Shirley Wilcher for their technical assistance.

The action of dactinomycin on cellular RNA synthesis and vaccinia virus multiplication in HeLa, mouse embryo (ME), African green monkey kidney (GMK) cells in culture were compared. Approximately a 10-100 fold higher concentration of dactinomycin was required in GMK cells as compared with HeLa cells and ME to equivalently suppress the cellular RNA synthesis and vaccinia virus multiplication. Also, dactinomycin was approximately 100-fold less toxic for GMK. The higher concentrations of dactinomycin required to inhibit GMK as compared with HeLa cells is probably due to a lower uptake of the antibiotic by the GMK cells.

Human erythrocyte inorganic pyrophosphatase rapidly lost activity by incubation above 37°C. The enzyme was protected against heat inactivation by pyro-phosphate, magnesium and cysteine. Dilute enzyme preparations were more labile than concentrated ones. The reaction catalyzed by pyrophosphatase was inhibited by nucleotide triphosphates. Inosine triphosphate served as substrate for stroma-free hemolysates.

The distribution of histamine and mast cells in the fundic, corporal and antral mucosa of the canine stomach was assayed. Twelve areas from each of 4 stomachs were studied. Mast cell profiles of these areas showed the cell population to be similar in all mucosal areas, although the greatest number of cells occurred deeper in the glandular mucosa of the antrum than in the glandular mucosa of the fundus and corpus. In contrast, the amount of histamine in the antral mucosa (73.3 ± 4.9 μg/g) was roughly one half of that noted in fundic and corporal mucosa (133.6 ± 1.4 μg/g), a difference which is statistically significant, (p<.05). One possible explanation for these observations is that the major portion of corporal and fundic histamine is not associated with mast cells.

Experiments were performed on segments of terminal guinea-pig ileum utilizing differential alpha and beta adrenergic receptor site blocking techniques. Phenyl-ephrine caused a contraction of this segment of guinea-pig intestine which resembled a pure alpha receptor site response. Both epinephrine and noreinephrine produced biphasic responses which follow a pattern of alpha excitation followed by beta inhibition while isoproterenol elicited a pure beta inhibitory action. The use of phenoxybenzamine as an alpha blocking agent abolished all excitatory responses while augmenting beta actions. Propranolol, through beta adrenergic receptor site blockade, potentiated alpha excitatory actions. The results are discussed in terms of the guinea-pig terminal ileum possessing an unique alpha adrenergic receptor mechanism which is excitatory in nature.

The authors thank Dennis Townsend and Sarah Irvine for technical assistance.

By radioimmunoelectrophoresis, antibodies associated with γ₁, γ₂ and γM globulins and 2 unidentified components, U₁ and U₂, were detected in rat antisera to BSA and BGG. When rats were immunized with these antigens adsorbed to bentonite or included in complete Freund's adjuvant, the sequence of the immune response in rats involves first the formation of γ₁ antibody followed upon further immunization by γ₂ antibody. Generally, γM and the unknown components, U₁ and U₂, only appeared in hyperimmune sera.

DNA, isolated by 2 methods from purified A. marginale, was found to possess average molar ratios of A/T and G/C of 1.9 and 2.65, respectively. There was also a lack of increase in hyperchromicity of isolated DNA in thermal denaturation studies. Attempts to isolate RNA or to detect U were unsuccessful.

The results of these studies show that lipogenesis is enhanced in liver and adipose tissue of intact rats as a consequence of meal-feeding. Fatty acid synthesis was approximately 200-fold higher in adipose tissue and 9-fold higher in liver of meal-fed as compared to nibbling rats. Increases in fatty acid radioactivity in adipose tissue, liver and serum were observed between 6 and 9 hours after the initiation of the meal. A possible explanation for these changes is proposed. Serum glucose and serum liver total lipid and cholesterol levels were also determined and no significant alterations due to meal-feeding were noted. From the observed rates of glucose-U-¹⁴C incorporation into liver and adipose tissue fatty acids an estimate was made of the relative importance of these tissues as sites of fatty acid synthesis. These calculations suggest that in the nibbling rat 50-90% of the fatty acids are synthesized in adipose tissue whereas when fatty acid synthesis is stimulated by meal-feeding, adipose tissue apparently accounts for about 95% of the total fatty acids synthesized.

The administration of 1 to 5 mg of cortisol to 1 kg rabbits markedly suppressed interferon production by E. coli endo-toxin. Single doses of cortisol, up to 25 mg, did not decrease circulating interferon levels following inoculations with Newcastle disease virus. This type of interferon was only partially suppressed with multiple injections of 250 mg. Thus, administered cortisol inhibited more readily the production of endotoxin-in-duced interferon than the virus-induced counterpart. Adrenalectomy markedly potentiated the production of interferon by endotoxin. Adrenalectomized rabbits inoculated with endotoxin produced serum interferon to a mean titer of 1:388, while the mean titer in control animals was 1:28, suggesting that endogenous steroids suppress the interferon esponse to endotoxin.

Mrs. Linda Carcione offered excellent technical assistance. One of us (Miss Catherine DeAngelis) was a recipient of a summer research fellowship under NIH grant 5416-05.

Satisfactory monolayer cultures were produced after freezing bovine embryonic kidney (BEK) and chicken embryo fibroblast cells in the presence of 7.5% DMSO in the storage medium and storage for varying lengths of time in liquid nitrogen vapor. The sensitivity of such cultures to selected viruses remained undiminished after 6 months of such storage. BEK cells frozen after initial growth as primary monolayer cultures generally provided satisfactory mono-layer cultures within a relatively short time (2 to 4 days) as compared with BEK cells which were frozen after fresh procurement from tissues (5 to 8 days).

The authors wish to acknowledge the excellent technical assistance of Mr. Horace C. Turner and Mr. Monty Bell.

Unilateral renal hypertension was produced in rats by constricting the aorta just above the ostium of the left renal artery. This caused a sharp reduction in renal arterial pressure which usually remained below 80 mm Hg for 1 to 2 weeks. During this interval the renal vein blood contained a potent vasopres-sor agent and an elevated content of renin. By 2 weeks the left renal arterial pressure had returned to normal levels in most animals and this was associated with disappearance of the vasopressor agent from renal vein blood as well as a marked reduction in the output of renin. Not a single rat was encountered in which a low renal arterial pressure persisted indefinitely or in which the kidney continued to produce excess vasopressor material or to discharge an increased amount of renin. These findings indicate that(1) in unilateral renal hypertension the renal arterial pressure probably governs the release of renin by the kidney and that(2) low renal arterial pressure and hence increased activity of the renin-angiotensin system are limited to the early or acute stage of renal hypertension.

The fatty acid composition of L-strain mouse fibroblasts growing both in serum and in lipid-free synthetic medium was measured. Linoleic acid comprised about 17% of the total fatty acids in cells grown on serum-supplemented medium and about 6% in cells grown for prolonged periods in lipid-free chemically defined medium. In contrast to other fatty acids however linoleic acid was not synthesized from C¹⁴-acetate added to the growth medium. The most probable explanation of these anomalous findings is that cells under deficiency conditions can conserve traces of linoleic acid which are present below detectable levels in the culture environment. No synthesis of positional iso-mers of linoleic acid similar to that which may occur in linoleic acid deficiency in vivo was observed.

The kinetics of inhibition by acetazolamide and sulfanilamide of the formation of carbon dioxide from bicarbonate, catalyzed by partially purified carbonic an-hydrase preparations from bovine erythrocytes, were those of competitive inhibition. This is contrasted with the non-competitive kinetics of sulfonamide inhibition of a number of carbonic anhydrase-mediated hydration reactions. K₁ for acetazolamide was 8.6 × 10^-8 M while that for sulfanilamide was 1.8 × 10^-5 M; these values are similar to those reported for inhibition of CO₂ hydration in the presence of bovine erythrocyte enzyme.

Note added in proof. From other types of evidence, J. E. Coleman (personal communication) has written a reaction scheme similar to that presented here.

To investigate a possible relationship between fat emboli and hypoxic decompression, rabbits were exposed to simulated altitudes of 30,000 feet and 60,000 feet in a decompression chamber. Normal rabbits, cholesterol-fed rabbits, and ethionine-treated rabbits were decompressed and autopsied, and a search was made for fat emboli in the lungs. None of the animals developed a significant number of emboli compared to control animals that were similarly treated but not decompressed. These data suggest that hypoxic decompression in the rabbit will not result in the formation of fat emboli, even in the presence of fatty liver or hyperlipemia. The factors responsible for fat emboli in fatal human decompression sickness remain unclear.

Pulmonary hyaline membranes (HM) described in respiratory distress syndrome due to hyaline membrane disease of infants (HMD), were shown to consist primarily of fibrin (1,2). Several investigators reported deficiencies in the fibrinolysin system of infants suffering from this disease (3-7). We have proposed earlier that plasminogen develops rather late in ontogeny thus leaving premature infants without defense against pulmonary fibrin deposition which may occur from pulmonary exudates developing in relation to birth trauma (8,9). A pathologic entity similar to HMD can be induced in guinea pigs by prolonged exposure to high concentrations of oxygen and aerosolized human amniotic fluid(10,11). The purpose of this study was to investigate possible changes which may occur in the fibrinolysin system during the development of experimental HMD.

Materials and methods. English smooth hair male guinea pigs of 250-350 g body weight from our own colony were used. The animals were kept in air conditioned quarters and allowed Purina guinea pig pellets, green salad leaves and water ad lib.

Hyaline membranes (HM-s) were produced by the following method. Exposure to 95% oxygen was undertaken in specially constructed plastic cages, through which gas flow was continuously maintained at a rate of 5 lit/min. Oxygen concentration in the chamber was periodically checked using a Beckman Model D2 oxygen analyzer. Human amniotic fluid was nebulized into the chamber from a 24850S N.C.G. Nebulizer at a rate of 10cc/ hour using oxygen as propellant. All experiments were performed at atmospheric pressure. Human amniotic fluid was immediately frozen after collection and stored at −70°C.

Thromboplastic potency was determined before use and only batches with significant activity employed. Commercially available bovine amniotic fluid showed little thromboplastic activity.

All guinea pigs subjected to such combined treatment died; the mean time of death was 4.6 days.

Iron excretion in sweat from the forearm of normal subjects and patients with iron overload is similar, with values in the lower range of normal. Deferoxamine has no appreciable effect on sweat iron concentration.

The author wishes to express his appreciation to Dr. Elmer B. Brown for encouragement and valuable criticism during this work, and to Mrs. Barbara Chipman for excellent technical assistance.

An African green monkey kidney cell line (Vero) has been found to support growth of Tacaribe virus. Cytopathic effects (CPE) were pronounced and appeared within 7 days after inoculation with 10^-5 dilution of the virus. They were reproducible in 10 serial subcultures. Tacaribe virus also produced distinct plaques with sharp boundaries in monolayer cultures of Vero cells under agar overlay. A linear relationship was observed between plaque counts and virus concentration. The development of CPE and plaques was specifically inhibited by Tacaribe virus immune rabbit serum, but not by Junin or Machupo virus antiserum. Virus assays obtained by titration endpoints of CPE or by plaque counts in tissue culture were comparable to those obtained by titration in suckling mice. When the cultures were infected with <0.001 PFU/cell, virus yields reached their peaks within 7 days.

A potentially lethal dermatitis observed in chicks fed a high fat (20% vegetable oil) diet can be attributed to the external application of the oil. Topical application of both vegetable and mineral oils to young chicks produced a severe hyperkerato-sis and high mortality within a few days. Mortality was largely prevented and skin damage substantially alleviated by including antibiotics in the oil and minimizing the number of environmental microbes. At least part of the topical fat toxicity syndrome can be attributed to secondary microbial infection.

The effect of section of the corpus callosum on cortical after-discharge patterns elicited by stimulation of the ec-tosylvian cortex in the cat was examined. Such a procedure had no apparent effect on any parameter of cortical after-discharge examined.

The authors gratefully acknowledge the technical assistance of Mr. W. Hardwick.

1. Cells from the thoracic duct of Lewis strain rats when injected into syn-geneic neonatally thymectomized rats are capable of reconstituting the host rats so that they are resistant to polyoma virus-induced tumors and can undergo positive delayed hypersensitivity reactions and Arthus reactions. 2. The protection afforded against polyoma-induced tumors is correlated with the number of small lymphocytes in the inoculum and not with the number of large lymphocytes. That this may be related to some other difference than mere size between cells found in the first day after cannulation and cells drained several days after cannulation cannot be ruled out. 3. Cells from spleens of normal Lewis rats are less effective than equal numbers of thoracic duct lymphocytes in reconstituting thymectomized rats. 4. Thoracic duct lymphocytes from neonatally thymectomized Lewis donors are not only much less numerous but also are much less effective on a per cell basis than lymphocytes from normal animals in reconstituting thymectomized hosts, thus demonstrating a qualitative as well as quantitative difference in the lymphocyte population. 5. Functional restoration is not correlated with histological restoration. 6. The possible mechanism of reconstitution is discussed.

I am greatly indebted to Dr. L. W. Law and Dr. R. C. Ting, Laboratory of Biology, NCI, for very helpful advice. I wish also to acknowledge the technical assistane of Mr. Donald Foor.

Anti-rat hemagglutinins and anti-human gamma globulin factors were demonstrated in sera of guinea pigs and rabbits exposed to repeated thermal injury. Similar factors were also found in sera of some patients with severe skin burns.

The decapeptide, lysyl brady-kinin (Kallidin I) was more effective in increasing vascular permeability in the skin of guinea pigs than was the nonapeptide, bradykinin, or Kallidin II. Bradykinin has been reported to be more active in some other assays measuring pharmacological effects than lysyl bradykinin. All other analogues of bradykinin tested were less active than bradykinin in the permeability assay with the exception of glycyl bradykinin, another decapeptide, which had an equal effect. While an inactive analogue of bradykinin did not block increased vascular permeability induced by a later injection of bradykinin, two active compounds, lysyl bradykinin and bradykinin inhibited the effect of a subsequent injection of either one into the same site.

Synthetic bradykinin BRS-640 was generously provided by Dr. Harry Baird, Sandoz Pharmaceuticals, Hanover, N. J. Dr. Ernest D. Nicolaides generously provided bradykinin analogues, which were synthesized in the Research Laboratories of Parke Davis & Co., Ann Arbor, Mich. Dr. Miklos Bodanszky Dept. of Chemistry, Western Reserve University, has provided helpful criticisms of this manuscript.

Experimental allergic encephalo-myelitis (AE) and adjuvant arthritis (AA) were induced in male Lewis rats. 6-Mercapto-purine and cortisone inhibited both AE and AA, but phenylbutazone and aspirin were effective only in AA. The different effects of these immunosuppressive and anti-inflammatory drugs on clinical signs, organ and body weights, and hematologic changes in AE and AA suggest either that the pathogenesis of the two models is dissimilar or that the acute inflammatory response is of greater etiological importance in arthritis. AE and AA should be useful for differentiating the im-munosuppressive and anti-inflammatory activities of drugs.

1. Mice maintained on an 8% casein diet are capable of synthesizing drug metabolizing enzymes during the period of rapid body growth. 2. Mice maintained on this diet exhibit a prolonged hexobarbital sleeping time at all periods after one week of experimental feeding. 3. The increased sleeping time seen during the earlier period of experimental feeding may result from a less active TPNH generating system than is present in controls. 4. The increased sleeping time seen during the fourth to fifth week on the 8% casein diet probably results from an actual decrease in the level of drug metabolizing enzyme and in cofactor level, as well as from a lower level of body fat in these animals.

An experimental model of both acute and chronic viral valvulitis was studied by direct immunofluorescent antibody technique with parallel histopathologic observation. Coxsackie B4 virus antigen was visualized in the affected valves and mural endocardium of acute infections and remained in situ even after the tissue developed scarring. The antigen was restricted to the stroma of the infected valves in the early stages and became more discretely localized in the endo-thelial cells at a later period. The intense, specific fluorescent reaction at the base of the affected valves provided evidence of valvular involvement by direct extension from adjacent myocardial lesions. Involvement of valves and mural endocardium of right side of the hearts was more frequent, especially in the early stages of infection. The studies show that the coxsackievirus B4 can produce valvulitis in mice as well as pericarditis, myocarditis and endocarditis.

Rubella hemagglutinin of high titer was prepared by alkaline extraction of infected BHK-21/13S cells grown in suspension culture. After this treatment, soni-cation or tween-ether extraction increased hemagglutinin titer 4-16-fold. Optimal conditions for hemagglutinin titration using eryth-rocytes from 1- to 3-day-old chicks included a low temperature and a pH of 6.2. A limited number of stability tests indicated that alkaline extracted hemagglutinin was stable at — 70°C and +4°C. The hemagglu-tinin was used to detect rises in HI antibody in paired serum specimens. Hemagglutination inhibition antibodies were present in titers of 1:40′ to 1:320, 2 to 3 years after rubella infection.

The supervision of all tissue culture preparation by Mrs. Anna Hall and the excellent technical assistance of Miss Marianne Davis and Miss Ludie Holland are gratefully acknowledged.

Alkaline extracted rubella complement fixing antigen was prepared from infected BHK-21/13S cells grown in suspension culture. Although antigen potent enough to ensure optimal dilutions may not have been used, the complement fixing antibody titers of convalescent phase specimens were considerably higher than they were when commercial cell pack antigen was used. They were also higher than the titers reported earlier with the use of other types of rubella complement fixing antigens. The alkaline extracted antigens were stable at —70°C, —20°C, and +4°C.

The excellent technical assistance of Miss Marianne Davis, Mrs. Carol Eason and Miss Ludie Holland is gratefully acknowledged.

The influence of substrates in serum ultrafiltrates of uremic individuals on isolated rat liver mitochondria has been determined. A substance or substances present in such an ultrafiltrate does uncouple oxi-dative phosphorylation, but only at the phos-phorylation site linked to the respiratory chain between NADH and cytochrome b. The inhibiting substance is cationic at pH 8.0 and is soluble in acidic ether.

A preliminary survey for tumorbearing fish among sole caught in waters of Wakasa Bay area, Japan was carried out. Five sole, one Limanda herzensteini and 4 Hippoglossoides dubius, with skin tumors were obtained. Histological observations of the lesions in both species revealed marked proliferation of epithelial elements accompanied with increase in fibrous structure of connective tissue. In addition marked increase of melanin-containing cells was observed in tissue sections of growth of L. herzensteini with darker appearance. The gross and microscopic findings of H. dubius resembled descriptions of the epithelial papillomas reported by other authors to occur among flathead sole populations near Friday Harbor, Washington.

The percutaneous, direct method for determination of the blood pressure was applied in the cat, without and with anesthesia. In one group of 5 males and 5 females, single determinations were made, to determine the feasibility of the method. In these animals the blood pressure was measured by 2 means—direct mean pressure with a mercury manometer, and systolic and dia-stolic pressures with a strain-gauge manometer. In this group the single pressures were generally high, and usually higher in the males. In another group, 2 males and 2 females, determinations were made weekly for 7 to 24 weeks. In these cats the initial pressures were also high, but similar in both sexes, but, in all the animals, gradually decreased and became stabilized at a lower level in about 4 weeks. In one male and in one female the production of unilateral renal ischemia, due to moderate constriction of the main renal artery, was followed by the development of hypertension, and, as in other species, the exclusion of the ischemic kidney of one of the hypertensive cats resulted in a prompt return of the blood pressure to the lowest prehypertensive level.

Over 300 compounds have been reported in human urine (1-4). These include inorganic compounds, organic acids, sugars, amino acids, purines and related compounds, enzymes, hormones, vitamins and their metabolites, estrogens, and many other organic compounds. The quantities of these constituents found in individual urine samples represent a wealth of information that can be used to evaluate body functions. Such quantitative data have been collected on a limited basis for many years and, as a result, many molecular constituents have been shown to have pathological significance. For example, a high uric acid content is associated with gout(5) and some leukemias (6); a high urocanic acid content is sometimes found in conjunction with asthma(7); large amounts of phenolic and indole amines appear to be excreted by schizophrenics(8), etc.

The Molecular Anatomy (MAN) program at Oak Ridge National Laboratory(9) is concerned with the description, at the molecular level, of the structure and organization of cells and tissue. One of the aims of this program is to develop high-resolution, automated systems for the analysis of low-molecular-weight substances found either free in cells or as constituents of macro molecules. A logical extension of this work is the further development of such analytical systems for use with human body fluids. This laboratory is now developing an automatic, high resolution analytical system for quantitatively determining the molecular constituents of human urine.

The approach is to modify and expand the capabilities of an existing nucleotide analyzer (10) for use as a urine analyzer. Initial results indicate that this concept is feasible; i.e., a high-pressure, high-resolution modification of the nucleotide analyzer has resolved over 100 chromatographic peaks of ultraviolet-absorbing constituents from a 2-ml urine sample. Twelve of these peaks have been tentatively identified.

TSR estimation of Sprague-Dawley-Rolfsmeyer strains of rats at 55 and 85 days of age was found to be highly significantly reduced by 22.8% and 14.8% respectively with subcutaneous injection of 50 μg melatonin/rat at 55 days, and 75 μg/ rat at 85 days but not by 100 μg/rat at 115 days of age. These results indicate that melatonin depresses TSR but the effect appears to be reduced with advaancing age. Feed consumption was reduced 8.76%, a nonsignificant reduction, in 115-day-old rats injected s/c with 100 μg melatonin/day/rat for a period of 12 days. The effect may be cumulative due to increasing doses of melatonin during each TSR estimation at monthly intervals. At the end of the experiment, the mean ovarian weight showed a highly significant reduction of 30.1% and the pituitary weight of 34.6% in comparison with the control group. In contrast, the mean adrenal weight showed a highly significant increase of 22.7%.

Injection of heparin into rats induced an increase in lipase activity of the plasma and a decrease in lipase activity of the tissues. The recovery of lipase levels in the tissues was observed after 2 to 3 hours. Before and after heparin injection the ratio of lipase activity inhibited by protamine sulfate and NaCl to lipase activity resistant to these inhibitors was not constant. After heparin injection increased activity of β-monoglyceridase in the tissues was observed. The tissues (of the No-Heparin-Injected rats) treated with epinephrme exhibited significant increases in lipase activity. However, no marked increase in lipase activity was observed in the tissues (of Heparin-Injected rats) treated with epi-nephrine.

DNA measurements in normal human anterior pituitary glands reveal a constant concentration per unit weight and increased DNA in larger glands. This suggests that new cell formation (hyperplasia) is responsible for increased gland size.

Two enterovirus-like agents were isolated in human amnion (FL line) cells from rectal swabs taken from healthy children during a longterm epidemiological study of viral infection in a semi-closed community during 1960-61. Of the first type (Hu 504), 26 representatives were isolated, of the second (Hu 659), 175. Both viruses were shown to exhibit the properties typical of human Cox-sackie A type enteroviruses. They were shown to exhibit the properties typical of human Coxsackie A type enteroviruses. They were shown to be prime strains of Coxsackie A Type 17 virus, with no apparent (Hu 504) or erratic (Hu 659) pathogenicity for suckling mice. Serological studies in a normal population group from the Pittsburgh area failed to show the presence of antibodies against either of these viruses. No conclusion can be drawn concerning the pathogenicity of these viruses for humans. The Mill virus, isolated by Murphy in Australia in 1963 from children suffering with acute diarrhea, appears to be identical with the Hu 659 virus.

The expert technical assistance of Miss Dianna Bellas and Miss Eileen Torretti is gratefully acknowledged.

The present results with insulin seem to indicate that the hormone, at the doses used in the present work, is without local and systemic effect on the intercellular transport of metabolites, at least if tested by the rate of intradermal diffusion of Evans blue dye.

The authors wish to thank Mr. Donald E. Hinton for his valuable assistance.

A series of 9 corticosteroids dissolved in ethanol were studied in the isolated cat papillary muscle preparation maintained at 27°C in a reduced calcium buffer. Only aldosterone exerted a significant positive inotropic effect. The magnitude of this positive inotropic effect was larger than previously observed at 37°C in a normal calcium buffer. The ethanol was markedly depressant to the papillary muscles. Hydrocortisone and dex-amethasone, in aqueous solutions, also exerted positive inotropic effects, whereas prednisolone and methylprednisolone were ineffective. Lowering the sodium or the potassium concentration of the medium did not result in significant inotropic responses to aldosterone or hydrocortisone.

Nine normal individuals and 6 patients with varying levels of renal insufficiency were given intravenous infusions of creatinine. The endogenous creatinine clearance:inulin clearance ratio in the normal subjects was 1.11. The ratio of the exogenous creatinine clearance to inulin clearance rose quickly to 1.73 as soon as the creatinine infusion was started, then gradually fell to 1.40 after ninety minutes of infusion. The patients with renal insufficiency had an initial ratio of 1.50 and a late rise to 1.71 after ninety minutes. It seems most likely that a small increment in the serum creatinine concentration can cause an increased effectiveness of the tubular mechanism for creatinine secretion. An alteration in the serum creatinine resulting in enhanced tubular transport, or reducing apparent serum creatinine concentration without affecting filtration, is less probable. The high pre-infusion ratio in patients with renal insufficiency, and the failure to respond to exogenous creatinine, suggests that the mechanism for increased tubular excretion may continue to function as long as the stimulus of an elevated serum creatinine persists.

When increasing amounts of thyroxine are administered to rats otherwise given an invariable dose of TRF, a dose of thyroxine is found to completely inhibit the TSH-releasing activity of that dose of TRF. Conversely, when increasing amounts of TRF are administered to animals pretreated with an invariable quantity of thyroxine known to inhibit the TSH releasing activity of several doses of TRF, a dose of TRF is eventually reached that will overcome the inhibitory effect of that amount of thyroxine. Neutron activation of a highly purified preparation of hypothalamic TRF failed to reveal presence of iodine in the TRF molecule.

Microscopic examination revealed that hearts of arctic ground squirrels killed in the field (noncaptive) were free of myocardial lesions but numerous areas of focal necrosis were found in the hearts of animals held captive for 2, 3, or 4 weeks. The etiology of these degenerative changes may be associated with the stress of captivity, but remains to be determined.

The authors thank Robert E. Becker of the USAF Arctic Aeromedical Laboratory, Fort Wain-wright, Alaska, for supplying the ground squirrel hearts.

γG-immunoglobulin, obtained from rabbit anti-bovine serum albumin serum, was treated with pepsin and subsequently reduced and alkylated to yield fractions containing 5S and 3.5S Fab fragments, respectively. The 5S fraction had precipitating antibody activity and induced passive cutaneous anaphylaxis (PCA) in mice. Such sen-sitization has not been demonstrated in the guinea pig. With respect to the dosage and the length of the latent period required, there was a marked difference in the abilities of the 5S antibody fractions and the γG-antibody to provoke PCA in the mouse. The 3.5S Fab fragments possessed “univalent” antibody activity but failed to induce PCA. However, 3.5 S Fab fragments inhibited the PCA reaction in the mouse, but failed to block the reaction in guinea pigs. Hybrid 5S fragments had the same properties as Fab fragments. These results point out additional differences between the PCA reaction of guinea pigs and mice. The findings also show that PCA in the mouse demands functionally bivalent antibodies.

We wish to express our appreciation to Dr. T. B. Tomasi, Jr. and Miss Jane Wark (formerly of the Dept. of Exp. Med., University of Vermont) for perfoming the analiytical ultracentrifugation and to Mrs. Patricia McEntee for technical assistance.

Aerosols of adenovirus types 4 and 7 and parainfluenza 3 were tested for biological stability as a function of relative humidity. The levels tested were 20%, 50%, and 80%. Both adenoviruses exhibited maximum stability at 80% RH. Parainfluenza on the other hand exhibited a minimum biological decay rate at 20% RH. Studies were carried out with aerosols having mass median diameters of about 2 μ.

The authors wish to cite the excellent technical assistance of Joseph W. Dominik and John F. Brewer of Fort Detrick and of Martin Schneider of Walter Reed Army Inst. of Research. The statistical analyses by Dr. N. Bohidar of Fort Detrick are also acknowledged.

The delay of passage of a charcoal meal through the mouse small intestine by morphine was antagonized by methyser-gide (UML), a potent inhibitor of 5-hydroxy-tryptamine (5-HT). Treatment of mice 16 hours prior to use with 5.0 mg/kg reserpine failed to modify the intestinal response to morphine. The effects of atropine methyl-bromide (AMB) on morphine and 5-HT were similar; treatment with AMB enhanced the delay in intestinal transit produced by these agents. Treatment of mice with physostigmine failed to alter the intestinal effects of either 5-HT or morphine. The data are interpreted as adding support to the hypothesis that some of the actions of morphine on the mammalian small intestine may be mediated by 5-HT.

The authors are indebted to Mrs. Sharon Heintz for her excellent technical assistance.

1. A working hypothesis concerning the nature of serum sickness involving a dissection of the antigen into “antigenic” component and “combining” component of the antigen is presented. 2. Evidence is presented that injection of aggregated BSA in amounts capable of inducing an immune response does not lead to the renal lesions of serum sickness in the rabbit. If, however, this stimulus was combined with subsequent administration of soluble BSA serum sickness nephritis occurred. 3. BSA centrifuged for 5 hours at 105,000 × g had lesser capacity to induce serum sickness than did an unmodified BSA. 4. These observations are consistent with the view that the production of serum sickness nephritis can be dissected into two separate components which can be separately manipulated.

The technical assistance of Miss A. Opstad and Mrs. L. Lang is gratefully acknowledged.

Further studies of 2 higher plants (Narcissus tazetta L and Magnolia kobus DC) with antiviral action against lymphocytic choriomeningitis infection in mice have been reported, and the activity of a third substance, propionin, added. The anti-LCM activities of the 3 natural products have been compared with methotrexate.

The authors are indebted to Dr. H. A. Chirigos, Nat. Inst. of Health, for supplying methotrexate and to Dr. Ralph F. Anderson, International Minerals & Chemical Corp., for supplying crudes containing propionin.

The carcinogenic metabolite, 2-amino-1-naphthol, of the bladder carcinogen 2-naphthylamine was air oxidized at pH 7.2 to produce many products separable by thin layer chromatography. The ether extract contained one major product which was identified as 2-amino-1,4-naphthoquinone.

Separation of supra-lethally irradiated tumor cells has been carried out to determine their effectiveness in reducing tumor latency and enhancing tumor takes. These experiments indicate that the most pronounced stimulation comes from the centrifugal fraction containing the “nuclei” from supra-lethally irradiated cells. The degree of stimulation by the “nuclear” fraction closely parallels the stimulation by whole radiation killed cells. The “cytoplasmic” fraction was also capable of augmentation of tumor survival and growth, however, less than either the “nuclear” fraction or whole radiation-killed cells. Disruption of the membranes of whole cells or “nuclei” by sonication eliminated the tumor growth stimulation. A tentative mechanism is proposed for the action of radiation killed cells, “nuclei,” and “cytoplasm” in enhancing growth and survival of transplanted isologous tumors.

Thirty-seven percent of turkeys fed DES died from aortic ruptures, while 69% of poults fed DES and thiouracil died from the disease. Total plasma lipid and cholesterol values were elevated by both treatments, but more so by DES alone. Turkeys fed DES or DES-thiouracil were hypotensive, but systolic blood pressure was lowest in turkeys fed the 2 combined drugs. There was pronounced hypertrophy of the thyroid gland of turkeys fed DES-thiouracil or thiouracil, and mild hypertrophy of the gland of poults fed DES. Body weights were depressed and the diameter of the lumen and thickness of the wall of abdominal aortas appeared to be reduced by DES-thiouracil feeding, as compared to feeding of DES alone.

The technical assistance of J. W. Carlisle, C. J. Miller, and L. Mallard is acknowledged.

Approximately 80% of injected aminonucleoside-8-C¹⁴ is excreted in the urine and feces within 24 hrs by the rat. The principal urinary products are unchanged amino-nucleoside, the monodemethylated analog, and allantoin. Daily administration of aminonu-cleoside for 12 days did not influence the course of its own metabolism.

It has been suggested that many of the biodegradation reactions which inactivate drugs and foreign compounds are essentially absent in fishes(1), since the fish and its surrounding medium represented in essence an infinitely large dialysing system. More recent evidence, however, indicates that a number of the detoxication reactions observed in mammals also are operative in fishes(2). In this communication we report studies on the metabolic alteration of catechoiamines. For purposes of comparison, the presence of a number of microsomal detoxication enzymes was determined in some of the tissues studied.

Materials and methods. The tissues were excised from sharks anaethesized with tricane methane sulfonate (Sandoz MS 222) and excised from sharks anaesthetized with tricane or were frozen (in dry ice) for later use. Unless otherwise noted, preparation of the enzymes was as follows: the tissue was homogenized first in a blender with 3 volumes of 1.15% KCl and then re-homogenized in a tight fitting glass-Teflon Potter-Elvehjem homogenizer. In the case of the liver, the homogenates were centrifuged for 10 minutes at 2,000 rpm and the large fatty layer was removed. Subsequently, the homogenates were centrifuged at 9,000 rpm for 10 minutes to obtain the so-called microsomal and soluble supernatant fractions (9,000 × g fraction). Microsomes were sedimented from this fraction by centrifugation at 30,000 rpm for 60 minutes, resuspended in 1.15% KCl and sedimented again. Catechol-O-methyl trans-ferase was estimated by the method of Axel-rod et al(3) using S-adenosylmethionine-C¹⁴ (New England Nuclear, 52.6 mc/mM) as the methyl donor. The reaction mixture was adjusted to pH 7.9-8.0 with either phosphate or Tris buffer and incubated in a final volume of 0.5 ml for 60 minutes at 25°C. The reaction was stopped by the addition of Na₂B₄O₇ (pH 10.0) and the methylated products were extracted with toluene : isoamyl alcohol (3:2;v/v).

A rapid agar slide method for localization and visualization of hyaluronate lyase (hyaluronidase) has been described. This procedure is based on enzymophoretic techniques developed for localization of mammalian enzymes following agar gel electro-phoresis. Differences in electrophoretic mobility between staphylococcal, streptococcal and bovine testicular hyaluronidase have been demonstrated. The use of enzymophoresis in agar for study of bacterial enzyme systems has been discussed as a valuable procedure for enzyme analysis during purification and characterization studies.

(1) A method for producing a suspension of pure mitotic chromosomes of Chinese hamster cells with insignificant contamination by interphase nuclei or their fragments is described. The yield obtained is high and the chromosome morphology is well preserved. (2) H³ thymidine label from such chromosomes became specifically incorporated into the chromosomes of fresh Chinese hamster cells and of human leucocyte cultures. No gross alteration in the chromosomes of the receptor cells occurred, and uptake of label was inhibited by the presence of added carrier thymidine in the medium, in an amount insufficient to inhibit DNA synthesis. Therefore, most of the incorporation appears to have involved previous degradation of the DNA at least to the level of free nucleotide.

The authors gratefully acknowledge the helpful suggestions of Dr. Theodore T. Puck, Dr. Sherman M. Weissman, and Dr. Gordon Allen, as well as the technical assistance of Mrs. Gloria E. Johnson.

The effect of drying on the loss of viability of Blastomyces dermatitidis in sterile soil is reported. 1. An average of 90% loss of viability occurred after 5 days storage at room temperature in cotton plugged tubes or in a desiccator. The loss increased to 95% after 11 days or longer. Aliquots of this soil mixed and stored for 10 days in a humidity chamber at room temperature showed a 2-fold increase in viable particles. 2. The loss of viability from storage of soil in tubes after 3 days was 75% with an increase to a 90% loss by 5 days. A 95% loss after 25 days when stored at room temperature or in a desiccator was observed. When soil was placed in a humidity chamber and stored at 4°C for 25 days, only 14% loss in viability occurred. 3. Although B. dermatitidis in soil may lose 90-95% viability after 5-10 days at room temperature, longer storage of up to 90 days does not affect the remaining 5% viable particles. Little change was noted in soil suspended in saline at 4°C after 7 days, but after 30 days, a 95% loss of viable particles was detected. 4. The degree of loss of viability appeared to be correlated with water loss by evaporation.

Freshly isolated frog gastric mucosa secretes hydrochloric acid, under appropriate conditions, in the absence of added his-tamine. However, if the mucosa is incubated with histamine-free media, the acid secretory rate decreases to a very low rate after approximately 16 hours, the p.d. increases, and the short-circuit current declines. Addition of 10^-4 M histamine to the nutrient medium bathing the serosal side restores acid secretion to about 80% of the initial rate; there is little change in p.d. and an increase in short-circuit current is observed. No difference exists in the ATPase activity of the resting and hista-mine-stimulated mucosa; the ATP level of the stimulated mucosa, on the other hand, is slightly greater than that of the resting mucosa. The procedure of preincubation of the mucosa in histamine-free media has made it possible for the first time reproducibly to obtain resting mucosae which can be stimulated by added histamine.

The effect of NH₄Cl on viral penetration was measured in chorioallantoic tissue in vitro by neutralizing absorbed virus with antiserum and determining the subsequent 24 hour yield of hemagglutinins. In some experiments conditions for single cycle replication were obtained by leaving the antibody in the supernatant during the entire period of growth and then measuring cell-associated hemagglutinins. Although some reduction of the amount of penetrated virus was demonstrable one hour after adding virus and NH₄Cl this later became insignificant. The prevention of viral penetration by NH₄Cl was considered too inefficient and irregular to account for the over-all inhibitory effect of NH₄Cl on viral replication. Inhibition by NH₄Cl of a type B (Lee) strain was comparable with that for Type A and reversibility of inhibition was demonstrated.

Responses of the coyote to E. coli endotoxin have been studied in animals intravenously anesthetized with sodium pentobarbital. Major results reveal portal hypertension concomitant with systemic hypotension; hemoconcentration; depressed liver function; hepatic, renal and adrenal congestion; intestinal necrosis and gall bladder edema. The liver appears to be the primary “target organ” of endotoxin in regard to hemodynamic, histological, and functional abnormalities. The coyote is more resistant to endotoxin than its domestic counterpart in terms of changes in hemodynamics and survival ability.

The influence of dietary factors and gastrointestinal disturbances on the prothrombin response to coumarin anticoagulants has often been reported (1). Relatively short periods of starvation can induce significant exaggerations in the response to these agents (2). Whether this effect is a function of low caloric intake, or an acute deficiency in a specific nutritional component is not known, The present study compares the effects of a balanced diet, starvation and diets free from carbohydrate or protein on the prothrombin response to the anticoagulant, acenocoumarin, in guinea pigs. The data indicate that modest amounts of dietary protein will prevent the exaggerated response resulting from short periods of starvation, whereas an equivalent amount of carbohydrate will not.

Methods. Non-albino male guinea pigs weighing 250-350 g were fed Rockland Guinea Pig diet (Tekland Inc., Monmouth, Ill.) supplemented with lettuce and water ad libitum until initiation of the experiment. Each animal consumed 25 to 40 g per day. On experimental days 1 through 3, feeding was confined to the pattern described below. Special mixes were prepared by General Biochemicals, Chagrin Falls, Ohio, and contained 15% nonnutritive fiber, 7.3% corn oil, and 9.3% salts, vitamins, and minerals. The carbohydrate mix included 15.0% corn starch, 10.3% sucrose, and 42.8% glucose, while the protein mix included 68.1% casein. Diets were fed by hand in the form of 10 capsules (Parke, Davis No. 3) twice a day, each containing 200 mg of pulverized food (total of 4 g per day) and was regularly observed to be consumed in toto. Acenocoumarin (25 mg/kg i.p.) was administered on days 2 and 3, and animals were sacrificed on the morning of day 4. Cardiac blood specimens were obtained and studied for clotting activity by methods previously described(2).

Asiaticoside, a triterpene glyco-side from Centella asiatica, was tested by local application for its effect on the tensile strength of linear skin wounds in the rat.

A series of experiments has been presented in which it was shown that crude extracts of human plasma contain a material that stimulates villous motility. It was not possible to distinguish a difference between the activities of crude mucosal, plasma, and urine preparations although it was suggested that there may be concentration differences.

Refined preparations of villikinin showed a similarity to mucosal and urine extracts in activity as well as mobility. However, it was not possible to demonstrate antivillikinin in preparations other than those from intestinal mucosa. The activity of villikinin was different from that produced by histamine, 5-hydroxytryptamine and bradykinin. It is felt that the data presented clearly demonstrate the presence of villikinin in the plasma and that plasma villikinin is similar to mucosal and urovillikinin.

The authors wish to thank Mr. David Alford and Mr. Philip Arnold for their technical assistance in the performance of the experiments.

Rats were separated into 2 groups—long-sleepers and short-sleepers—on the basis of their response to a dose of hexo-barbital. The drug-metabolizing activities of the hepatic postmitochondrial supernatant fraction from long-sleepers were less than those from short-sleepers with respect to hexo-barbital, acetanilide, aminopyrine, and o-nitro-anisole, each of which represents a different drug-metabolism pathway. This trend was observed in all 5 strains of rats examined. After phenobarbital pretreatment, this difference between the two groups was no longer observed in one strain studied. The strain differences in drug-metabolism activities were not as great in the rat as those reported in various rabbit strains.

The incidence of seizures caused by exposure to and withdrawal from CO₂ concentrations ranging between 5 and 50% was determined in adult rats and immature rats less than one month old. It was found that neither exposure nor withdrawal seizures could be elicited in animals less than 16 days of age, even though the same CO₂ atmospheres produced seizures in older animals. It was concluded that the lack of response of very young rats to CO₂ was primarily a manifestation of the general central nervous system immaturity of the infant rat, even though other factors, e.g., lack of CO₂ equilibration, might also affect the CO₂ response.

A sensitive method of assay for Mengovirus RNA has been investigated using an agar-L cells suspension plaque technique as previously described for poliovirus RNA-HeLa cells system. The activity of polybasic substances (DEAE-dextran and Poly-L-Ornithine) at different concentrations also has been studied. Both compounds cause an increase of infectious titer of Mengovirus RNA at a rate depending on their respective concentrations. When used at the optimal concentrations, DEAE-D shows enhancing activity of about one thousand times and PO of one hundred times with respect to the control conditions. When the drugs were used in combination, no synergistic effect was evident. The highest infectious titer (10^7.2 PFU per ml) was obtained using DEAE-D at a concentration of 800-1600 μg per ml as diluent fluid for the infectious RNA preparations.

The authors would like to acknowledge the valuable suggestions and help by Dr. Gebhard Koch and Dr. Hilton B. Levy.

One-to 5-day old chicks were exposed to a 10°C ambient temperature and cloacal temperatures Were recorded every 15 minutes for one hour. Young chicks show a rapid development of thermoregulation the first 5 days after hatching, with significant changes apparent even during the first day and one-half. This rapid homeothermic development is correlated with age and cannot be explained by increases in body weight, food intake, or insulation.

Caprochlorone demonstrates clear-cut or suggestive activity in cell culture systems against 15 myxoviruses and vaccinia, while 9 other virus strains are insensitive. The action mechanism of caprochlorone against influenza A/WSN, A/PR8, and B/Maryland was compared with that of 1-adamantanamine · HCl, ammonium phosphate, viral antiserum, and acidification. The time at which viral replication could be inhibited, up to 15 minutes after addition of virus, was similar in all cases. This inhibition resulted from failure of virus to penetrate the cell membrane as indicated by sensitivity of virus to neutralization by antiserum even 2 hours after addition of virus and inhibitor. Reversal of inhibition of A/WSN and A/PR8-infected cultures treated for 2 hours is accomplished by removing the compounds and replacing with medium; at this time, acidification inactivates A/WSN, whereas A/PR8 is refractory. Inhibition of B/Maryland-infected cultures with ammonium phosphate is reversed by removal of compound after 2 hours, but resistance to acidification is shown. By contrast, caprochlorone or I-adamantanamine · HCl inhibition of B/Maryland-infected cultures could not be reversed. In addition to inhibition of cell penetration by virus, capro-chlorone delayed release of newly formed virus from the cell, whereas l-adamantana-mine · HCl and ammonium phosphate did not.

We wish to acknowledge the excellent technical assistance of Edward Flannery, Janice Wagner, and especially Barbara Zappala.

The response of guinea pigs to dietary cholesterol is unique among small animals. Instead of developing cardiovascular lesions, the guinea pig becomes acutely anemic. The anemia is accompanied by enlarged liver and spleen, and fatty infiltration of liver, spleen, lung, and kidney. Most of the organs showed striking pathological changes when examined microscopically. The liver showed lipid globules, erythropoiesis, spotty necrosis, accumulation of hemosiderin and cholesterol, and diffuse regeneration. Splenomegaly was due to erythropoietic proliferation, red cell congestion and, to a lesser degree, to fatty deposits. The primary change in the adrenal was a selective fatty infiltration of the zona fasciculata. The lungs showed fat globules in the histiocytes as well as fat emboli in the capillaries. The testes were small and poorly developed. Spermatogenesis was greatly impaired. The bone marrow was hyperplastic, and the kidney showed glo-merulosclerosis. The pancreas and heart were unaffected. The changes observed resulted primarily from alterations in the cellular structure caused by the accumulation of cholesterol, and secondarily from impaired liver function and the effects of the hemolytic process.

Rabbit spleen cells producing antibodies against mouse immunoglobulin antigens were detected by a modification of the hemolytic plaque technique. The peak cellular and serologic response occurred 7 to 8 days following primary immunization and had subsided completely by the fifteenth day. The absence of spontaneous background distinguished protein reactive PFC from those reactive with erythrocyte antigens.

We thank Mr. B. Harrell for expert technical assistance and Dr. M. Landy for useful suggestions in preparation of this manuscript.

The erythrocytic AT Pase activity of uninfected calves and calves infected with Anaplasma marginale was determined. Samples from infected animals showed increased ATPase activity during the initial appearance of marginal bodies in the erythro-cytes and at the peak of infection. In view of this increase the possibility of enzyme activity in the Anaplasma bodies per se was investigated. Marginal bodies were purified and subjected to sucrose density-gradient sedimentation procedures. It was demonstrated that fractions containing purified marginal bodies possessed significant ATPase activity. The possible relationship between this activity and the pathology of anaplasmosis was discussed.

The influence of temperature on the maximum biliary excretion of bilirubin and sulfobromophthalein was studied in anesthetized rats and mice. Experimental techniques commonly employed in maximum biliary excretion studies were shown to significantly alter normal thermoregulatory mechanisms. A decrease in body temperature significantly decreased the bilirubin transport maximum in both rats and mice. A loss of body temperature in the rat also produced a significant decrease in the BSP transport maximum. In both rats and mice bile flow showed a diminution corresponding with the decrease in rectal temperature. The results emphasize the importance of monitoring body temperature during the course of experiments employing maximum biliary excretion as an endpoint.

The authors wish to acknowledge with gratitude the excellent assistance of Mrs. Sharon Shriver.

In volunteers the sequential appearance of the immunoglobulin classes of neutralizing antibody formed as a result of infection with A₂/Bethesda/10/63 influenza was studied. The antibody appeared in the IgG class and no significant antibody in the IgM or IgA classes was found. It was particularly notable that no IgM antibody could be detected 7 days after inoculation. The pattern of antibody against A₂/Japan/305/57 was nearly identical to that formed against the inoculum strain. Antibody rises were also noted against A/PR8 and A₁/FM1 influenza viruses.

Tests were performed on baseline and periodic blood samples obtained from rabbits treated with intramuscular injections of Triton WR-1339. The ensuing hyper-lipemia was characterized by marked elevation in total fatty acids, triglycerides, cholesterol, lipid phosphorus, and total lipids. Paper electrophoresis revealed a concomitant increase in β-lipoproteins. Lipid mobilizer (LM) also increased strikingly, as was determined indirectly by testing plasma aliquots for inhibition of heparin activated clearing lipase. On the basis of these and related studies, is is suggested that Triton hyper-lipemia is produced, at least in part, through sustained release of increased amounts of LM which mobilize triglycerides to the portal circulation. Furthermore, Triton modifies metabolic activity of the liver so that post-hepatic hyperphospholipidemia and hypercholester-emia ensue.

In many experimental procedures involving rats or mice, regularly timed blood specimens must be obtained. The use of the tail veins as a source of this blood necessitates incapacitating the animal in either elaborate restraining devices or wrapping them in toweling. Both techniques are often traumatic to both animal and experimenter. A simple device is described which alleviates this problem.

An ordinary low-density narrow-mouth polyethylene bottle is cut with a razor blade as in Fig. 1. Enough of the bottle is left intact to allow the rat to brace his hind legs. Also an injection port may be cut in the side if necessary. The animal needs very little inducement to enter the bottle and frequently will walk in to it of its own accord if the bottle is merely placed before it. Once the rat is in with its tail still out, a wide piece of adhesive tape is used to close the entrance. The size of the bottle should be commensurate with the size of the rat; an 8 oz bottle will conveniently hold a 200 g rat.

Once within the bottle, the rat cannot turn around, sits comfortably with his nose in the neck of the bottle thus breathing freely, and is handled easily without being agitated or aroused, Intraperitioneal injections can be given easily through a port cut in the side of the bottle. If the experiment is terminal, the bottle cap, containing a piece of cotton saturated with chloroform, my be screwed on, to kill the animal. The device is sufficiently inexpensive so that both rat and bottle may be disposed.

A comparison of the total number of neutrophils and neutrophil precursors within the humerus of germfree and conventional mice revealed no statistically significant difference between the two groups. The distribution of marrow neutrophils with respect to morphologic type and the number of neutrophils in mitosis were similar in the two groups. There was, however, a slight, but significantly, lower concentration of blood neutrophils in germfree mice as compared to controls. Removal of mice from the germfree environment was associated with a rapid decrease in the number of mature and relatively mature neutrophils in the marrow and with a subsequent increase in potentially mitotic, immature neutrophils, following which the marrow returned to normal. Germfree mice responded to endotoxm administration with an increase in rate of release of neutrophils from the marrow, as do normal mice. It was concluded that in this model system the presence or absence of microorganisms in the environment could not be assigned a major role in regulating granulocytopoiesis.

We are indebted to Mrs. Vreni Bithell and Miss Lona Bindbeutel for technical assistance.