On the reaction kinetics in water of 1,3-propane sultone and 1,4-butane sultone: a comparison of reaction rates and mutagenic activities of some alkylating agents. To determine correlations between the biological action pattern and chemical reactivity of alkylating agents, the rate constants for reactions of 1,3-propane sultone and 1,4-butane sultone with a series of nucleophiles at 37 degrees C have been determined. Previously published data on the mutagenicity of the two sultones and of some alkyl methanesulfonates and dialkyl sulfates towards Schizosaccharomyces pombe have been used in the evaluation of the dependence of mutagenic effectiveness on chemical reactivity. It is of interest to note that the mutagenic effectiveness of the two sultones, if expressed per alkylating event at a certain low nucleophilicity is the same as that of e.g. methyl methanesulfonate and ethyl methanesulfonate.


The sensitivity of HeLa and Chinese hamster (ovary) cells to methylene dimethane sulphonate. HeLa and Chinese hamster (ovary) cells were exposed in vitro to methylene dimethanesulphonate (MDMS) and their survival of colony-forming ability was assayed in monolayer culture. Asynchronous cultures were exposed to the drug for the whole culture period but cell survival was complicated by the toxicity of formaldehyde which is a final breakdown product of the drug. A short treatment schedule of 15 min within the hydrolytic half life of the agent was therefore employed and the response of synchronous cultures of HeLa cells was then assayed throughout the course of the cells cycle. Cells were most sensitive at the beginning of the DNA synthetic phase (early-S) and most resistant at the end (late-S).


Mechanism of the inhibition of transcription by PR toxin, a mycotoxin from Penicillium roqueforti. PR toxin, a mycotoxin synthesized by Penicillium roqueforti, impairs the transcriptional process in liver cells; the two main RNA polymerase systems (enzymes A and B) are affected by PR toxin. The toxin does not require an enzymic conversion before interfering with in vitro RNA synthesis. Addition of ammonium sulphate completely prevents the inhibition of transcription by PR toxin. In vitro results, using RNA polymerase purified from E. coli, suggest that PR toxin impairs the activity of the RNA polymerase itself. Regarding the step of the transcription process affected, it is shown that PR toxin inhibits both initiation and elongation of the polynucleotide chain.


Binding of thiols to microsomal cytochrome P-450. Lipophilic thiol compounds interact spectrally with liver microsomes from phenobarbital-pretreated rats by formation of unusual optical difference spectra with peaks at 378, 471, 522 and 593 nm in the oxidized state. The binding kinetics were biphasic. The EPR spectrum of cytochrome P-450 was slightly modified but the magnitude of the low-spin signal was unchanged. n-Octanethiol competitively displaced metyrapone and n-octane from the active site of cytochrome P-450. Other thiols behaved similarly with variations in the magnitude and the affinity of the binding process. Tertiary thiols caused the formation of the high-spin cytochrome P-450 substrate complex, and model studies with myoglobin revealed that steric hindrance prevented the liganding of the tertiary thiol group to the ferric cytochrome P-450. Addition of thiols to dithionite reduced microsomes resulted in relatively small spectral changes with maxima at 449 nm typical for ligand complexes of the ferrous cytochrome. It was concluded that lipophilic thiols can be bound as ligands by at least two species of oxidized cytochrome P-450 which represent, however, not more than about one fifth of the total cytochrome P-450 content in liver microsomes from phenobarbital-pretreated rats.


Copper toxicity: evidence for the conversion of cupric to cuprous copper in vivo under anaerobic conditions. We have determined the toxicity to cells of Escherichia coli B of cupric copper applied under aerobic and anaerobic conditions in two ways. The growth of cells in liquid medium incorporating cupric copper shows differential inhibition, comparing aerobic and anaerobic conditions--toxicity being greater under anoxia. The growth of colonies upon agar plates incorporating cupric copper does not show such a differential effect. We conclude that colonies on plates are largely anoxic even when incubated aerobically. EPR spectra of cells obtained at various times after application of cupric copper under anoxic conditions indicate the conversion of a considerable proportion of the Cu(II) to a non-paramagnetic species, probably Cu(I). We demonstrate that Cu(I) is more toxic than Cu(II) to cells when applied under anoxic conditions and conclude that the difference in toxicity of Cu(II) applied to cells under aerobic and anaerobic conditions results from the greater extent of reduction of Cu(II) to Cu(I) under anaerobic conditions.


Arenavirus taxonomy: a review.

Arenavirus taxonomy: a review. Despite a late beginning, the construction of the arenavirus taxon and its placement in the scheme of the International Committee on Taxonomy of Viruses has now been completed. The bringing together of the member viruses has already provided valuable indications of promising laboratory and field study approaches; in the future this classification will contribute further to our understanding of the natural history and disease processes of the human pathogens of the group.


Morphology and morphogenesis of arenaviruses. Arenaviruses have unique structural characteristics; they are pleomorphic, have a mean diameter of 110-130 nm, and consist of a membranous envelope with surface projections surrounding an interior containing ribosomes and filaments. Virus particles bud from plasma membranes of infected cells and in many cases large intracytoplasmic inclusion bodies are formed. These characteristics allow generic identification, but not differentiation of individual viruses. Ultrastructural identification of virus particles and pathological processes in infected tissues of man and experimental animals is important in understanding the nature of arenaviral pathogenesis Such identification also contributes to our understanding of the mechanisms of viral shedding and transmission in reservoir host species.


Antigenic properties of the arenaviruses. Arenaviruses are known to show antigenic relationships in the complement-fixation (CF) and fluorescent antibody tests but not in the neutralization test. The humoral response to some of the arenaviruses is characterized by a dissociation between the antibodies determined by the CF test and those determined by the neutralization test and also by its late appearance following natural infection. Investigations are reported showing that, as regards the CF test, Junin, Machupo, Amapari, and Tacaribe viruses are closely related, while LCM and Lassa viruses are distantly related to each other and to the remaining viruses in the group. Studies by agar gel diffusion and precipitation have shown complete specificity except among viruses that are very closely related in the CF test. Observations with sera from persons infected with Lassa fever virus show that CF antibodies rarely appear before the 18th day after onset; they can persist, with diminishing titres, for up to 6 years.


Antigenic relationships of lymphocytic choriomeningitis virus and Tacaribe virus in the indirect haemagglutination test. The indirect haemagglutination test has been developed for use with LCM and Tacaribe viruses employing antibody-sensitized erythrocytes. Both in this test and in the indirect haemagglutination inhibition test cross-reactions between LCM and Tacaribe viruses were revealed. This opens new perspectives for group and species identification of arenaviruses.


Infection of wild and laboratory animals with Machupo and Latino viruses. Natural infection with Machupo and Latino viruses occurs only in the cricetine rodent Calomys callosus. Machupo virus induces fatal infection in suckling mice and hamsters, and in adult guinea-pigs, marmosets, and rhesus monkeys. Latino virus kills only suckling hamsters; it produces chronic but non-viraemic infection in Calomys rodents.Machupo virus, in contrast, induces a viraemic immunotolerant infection in suckling Calomys, and a split response in animals more than 9 days of age. Tolerant infection is associated with haemolytic anaemia and splenomegaly, lesions not observed in animals able to clear viraemia and produce circulating neutralizing antibodies. Experimental increase in the fraction of tolerant response was obtained by decreasing the virus dose or by phenotypic inbreeding of rodents. Long-term effects of tolerant infection included mild runting, decreased survival time, and almost total sterility among females, largely caused by fatal virus infection of embryos.


Tamiami virus infection in mice and cotton rats. Tamiami virus produces a lethal encephalitis in suckling mice, and the illness is mediated, at least in part, by cellular immunity. Infection of extraneural organs, including lymphoid organs, is limited. The same virus produces a widespread infection in its natural host, the cotton rat, but neither symptomatic illness nor cytopathology results. Since antibody is produced, as in the murine infection, suppression of cellular immunity to the virus may be responsible for the non-cytolytic infection. Lymphoid tissue is extensively infected in the cotton rat and a relationship between this lymphotropism and immunosuppression is suggested.


Experimental biology and pathogenesis of Junin virus infection in animals and man. A fatal disease resembling Argentine haemorrhagic fever of man has been produced in guinea-pigs and mice by inoculation with Junin virus. Infected guinea-pigs show macroscopic and microscopic haemorrhagic lesions, marked bone marrow changes, decreased leukocytes and platelets in the peripheral blood, and impairment of immunological response. This response permits differentiation between pathogenic (XJ) and attenuated (XJ Cl(3)) strains. Guinea-pigs inoculated with the XJ Cl(3) strain develop an inapparent infection accompanied by slight haematological changes, the appearance of antibody, and protection against challenge with the pathogenic strain. The attenuated strain has been used successfully as an immunizing antigen in 636 human volunteers. Guinea-pigs infected with Tacaribe virus show cross-protection against Junin virus, with the presence of heterologous neutralizing antibodies. Suckling mice infected with Junin virus develop a typical viral encephalitis; the pathogenicity of the virus decreases with increasing age of the mice. Experiments with thymectomized mice and with mice treated with antithymocyte serum suggest that the pathogenicity of Junin virus in this host is related to the integrity of the thymus-dependent immune system. There is evidence that humoral antibodies do not play any role in the development of the encephalitic lesions but rather protect mice against Junin virus infection. A recent serological survey among laboratory workers and inhabitants of the endemic area has demonstrated the presence of inapparent infection with Junin virus.


Pathogenesis of Machupo virus infection in primates. Experimental Machupo virus infection of rhesus and cynomolgus monkeys produced a severe illness consisting of an initial clinical phase and a later neurological phase. Cumulative mortality during the two phases was 80% and 95% respectively. Attempts to alter the pathogenesis with decomplementation or immunosuppression resulted in earlier deaths of the monkeys.


The structure of rodent faunas associated with arenaviral infections. The biogeographical examination of rodent faunas associated with arenaviruses reveals two distinct patterns. Lymphocytic choriomeningitis (LCM) virus is associated primarily with a single murid species, Mus musculus, although it is also known to cause laboratory infections in other species. On the other hand, the arenaviruses from the Western hemisphere are associated exclusively with a large and diverse group of cricetid rodents. Studies to date, although limited, have not demonstrated their association with any other rodent groups, although in South America alone at least twelve other rodent families are known. Evidence at the present time indicates that Lassa virus is only associated with a common African rodent, Mastomys natalensis. From this limited evidence it is as yet difficult to determine whether Lassa virus will follow the pattern of the South American arenaviruses, most of which are known from several species of rodents, or that of LCM virus, which appears to be associated with only a single rodent species. In this paper, the history and structure of South American, Eurasian, and African rodent faunas are described.


Rodent control programmes in areas affected by Bolivian haemorrhagic fever. Bolivian haemorrhagic fever (BHF) caused by Machupo virus is acquired by contact with the excretions and secretions of Calomys callosus, an indigenous cricetine rodent which is preadapted to peridomestic habitats. It competes successfully with Mus musculus, but not with Rattus rattus. A successful disease control programme has functioned in Beni Department since 1964. It is based on trapping surveys and the detection of splenomegaly in Calomys rodents as an index of chronic virus infection. Mass trapping and poisoning are used initially, and regular trapping is employed to control Calomys populations in towns where disease has occurred. More than 1000 cases of BHF were recorded from 1960-1964, but less than 200 in the past 10 years. The cost of this programme is approximately $30 000 annually.


Protection of monkeys against Machupo virus by the passive administration of Bolivian haemorrhagic fever immunoglobulin (human origin). Bolivian haemorrhagic fever immunoglobulin of human origin, given either prior to or shortly after experimental infection with Machupo virus, protected rhesus and cynomolgus monkeys against initial clinical illness. Some survivors developed severe neurological signs 30-47 days after virus inoculation and died 4-6 days later. Results from one of the experiments suggested that the development of neurological signs was associated more frequently with high doses of immunoglobulin than with intermediate or low doses.


Status of arenavirus vaccines and their application. A limited but definite need exists for vaccines against Lassa, Junin, and Machupo viruses. Medical and laboratory personnel, as well as defined high-risk population groups, require protection from these highly virulent agents. To date little work has been done on inactivated vaccines for these viruses. A live attenuated Junin vaccine has been tested successfully in more than 600 persons, and a high-passage Machupo virus strain has protected rhesus monkeys against lethal infection produced by a homologous field strain.Work has been initiated on possible heterologous protection induced by infection or antigenic stimulation with arenaviruses not pathogenic for man. Crucial for the eventual development of effective vaccines are the construction of more maximum security laboratories and the further elucidation of the experimental and natural biology of the agents in lower animals and man.


[Action of an ATP analog, adenosine 5'-hyophosphophosphate in the reactions catalysed by hexokinase and fructose-6-phosphate kinase]. Adenosine 5'-hypoposphate phosphorylates glucose and fructose 6-phosphate in the presence of hexokinase and fructose 6-phosphate kinase respectively. It behaves as a competitive inhibitor versus ATP in the hexokinase reaction. Its affinity for the two enzymes is similar to that of ATP, the maximal velocities being however much lower.


[Possible role of cell contact in the control of a differentiation program in Dictyostelium discoideum]. Synthesis of cAMP-phosphodiesterase falls at the end of the aggregation phase in Dictyostelium discoideum. Exogenous cAMP pulses, known to induce they synthesis of that enzyme during the course of the aggregation process, do not prevent the shut off of enzyme synthesis. Specific intercellular contacts which form at the end of aggregation seem to be required for the inhibition of enzyme synthesis.


[Demonstration of nucleotide pyrophosphatase and phosphohydrolase activities in normal lymphocyte of Balb/c mice and absence of these activities in YC8 tumor cells and in lymphocytes of animals with YC8 lymphoma]. Saline extraction of tumor cells from YC8 lymphoma transplanted in Balb/c Mice, of lymphocytes from animals bearing this tumor and of lymphocytes from control animals, allowed us to show important hydrolytic activities towards UDP-[(14C)]-galactose and galactose-[(14C)]-1-phosphate in normal lymphocytes. These activities disappear in lymphocytes from Mice bearing this tumor and in tumor cells themselves.


[Induction of dolykaryocytes by vesicular stomatitis virus in XC rat cells]. V.S.V. induced polycaryocytes in rat embryonic fibroblasts, transformed by the Prague strain of Sarcoma Rous (XC cells). This fusion is strictly dependent on the expression of the viral genome and is probably due to the incorporation of viral antigens in the cell membrane. The integrity of cellular RNA synthesis is however not required. The fusion is probably due to a membrane structure characteristic of these transformed cells.


The role of cyclic adenosine 3', 5'-monophosphate and calcium in the regulation of contractility and glycogen phosphorylase activity in guinea pig papillary muscle. We studied the relationships between the positive inotropic effects of isoproterenol, increased frequency of contraction or paired electrical stimulation, and cyclic AMP concentration and phosphorylase activity in isolated guinea pig papillary muscles. The minimum concentration of isoproterenol (10 nM) that augmented isometric force development increased cyclic AMP concentration. However 100 nM isoproterenol was required to increase the phosphorylase activity ratio (-AMP/+AMP) from 0.15 +/- 0.03 to 0.25 +/- 0.03. After addition of 1 muM isoproterenol to the bath, cyclic AMP increased within 0.5 minute from 0.58 +/- 0.03 to 1.04 +/- 0.13 mol/kg (wet weight), peak contractile force was elevated 2-fold at 1 minute, and the phosphorylase activity ratio rose to 0.40 +/- 0.02 in 4 minutes. Although an increase in contraction frequency (6/min to 36/ min) and paired stimulation produced more than a 3-fold increase in peak contractile force, there were no changes in cyclic AMP and phosphorylase activity. The cyclic AMP concentration during diastole was 0.60 +/- 0.04 and in midsystole, 0.55 +/- 0.03 mumol/kg. Anoxia increased the phosphorylase activity ratio from 0.19 +/-0.02 to 0.41 +/- 0.04 without elevation of cyclic AMP concentration. Removal of Ca2+ from the bathing medium prevented active force development and the anoxic increase in phosphorylase activity, but did not prevent the isoproterenol-induced increase in cyclic AMP and phosphorylase. These results suggest that cyclic AMP is a factor in the catecholamine-induced enhancement of inotropic state. However, it does not appear to play a role in the maintained augmentation of inotropic state produced by increased contraction frequency and paired stimulation, nor does the concentration of the cycle nucleotide appear to vary during the contraction cycle or during anoxia. Extracellular Ca2+ is required for contraction, the positive inotropic aciton of catecholamines and phosphorylase b to a conversion by anoxia.


Swine aortic smooth muscle in tissue culture. Some effects of purified swine lipoproteins on cell growth and morphology. Smooth muscle cells (SMC) were grown from inner media explants of swine aorta and used as a model for studying the role of lipoproteins in atherogenesis. These cultured cells retain the characteristics of SMC through multiple passages. Cell growth curves, in time, were obtained by using standard counting techniques, SMC grew slowly (0.019 cycle/day) in modified Dulbecco-Vogt medium supplemented with 1.5% swine serum. Purified lipoproteins were prepared from three normolipidemic and two hyperlipidemic (cholesterol-fed) swine. When the medium of 84 growth experiments was supplemented with these lipoproteins, SMC growth rate increased linearly with lipoprotein cholesterol concentration up to 10 mg/dl. At 10 mg/dl of lipoprotein cholesterol, very low density lipoproteins (VLDL) increased growth rate 7.2-fold (P less than 0.01); low density lipoproteins (LDL) 5.7-fold (P less than 0.01); high density lipoproteins (HDL2) 3,4-fold (P less than 0.02); and HDLc, and lipoprotein appearing in the hyperlipidemic swine, 3.0-fold (P less than 0.01). Addition of 10% lipoprotein-free serum stimulated growth rate 6.0-fold (P less than 0.01). There was no difference between normo- and hyperlipidemic lipoproteins with respect to cell growth rate. Factors present in the ultracentrifugal bottom, and factors appearing during the platelet release reaction, were shown to contribute to the SMC growth response. Morphological alterations characteristic of intimal foam cells occurred in SMC grown in VLDL at triglyceride levels in excess of 15 mg per 100 ml. Thus there are distinct parallels between SMC response in this model in vitro and atherogenesis in vivo.


The effect of severe trauma on urinary cyclic AMP in children of different ages. Under routine clinical conditions the urinary excretion of cyclic AMP was studied in 97 healthy children (3 months to 16 years old) and 65 child victims of various forms of trauma and proved to be age-dependent. The mean admission cAMP urinry levels for children of all ages were increased by 44.8% and were time dependent. In 26 injured children (3 months to 9.5 years old) the excretion increased to 12.10 +/-1.29 mumol cAMP/g creatinine as compared to 8.06 +/- 2.25 mumol cAMP/g crEAtinine (50.0%; P less than 0.01) and in 38 children (9.5-16 years) it rose to 6.43 +/- 4.12 from 4.72 mumol +/- 1.10 (36.2%; P less than 0.001). In 23 victims admitted within the first two hours after trauma the cAMP levels were elevated by 31.25% in contrast to 49.2% elevation in those admitted from 2 to 24 h after trauma. In a five-day follow-up of a group of 15 surviving patients the highest excretion of cAMP was found on the first day. It was followed by a steep decline until the values plateaued on the third day at values slightly elevated above control. Compared with the fifth day, the first day excretion expressed as mumol cAMP/24 h was enhanced by 92.3% (P less than 0.01) and as a ratio to creatinine (muol/g creatine) by 52.24% (P less than 0.05. In comparison with injured adults studied previously under analogical conditions, the cAMP excretory response to trauma in children differs in higher intensity (P less than 0.01) and in the nature of the daily pattern starting with the third day. It is pointed out that the intensity of the cAMP response to pathological stimuli may be age-dependent also in other clinical conditions.


Apolipoprotein of the low density lipoprotein of human plasma: structural study in familial hyperbetalipoproteinemia. Isolation of the low density lipoprotein (LDL) from human plasma was accomplished within a narrow density interval (1.030-1.045 g/ml). After covalent modification by maleic anhydride of the protein moiety and delipidation it was possible to fractionate, by ion-exchange chromatography, up to 35% of the protein into three peptides with distinctive amino acid compositions. These fractions appeared to be identical in normal individuals and in patients with familial hyperbetalipoproteinemia (FH). Our findings provide further evidence that in FH the abnormal catabolism of the protein moiety of LDL is not related to a modification of its structure.


Radioimmunoassay for human plasma apolipoprotein B. We have developed a simplified double antibody radioimmunoassay for human apolipoprotein beta. The purified antigen, a narrow density subclass of beta lipoprotein (d 1.030-1.050 g/ml; was isolated by a combination of gel filtration and ultracentrifugationmthis material gave a single immunoprecipitin arc  on crossed immunoelectrophoresis into an antibody to whole human serum. The antigen was radiolabelled using iodine monochloride at pH 10. The iodinated antigen was indistinguishable immunochemically from native material and eluted as a single radioactive peak from a Sephadex G-200 column. The lower limit of sensitivity of the assay was 15 ng protein, and the working range, 15-200 ng. The mean apolipoprotein B level (+/-1 S.D.) in 128 healthy control subjects was 86.6 +/- 29.6 mg/100ml and is in agreement the values published by other workers using unmodified assays.


Functional hypoparathyroidism and parathyroid hormone end-organ resistance in human magnesium deficiency. Hypocalcaemia is a well-recognized manifestation of magnesium deficiency. We have studied seventeen patients with this syndrome in an attempt to determine the pathogenesis of the hypocalcaemia. Mean initial serum calcium concentration was 5-6 mg/dl and mean initial serum magnesium concentration was 0-75 mg/dl. Serum immunoreactive parathyroid hormone (IPTH) was measured in sixteen patients in the untreated state. Despite severe hypocalcaemia, serum IPTH was either undetectable (less than 150 pg/ml) or normal (less than 550 pg/ml) in all but two patients. Serial measurements made during the initial 4 days of magnesium therapy in four patients showed an increase in serum IPTH within 24h, but a delayed increase in serum calcium, which required approximately 4 days to reach normal values. The effect of the rapid normalization of serum magnesium on serum IPTH and serum calcium concentration was studied in three patients. Within 1 min after 144-300 mg of elemental magnesium was administered i.v., serum IPTH had risen from undetectable to 3600 pg/ml and 1725 pg/ml in two patients and from 425 pg/ml to 937 pg/ml in the third. Serum calcium concentrations were unchanged after 30-60 min. These data provide evidence for impaired parathyroid gland function in most of the magnesium deficient patients. The rapidity with which serum IPTH rose in response to magnesium therapy indicates that this may reflect a defect in parathyroid hormone (PTH) secretion rather than its biosynthesis. The failure of serum calcium concentration to increase during the initial days of magnesium repletion, at a time when serum IPTH concentrations were normal or elevated, suggests end-organ resistance to PTH in these patients. The renal response to PTH was examined in two magnesium deficient patients by measurement of urinary cyclic AMP excretion following administration of parathyroid extract. In both patients there was a minimal increase in urinary cyclic AMP concentrations. In contrast, when the hepatic response to glucagon was tested on the same patients by measurement of plasma cyclic AMP concentrations following administration of glucagon, normal increases were observed. These results suggest that adenylate cyclase systems of various organs may be affected differentially by a state of magnesium deficiency. It is suggested that magnesium deficiency may result in defective cyclic AMP generation in the parathyroid glands and in the PTH target organs. This could be the principal mechanism operative in both impaired PTH secretion and end-organ resistance to PTH which together contribute to the development of hypocalcaemia.


Starvation in man.

Starvation in man. Starvation entails a progressive selection of fat as body fuel. Soon after a meal glucose utilisation by muscle ceases and fatty acids are used instead. Ketoacid levels in blood become elevated over the first week, and the brain preferentially uses these instead of glucose. The net effect is to spare protein even further, as glucose utilisation by brain is diminished. Nevertheless, there is still net negative nitrogen balance, but this can be nullified by amino acid or protein supplementation. Insulin appears to be the principal regulatory hormone. Recent data suggest that decreased levels of active T3 may play a role by sparing otherwise obligated calories by decreasing metabolic needs.


Adenine arabinoside (ARA-A) blocked HSV-2 infected human skin fibroblasts as targets for the detection of serum mediated and cellular cytotoxicity in patients with cervical carcinoma. Human skin fibroblasts remained viable after infection with herpes simplex virus type 2 (HSV-2) if grown after infection in the presence of adenine arabinoside (ARA-A); The cells were specifically killed by normal human lymphoid cells in the presence of HSV-2 convalescent or rabbit immune serum, and this cytotoxicity could be blocked by a crude HSV-2 antigen preparation. Antibodies capable of mediating such cytotoxicity against HSV-2 induced cell surface antigens could be detected in four out of eleven patients with carcinoma of the uterine cervix. Ten patients were tested for the presence of direct cellular cytotoxicity against HSV-2 infected, ARA-A blocked target cells and none was found.


Leucocyte capillary migration: an adherence dependent phenomenon. To determine the mode of action of leucocyte inhibitory factor (LIF) on polymorphonuclear leucocyte (PMN) migration out of capillary tubes, this phenomenon has been compared with PMN adhesion to cotton wool columns. Colchicine and vinblastine sulphate had no effect on either of these processes but cytochalasin B caused marked inhibition of PMN migration and increased PMN adhesion. The cytochalasin B effect could be reversed in the presence of drugs known to increase intracellular levels of cyclic AMP. For inhibition of PMN migration by LIF, Mg2+ but not Ca2+ was essential. LIF not only inhibited PMN migration but also increased PMN adhesion to cotton wool columns. LIF inhibitory activity on PMN migration was not affected by colchicine or vinblastine. Leucocyte migration and adhesion depend on similar mechanisms, and are both influenced by LIF which probably acts by affecting micro-filament function.


Colestipol in familial type II hyperlipoproteinemia: a three-year trial. Colestipol, a new bile acid sequestrant polymer, has been shown to lower the serum cholesterol level more than 30% in 13 patients with familial type II hyperlipoproteinemia. Placebo for 6 wk was followed by colestipol for periods up to 36 mo. A slight but not significant increase of serum triglyceride concentrations was observed during the first 18 no, but they returned to values under the baseline level thereafter. No signs of impaired intestinal fat resorption were noted. Side effects were primarily gastrointestinal (mild and transient constipation). Colestipol seems to be an effective and safe drug in the treatment of the famiial type II hyperlipoproteinemia, without escape phenonmenon.


Malignant fibrous histiocytoma associated with bone infarcts: report of a case. A caisson worker with symmetrical bone infarcts in the tibiae demonstrated a malignant transformation of one of the bone infarcts with wide-spread metastases to the lungs and viscera. Histologically, the tumor was found to be a malignant fibrous histiocytoma and despite amputation, the patient died approximately one year following the diagnosis. This case lends credence to the concept of a cause and effect relationship existing between bone infarcts and the subsequent development of bone sarcoma.


Tumors associated with metallic implants in animals. Eight cases of malignant tumors originating in close proximity to various metallic surgical implants which were used in the treatment of common canine and feline fractures are presented. There were 5 osteosarcomas, one fibrosarcoma and 2 undifferentiated sarcomas with the characterisitcs of malignant histiocytomas. All 8 occurred in the md shaft of the femur, humerus or radius, a most unusual location considering that almost all primary canine osteosarcomas have been found tramedullary pins, 2 with intramedullary Steinmann pins (one with cerclage wires) and one with an AO plate screws. Seven of the fractures treated had united and only one pseudoarthrosis was present where the cerclage wires were used. All implants used showed corrosion and had been in place for 6 months to 6 years prior to tumor discovery. None was infected.


Issues in developing quality aftercare clinics for the chronic mentally ill. Two large aftercare clinics were established to provide treatment and rehabilitative care for a chronic mentally ill population requiring neuroleptic drugs. The clinics have evolved rapidly and expanded their service over the past 3 years but have required constant monitoring and modification in order to provide quality as well as quantity care in the community setting. These modifications are described and the importance of further community-based investigation in the rehabilitation and treatment of the chronic mentally ill is stressed.


Practical guidelines for the community treatment of markedly impaired patients. A sound technology does not yet exist for successfully treating in the community those patients traditionally treated by public mental hospitals and aftercare programs. The current paper attempts to advance this technology by presenting practical guidelines based on the empirical work of the writers and others. Recommendations include a focus on the teaching of coping skills, an in vivo site of treatment, an assertive approach, and use of a variety of social learning techniques. Staffing and monetary needs for community treatment are also discussed.


Newer aspects of parathyroid chemistry and physiology. We have attempted to integrate in this review many recent studies on the formation, packaging and secretion of parathormone by the parathyroid gland. We have shown that substantial new insights have been gained into this complex cellular operation. Continuing investigation should clarify many of the outstanding questions regarding function of the parathyroid in their central role in calcium homeostasis.


Immunoglobulin concentrations and bovine enterovirus inhibitors in fetal bovine fluids. Fluids from 53 bovine fetuses ranging in age from 90 to 240 days were examined for immunoglobulin G (IgG) and immunoglobulin M (IgM) and neutralizing activity to ten bovine viruses. Non-specific inhibitors to bovine enteroviruses were found in serum, allantoic, and amniotic fluids of most samples tested. In most cases, serum IgG were within normal values. Neither IgG nor IgM was detected in amniotic fluids, whereas 2 samples of allantoic fluid contained traces of IgG.


Bone pathology in hypervitaminosis D an experimental study in young pigs. Five groups of 4 weanling pigs were fed a diet with 1.2% calcium and 1.0% phosphorus for 8 weeks with vitamin D3 at 1, 5, 25, 125 and 625 times the recommended levels, respectively. Hypercalcemia and hypophosphatasemia developed rapidly and persisted in Group 5 and developed more slowly but steadily in Group 4. Increasing levels of vitamin D3 influenced progressively and negatively the activity of resorbing osteocytes with osteopetrosis in Groups 2 and 3 and with osteonecrosis in Group 5. Atrophy of osteoblasts further contributed to the osteopenia in Group 5. Cartilage growth activity was arrested in Group 5. The negative effect on the resorbing osteocytes, which finally lead to death of the cells, was ascribed directly to vitamin D3 toxicosis since hypoparathyroidism and hypercalcitonism, both resulting from hypercalcemia, are not known to induce osteonecrosis. Since hypercalemia was finally as severe in Group 4 as in Group 5 and since there was soft tissue calcinosis only in Group 5, the calcinosis was always considered dystrophic, an interpretation supported by the observation that degenerative histologic changes preceded soft tissue calcinosis.


Use of cell cultures with persistent virus infections to test the efficacy of antiviral compounds. BHK-21 cells persistently infected with either vaccinia or foot and mouth disease virus were used to study the efficacy of antiviral compounds. The results of the persistent infection cell culture (PICC) test were compared with those obtained by the plaque reduction (PR) test. The comparison showed that: (1) the PICC test is more informative than the PR test; (2) stimulative as well as inhibitory activities of compounds are detectable, and (3) since the PICC test can be carried on for several weeks or even months this test is especially well suited to study the problem of drug resistance in cell cultures.


[Sitosterol in familial hyperlipoproteinemia type II. A randomized double-blind cross-over study]. The effect of beta-sitosterol on the lipid and lipoprotein level was evaluated in a randomised double-blind cross-over trial in 24 patients with primary familial type II hyperlipoproteinaemia over a period of 16 weeks. All patients completed the trial, however 10 of them had to be excluded from the evaluation due to fluctuations of their body weight or unreliable drug intake. Sitosterol lowered the total cholesterol level by 12.5% (P less than 0.01) from 9.96 mmol/l (3.69 g/l) to 8.37 mmol/l (3.23 g/l). The LDL-cholesterol level was lowered by 19.5% (P less than 0.05). The sitosterol concentration in plasma was consistently lower than 0.3% of total cholesterol. No side effects or tachyphylaxis was observed in the course of the trial. A return to normal of an increased serum cholesterol level by a combination of a lipid lowering diet and sitosterol monotherapy was only achieved in one patient.


[Poliomyelitis in the German Federal Republic 1974-1975 (author's transl)]. The virological surveillance of poliomyelitis in 1974-1975 led to the detection of specific characteristics of wild viruses in 57 out of 221 cases of poliovirus isolation. The disease symptoms were typical for poliomyelitis in 36 of these cases, less characteristic in 13. Polio wildvirus was isolated 8 times from the surroundings of the patients. In 36 foreign patients and one German the causative agent was imported from an endemic area. In these cases and in a further 7 German patients the disease was sporadic. In contrast 5 cases in German children in late autumn 1975 constitute a local epidemic of poliomyelitis.


Interaction between thyroid hormones and erythrocyte membranes: competitive inhibition of binding 131 I-L-triiodothyronine and 131 I-L-thyroxine by their analogs. Molecular structural characteristics of thyroid hormones which influence binding to the erythrocyte membranes were investigated by competitive binding experiments. The ability of thyroid hormone analogs to displace 131 I-L-thyroxine and 131 I-L-triiodothyronine from the membranes was considered evidence of their competitive binding. The diphenyl ether linkage (thyronine) was essential as compounds with a single aromatic ring were weakly competitive. The presence of three iodine atoms at 3, 5 and 3' positions on thyronine was optimal for maximal competitive binding. There was weak competitive binding of analogs if chlorine or bromine was substituted for iodine. The alanine side chain was required for optimal binding as N-acetyl-l-thyroxine and various deaminated analogs were poor competitors compared to T4 and T3. L-isomers of T4 and T3 showed greater competitive binding to erythrocyte membranes than the corresponding d-isomers.


Failure of 1, 25 dihydroxycholecalciferol (1, 25-(OH) 2-D3) to modify cyclic AMP levels in parathyroid hormone-treated and untreated bone cells. Treatment with 1, 25-(OH) 2-D3 (1 ng/ml-25 ng/ml) for periods ranging from 2.5 min. to 60 min. did not alter cyclic AMP levels in bone cells isolated from periosteum-free rat calvaria, or in cells isolated from rat periosteal tissues. 1, 25-(OH) 2-D3 failed to modify the acute increases in cyclic AMP elicited by PTH (10 ng/ml-1 ug/ml). Two separate 1, 25-(OH) 2-D3 preparations, biologically active in other systems, were ineffective under a wide variety of experimental conditions. These results suggest that 1, 25-(OH) 2-D3 is not an acute modulator of cyclic AMP metabolism in PTH-treated and untreated bone cells.


Regulation of cyclic adenosine 3', 5' -mono-phosphate dependent protein kinase of rat ovarian cells by luteinizing hormone and human chorionic gonadotropin. Regulation of cAMP dependent protein kinase activity from rat ovarian cells has been studied in response to luteinizing hormone and human chorionic gonadotropin. Treatment of cells with human chorionic gonadotropin in concentration range of 2.5ng-1000ng/ml resulted in increased accumulation of cAMP,activation of protein kinase followed by the stimulation of progesterone synthesis. A sixfold increase in the activity ratio, defined as the ratio of protein kinase stimulated in situ to that maximally stimulated in vitro by exogenous cAMP, was observed with 1ug/ml of hCG. This concentration of hormone also produced a ten-fold increase in cAMP and a thirty-to forty-fold increase in progesterone synthesis. Protein kinase activation was specific for LH and hCG, as other polypeptide hormones were without any appreciable effect. The stimulation of protein kinase persisted even after the elevated cAMP level began to fall. It appears that the activation of protein kinase is an obligatory early event that mediates an increase in gonadotropin stimulated progesterone synthesis.


Cytoplasmic glucocorticoid receptors in the developing small intestine of the rabbit fetus. Glucocorticoid binding and alkaline phosphatase activity in the small intestine of the fetal rabbit were studied to investigate the relationship of glucocorticoid receptors and the development of the tissue. In the cytosol fraction, the binding of (3H)dexamethasone involves a macromolecule with high affinity (Kd = nM) for the hormone and a limited number of binding sites (saturable at a hormone concentration of 10 nM). That the binding reaction involves a protein and sulfhydryl groups was demonstrated by the absence of binding of the steroid in the presence of Pronase and sulfhydryl blocking reagents. In sucrose density gradients, the complexes have sedimentation coefficients of about 4S and 7S at low ionic strength, but only 4S at high ionic strength (0.4m KCl). The binding protein is thermolabile, and is stabilized by complexing with the hormone. The ability of different steroids to compete with (3H)dexamethasone for the binding sites correlates well with their glucocorticoid potency. During development of the fetal rabbit small intestine, the total number of glucocorticoid-binding sites in the cytosol increases in parallel with the increases in the tissue weight until term. However, the concentration of the binding sites (pmol/mg cytosol protein) is maximum at day 25 of gestation, followed by a decrease to the adult level within a few days after birth. Alkaline phosphatase activity is first detectable on day 25 of gestation and increases rapidly thereafter. These observations suggest that there may be a temporal relatioship between the development of the fetal rabbit small intestine, as reflected in the alkaline phosphatase and the levels of glucocorticoid receptors in the cytosol.


Testosterone-binding protein in reproductive tracts of fetal rats. Testosterone-binding proteins may mediate the induction of Wolffian duct differentiation by testicular testosterone. The presence of such protein(s) was sought in reproductive tracts of 14.5-21.5-day-old fetal rats. Supernatant fractions (127,000 x g) were equilibrated with [3H]T) +/- radioinert testosterone in Tris - HCl:EDTA buffer, pH 7.4, (approximately 0.1 mg protein/0.5 ml) at 4 C for 16 hours. Bound and free (3H)T were separated by charcoaldextran adsorption or Sephadex G-100 gel filtration. The results with 14.5-15.0-day-old tracts were: a) specific binding to protein was saturated with increasing (3H)T concentration; b) Scatchard plot analysis indicated the presence of a single class of binding sites with high affinity (apparent Kd = 2 nM) and limited capacity (approximately 16 fmol/mg protein) for (3H)T; c) specific uptake was limited to (3H)T and (3H)5 alpha-dihydrotestosterone; d) (3H)T uptake by the tract supernatant was tissue-specific; e) pronase treatment abolished binding capacity for (3H)T; f) bound radioactivity consisted solely of (3H)T; and g) the mesonephric and ductal segment of the genital tract specifically binds (3H)T. The data demonstrate binding protein(s), specific for testosterone and possibly dihydrotestosterone, in the genital ducts of 14.5-15-day-old fetal rats. (3H)T binding to genital duct supernatants from male but not from female fetuses increased about 5-fold between 14.5 and 20.5 days of gestation. Upon Sephadex G-100 gel filtration, radioactivity was confined to the macromolecular fraction appearing in the void volume. Nuclear fractions, obtained from intact ducts incubated with (3H)T at 30 C but not at 0 C contained radioactivity. These observations are compatible with the existence of a cytoplasmic testosterone receptor or carrier protein aggregate. We have thus concluded that testosterone-binding proteins are present in the genital ducts of rat fetuses and that, in the male, their concentrations increase with progressive Wolffian duct differentiation.


1alpha,25-dihydroxyvitamin D3-like component present in the plant Solanum glaucophyllum. The ability of an aqueous extract prepared from dried leaves of the South American shrub Solanum glaucophyllum to interact with a chick intestinal receptor system highly specific for the biologically active form of vitamin D, 1alpha, 25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), was monitored under both in vivo and in vitro conditions. The pretreatment of chicks with the S. glaucophyllum extract reduced the amount of subsequently administered radioactive 1alpha, 25-dihydroxyvitamin D3 localized in the intestinal chromatin fraction by 90% when compared with vitamin D-deficient controls. In addition, the active principle in the aqueous extract was found to compete effectively with 1alpha, 25-(OH)2D3 in a reconstituted intestinal receptor system in vitro. The plant factor(s) was found to be as effective as 1alpha, 25-(OH)2D3 in decreasing the renal 25-hydroxycholecalciferol-1alpha hydroxylase activity levels when administered to vitamin D-deficient chicks. By means of the in vitro intestinal receptor assay, it was possible to estimate that there are approximately 10 to 60 mg of the biologically active material in one kilogram of dries S. glaucophyllum leaves. These data indicate that the plant factor(s) may be a structural analog of 1alpha, 25-(OH)2D3 and suggest the feasibility of its isolation.


Stimulation of testosterone production in isolated rabbit thecal tissue by LH/FSH, dibutyryl cyclic AMP, PGE2alpha, and PGE2. The capacities of isolated rabbit theca and granulosa cells to secrete testosterone were studied in vitro. Large Graafian follicles (1-1.5 mm in diameter) were dissected intact from the ovaries of adult estrous rabbits. Granulosa cells from 4 follicles (50,000 cells) and theca tissue (16 pieces per dish, equivalent to 4 follicles) were cultured separately for 6 days either as controls (without exogenous hormones) or with one of the following agents: 1 lU/ml LH/FSH (Pergonal), 10-3M dibutyryl cyclic AMP (Bu2cAMP), 1 mug/ml prostaglandin F2alpha (PGF2alpha), or 1 mug/ml prostaglandin E2 (PGE2). The media were collected every 2 days, and the testosterone (T) was measured by radioimmunoassay. The control cultures of granulosa cells secreted small amounts of T (700 +/- 317 pg/culture: mean +/-SE) during the first 2 days in vitro, and the addition of LH/FSH, Bu2cAMP, PGF2alpha, or PGE2 did not significantly stimulate T production. After 2 days in vitro, very little T (greater than 200 pg/culture) was produced by control and prostaglandin-treated granulosa cells, whereas those incubated with LH/FSH and Bu2cAMP maintained their initial T production rates. Theca control cultures produced 3 +/- 0.4 ng of T (mean +/- SE) during the first 2 days in 13.6-fold by LH/FSH, 3.6-fold by Bu2cAMP, and 3-fold by PGF2alpha and PGE2- T was not detected in theca cultures after 2 days except in those treated with LH/FSH or Bu2cAMP, which produced 1.5 +/- 0.5 and 1.6 +/- 0.3 ng of T, respectively, at 4 days (mean +/- SE). These results suggest that under the present conditions, pieces of rabbit thecal tissue have a greater capacity to produce T de novo than do isolated granulosa cells, and indicate that T production is transiently stimulated by LH/FSH, Bu2cAMP, PGE2alpha, and PGE2.


Endometrial content of nuclear estrogen receptor and receptivity for ovoimplantation in the rat. The purpose of this study was to provide information concerning the amount of nuclear binding sites for estrogen in endometrial cells during the first week of pregnancy in the rat. The "insoluble" (high-salt non-extractable) binding during the first four days of pregnancy follows a circadian rhythm, with a maximal value at midnight, and a minimal one a noon. By day 5, the amount plateaus at a minimal value. The number of estradiol binding sites of the soluble (high-salt extractable) nuclear fraction shows a similar pattern. All the necessary elements for the estrogen-dependent induction of endometrial receptivity for nidation appear, therefore, to be present already by day 3, but the turning on of the endometrial response to estrogen on day 4 seems to depend on additional factor(s).


Changes in (125I) labeled human chorionic gonadotropin (hCG) binding to porcine granulosa cells during follicle development and cell culture. The specific binding of (125I)iodo human chorionic gonadotropin to porcine granulosa cells isolated at two stages of follicle maturation was quantitated immediately after harvest and following 6-7 days of culture. Both porcine LH (pLH, LER 786-3) and hCG inhibited (125I)iodo hCG binding, while pFSH (ler-1132) did not compete for hCG with granulosa cells for 24-48 hours at 37 C did not alter its subsequent ability to bind to fresh cells. The binding of (125I)iodo hCG was a rapid process which was not readily reversible. Scatchard analysis of the equilibrium binding data resulted in linear plots showing the hCG binding capacity of highly differentiated (HD) cells, harvested from large preovulatory follicles, to be significantly greater than that of moderately differentiated (MD) cells isolated from smaller luteal phase follicles, both before (794 vs 93 pmoles/g; P less than 0.001) and after (157 vs 5 pmoles/g; P less than 0.001) culture. A decline in binding capacity occurred in both cell groups during culture and was associated with a significant decrease in progestin production. In contrast, the hCG binding affinity of the granulosa cell was equivalent at both stages of follicle development and remained unchanged after 6 days of culture (mean apparent Kd = 2.9 x 10-10M; n=27. These data indicate that porcine granulosa cells contain a homogeneous class of LH receptors whose number, but not affinity, increases during follicle maturation in vivo. The loss of receptors in vitro suggests the absence of some factors(s), as yet unidentified, critical to the maintenance and development of the receptor population.


Biochemical characterization with parathormone and calcitonin of isolated bone cells: provisional identification of osteoclasts and osteoblasts. Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately two-fold increases in cAMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activites were measured in the two types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase, and general protein synthesis. Although each of these activites was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase were higher in te PT cells. Parathormone stimulated acid phosphatase and hyaluronate synthesis by 100-200% only in the CT cells; in inhibited alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase by 75-90% only in the PT cells. Calcitonin alone had no effect on any of these activities other than cAMP production, but in inhibited the action of parathormone in the CT cells. The sensitivities, time courses of development,and magnitudes of these hormonal effects were similar to those observed previously in intact calvaria, indicating that the isolated cell system is a reliable model for the study of bone metabolism. Based on the metabolic responses of the cells, we postulate that the CT type of populations is enriched in osteoclasts and, possibly, osteocytes, and the PT type of population is enriched in osteoblasts.


Effects of excess iodide and other anions on thyroid hormone secretion in normal or hypophysectomized rats treated with graded doses of thyroid hormone. In order to obtain further information about the stimulatory action of excess iodide on thyroid hormone secretion in thyroxine (T4)-treated rats, experiments were performed in hypophysectomized rats, or rats treated with graded doses of T4 or triiodothyronine (T3).T3 as well as T4 played a permissive role in the production of the iodide effect in normal animals, but T3 was more effective than T4. Excess iodide stimulated thyroid hormone secretion in hypophysectomized animals, this finding being compatible with the hypothesis that, by inhibiting TSH secretion, T3 and T4 produced a condition in which excess iodide stimulated thyroid hormone secretion in intact rats. However, T4 played an additional role in thyroid hormone secretion by acting directly on the thyroid. In hypophysectomized animals, small doses of T4 stimulated thyroid hormone secretion, and this action was additive to that of excess iodide, whereas large doses of T4 were inhibitory and reduced the effectiveness of excess iodide. The stimulatory action on thyroid hormone secretion was specific for iodide and was not shared by other anions. The action of excess iodide was blocked by methimazole. We suggest that excess iodide stimulates thyroid hormone secretion by increasing intrathyroidal concentrations of cyclic AMP in the absence of TSH, and that this increase in cyclic AMP concentration is blocked by methimazole.


A cytoplasmic estradiol receptor in rat testicular tissue. Testicular cytosol from Sprague-Dawley rats was shown to possess an estradiol binding component which exhibited the properties of a receptor. Specific binding of estradiol by the receptor was shown, at 4C in vitro, to reach equilibrium by 6 h. That level of binding was maintained at a plateau for 18 h. Estradiol binding was demonstrated to increase linearly as a function of testicular cytosol concentration ultilized. The Ka at equilibrium was determined as 4.07  .05 x 1010M-1, while the number of binding sites for whole testicular tissue from mature rats was 1.16 x 10-14 +/- 0.061 moles/mg cytosol protein. Sucrose gradient sedimentation analysis of the cytosol receptor revealed that the major peak of radioactivity migrated in the 8S region. Enzymatic treatment of the cytosol demonstrated that the binding component was protein in nature. Heat treatment, 70 C for 10 min, resulted in 83% loss of the receptor activity. Storage in liquid N2 (for as long as 60 days) was shown to maintain receptor activity. Steroid specificity studies revealed that other estrogens were competitive with 17beta-estradiol for binding sites, but progestins, testosterone, and dihydrotestosterone did not affect estradiol binding. Tissue specificity studies showed that the capacity of testicular cytosol to bind estradiol was much greater than that of several non-target organs. Ontogenically, the cytoplasmic estradiol-binding capacity/testis was low from 7 to 21 days of age, but rose dramatically between 3 and 7 weeks of age, and an apparent plateau was reached at 15 weeks. The injection of unlabeled 17beta-estradiolin vvio was shown to deplete the cytosol of estradiol binding sites as determined in vitro. Depletion was rapid, occurring within 30 min, and the cytoplasmic estradiol receptors remained low for a period of 4 h. At 6 h, replenishment has occurred and control levels of cytoplasmic receptors were restored by 12 h. These results suggest that an estradiol receptor is present in testicular tissue of the rat, and that binding capacity is maximum in the mature testis.


Further characterization of a rat ovarian testosterone receptor with evidence for nuclear translocation. We have recently described receptor like testosterone (T)-binding protein in the cytosol of ovaries from estrogen-primed hypophysectomized immature female rats (HIFR). This binding protein is thermolabile, saturable, specific for T, and sediments at 7-8 S on sucrose gradients. We now report the further characterization of this protein. It is present in the cytosol of isolated granulosa cells, has a molecular weight of 240,000, a frictional ratio of 1.8, and a mean Stokes radius of 73 A. The binding protein is acidic and cysteine residues are necessary for binding. We have also demonstrated the selective nuclear accumulation of T by granulosa cells after the in vivo administration of (3H)T to estrogen primed HIFR. After the in vitro incubation of the ovaries with (3H)T, a thermolabile protein specific for T was extracted from nuclei under high-salt conditions. The cytosol and nuclear T-binding proteins described in this report have many characteristics of an androgen receptor, and may play a role in the regulation of granulosa cell proliferation during preantral follicular growth.


Hormonal stimulation of alpha-amylase synthesis in porcine pancreatic minces. Minces of porcine pancreas maintained a linear rate or protein synthesis for more than 4 h with similar kinetics in the presence of absence of hormones. alpha-Amylase protein, quantitated by using glycogen percipitation, SDS polyacrylamide gel analysis, and radioimmunoassay, was found to represent 6-7% of the total protein content of the minces. The addition of prostaglandin E1 (10-7M), or a pancreozymin preparation (3-10 Crick-Harper-Raper units/ml) to the incubation media produced within 60 min an increase in alpha-amylase protein above that stored in the tissue 10- to 20-fold greater than that observed in control minces. The addiction of cAMP analogs (10(-3)M) to the medium produced a more variable stimulation of alpha-amylase content. Quantitation of the incorporation of radiolabeled amino acid into alpha-amylase protein with simultaneous determination of the specific activity of the precursor pool suggested that these agents produced at least 3- to 5-fold stimulation of de novo alpha-amylase synthesis. This stimulation was not the result of decreased rates of alpha-amylase catabolism. The addition of actinomycin D (20 mug/ml), a concentration inhibiting more than 93% of new RNA synthesis, failed to alter the stimulation of alpha-amylase synthesis by these agents. Apparent hormonal stimulation of non-alpha-amylase protein synthesis was also observed.


16Beta-hydroxydehydroepiandrosterone: the dichotomy between renal receptor binding and urinary electrolyte activity. Excessive production of 16beta-hydroxydehydroepiandrosterone (16beta-OH-DHEA) has been suggested as a cause of low-renin essential hypertension. The mineralocorticoid effect of 16beta-OH-DHEA was reported to be one-fortieth that of aldostereone in the rat bioassay. Using kidney slices from adrenalectomized rats, the affinity of 16beta-OH-DHEA and a series of related compounds for mineralocorticoid receptors has been determined. In studies done at both 4 C and 37 C, the affinity of 16beta-OHDHEA for mineralocorticoid receptors was found to be less than 0.1% that of aldosterone (P less than 0.01). Various related steroids and/or potential metabolites similarly showed negligible affinity for the aldosterone receptor. In addition, In addition, 16beta-OHDHEA showed no significant affinity for renal dexamethasone-binding sites (Type II glucocorticoid receptors), corticosterone-binding sites (Type III glucocorticoid receptors), dihydrotestosterone binding sites, or estradiol binding sites. In in vivo competition experiments, the concurrent administration of 50 mug deoxycorticosterone reduced (3H)aldosterone binding to 20-30% of control levels; 50 mug 16beta-OH-DHEA did not compete for (3H)aldosterone binding sites. In in vivo bioassay electrolyte excretion was found-in contrast to that of aldosterone-to be variable. Within a given group, certain rats reproducibly responded to 16beta-OH-DHEA by sodium retention and kaliuresis; in others no response was observed. In vitro binding studies comparing "responders" with "non-responders" demonstrated that in neither group did 16beta-OH-DHEA have significant affinity for renal mineralocorticoid receptors. Accordingly, the mechanism whereby 16beta-OHDHEA produces changes in urinary electrolyte excretion appears independent of classical mineralocorticoid effector mechanisms. The conditions under which this effect is seen await eludication.


Gastrin in the perinatal rat pancreas and gastric antrum: immunofluorescence localization of pancreatic gastrin cells and gastrin secretion in monolayer cell cultures. The presence and development of immunoreactive gastrin (IRGa) in the fetal and neonatal pancreas and pyloric antrum of the rat were studied. IRGa appeared in both organs at least as early as the 16th day of fetal life. Antral IRGa increased rapidly and continuously in the neonatal period, while pancreatic IRGa concentration increased and was maintained at a relatively constant level from days 5 to 35. Monolayer cell cultures of the neonatal rat pancreas were used to evaluate the role of cyclic AMP mediated release of gastrin. The addition of N6,O2'-dibutyryl cyclic AMP (4 mM) or theophylline (4 mM) to the culture medium induced significant release of gastrin. The stimulation of adenylate cyclase with cholera toxin (10 ng/ml) also resulted in significant gastrin release. Long-term cultures (18-24 days) were shown to release gastrin continuously at a relatively constant rate. The cellular localization of pancreatic gastrin in 7-day-old cultures was performed by immunological techniques, using fluorescein-labeled antibodies to gastrin. The gastrin-containing cells were located at the periphery of most of the endocrine cell clusters. Immunofluorescence techniques for insulin and glucagon also showed that the alpha cells had a similar peripheral distribution, although they were more frequent in number. In contrast, insulin-containing cells were numerous and were present in all areas of the endocrine cell clusters. The studies support the following conclusions: a) Gastrin is present in the rat pancreas, even as early as late fetal life; b) Gastrin-producing cells are present and functionally competent in monolayer cell cultures of the neonatal rat pancreas for prolonged periods of time (24 days); c) Gastrin is released from these cells when intracellular levels of cyclic AMP are increased; d) By immunofluorescence methods, the gastrin-producing cells in pancreatic cell cultures are found to be located at the periphery of the endocrine cell clusters.


A high-affinity estrogen-binding protein in rat placental trophoblast. A high-affinity, low-capacity estradiol-binding molecule (RE) has been demonstrated in the basal zone trophoblast (BZT) of the pregnant rat. On day 11 of pregnancy (day 0 = first sperm-positive day) RE is present in BZT cytosol, where it has a ka of 1.2 X 10(6)M-1 sec-1, t1/2 = 12.7 min, at 20 C. The Kd, under similar conditions, consists of 2 components, 1.3 X 10(-4) sec-1, t1/2 = 90 min, and 5.9 X 10(-5) sec-1, t1/2 = 196 min. When one uses the faster component, the equilibrium constant, Kd, obtained from kd/ka is 1.1 X 10(-10)M, in close agreement with that obtained from Scatchard analysis of specific estradiol (E2) binding at 20 C. On day 11 there were approximately 12,000 sites/cell in BZT cytosol. Scatchard analysis of nuclear RE on day 11 indicated a Kd of 1.85 X 10(-10)M and approximately 21,000 sites/nucleus, but, in day 15 BZT, nuclear RE was undetectable. Neither cytosol nor nuclei prepared from placental labyrinthine zone (LZT) tissue (fetal placenta) showed evidence of high-affinity, low-capacity E2 binding. Sucrose density gradient analysis on 5-20% linear gradients showed the cytosol RE to be approximately 4S whether in high or low-salt conditions. When measured against binding by 3H-labeled estradiol (*E2), the cytosol BTZ RE was competed for strongly (80-90%) by estrone, estriol, diethylstilbestrol, and estradiol-17alpha at 200 times excess. Nafoxidine-HCl, also at 200X excess, competed to approximately 50%. Corticosterone, progesterone, testosterone, dehydroepiandrosterone, and pregnenolone did not compete. The hormone specificity of nuclear BZT RE was similar to that of the comparable cytosol RE with the exception that nafoxidine did not compete. This was probably due to differences in kinetics, nafoxidine requiring a longer time to reach equilibrium than the other estrogens. The size of the nuclear RE by sucrose density gradient analysis was approximately 2S by KCl extraction (which was inefficient) or 4S by trypsin extraction. We conclude that the BZT of the day-11 rat placenta contains an estrogen-binding molecule with many of the attributes of a true receptor.


Induction of follicle-stimulating hormone (FSH) receptors in rat ovaries by estrogen priming. Estrogen pretreatment of hypophysectomized immature rats stimulated granulosa cell proliferation and enhanced FSH binding to the ovary in vivo. The present studies were undertaken to ascertain whether estrogen pretreatment altered the number of specific FSH receptor sites per ovarian cell, resulting in increased FSH binding. Cell suspensions were prepared from the ovaries of groups of rats pretreated with graded doses of 17beta-estradiol. The isolated cells contained specific FSH receptors with high affinity for FSH. The results of these studies clearly showed that the number of specific FSH receptors per ovarian cell was unaffected by pretreatment with graded doses of 17beta-estradiol or with diethylstilbestrol. The enhanced in vivo FSH binding was solely the result of estrogen-induced proliferation of granulosa cells with a fixed number of specific FSH receptors per cell.


Lactogenic and growth hormone-like activities in pregnancy determined by radioreceptor assays. Factors with growth hormone and lactogenic activities have been identified in serum samples and placental extracts of 9 species using a radioreceptor assay for prolactin or lactogens (RRA-PRL) and one for growth hormone-like activity (RRA-GH). The concentrations of placental lactogen (PL) and growth hormone-like activity (GHLA) in pregnancy were different in each species. The levels of PL began to rise at or before mid-pregnancy and either remained elevated until term (hamster, goat, sheep, monkey, and human), declined gradually after reaching peak concentrations just beyond mid-pregnancy (guinea pig), or had 2 peaks of activity (mouse, rat). The peak concentrations in different species ranged from 350 ng/ml in the cow to 45,000 ng/ml in monkey serum samples. The concentrations of GHLA were similar to PL levels in the guinea pig, goat, cow, and monkey but were lower than PL levels in the sheep and human. The maximal concentrations of PL and GHLA in placental extracts varied from 1 mug/g in guinea pigs to 200 mug/g in sheep. The ratio of lactogenic/growth hormone-like activity of both serum and placental extracts varied greatly among the various species. Gel filtration studies of serum samples  revealed that PL and GHLA in the hamster, mouse and rat had different elution volumes, whereas, in the remaining species, the two activities co-eluted.


Effects of vitamin D and its metabolites on calcium transport in the diabetic rat. We studied diabetic rats, 5 days after streptozotocin injection, and matched controls to determine whether depressed duodenal calcium absorption associated with uncontrolled diabetes in the rat would respond to vitamin D or its metabolites. At the appropriate time following the intravenous injection of 0.25 mug of either vitamin D3, 25-hydroxycholecalciferol (25-OHD3), 1,25-dihydroxycholecalciferol (1,25-OH)2D3), or 1alpha-hydroxycholecalciferol (1alpha-OHD3) to half of each diabetic and control group, calcium transport was evaluated using everted duodenal sacs with 0.4 mM40Ca and tracer 45Ca on both mucosal and serosal surfaces. All agents stimulated duodenal calcium absorption in controls. Diabetics responded only to 1,25-(OH)2D3, the metabolite that acts directly on the duodenum, and to its synthetic analog, 1alpha-OHD3. 1alpha-OHD3 is activated to 1,25-(OH)2D3 by 25-hydroxylation in the liver; 25-OHD3 must be 1alpha-hydroxylated in the kidney to be active. The stimulation of duodenal calcium absorption in diabetic rats by 1alpha-OHD3, but not by either vitamin D3 or 25-OHD3, is most consistent with a defect in vitamin D metabolism at the 1alpha-hydroxylation step in the kidney.


Concerning the hormonal regulation of androgen binding protein in rat testis. Androgen binding protein (ABP) was measured in testis following an acute injection of FSH to ascertain whether this protein could serve as an endpoint marker of FSH action in the Sertoli cell. A single intravenous injection of oFSH (200 mug NIH-S-10) resulted in a rapid stimulation of ABP activity in testis of either 10- or 14-day old rats. Maximal increases were noted by 2 h in both cases (undetectable to 0.9, and 0.3 to 1.5 pmol ABP/mg protein in 10- and 14-day old rats, respectively) and by 4h ABP activity had again returned to control values. Although FSH failed to acutely stimulate ABP in 60-day-old rats, hypophysectomy of these animals resulted in a return of sensitivity within 3 days. The acute stimulation of ABP by FSH was also shown to be dependent upon both the route of hormone administration and the dose of FSH. Finally, the rapid decrease in ABP activity following FSH could be prevented by injection of a second dose of hormone. ABP activity was also increased by intratesticular injection of an analog of cyclic AMP, 8-bromo cAMP. This response was also dose-dependent and the time course of response was indistinguishable from that resulting following FSH. A test of nucleotide specificity revealed that any adenine nucleotide would stimulate ABP, whereas guanine compounds were ineffective. Peptide hormone specificity was next examined. It was determined that 200 mug of a variety of crude pituitary hormone preparations (LH, GH, PRL and ACTH) were stimulatory. However, when highly purified hormones were utilized, hFSH (LER-1577) did not increase testicular ABP whereas 1 mug of oLH (Papkoff) was maximally stimulatory. Further studies revealed that all compounds (including peptide hormones and nucleotides) which resulted in acute elevation of ABP activity also increased the intratesticular concentration of testosterone. Moreover, a single ip injection of testosterone produced a steroid specific stimulation of ABP which reached maximal levels within 1 h. These findings suggest that the acute regulation of ABP activity in the testis may be a result of the intratesticular concentration of testosterone and not due to a direct effect of FSH as had been previously hypothesized.


Reductions in glucocorticoid inhibition of glucose oxidation and presumptive glucocorticoid receptor content in rat adipocytes during aging. Exposure of adipocytes to glucocorticoid hormones in vitro causes inhibition of glucose transport and metabolsim. Maximal inhibition of glucose oxidations is reduced from 42-50 to 22-25 to 5-8% in young, mature, and senescent rat adipocytes, respectively. Percent values also reflect absolute reductions since basal levels of glucose oxidation per cell are constant at all ages. Adipocytes of CD strain rats continue to increase in size throughout their lifespan, while cell size remains constant during the latter 80% of the Wistar adipocyte lifespan. Thus, cellular age, as well as possibly size, seems to be associated with these changes since they occur in adipocytes of both strains. Concentrations as well as absolute numbers of presumptive glucocorticoid receptors per cell are progressively reduced during maturation and aging of adipocytes in both rat strains. Glucocorticoid effects are known to require about 2 h and can be blocked by various antimetabolites during this period, reminiscent of classical steroid receptor-mediated responses. Thus, gradual loss of glucocorticoid receptors from adipocytes during maturation and aging may be related to progressively decreased glucocorticoid responsiveness.


Distribution binding sites for the progesterone receptor within chick oviduct chromatin. Chromatin from the oviducts of estrogen-treated chicks was sheared and fractionated on sucrose gradients. This resulted in the production of several chromatin fractions which differ in their sedimentation properties, protein composition, the number of acceptor sites for the progesterone-receptor complex, and the ability to serve as a template for RNA synthesis. The pellet chromatin fraction shows an enhanced ability to bind the progesterone-receptor complex in vitro and in cell-free systems. Kinetic analysis indicates that the majority of the acceptor sites for the progesterone-receptor complex are located in the pellet chromatin fraction which is lowest in template activity. The sites may be important loci for initiating the changes in RNA synthesis following the exposure of target cells to steroid hormones.


Adenosine-mediated stimulation of bone cell adenylate cyclase activity. Adenosine rapidly stimulated adenylate cyclase activity but did not modify cyclic AMP degradation when added to a particulate fraction prepared from isolated bone cells. The effect of adenosine was one-half maximal at 5-10 micronM, and was not mimicked by 5' AMP, inosine, guanosine, uridine, adenine, or ribose. Basal and adenosine-stimulated adenylate cyclase activites were directly proportional to the concentration of particulate protein in the assay system. Theophylline decreased the degree to which adenosine stimulated adenylate cyclase activity, whereas another phosphodiesterase inhibitor, RO-20-1724, failed to iiinfluence the effect of adenosine. Adenosine itself, and not a metabolite of adenosine is the stimulator of adenylate cyclase, since it was neither phosphorylated nor deaminated appreciably by the particulate fraction. The particulate fraction did not convert substrate ATP to adenosine in amounts sufficient to enhance adenylate cyclase. The stimulatory effect of adenosine was maximal at 1.2 mM Mg2+, declined with increases in the Mg2+ concentration, and was replaced by inhibition at 20 mM Mg2+. At 2.4 mM Mg2+, basal adenylate cyclase activity peaked at 1.1 mM ATP, and was inhibited by higher ATP concentrations. The magnitude of adenosine stimulation was greater at inhibitory concentrations of ATP than at concentrations which yielded maximum activity. The results suggest that the previously demonstrated ability of adenosine to increase cyclic 3'5' AMP levels in intact bone cells stems from its effect on adenylate cyclase. Adenosine may act by modifying the regulatory nfluence of free Mg2+, uncomplexed ATP, (or both), on adenylate cyclase. Theophylline appears to interfere with the action of adenosine by a mechanism which is distinct from its capacity to inhibit cyclic AMP phosphodiesterase activity. (Endocrinology 99:901,1976)


Effect of glucose on adenosine 3', 5'-monophosphate levels in rat pancreatic islets. Time course of the changes in insulin release and cyclic AMP levels in isolated rat islets incubated in media containing 5 or 16.7 mM of glucose were followed. The higher glucose concentration caused a slight but significant increase of cyclic AMP levels after 10 min incubation, but not 5 min incubation, whereas the stimulation of insulin release by 16.7 mM of glucose was apparent in both incubation times. Theophylline increased cyclic AMP levels markedly but did not stimulate insulin release when the glucose concentration was 5 mM. A slight augmentation by theophylline of insulin release was observed in the incubation medium containing 16.7 mM glucose. All these findings suggest that the elevation of cyclic AMP in islets may not play a role for the initiation of the insulin release induced by glucose, though it may act to modulate the glucose effect.


Direct evidence on incorporation of the receptor into the nuclei of rat ventral prostate in the form of the complex with dihydrotestosterone. Cytosol 9S receptor was prepared from the supernatant fluid at 105,000 X g of rat prostate homogenates by a Sephadex G-100 gel chromatography, and was labeled with 131I. The 131I-labeled 9S receptor retained the activity of forming a complex with 3H-dihydrotestosterone, similarly to the intact cytosol receptor, when examined by a sucrose density gradient centrifugation. When the 131I-labeled cytosol 9S receptor was incubated with isolated prostatic nuclei in the presence of 3H-dihydrotestosterone, it was found that the 131I-labeled receptor was directly incorporated into the nuclei in a form of the complex bound to 3H-dihydrotestosterone. 131I-Labeled receptor-3H-dihydrotestosterone complex which was incorporated into the nuclei was extracted with 0.5 M KCl solution, and the nuclear complex sedimented with a velocity of 5S. The incorporation of 131I-labeled receptor-3H-dihydrotestosterone complex into the nuclei increased along with the raised temperature of incubation, whereas association of the complex with the chromatin reached maximum at 35 degrees C and then decreased gradually beyond this temperature. In the time course study, either the incorporation of the complex into the nuclei or the association with the chromatin reached the maximal levels within 10 min and leveled off up to 60 min. On the other hand, 131I-labeled serum protein was far less efficiently incorporated into the nuclei than the radioiodinated 9S receptor, and association of the serum protein with the chromatin was limited to a very little extent. The 131I-labeled 9S receptor-dihydrotestosterone complex associated with the chromatin was found to be preferably distributed into the non-histone protein as well as the DNA itself of the chromatin. The radioactivity lost by dehistonization of the chromatin was almost negligible. Furthermore, the cytosol 9S receptor fraction bound to 3H-dihydrotestosterone was purified about 100 fold by the two consecutive column chromatographies. The partially purified receptor fraction which was labeled with radioiodine incorporated mostly into non-histone protein and DNA fractions.


Cushing's syndrome due to huge adrenocortical multinodular hyperplasia. A case of Cushing's syndrome due to huge adrenocortial multinodular hyperplasia who was shown to be hyperresponsive to ACTH administration, unresponsive to metyrapone administration and resistant to dexamethasone high dose suppression was reported. After two years' duration of his symptoms, the multinodular adrenals weighing 161 g in total were removed by bilateral adrenalectomy which abolished his symptoms. Postoperatively, plasma ACTH rose gradually to above normal levels, suggesting the presence of primary disorder in the hypothalamus-pituitary axis.


Some biological properties of human chorionic follicle stimulating hormone. The biological properties of human chorionic FSH (hCFSH) for rat ovaries were investigated. Highly purified hCFSH had similar response to the ovarian augmentation test as bovine FSH and significantly enhanced 3H-thymidine uptake by granulosa cells and theca cells in the ovary of hypophysectomized rat. In contrast, highly purified hCG little responded to the ovarian augmentation test and had no effect on 3H-thymidine uptake by the ovary. These results indicate that hCFSH may promote the follicular growth of ovary resulting from granulosa cell proliferation and its enlargement. In addition, freshly harvested porcine granulosa cells were employed in an in vitro system to investigate specific binding of hCFSH to ovarian receptor. Radioiodinated hCFSH (125I-hCFSH) and hCG (125I-hCG) were respectively incubated with cell suspensions. Binding of these hormone preparations was proportional to the cell number and increased with the time of incubation through 120 minutes. The binding ability of 125I-hCFSH to the cells was greater than that of 125I-hCG. Increasing concentrations of unlabeled hCFSH in the incubation mixture progressively inhibited the uptake of 125I-hCFSH by granulosa cells. Unlabeled hCG was not able to compete with 125I-HCFSH binding. The similar phenomenon to inhibit the binding of 125I-hCG to the cells was also recognized in the presence of unlabeled hCG. These findings suggest that granulosa cell has at least two different types of receptor sites: one for hCFSH and the other for hCG.


Changes in cyclic AMP level of rat thyroid by acute and chronic stimulation of thyrotropin in vivo. To assess a possible postmortem change in the level of cyclic AMP, the thyroids were snap-frozen at various time intervals after removal. Rats were fed a low-iodine diet (LID) with PTU for 2 weeks and a week after PTU discontinuation (PTU withdrawal). In all cases, the cyclic AMP level tended to increase as time elapsed from removing till fixing the thyroids, but in the PTU withdrawal group, the level was rapidly increased 2-fold after 5 min. In an acute experiment, the thyroids were removed under anesthesia and frozen rapidly. Intravenous administration of ovine thyrotropin (250 mU) and TRH (500 ng) brought about a rapid increase in the thyroidal level of cyclic AMP to 40% to 20% over the control level. Two weeks after PTU treatment, circulating thyrotropin was increased to a maximum of 19-fold and a further enhancement ("rebound") was observed after PTU withdrawal. PTU treatment led to an increase in the thyroidal level of cyclis AMP pre mug DNA to 60% over the control value. Following PTU withdrawal, the rise in the level of cyclic AMP returned to the normal level. However, there was no change in the concentration when it was expressed as per mg wet tissue weight. Therefore, the increase in the thyroidal concentration of cyclic AMP per mug of DNA may be due to an increase in volume of the follicular cells.


Biological activity of synthetic corticotrophin with short chain lengths in the rabbit. The andrenocorticotrophic effect of a synthetic substituted adrenocorticortophic hormone (ACTH), alpha-1-18NH2-D-Ser1-Lys17, 18-ACTH (CIBA 47, 795-Ba) has been compared with that of alpha1-24-ACTH (tetracosactide), alpha1-24-ACTH depot (tetracosactide depot) and alpha1-18NH2-Gly1-ACTH (giractide) in the rabbit. Plasma corticosteroid levels after salin injection were higher in the afternoon than in the morning. The highest value was observed at 3 p.m. 41,795-Ba, either given intravenously or intramuscularly, was shown to be the most potent peptide followed by tetrocosactide depot and was 15 times more potent than tetracosactide and giractide in steroidogenic activity in the rabbit. The intravenous administration of 41,795-Ba caused more sustained stimulation of the adrenal cortex than the intramuscular injection. These results reveal the diurnal variation pattern of the pitutitary-adrenal axis of the rabbit similar to that of the rat and also confirm the finding that alpha1-18NH2-Dser1-Lys17, 18-ACTH is a potent adrenocorticotrophic peptide without the addition of any agent to delay its absorption in the rabbit.


[The fine structure of the rat pineal and the influence of anti-androgens and castroation]. The pineal body, along with the hypothalamus-hypophysis system, is in the centre of sexual hormone regulation and, in addition to other functions, develops an antigonadotropic action through its organ specific hormone (melatonin). In order to clarify further open questions and to analyse more closely the morphology of the fine structure of the organ, light and electron microscopic studies were made of the pineal bodies of sexually mature male rats after hormonal castration by the administration of cyproterone and cyproterone acetate. The findings were compared with results obtained in the pineal bodies of surgically castrated animals of the same strain. Epiphysectomy was performed 3, 6, 9 and 12 weeks after castration or application of antiandrogen. In the pineal bodies, "light" and "dark" pineal cells and an interstitial cell form could be detected electronmicroscopically. The interstitial cells are found localised near the vessels in particular; their ramifications reach into the perivascular cleft. The light pineal cells preponderate and react essentially in the series; they are therefore considered as the really active form of parenchyma cells. Increased cell activity is already observed three weeks after treatment with antiandrogen: the nucleoli are enlarged, the ribosomes, the mitochondria and ergastoplasm are increased, the endoplasmic reticulum quantified and extended, and also the Golgi regions. The cells are consequently enlarged. Lysosomes also appear which frequently enter the liposomes. The changes in the liposomes after application of antiandrogen are remarkable. Initially they are evacuated, partially drawn out. Later the liposomes are enlarged and increased and often fill the cell body. These pineocytes form an appendage to the castration cells of the hypophysis. The liposomes are in a very close spatial, formal genetic relationship to the Golgi apparatus and to the rough walled reticulum. The larger liposomes apparently arise also through the confluence of smaller ones. Three structural elements of the liposomes could be indentifed: a homogenous, a lamellar and a granular component. The fine morphological reactions are most marked after cyproterone acetate. For the first time, bundles of "microtubuli" are described, the significance of which is not yet clear. They probably arise from the endoplasmic reticulum, they are only found after cyproterone acetate and are presumably due to the gestagen component of the cyproterone acetate. These structures have not previously been observed, either in pineal bodies or in other organs. The structures found after antiandrogen are not so outstandingly recognisable after surgical castration. The biological differences of the surgical compared with hormonal castration therefore seem to be reflected in the cell picture of the pineocytes. Consequently, the pineal body, after treatment with antiandrogen, shows cytostructural changes similar to those of increased anabolism...


[The cortisol concentration in plasma of newborn piglets and after ACTH loading]. In plasma of newborn piglets 12.6 +/- 2.9 mug cortisol/100 ml was estimated radioimmunologically. During the first 24 hours of life the concentration decreases to 1/3 of the starting level. After application of 5 IU ACTH/kg body weight, there was in newborn as in 5-day-old piglets an increase in the concentration of cortisol, while an influence on the level of glucose in plasma could not be detected in 0-day-old animals.


Prevention of dietary induction of rat liver glucose-6-phosphate dehydrogenase by cyclic adenosine 3'.5'-monophosphate and its elimination by glucose prefeeding. Glucagon, epinephrine and cyclic adenosine 3',5'-monophosphate (cyclic AMP) prevented the induction of liver glucose-6-phosphate dehydrogenase (G6PD) by refeeding a glucose-casein mixture to starved rats. The prevention by these agents occurred without any change in the amount of diet consumed. When the injection of cyclic AMP and the refeeding of glucose-casein diet were initiated simultaneously, there was an inhibiton of G6PD induction depending upon the dose and frequency of cyclic AMP administration during the period of refeeding, while when cyclic AMP was given later than 12 h of the refeeding, the lag period for induction of this enzyme, there was no preventive effect. A glucose prefeeding was also found to counteract the inhibitory effect of cyclic AMP on G6PD induction by the subsequent glucose-casein refeeding. The present data together with the elimination of actinomycin D effect by the glucose prefeeding suggest that the inhibitory effect of cyclic AMP on the induction of G6PD dehydrogenase is exerted at the level of transcription.


An approach to the anabolic action of androgens by an experimental system. As skeletal muscle is more developed in the male than in the female, it is supposed that androgens might be responsible for myotrophic or anabolic action. In this respect, many studies were made to try to answer the question: are androgens (or some of their metabolites) responsible for myotrophic action and by what mechanism? Do they act directly on skeletal muscle as on other target organs, or have they an indirect action on muscle, perhaps through a secondary stimulant (for instance synthesized in the liver)? Evidence is now presented that, in the rat's skeletal muscle, androgens likely act through the binding to a cytosoluble receptor, as they do in the ventral prostate. This receptor has analogous properties to all other androgen receptors. It is a proteinaceous (pronase sensible) "8S" component binding testosterone and androstanolone with high affinity and small capacity; it does not bind androstanediols. This finding and the increased incorporation of 3H-thymidine in nuclei and increased protein synthesis obtained in muscle cell culture after action of testosterone favour the concept that muscular cells are direct targets of androgens in skeletal muscles. Presently, the steroid specificity of receptor binding cannot be assessed quantitatively with crude cytosol preparation. While in ventral prostate, androstanolone has a higher affinity, the binding experiments have not yet indicated in muscle if the higher affinity of testosterone is related to differential binding of the two steroids, or to the complex effects of enzymes present in the extracts. In fact, evidence was obtained for 5alpha-reductase and 3alpha,beta-hydroxysteroid reductase activities under the same experimental conditions as for binding determinations. Therefore, the apparent antrostanolone binding in muscle could be lowered by transformation into androstanediols not binding to receptor, or the increase of apparent testosterone binding due to transformation into androstanolone. So the problem of whether testosterone or androstanolone or another natural steroid is the most effective myotrophic hormone in rat skeletal muscle remains unsolved. However, this animal model allows the study of certain interesting aspects of action of androgens on muscle. When receptor preparations are partially purified and not contaminated by metabolizing enzymes, different natural or synthetic steroids can be tested as to their affinity and anabolic effectiveness in muscle. It would be of pharmacological interest if receptor diversity made it possible to distinguish myotrophic action from virilizing activities. This in vitro system allows studying the mechanism of action of molecules which could have in vivo an anti-androgen effect and it is remarkable that radioactive testosterone and androstanolone can compete for receptor binding by an excess of estradiol, progesterone and cyproterone acetate...


Encephalitis caused by louping ill virus in a group of horses in Ireland. An outbreak of LI infection in a group of free range horses is described. Three of 4 horses displayed signs of CNS disturbance and 2 of these died after illnesses ranging from 2-12 days duration. In both cases a variable degree of viral polioencephalomyelitis was observed. A virus antigenically indistinguishable from a reference strain of LI virus was isolated from the brain and cervical cord of a 3 y.o. draft mare. Serum samples obtained from 3 of the horses contained HI, CF, precipitating and neutralising antibodies to LI virus, with a rise in antibody titre being demonstrated in 2 animals.


Efficacy of Japanese encephalitis vaccine in horses. The efficacy of Japanese encephalitis vaccine in horses has been described from the effect of mass vaccination on the local prevalence of the disease in horses in each district of Hokkaido, Japan.


Binding of a metyrapone spin label to microsomal cytochrome P-450. Probing of the active site of microsomal cytochrome P-450 was carried out with a spin label derived from 2-methyl-1,2-bis(3-pyridyl)-1-propanone (metyrapone). Its optical binding spectra to cytochrome P-450 resemble the spectra with metyrapone. The electron paramagnetic resonance spectrum of the spin label in microsomes indicates binding with strong immobilization. Since the apparent optical and EPR binding constants agree very well (Ks approximately 2-10(-5) M), and metyrapone is found to displace the spin label, we conclude, that the spin label binds to the active site of cytochrome P-450. Addition of detergents or an increase in temperature mobilizes the bound spin label slightly. The EPR signal accounts for only 60% of the bound spin label due to its dipolar interaction with the low-spin ferric heme of cytochrome P-450. From this finding, the distance between nitroxide and iron is evaluated to be 11 A. This supports the model that one of the pyridine nitrogens of metyrapone is coordinated to the iron of cytochrome P-450. The bound spin label shows virtually no interaction with ferricyanide as if the active site of cytochrome P-450 is a hydrophobic pocket not accessible to ions.


Solubilization and purification of rat liver microsomal 1,2-diacylglycerol: CDP-choline cholinephosphotransferase and 1,2-diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase. 1. The procedure, which involved 2-step sonication of microsomes at pH 7.4 and then at pH 8.5 in the presence of sodium deoxycholate and subsequent dialysis, resulted in 4-5-fold purification of choline-phosphotransferase and ethanolaminephosphotransferase with the yield of 40-50%. 2. Ethanolaminephosphotransferase was further purified 8.5-fold over microsomes by sucrose density gradient centrifugation of the partially purified preparation, while cholinephosphotransferase activity was considerably lost during this procedure. No separation of the two transferases from each other was achieved at this step. 3. Cholinephosphotransferase required Mg2+ as cofactor, and microsomal phospholipids for its maximal activity. On the other hand, Mn2+ was more effective than Mg2+ as cofactor for ethanol aminephosphotransferase, and this enzyme was inhibited by microsomal phospholipids. 4. Both transferases were stimulated several-fold by sodium deoxycholate and also showed similar optimal pH ranging from pH 8.0 to 8.5. 5. Km values for 1,2-diacylglycerol emulsion were 81.0 muM for cholinephosphotransferase and 63.0 muM for ethanolaminephosphotransferase, respectively. CDP-choline and CDP-ethanolamine competitively inhibited, with the same Ki value (both 350 muM), ethanolaminephosphotransferase and cholinephosphotransferase, respectively. The Ki values obtained were much greater than the corresponding Km values for the cytidine substrates (36.4 muM for CDP-choline and 22.0 muM for CDP-ethanolamine). 6. The partially purified enzymes were further treated with Triton X-100. When enzyme activities were assayed with Mg2+, cholinephosphotransferase, although considerably inactivated, was partially separated from ethanolaminephosphotransferase by sucrose density gradient centrifugation of Triton-treated preparations. Furthermore, cholinephosphotransferase (but not ethanol-aminephosphotransferase) itself was partially separated into Mg2+ -requiring and Mn2+ -requiring components. In contrast, ethanolaminephosphotransferase assayed with either Mg2+ or Mn2+ formed a single peak together with Mn2+ -requiring cholinephosphotransferase.


Cell-cycle dependence of two nuclear histone kinase enzyme activities. Growth-associated histone kinases have been extracted from Physarum polycephalum nuclei and resolved into two components by ion-exchange chromatography. The two component activities have different substrate specificities and different times of appearance in the cell cycle. It is proposed that the enzyme(s) phosphorylate H1 histone in vivo in G2 phase, possibly sequentially in time at different sites in the H1 amino acid sequence.


Studies on the structure and function of chick-oviduct chromatin. 2. Biochemical characterization of two chromatin fractions isolated by ECTHAM-cellulose chromatography. Chromatin prepared at various stages of hormone-mediated development of the chick oviduct was investigated for the relative proportions of transcriptionally active (fraction I) and repressed (fraction II) fractions by ECTHAM-cellulose chromatography. During primary stimulation with estrogen, the amount of chromatin DNA in fraction I plotted as a function of time of stimulation showed a bell-shaped profile, similar to the profile obtained earlier for the number of chromatin sites available to RNA polymerase for initiation of RNA synthesis. Chromatin form a transcriptionally inactive system, hen erythrocytes, eluted mainly (98%) as fraction II. The transcriptionally active fraction I of estrogen-stimulated oviduct contained a 4-fold greater RNA polymerase II activity than was found in fraction II. This could be explained by a differential inhibition of RNA polymerase activity in fraction II since enzyme preparations extracted and purified from both chromatin fractions showed equal activities. In support of this finding, fraction I eluted from ECTHAM-cellulose showed a 4-fold greater concentration of rifampicin-resistant RNA chain initiation sites as compared to fraction II. When chromatin from oviduct mince incubated with labeled progesterone and 17 beta-estradiol and was chromatographed on ECTHAM-cellulose, the transcriptionally active fraction also contained a 4-fold greater concentration of bound hormone (per weight DNA) as compared to the repressed fraction.