Experimental Biology and Medicine

The effects of ip injections of phenobarbital (100 mg/kg), phenylcycli-dine (Sernylan; [l-(l-phenylcyclohexyl)-piperidine-HCl] (1 mg/kg), and ketam-ine (Ketaset; [dl(2-O-chlorophenyl)-2-(methylamino)cyclohexanone- HC1] (1 mg/ kg) on lipid metabolism in rats were compared. This study was undertaken to determine whether the two sedatives currently used in primates share any of the undesirable effects of phenobarbital on lipid metabolism. All three compounds were administered to male Wistar rats for 6 days. Phenobarbital was hepatomegalic, stimulated 7α hydroxylation of cholesterol, and inhibited cholesterol synthesis by rat liver slices from mevalonate, but not acetate. The two other sedatives exhibited effects very similar to those observed in the controls. From our work in rats it is concluded that the use of Sernylan or Ketaset for sedation of nonhu-man primates will not significantly affect these parameters of lipid metabolism.

The biologic roles of guanosine 3′,5′-monophosphate (cyclic GMP) and adenosine 3′,5′-monophosphate (cyclic AMP) in the secretion of lysosomal enzymes from, and in phagocytosis by, human neu-trophils were studied. Contact between neutrophils and particulate immunologic reactants results in both phagocytosis of the particles and secretion of lysosomal enzymes. These cellular events are accompanied by the accumulation of cyclic GMP and require the presence of extracellular calcium. Acetylcholine, pilocarpine, and cyclic GMP enhance, whereas epinephrine, cyclic AMP, and/or dibutyryl cyclic AMP inhibit, both phagocytosis and lysosomal enzyme secretion. The stimulatory action of cholinergic agents and the inhibitory action of epinephrine are accompanied by the accumulation of cyclic GMP and cyclic AMP, respectively, in human neutrophils. The data suggest that cyclic GMP mediates whereas cyclic AMP inhibits the major functions of human neutrophils. Moreover, by virtue of their effects on cyclic nucleotide accumulation, autonomic neurohormones are capable of modulating human neutrophil function.

Activity of lysyl oxidase, an enzyme responsible for production of alde-hydic precursors for lysine-derived collagen crosslinks, was measured in tibial meta-physes from chicks receiving different dietary levels of vitamin D and Ca for 2 weeks after hatching. Enzyme activities were increased twofold in D-deficient chicks compared to activities from chicks receiving control levels of vitamin D. Addition of Ca to the D-deficient diet had no effect on lysyl oxidase activity. It is suggested that vitamin D may play a role in the age-related decrease in lysyl oxidase activity that normally occurs in chick bone.

We thank Unni Kristoffersen, Catherine Lane, Rachel McNeil, and Lola Rigby for their technical assistance, and Jean Cochran for typing the manuscript. This investigation was supported by NIH Research Grant Number DE 02668 from the National Institute of Dental Research, and by NIH Grant Number RR 05333 from the Division of Research Facilities and Resources. One of us (W.G.) was supported by NIH Postdoctoral Fellowship Number DE 01658 during this study.

Myocardial slices from the left ventricle of cat hearts were incubated in Krebs-Henseleit buffer containing 10 mM glucose which were gassed with 95% O₂ and 5% CO₂. Tritiated dexamethasone (DEXA 0.171 mM) or methylprednisolone (MP 0.805 mM) was added under varying conditions of temperature, pH, and in the presence of various metabolic inhibitors. Glucocorticoid uptake by myocardial tissue was found to be temperature-dependent, pla-teauing after 60 min of incubation at 0.21 μmole of DEXA/g of tissue and 0.95 μmole of MP/g of tissue at 37°. Steroid uptake was retarded about 50% at 0°. Increasing the concentration of either steroid resulted in a linear increase in the uptake of that steroid. Incubation in the presence of metabolic or SH-inhibitors produced no significant changes in glucocorticoid uptake. Optimal glucocorticoid uptake occurred between pH 7.3-7.4. Thus, myocardial cells appear to be able to take up large quantities of glucocorticoids, but the uptake process does not appear to be energy-dependent.

Opiate administration has profound effects on neurotransmitter metabolism and neuroendocrine functions. Since cAMP is an intracellular metabolite common to the actions of many of the involved compounds stimulating distinct and specific membrane receptors, cAMP concentrations representative of in vivo conditions in heroin-treated and control animals were determined. Rats were sacrificed by immersion in liquid nitrogen 30 min after heroin injection. Tissue samples, dissected without thawing, were assayed by radioimmunoas-say to determine cAMP/mg of protein. Comparisons between treated animals and sham-injected controls revealed significant cAMP concentration increases in hypothal-amus, cerebral cortex, cerebellum, adrenal medulla, adrenal cortex, and liver (P < 0.005). Cyclic AMP concentrations in heart, skeletal muscle, kidney, and fat were not significantly different from controls. The increased tissue cAMP concentrations associated with the altered physiology of heroin administration apparently represent a pattern of integrated responses to the combination of opiate receptor binding and effects on neurotransmitter metabolism and circulating hormones.

These studies were begun while the author was in the Division of Biochemistry of the Walter Reed Army Institute of Research, with the technical assistance of Mr. S. West, and the statistical guidance of Ms. Helen Sing.

Administration of methyl-dopa (100 mg/kg, orally twice daily for a period of 3 days) to mongrel dogs produced significant reductions in blood pressure and heart rate. The hypotensive effect of the drug was due to a reduction in peripheral resistance. Methyldopa treatment also produced a significant decrease in coronary vascular resistance. Studies on the left ventricular function indicated that treatment with methyldopa does not compromise the ability of the myocardium to respond to an increased work load. Thus, the beneficial effect of this agent on the myocardial circulation, together with its lack of any detrimental effect on the cardiac function suggest that methyldopa may be an effective agent for the control of hypertension.

The generous supply of methyldopa by Dr. C. A. Stone of the Merck Institute of Therapeutic Research, West Point, Pennsylvania, is greatly appreciated. The authors wish to thank Dr. Regis R. Vollmer for his help; and Ms. Barbara Lewis for excellent preparation of this manuscript. This study was partly supported by USPHS Grant No. HL 17401.

By the fifth week of restricting water intake at 1100 to a 15-min period per day, estrous cycle lengths had shifted in most animals (31/40) from 4 to 5 days with the 5-day cycles usually consisting of 3 days of diestrus. In the 5-day cycling rats, plasma corticosterone peaked at 1100 and also at 1900 hr, the proestrous surge in plasma LH was not significantly changed, although the variation in individual animals was greater and the prolactin surge was prolonged. Similar prolonged cycles followed water restriction at 1800 hr with animals returning to regular 4-day cycles when given water ad libitum. These results suggest that the alteration of the normal corticosteroid rhythm and/or changes in prolactin secretion may account, in part at least, for the changes noted in cycle length after restricting the water intake.

The authors thank Miss Katherine Bangs, Mr. Mike Profit, and Mrs. Frances Smith for valuable technical and secretarial assistance, and Mr. Bob McAlister for drawing the figures. We would also like to express our thanks to Dr. Anna Taylor and Berilyn Branch for their assistance in the Fluoro-metric determinations.

Renal cortical blood flow and superficial cortical blood flow were measured in chronic sodium-loaded, sodium-deprived, and normal rats. Neither total renal cortical blood flow or distribution of cortical blood flow was different among the three groups of animals. Alterations in the amount of sodium excreted, therefore, are not related to alterations of renal cortical blood flow distribution.

Effects of epinephrine, serotonin, and KC1 on development of isometric tension were studied in strips of rat femoral and tail artery smooth muscle. Striking differences were detected between the preparations. Tension development in tail artery smooth muscle was greatest with epinephrine, intermediate with serotonin, and least with K⁺. In femoral preparations tension developed was greatest with serotonin. Smaller but comparable contractions were elicited with epinephrine and K⁺. Theoretical and experimental dose-response curves for epinephrine and serotonin agreed closely, whereas curves for K⁺ differed markedly. The relationship between fractional response and fractional stimulus was hyperbolic for epinephrine and serotonin but sigmoid for K⁺. The Hill coefficient for serotonin was about 1, slightly smaller than 1 for epinephrine (0.5 to 0.8) and significantly greater for K⁺ (4 to 5). These findings show that responses elicited with either epinephrine or serotonin are mechanistically consistent with receptor occupancy theory. They suggest that specific receptors for K⁺ exist and that they may involve positive cooperative interactions similar to those described for receptor mechanisms in electro-plax.

Spontaneous contractions were elicited by the vibration of small amplitude (microvibration) imposed upon the quiescent strips prepared from the ureter and portal vein of dogs. An acceleration of the existing rhythm in the spontaneously contracting strips was also brought about by microvibration. Frequencies and amplitudes of microvibration ranged from 1 to 90 Hz and from 50 to 150 μm, respectively. Imposed cyclic strains were less than 1% at their maximum. Up to the amplitude of 150 μm, the greater the amplitude, the faster the induced rhythm. Stepwise increase of the frequencies up to 30 Hz brought about a gradual acceleration of rhythmicity. At frequencies of above 30 Hz, no appreciable frequency-dependent differences were observable among the responses induced. Local adrenergic mechanism proved to have nothing to do with these effects.

Mice infected intravenously with vaccinia virus develop characteristic lesions over the entire tail surface. This experimental virus infection presents a highly sensitive and reliable model for evaluating the antivaccinia activity of antiviral compounds. Ara-C (1 -β-D-arabinofuranosylcytosine), ribavirin (1-β-D-ribofuranosyl-1,2,4-tria-zole-3-carboxamide), IUdR (5-iodo-2′-deoxyuridine) as well as two novel analogs of IUdR, EtUdR (5-ethyl-2′-deoxyuridine), and NCSUdR (5-thiocyanato-2′-deoxyuri-dine), were found to inhibit the formation of vaccinia tail lesions, when administered in-traperitoneally once daily for 7 days starting immediately after virus infection. The order of (decreasing) activity was: ara-C > IUdR > NCSUdR > ribavirin > EtUdR. Various drug combinations, involving IUdR + ara-C, NCSUdR + ara-C, NCSUdR + IUdR, NCSUdR + ribavirin, etc., were evaluated, but none proved more efficacious than either compound administered alone.

This investigation was supported by grants from the F.G.W.O. (Fonds voor Geneeskundig Wetenschappel-ijk Onderzoek) and the K.U.L. (Katholieke Universi-teit Leuven) “Fonds Derde Cyclus.” M. L. was supported by a postdoctoral fellowship of the K.U.L. “Fonds Derde Cyclus.” We thank Dr. H. A. Jolley (Cyanamid International Co., Pearl River, New York) and Dr. F. Dehaen (Cyanamid Benelux, Lederle Laboratories Division, Brussels) for the generous supply of ribavirin.

Rabbits were infected or rein-fected intradermally in multiple sites with BCG. One day before the BCG infection they were injected iv with a single pulse of tritiated thymidine (³HT). At various times thereafter, the BCG lesions were biopsied and evaluated for ³HT-labeled mononuclear cells (MN), which are mostly macrophages but include some lymphocytes. Periodically Old Tuberculin (OT) was injected intradermally, creating MN traps, which were biopsied and evaluated 1 day after their onset. These traps monitor the population of ³HT-labeled MN in the blood stream that readily enters sites of inflammation.

During the first 5 days after the ³HT pulse, a higher percentage of labeled MN accumulated in tuberculin traps on rabbits reinfected with BCG than in traps on rabbits with primary infection. The high percentage of labeled MN in the former was probably due to the more intense inflammation caused by preexisting tuberculin hypersensi-tivity. By 8 days after the ³HT pulse, a lower percentage of labeled MN had accumulated in BCG lesions of reinfection than in primary BCG lesions. Evidently the presence of delayed hypersensitivity increased the turnover of MN in tuberculous lesions. By 15 days after the ³HT pulse, the percentage of ³HT-labeled MN was much higher in BCG lesions than in 1-day traps, suggesting that such MN had accumulated in the lesions over a period of several days.

Sera from inbred SPF White Leghorn chickens at 6 weeks or 19-22 months of age did not have anti-OA agglu-tinins as determined by the passive HA reaction; 2-day-old chicks also lacked the anti-OA activity. Inbred chickens kept in SPF isolators for 6 weeks, then transferred to a conventional environment for 7 weeks, produced anti-OA agglutinins. Inbred White Leghorn adult chickens conventionally reared had a high anti-OA agglutinin titer. It appeared that natural anti-OA agglutinins are elaborated in response to environmental pathogens in certain lines of chickens. The titer in the individual chicken appeared as early as 10 weeks and was maintained until old age. The presence of natural anti-OA agglutinins did not prevent egg-laying but appears to have delayed it.

The HA reaction is specific and can be absorbed.

We wish to thank Dr. E. L. Hodapp for valuable assistance during the course of this investigation, and Dr. L. R. Draper for critical review of the manuscript.

Renal artery constriction (RAC) to 30% of normal flow for 12 hr in the unilaterally nephrectomized dog produced a marked increase in both erythropoi-etin titers and prostaglandin E (PGE) levels in the blood. In dogs pretreated prior to RAC with indomethacin, a potent inhibitor of prostaglandin synthetase, there was no significant increase in either PGE or eryth-ropoietin levels as compared to zero-time control values. These data suggested an involvement of renal PGE in the generation of erythropoietin following RAC.

The authors gratefully acknowledge the excellent technical assistance of Mr. Jesse Brookins, Ms. Patricia Dargon, Mr. Rene Stiaes, and Mrs. Jacqueline Frailey.

Melatonin (12-100 μg/day) administered via subcutaneous Silastic implants prevented or suppressed light-induced testicular recrudescence in adult golden hamsters. In addition, melatonin (150 μg/day) induced marked testicular regression in sexually mature hamsters maintained on photostimulatory long days. These results clearly establish that exogenous melatonin can inhibit gonadal function in adult male hamsters.

The authors express their appreciation to Donald W. Carroll for expert assistance provided throughout this study.

Hypothalamic-pituitary control of prolactin and LH secretion was tested in young (4-6 months) and aged (22-30 months) male Long-Evans rats given l-dopa, methyl dopa, LHRH, or stress treatments. Pretreatment serum LH levels were consistently higher in young than in the aged groups. The increase in serum LH after LHRH injection was only about half as much in aged as compared to young control males. Although acute stress caused a prompt increase in serum LH in young male rats, this treatment was without effect in the aged group. Methyl dopa treatment stimulated serum prolactin secretion in both young and old rats. Although L-dopa treatment caused a reduction in serum prolactin in both age groups, the sensitivity, magnitude, and duration of the reduction was smaller in the aged rats.

Systemic administration of parachlorophenylalanine (PCPA, 100 mg/ kg sc on alternate days x two times), a blocker of serotonin (5-HT) synthesis, considerably decreased brain 5-HT and plasma prolactin (PRL) levels in young male rats. Intraventricular (IVT) administration of 5,7-dihydroxytryptamine (5,7-DHT, 200 μg/20 μl), a neurotoxic drug which destroys 5-HT nerve terminals, induced, 3, 12, and 30 days after treatment, a marked depletion of brain 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) and considerably reduced plasma PRL levels at each time interval. Feeding of rats for up to 4 days with a tryptophan (TP)-deficient diet, caused a depletion of brain 5-HT and 5-HIAA contents and did not modify plasma PRL levels. Addition of TP (2 g/kg of diet) to the TP-deficient diet resulted in increased brain 5-HT and 5-HIAA contents and significantly increased PRL levels. These data provide evidence for the role of the 5-HT system in the maintenance of tonic PRL secretion.

We are grateful for the technical assistance of Miss Tecla Ciapparelli and Mr. Giuseppe Tavasci, and to Miss Delia Deriu for skillful secretarial help. Dr. H. G. Baumgarten kindly supplied 5,7-DHT. The reagents for the rat PRL radioimmunoassay were a gift of the Rat Pituitary Hormone Distribution Program of the National Institute of Arthritis, Metabolism, and Digestive Diseases.

The effect of cycloheximide (1.5 mg/kg), a potent inhibitor of protein biosynthesis, on D-galactosamine (375 mg/ kg)-induced hepatic necrosis and hepatic triglyceride accumulation was studied in rats. Serum transaminase levels, 24 hr after D-galactosamine administration, were significantly reduced in animals treated simultaneously or 4 hr before D-galactosamine with cycloheximide, when compared to animals given D-galactosamine alone. Transaminase levels in rats given cycloheximide 4 hr after D-galactosamine were not reduced. Histological grading of hepatocyte necrosis showed a similar pattern of protection in the pretreated and simultaneously treated groups. Hepatic triglycerides were significantly reduced only in the latter group.

Fatality 48 hr after D-galactosamine administration was significantly less common in rats pretreated with cycloheximide when compared to rats given D-galactosamine without cycloheximide, and surviving animals in the cycloheximide pretreated group had a lower serum transaminase level, a lower necrosis score, and a reduced hepatic triglyceride level.

These data are consistent with the concept that protein synthesis is important in the pathogenesis of D-galactosamine-in-duced hepatotoxicity.

The presence of ethacrynic acid in the nutrient sulphate solution produces, generally in less than 3 min, a decrease in resistance accompanied by an increase in transmucosal PD. These results strongly suggest that the dominant effect of ethacrynic acid is not on the Na⁺ pathway of the nutrient membrane, but without further knowledge of the resistance of other ionic pathways, it cannot be inferred whether the effect predominates on the K⁺ pathway as in Cl^- media.

Alloantisera to H-2K, H-2D, and la antigens markedly inhibited the binding of EA but not FITC-IgG by the B cell Fc receptor. EA rosette formation approached normal levels when masked H-2 but not la specificities were allowed to cap on the membranes of B cells. β₂-μ coated SRBC were bound by the Fc receptor, and high concentrations of soluble β₂-μ, were found to moderately inhibit EA rosette formation while lower concentrations enhanced binding. The data support the concept of Fc/Ia identity, and they suggest that H-2K, H-2D, and the Fc receptor may be closely grouped on the membranes of B cells. Further, these observations suggest that the β-microglob-ulin associated with H-2 could serve to link T cells with the Fc receptor of B cells during the inductive phase of antibody synthesis.

Two cultured human glioblas-toma cell lines were exposed to graded doses of BCNU, and the sequential cytoxic and cytocidal changes were studied histolog-ically. Changes included cessation of mitotic activity, reduction in population, rounding and clumping of the cells, nuclear pyknosis, and cell death. The intensity of the changes was proportional to the dosage. With 3 daily doses of 18 μg/ml, the population in Tumor A-172 was only 20% of that in the controls, and 75% of the cells present appeared non-viable. This dosage is about four times that used in a single course in treating humans. After 3 doses at 60 μg/ml, cell death appeared complete, but transfer without additional treatment brought out 2-min foci of viable cells, with several mitoses. A more anaplastic glioblastoma, T98, was much less sensitive to BCNU, responding only by lowered population density, decreased mitoses and slight-to-moderate cytotoxicity in one experiment.

We would like to thank Annie Rue for her excellent assistance in culturing the cells.

Parotid salivary proteins from monozygotic twins, dizygotic twins, nontwin sibs, and unrelated male and female subjects of the same age range as the twins were separated by polyacrylamide gel slab elec-trophoresis at pH 9.0. Variability of the stained protein patterns increased in the order: monozygotic twins; dizygotic twins and nontwin sibs; unrelated subjects. It is concluded that genetic factors are the major contributors to variability of parotid salivary proteins among subjects.

The twins analyzed in this study and the zygosity determinations of the twins were made available through the cooperation of David T. Lykken, Department of Psychiatry, University of Minnesota.

The effects of scillaren and di-nitrophenol on bilirubin excretion by the perfused rat liver were studied. Both compounds inhibited bile flow, scillaren by 20 to 40%, and dinitrophenol by 60 to 80%. Bilirubin excretion was also impaired. However, the effect of scillaren on bilirubin excretion was less than that on bile flow, as indicated by an increase in the bile bilirubin concentration, whereas dinitrophenol had a greater effect on bilirubin excretion than on bile flow. Dinitrophenol also inhibited the hepatic removal of unconjugated bilirubin from the perfusate, probably because it impaired the initial uptake and/or storage of unconjugated bilirubin by the perfused liver.

Low level direct renal nerve stimulation increases renal tubular sodium reabsorption in the absence of changes in glomerular filtration rate, renal blood flow, or intrarenal distribution of blood flow. Blockade of this response with phenoxyben-zamine (or guanethidine) supports the interpretation that it is mediated by direct adre-nergic innervation of the renal tubule.

This work was supported by USPHS Grant Nos. HL 13765 and AM 15843, and Program Project No. HL 14388, and grants from the Iowa Heart Association. Mr. Zambraski was supported by a National Defense Education Act Fellowship. Drs. DiBona and Kaloyan-ides were Clinical Investigators, Veterans Administration. We are grateful to Smith, Kline and French Research Laboratories for a generous supply of phenoxy-benzamine.

The distribution of labeled mi-crospheres within the renal cortex was used to evaluate the influence of physiological amounts of antidiuretic hormone on intra-renal blood flow distribution in hypophysec-tomized dogs and in rats with hereditary diabetes insipidus. In both species, intravenous infusions of ADH caused a significant decrease in the ratio of inner to outer cortical blood flow. The change in blood flow distribution observed in the hypophysecto-mized dog with ADH was primarily a consequence of a decrease in inner cortical blood flow. No consistent changes in outer cortical blood flow were found. Also in the dog, glomerular filtration rates and electrolyte excretion rates (Na and K) increased following ADH. In contrast, ADH infusion into Brattleboro rats caused no change in glomerular filtration rate or excretion of Na and K.

I would like to thank Dr. Carl Hansen of NIH for kindly supplying our Brattleboro breeders. The excellent technical assistance of Mrs. Christine Eldon is gratefully acknowledged.

Swimming, if undertaken for 1 hr/day, 3 days/week for 6 weeks prior to treatment with DOCA, unilateral nephrec-tomy, and salt-loading, delayed the development of hypertension in rats. If the swimming were undertaken concurrently with or after initiation of treatment with the above hypertensive regime, then it was without effect on the level of blood pressure attained or the length of time required to attain a given blood pressure. Swimming in itself resulted in a significant increase in arterial blood pressure. Previous training, such as swimming, may delay the development of hypertension through an alteration in vascular structure or smooth muscle sensitivity. The increase in blood pressure noted in physically trained rats may be a consequence of the training regime itself acting as a stressor.

Caffeine has been found to potentiate the acute anti-inflammatory activity of aspirin, indomethacin, and phenylbuta-zone, but not the activity of sodium salicy-late or hydrocortisone, in the carrageenan pleurisy or hindlimb models of inflammation in the rat. The mobilization of inflammatory cells was not affected by aspirin in the presence or absence of caffeine.

The mild analgesia produced by aspirin was confined to a hyperalgesic test in which this drug was able to reduce inflammation and concomitant hyperalgesia and thereby produce an “apparent” analgesic effect. This “apparent” analgesia produced by aspirin was potentiated by caffeine. The mechanism responsible for the potentiated anti-inflammatory and mild analgesic activity of aspirin remains unknown since caffeine did not alter the plasma salicylate levels or prostaglandin synthetase inhibition produced by aspirin.

The authors wish to thank Dr. R. A. Maxwell and Dr. D. H. Namm for their advice and constructive criticism in the preparation of this manuscript.

Cells from two human pituitary tumors were grown in capillary culture units and prolactin production was measured. A single unit could produce 3 mg of prolactin over a 4-month period. The cultured cells responded to TRH exposure by increasing their rate of prolactin secretion. Cultivation of cells in capillary units could be the method of choice for reducing the shortage of human hormones.

The authors appreciate the suggestions of Drs. Pie-tro M. Gullino, Andreas Chrambach, and Walter Lov-enberg. We are indebted to Drs. Walter Lockhardt, Frank Neelon, and Tom Huff for obtaining the pituitary tissues. We also thank Mr. Norman Beaudry and Ms. Sharon McAvinue for technical assistance.

Total mercury concentration was measured by atomic absorption spectro-photometry in 100 maternal-umbilical cord blood pairs, 38 placentae, and 32 breast milk samples, all from patients at the University of Iowa Hospitals. The mean maternal and cord blood levels were 1.01 and 1.24 parts per billion (ppb), respectively. Though the difference between maternal and cord values was not statistically significant, the two were significantly correlated with each other, and maternal blood level increased significantly with maternal age. Mean placental and milk concentrations were 2.28 and 0.93 ppb, respectively. The mercury levels in this study were lower than those reported from elsewhere, probably reflecting a combination of methodological refinement and relatively low mercury exposure in a rural population whose diet is low in fish consumption. These low levels suggest that the risk of perinatal mercury damage is small in such a population.

Porcine follicle-stimulating hormone (pFSH) has been prepared from acetone dried pituitary glands. The acetone powder was initially extracted in pH 5 ethanol-acetate buffer (40% ethanol and acetate at 0.5 ionic strength) containing 0.02% PMSF. The gonadotropin rich fraction, obtained in the 80% ethanol precipitate, was dissolved in 0.12 M NH₄HCO₃, heated at 60° for 3 min, cooled, and centri-fuged. The supernatant was lyophilized for chromatography on QAE-Sephadex, then Sephadex G-100. The pFSH obtained has a biological activity of 15 x NIH-FSH-P-1 and 2.1% sialic acid. The amino acid analysis is characterized by high lysine, aspartic acid, and glutamic acid with notable amounts of threonine, proline, and half-cys-tine.

A study of the genital micro-flora in virgin female rats of reproductive age has demonstrated that alpha and nonhe-molytic streptococci, Pasteurella pneumotro-pica, diphtheroid bacilli, Staphylococcus epi-dermidis, and Proteus mirabilis are the predominant genital organisms. The pH of the rat vagina was demonstrated to be near neutrality. It is suggested that the higher pH in the rat vagina as compared to the human vagina may account for some of the differences between human and rat genital micro-flora.

A. J. Markovetz is the recipient of a Development Award-Research Career Program (5KO4 GM14681) from the National Institute of General Medical Sciences.

The assistance of Marilyn J. Ohm in identifying anaerobic bacteria for this study is gratefully acknowledged.

The effects of vaginal suppositories containing 1.0 mg of 15[S]15-methyl-PGF_2α on oviductal motility, egg transport, and fertility were determined in rabbits. Suppository treatment caused a significant increase (P < 0.02) in the amplitude of oviductal contractions, and a decrease in the frequency of contractions (P < 0.04). Altered oviductal motility persisted for an average of 2 hr after treatment. Treatment with 1, 2, or 3 suppositories at various times after ovulation caused a significant reduction in the number of eggs located in the oviducts (P < 0.025). There was, however, a great deal of variation in egg recovery in treated animals (range 0 to 100%). Treatment of mated rabbits during the time of tubal egg transport caused a significant reduction in the number of Day-12 implants (P < 0.05). The percentage of corpora lutea represented by Day-12 implants was similar to egg recovery rates in animals similarly treated. The treatment had no effect on fetal survival from Day 12 to 28 of pregnancy. The decrease in fertility caused by these vaginal suppositories is presumably due to the stimulatory effect on oviductal motility which accelerates tubal transport of the embryos into the uterus. Embryos that arrive in the uterus prematurely probably do not implant and degenerate or are expelled.

Rats fed a purified diet containing ascorbic acid developed hypercholesterolemia. Because rats do not require exogenous ascorbic acid, they may be comparable to humans who supplement their diets with ascorbic acid in capsular form. The amount of ascorbic acid in this experiment was equivalent to 82 to 630 mg of capsular ascorbic acid ingested by an average man and was well below the amount ingested by those in search of respiratory benefit. The data are consonant with those on humans consuming controlled diets.

Human skeletal muscle ho-mogenate has been shown to contain enzymes that catalyze the hydrolysis of L-leu-cyl p-nitroanilide and carbobenzoxygluta-myl-L-tyrosine, known substrates, respectively, for arylamidase and cathepsin A. The muscle arylamidase was found to be inhibited by p-chloromercuribenzoate. Addition of Co²⁺ resulted in slight stimulation of its activity. Neither ethylenediamine tetra-acetate nor thiol compounds had any appreciable effect on the enzyme. When compared to controls, no significant differences in muscle arylamidase levels were observed in patients with muscular dystrophies and certain selected neuromuscular diseases. Cathepsin A was, however, increased in muscles moderately affected by muscular dystrophy and denervating diseases.

A plasma culture system has been used successfully to grow and quanti-tate megakaryocyte colonies from mouse bone marrow following their staining for acetylcholinesterase activity in situ. Colonies averaging about six acetylcholinester-ase-positive cells appear with a peak incidence after 4 days in culture with a plating efficiency of one colony formed for every 104 nucleated cells plated.

In orally estrogen-primed animals, four times as much progesterone is required to bring about secretory changes comparable to those obtained following subcutaneous priming.

These findings might be interpreted as evidence that: (a) oral estrogen-induced proliferative changes are either different or inadequate, and (b) excess progesterone is conditio sine qua non for the induction of those proliferative changes susceptible of being transformed into secretory ones.

By sucrose density gradient ul-tracentrifugation and standard hemolytic complement assays, complement fixing activity of about 19 S and greater was found in blister fluids of one patient with pemphigus and three of five patients with bullous pem-phigoid. Control blister fluids, including experimentally induced blisters, lacked such activity. Complement fixing activity was apparent, however, in 6 of 10 pemphigus sera and in two of five bullous pemphigoid sera tested. The material present in both pemphigus and bullous pemphigoid appears to activate the classical complement pathway.

Twenty-eight to 133 days after hypophysectomy of the rat, somatostatin as revealed immunocytochemically was depleted from all segments of the median eminence and from the proximal part of the infundibular stem. A consistent change in the store of somatostatin in the OVLT could not be demonstrated.

Mice were found to be generally refractory to sc challenge with heat-killed cells of Pseudomonas aeruginosa and did not die unless unusually high concentrations were employed. Approximately 38.6% of the animals receiving a single, sublethal dose of 1 × 10¹⁰ dead cells developed black, crusty, necrotic skin lesions within 3 to 5 days. No major gross and histopathological changes were detected in internal organs. If the animals were sc administered sublethal doses of either live or dead cells of P aeruginosa 16 days prior to sc challenge, then the incidence of black lesions rose to 78.6 and 50% of the animals, respectively. Of several antineoplastic agents tested, only methotrexate significantly affected the 72-hr LD₅₀ resulting in a drop to 1.8 × 10⁹ from 3.4 × 10¹⁰ cells.

However, both methotrexate and actinomy-cin D decreased the incidence of the black lesions.

This investigation was supported by the Office of Naval Research Grant No. N-00014-69-0235, the National Science Foundation graduate traineeship Grant No. GZ 2033, and the National Institutes of Health general research support Grant No. 5 S01 RR05384.

Stimulation of pancreatic secretion by a fatty meal resulted in a nonpar-allel time-course of intrapancreatic amylase, lipase, trypsinogen, and chymotrypsinogen levels. Concomitant variations in the rate of pancreatic protein biosynthesis are observed. Those results support the hypothesis that nonparallelism in exportable enzyme levels is a consequence of a nonparallel regulation by the stimulus of the individual rates of enzyme biosynthesis.

The authors thank Prof. H. Sarles, Prof. J. Morisset, and Doctor O. Guy for helpful discussions.

The hypothesis that hypoxic pulmonary vasoconstriction is mediated directly by depolarization of the vascular smooth muscle was tested in anesthetized dogs. Pulmonary vascular responses to hy-poxia were first determined in eight dogs during 20-min exposures to 10% O₂. Each animal was then treated with verapamil (0.5 mg/kg, iv), to block transmembrane Ca²⁺ influx in an attempt to abolish the vasoconstrictor responses to hypoxia. The hypoxic exposures were then repeated, and the pulmonary vascular responses were compared to the control responses. Verapamil administration attenuated hypoxic pulmonary vasoconstriction, but did not abolish the responses to hypoxia. Pulmonary vascular resistance increased 87% during the control hypoxic exposure, but increased only 38% during hypoxia after verapamil. The response to another vasoconstrictor, prosta-glandin F_2α, was not reduced by verapamil, indicating a different mechanism of mediation. These results suggest that the pulmonary vasoconstrictor response to alveolar hypoxia, in the intact dog, involves trans-membrane Ca²⁺ influx, and are consistent with the idea that hypoxia acts primarily by directly depolarizing vascular smooth muscle, rather than acting indirectly through a chemical mediator.

Several methods of inducing experimental allergic encephalomyelitis (EAE) in rats were examined using different (i) rat strains, (ii) combinations of ence-phalitogen with different adjuvants, and (iii) sites of encephalitogen inoculation. The time course and severity of the ensuing diseases were determined and methods delineated for inducing a disease with limited variability and high incidence.

Omitting the mycobacterial component from the adjuvant eliminated the complication of adjuvant arthritis, which may develop after the appearance of EAE. En-cephalitogenic emulsions prepared with an equal volume of frozen guinea pig spinal cord (GPSC) and hexadecane or squalene, injected into two inguinal nodes or one foot pad of Lewis rats, provided two quick and easy ways to induce EAE. Emulsions of encephalitogen with Freund's complete adjuvant or hexadecane, injected into the ear, also induced EAE but lengthened the time between the antigen inoculation and clinical symptoms which accompany the onset of EAE disease. However, injection into the ear offers an advantage over the Newbould technique (direct instillation of encephalitogen in pre-exposed lymph nodes), since the animals can be confidently predosed with drugs which may reduce lymphoid mass. Effects of local inflammation on systemic drug metabolism are also minimized when using the ear route.

The studies were supported by the U.S. Public Health Service through Grant No. 15759. We are much indebted to Drs. Osamu Kohashi and Brian Newbould for helpful discussions, to Jack Fitzgerald and Rosemary Stremel for assistance in handling the animals, and to Mrs. Sheryl Lumas for preparing the manuscript.

Twelve heifers were exposed to 21° ambient temperature for 10 days, and then subjected to 4.5, 21, or 32° for 9 days in controlled environmental chambers. Serum prolactin (PRL) decreased linearly (P < 0.01; 0.6 ng/ml/°) as the temperature was reduced during the first day from 21 to 4.5°; serum PRL increased linearly (P < 0.05; 1.17 ng/ml/°) as the temperature was increased from 21 to 32°. Between Days 2 and 9 serum PRL averaged 2.6, 13.0, and 27.7 ng/ml (P < 0.05) at 4.5, 21, and 32°, respectively. Injection of thyrotropin-releasing hormone (TRH) caused serum PRL to increase within 5 min from 20.4 to 109.8 ng/ ml at 32°; at 21° serum PRL increased from 15.7 to 62.8 ng/ml, whereas at 4.5° serum PRL did not respond to TRH. Serum growth hormone (GH) averaged 4.0, 6.3, and 9.4 ng/ml at 4.5, 21, and 32°, respectively, but these means were not different (P > 0.10). TRH released GH at all temperatures tested, but the quantity released was unaffected by ambient temperature. Relative humidities of 50 and 90% did not significantly alter (P > 0.05) serum PRL or GH. We conclude that ambient temperature affects basal and TRH-stimulated concentrations of serum PRL but not GH in heifers.

The technical assistance of Eileen Bostwick, Larry Brock, and David Kreider is most appreciated.

The adjustments in total fluoride concentration in plasma, bones, liver, and muscle were examined when rats were given a diet of very low fluoride content following a dietary regimen of elevated fluoride intake. The animals received a diet containing 34 ppm of fluoride and water with 50 ppm added fluoride in the 28-day initial period and in the depletion period they were given a diet containing only 0.21 ppm of fluoride and distilled water. The findings indicated a 12-fold increase in the fluoride content of the humeri after 28 days of high-fluoride intake with a greater increment by the epiphyses than by the dia-physes. During 21 days of the depletion period the skeletal fluoride was reduced by only 7.7% indicating a marked retention of fluoride during processes of bone remodeling and growth. The plasma, muscle, and liver total fluoride contents were significantly increased at the end of the period of high-fluoride intake, but these concentrations were found to be restored to base-line levels in 3-7 days of the depletion period. By comparison of the distribution of total fluoride with injected radiofluoride between tissue and plasma waters, it was concluded that muscle and liver contain bound fluoride that does not exchange completely with ionic fluoride.

Hypoglycemic rats bearing insulin-secreting islet-cell adenomas produced by the combined action of streptozotocin and nicotinamide were treated with streptozotocin. Antitumor response was demonstrated by elevation of blood glucose, reduction in plasma and tumor IRI, and histo-pathologic changes in the beta-cell neoplasm. The rodent tumor model may serve as a predictive system for selection and investigation of mechanisms of action of future antitumor agents to be used in the treatment of malignant insulinoma in man.

The authors gratefully acknowledge the many helpful suggestions of Dr. David A. Cooney and Mrs. Ruth D. Davis of the Laboratory of Toxicology, DCT, NCI, without whose extended cooperation these studies would not have been initiated or completed; and Marie Gottlieb and Elizabeth Perry for valuable technical assistance.