Mannosidase II and the 135-kDa Golgi-specific antigen recognized monoclonal antibody 53FC3 are the same dimeric protein

Monoclonal antibodies are frequently used as organelle-specific markers without identifying the specific antigen recognized. We have purified the protein recognized by a Golgi-specific monoclonal antibody 53FC3 (Burke, B., Griffiths, G., Reggio, H., Louvard, D., and Warren, G. (1982) EMBO J. 1, 1621-1628). Peptide microsequencing suggested that this antigen is mannosidase II, which was confirmed by cross-immunoprecipitation with an anti-mannosidase II antibody and by precipitation of mannosidase activity with the monoclonal antibody. Mannosidase II was found to exist normally as a disulfide-linked dimer.     

The origin of the membrane convolute in degranulating platelets. A comparative study of normal and "gray" platelets

Thrombin-stimulated normal platelets contain a membrane system of dilated channels with openings to the exterior. Whether these membranes originate from the surface connected system (SCS), the alpha-granules or internalized portions of the plasmalemma has not yet been defined. The present study traces in series of ultrathin sections the rearrangement of these membranes during shape change, degranulation and internalization of surface membranes in washed normal and "gray" platelets upon the stimulation with thrombin (1 IU/ml). Cationized ferritin (CF) was used as a surface marker in order to recognize internalized portions of the plasmalemma. Within the first seconds after stimulation, both normal and gray platelets changed their shape by extrusion of the SCS membranes. Simultaneously they started to internalize surface membrane and formed surface membrane invaginations closely attached to the outer rim of the cytoskeletal sphere which developed during the internal contraction. CF was internalized in these invaginations. CF was not observed within the system of dilated channels of stimulated platelets, however. Thrombin-stimulated gray platelets showed a markedly reduced number of dilated channels or none at all. This observation may be due to the fact "gray" platelets are deficient in alpha-granules. It is concluded that the dilated system of membranes in degranulated normal platelets originates from membranes of the alpha-granules which have performed compound exocytosis.     

Genetic analysis of Tourette syndrome suggesting major gene effect.

Data on Gilles de la Tourette syndrome are analyzed by multiple threshold models in inheritance that incorporate sex effect. The polygenic-multifactorial model is rejected. Single major locus inheritance can account for the data, although many of the occurrences of Tourette are due to nongenetic phenocopies. In both models, males and females share a common genetic environmental liability, but the less prevalent sex, that is, females, has a higher genetic loading for the disorder. The predicted population prevalences in the single major locus model are 2.3% for males and 0.8% for females. The implications for genetic and biological research in Tourette syndrome are discussed.     

Purification and properties of a host cell protein required for poliovirus replication in vitro

A host cell protein required for poliovirus RNA-dependent RNA replicase activity in vitro has been purified several thousand-fold from an uninfected HeLa cell postmitochondrial supernatant. A single protein of apparent Mr = approximately 67,000 daltons and pI 6.3 is associated with this "host factor" activity. Poly(U)-Sepharose chromatography of the template-dependent replicase isolated from poliovirus-infected cells results in the complete loss of replicase activity if a salt gradient is used to develop the column. Host factor elutes early in the salt gradient and restores replicase activity to protein fractions eluted later in the gradient. The host factor, estimated to be present at 50,000-100,000 copies/cell, interacts physically with replicase.     

Genetic regulation of variable Vi antigen expression in a strain of Citrobacter freundii.

Certain strains of the genus Citrobacter exhibit a variable expression of the Vi surface antigen that appears to involve a special mechanism for regulation of gene expression. Two nonlinked chromosomal loci, viaA and viaB, are known to determine nonvariable Vi antigen expression in strains of Salmonella. To confirm the presence of analogous loci in Citrobacter and to ascertain whether either of them is involved in variable Vi antigen expression in this organism, donor strains were constructed from Citrobacter freundii WR7004 and used to transfer their Vi antigen-determining genes to ViaA- and ViaB- Salmonella typhi recipient strains. Vi antigen expression in C. freundii was found to be controlled by loci analogous to the Salmonella via genes. S. typhi recipients of the C. freundii viaA+ genes were restored to the full, continuous expression of the Vi antigen normally seen in S. typhi. Thus, the C. freundii viaA genes appeared to play no role in the variable expression of the Vi antigen. In contrast, S. typhi recipients of the C. freundii viaB+ genes exhibited the rapid, reversible alternation between full Vi antigen expression and markedly reduced Vi antigen expression that was seen to occur in the C. freundii parent. The C. freundii viaB locus was thus identified as the one whose genes are regulated so as to produce variable Vi antigen expression. Genes determining another C. freundii surface antigen, the synthesis of which is not affected by the mechanism regulating Vi expression, were coinherited with the C. freundii viaB+ genes. An invertible, insertion sequence element located within the C. freundii viaB locus is proposed to account for the regulation of variable Vi antigen expression.     

Immunohistochemical studies on electron transport proteins associated with cytochromes P-450 in steroidogenic tissues. II. Microsomal NADPH-cytochrome c reductase in the rat adrenal.

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P-450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum produced against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase technique and an indirect fluorescent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found to differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.     

Effects of acute and chronic restraint and estrus cycle on pituitary-adrenal function in the rat

Female Long-Evans hooded rats with 5-day estrus cycles were subjected to 4 hr of continuous restraint for either 1 or 20 days. On the last day of the stress regimen, plasma and adrenal corticosterone concentrations were determined and classified according to the stage of the estrous cycle. The results indicated that acute stress produced greater plasma corticosterone concentrations than controls only during estrus, whereas in response to chronic stress significant stress-induced increments were observed during estrus and proestrus. The results suggest that the estrous cycle influences the magnitude of the stress-induced increments for both acute and chronic stress. In addition, the pituitary-adrenal system did not show habituation to repeated administration of this stress, but sensitization was observed during proestrus.     

Interferon: effects on the immune response and the mechanism of activation of the cellular response.

The discovery of interferon in 1957 by Drs. Isaacs and Lindenmann led to major revisions in the concepts of man's defenses against viral infections. There are at least two types of interferon. Along with their antiviral properties, they have recently been shown to exert a suppressive effect on the humoral and cellular immune response; they affect both B and T lymphocytes. A variety of substances, including virus, polyribonucleotides, and mitogens for T lymphocytes, are good interferon inducers. T lymphocytes seem to be necessary for these inducers to exert their immunosuppressive effects. The immunosuppressive effects of interferon inducers suggests that interferons may be mediators of suppressor T lymphocyte effects. In the virus system, interferon does not exert its antiviral effects by direct action on the virus, but rather derepresses a cell gene that results in the production of an antiviral protein. This antiviral protein is probably the mediator of inhibition of virus replication. This is a complex sequence of events that results in the interaction of interferon with the cell membrane and the resulting production of the antiviral state in the cell. This review will examine the various steps of this involved process.     

NADPH-cytochrome P450 reductase.

The rate of reduction of cytochrome P450 in hepatic microsomes in the presence of NADPH has been measured with a dual wavelength stopped-flow spectrophotometer. The results obtained, with microsomes prepared from phenobarbital-pretreated rats, indicate that the reduction process is biphasic and most probably composed of two concurrent first-order reactions. The rate constant for the reduction of cytochrome P450 in the fast phase in the presence of ethylmorphine is 1.74 s^?1. Since approximately 50% or more of the cytochrome P450 is reduced in the fast phase under these conditions, the rate of reduction of cytochrome P450 is approximately 150 nmol min^?1 (mg of protein)^?1. Under similar conditions the rate of ethylmorphine N-demethylation is 8.6 nmol min^?1 (mg of protein)^?1. Thus the rate-limiting step in ethylmorphine N-demethylation cannot be the introduction of the first electron into cytochrome P450 by NADPH-cytochrome P450 reductase.     

The nature of the suppressive effect of interferon and interferon inducers on the in vitro immune response

Viral-induced interferon inhibition of the primary in vitro plaque-formong cell (PFC) response in the mouse (C57B1/6) involves a dynamic relationship between the nature of the antigen, the concentration of interferon added to antigen-stimulated cultures, and the time of addition of interferon relative to antigen addition. The PFC response to a thymus-dependent antigen (sheep red blood cells) was more easily suppressed by viral-induced interferon than was that to a thymus-independent antigen (E. coli 0127 LPS), both in terms of inhibitory concentrations of interferon and the time at which the interferon could be added to cultures after antigen and still inhibit the PFC response. These differential effects of interferon could be related to the difference in cellular requirements (B and T lymphocytes) of the two antigens. Interferon was effective in inhibiting the in vitro PFC response of antigen-primed spleen cells, indicating that it can block the response of memory lymphocytes. By using interferon inducers as inhibitors of the in vitro PFC response, it was possible to show that at least two antigenically distinct interferons may be involved in suppressing the immune response. It is known that one type of interferon is induced by virus and synthetic double-stranded polyribonucleotides. The other type is stimulated by antigen and T cell mitogens. A model is proposed to explain the nature of these interferon inhibitory effects in terms of mediation of immune suppressor cell effects.