Pathogenesis of viral hepatitis

The aim of our research is to use animal models to elucidate the molecular basis for viral clearance and liver disease in the pathogenesis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. The results herein discussed provide insight into immunological and virological processes that may lead to the development of new therapeutic strategies to terminate chronic HBV and HCV infections.     

ATP uptake in the Golgi and extracellular release require Mcd4 protein and the vacuolar H+-ATPase

Extracellular nucleotides signal via a large group of purinergic receptors. Although much is known about these receptors, the mechanism of nucleotide transport out of the cytoplasm is unknown. We developed a functional screen for ATP release to the extracellular space and identified Mcd4p, a 919-amino acid membrane protein with 14 putative transmembrane domains, as a participant in glucose-dependent ATP release from Saccharomyces cerevisiae. This release occurred through the vesicular trafficking pathway initiated by ATP uptake into the Golgi compartment. Both the compartmental uptake and the extracellular release of ATP were regulated by the activity of the vacuolar H+-ATPase. It is likely that the Mcd4p pathway is generally involved in non-mitochondrial ATP movement across membranes, it is essential for Golgi and endoplasmic reticulum function, and its occurrence led to the appearance of P2 purinergic receptors.     

Conditional cell ablation by tight control of caspase-3 dimerization in transgenic mice

Studying the effects of the loss of a specific cell type is a powerful approach in biology. Here we present a method based on the controlled activation of the apoptotic machinery. We expressed a modified caspase-3-containing chemical inducer of dimerization (CID)-binding sites in the livers of transgenic mice. In the absence of CID, no liver injury was detectable, underlining the absence of leakage in our system. In contrast, injection of the CID produced activation of the chimeric caspase-3, which led to a dose-dependent pure hepatocyte ablation with subsequent regeneration. This method is effective in both growing and nongrowing cells, and is therefore applicable to a wide range of cells and tissues. Moreover, because apoptosis has been described in numerous pathological circumstances, this system is useful for generating mouse models of human disorders as well as for studying the recovery or regeneration of tissues after cell loss.     

Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice

In this study we evaluated the ability of activated intrahepatic APCs to inhibit hepatitis B virus (HBV) replication in transgenic mice. Intrahepatic APCs were activated by administration of an anti-CD40 agonistic mAb (alphaCD40). We showed that a single i.v. injection of alphaCD40 was sufficient to inhibit HBV replication noncytopathically by a process associated with the recruitment of dendritic cells, macrophages, T cells, and NK cells into the liver and the induction of inflammatory cytokines. The antiviral effect depended on the production of IL-12 and TNF-alpha by activated APCs; however, it was mediated primarily by IFN-gamma produced by NK cells, and possibly T cells, that were activated by IL-12. Collectively, these results suggest that activated APCs can directly produce antiviral cytokines (IL-12, TNF-alpha) and trigger the production of other cytokines (i.e., IFN-gamma) by other cells (e.g., NK cells and T cells) that do not express CD40. These results provide insight into a hitherto unsuspected antiviral function of intrahepatic APCs, and they suggest that therapeutic activation of APCs may represent a new strategy for the treatment of chronic HBV infection.     

Regionaland Interspecies Differences in Brain Progesterone Metabolism

Metabolites of [³H]progesterone were studied in slices prepared from different brain regions of male rat, mouse, and monkey. The major metabolites were 5α-dihydroprogesterone (5α-DHP) and 3α,5α-tetrahydroprogesterone (3α,5α-THP) in rat brain slices, 5α-DHP and 20α- dihydroprogesterone (20α-DHP) in mouse brain slices, and 20α-DHP in monkey brain slices. In rat olfactory bulb slices, 5α-DHP represented 25.2 ± 3.3% of total radioactivity and 3α,5α-THP 17.5 ± 2.8%, whereas in rat medulla oblongata slices, 5α-DHP was 31.3 ± 3.5% and 3α,5α- THP 5.4 ± 1.5% of total radioactivity. In slices from other rat brain regions, both metabolites represented 12–20% of total radioactivity.-The highest metabolite content in mouse brain was also detected in olfactory bulb slices, where 5α-DHP represented 16.6 ± 4.6% and 20α-DHP 9.5 ± 2.3% of total radioactivity. In cortical and corpus callosum slices of monkey brain, 26.8 ± 4.4% and 2.4 ± 0.5% of total radioactivity, respectively, were converted to 20α-DHP, and less than 3% of total radioactivity could be attributed to any of the other metabolites detected. The 3α,α-THP content in both rat and monkey brain was below 1 nM, but increased in rat brain to 6.7 ± 2.5 nM after electroshock. Endogenous 3α,5α-THP might play an important role in the regulation of rat behavior through the modulation of GABA action on the GABA_A receptor. The significant interspecies differences in the brain progesterone metabolism should be considered in evaluating the functional role of neurosteroids in various species.     

Endogenous protein kinase inhibitors. Purification, characterization, and distribution in different tissues.

A thermostable inhibition of ATP-protein phosphotransferase (EC 2.7.1.37) (protein kinase) which is present in crude tissue extracts has been resolved by gel chromatography (Sephadex G-100) into two molecular forms. These two forms will be referred to as type I and type II inhibitor. The type I inhibitor (Mr approximately or equal to 24,000) is specific for cAMP-dependent protein kinase and corresponds to the inhibitor described earlier (Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fisher, E. H., and Krebs, E. G. (1971) J. Biol. Chem. 246, 1977-1985). The type II inhibitor (Mr approximately or equal to 15,000) competes for the enzyme with various substrate proteins (histone, alpha-casein, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). The type II inhibitor blocks protein phosphorylation catalyzed by several types of protein kinases (cAMP- and cGMP-dependent or cyclic nucleotide-independent protein kinases). The type II inhibitor from rat brain has been purified 1500-fold; this protein is thermostable, has acidic characteristics, and does not require Ca2+ ions for its activity. Different ratios and concentrations of type I and type II inhibitors of protein kinase are found in rat skeletal muscle, pancreas, cerebellum and corpus striatum, and in lobster tail muscle.     

Quantitative determination of nuclear pore complexes in cycling cells with differing DNA content

The number of pore complexes per nucleus was determined for a wide variety of cultured cells selected for their variable DNA content over a range of 1-5,6000. The pore number was compared to DNA content, nuclear surface area, and nuclear volume. Values for pore frequency (pores/square micrometer) were relatively constant in the species studied. When the pore to DNA ratio was plotted against the DNA content, there was a remarkable correlation which decreased exponentially for the cells of vertebrae origin. Exceptions were the heteroploid mammalian cells which had the same ratio as the diploid mammalian cells despite higher DNA content. The results are interpreted to mean that neither the nuclear surface, the nuclear volume, nor the DNA content alone determines the pore number of the nucleus, but rather an as yet undetermined combination of different factors. The surface and volume of vertebrate nuclei do not decrease with decreasing DNA content below a given value. The following speculation is suggested to account for the anomalous size changes of the nucleus relative to DNA content in vertebrates. Species with small DNA complements have a relatively large proportion of active chromatin which determines the limits of the physical parameters of the nucleus. The amount of active chromatin maybe the same for at least the vertebrates with low DNA content, At high DNA content, the nuclear parameters may be determined by the relatively high proportion of inactive condensed chromatin which increases the nuclear surface and volume.     

A eukaryotic carboxyl-terminal signal sequence translocating large hydrophilic domains across membranes

Yeast Golgi ecto-ATPase Ynd1p is an unusual type III membrane protein with the longest translocated N-terminus reported. Sequential deletion analysis reveals that translocation of this 500-residue-long hydrophilic domain across the membranes requires the C-terminal transmembrane domain of Ynd1p and its flanking regions. Additional studies indicate that the topogenic sequence of Ynd1p overrides the effect of a reverse signal-anchor sequence present at the N-terminus of Ynd1p, while it is not affected by a classic signal sequence at the N-terminus. When placed at the C-terminal end, the sequence can translocate large extracellular domains of two membrane proteins across the membranes. The data demonstrate the existence of a true eukaryotic C-terminal signal sequence.     

New Neurochemical Markers for Psychosis: A Working Hypothesis of Their Operation

Reelin (Reln) is expressed in specific GABAergic neurons in layer I and II of neocortex, and is secreted into the extracellular matrix where it surrounds dendrites, spines and neurite arborizations, and binds to integrin receptors located on post-synaptic densities of apical dendritic spines. Experiments in rodents (including wild type or reeler heterozygous mice) and non-human primates suggest the Reln secreted in the extracellular matrix of neocortex, via integrin receptors, modulates the function of the adaptor protein DAB1(drosophila disable-gene) homologous product) thereby participating in dynamic processes associated with plasticity changes in dendrites, dendritic spines and their synapses. A local protein synthesis at dendritic spines (ie the activity regulated cytoskeleton associated protein, Arc) probably acts as a signal for plastic modulatory activities in synapses operative in neural group interactions. A research strategy directed toward identifying specific neurochemical markers operative in the etiopathology of psychotic disorders lead to the identification of a downregulation (30-50%) of Reln and glutamic acid decarboxylase 67(GAD67) expression in prefrontal cortex and other brain areas of schizoprenia and bipolar disorder patients with psychosis. These downregulations were not due to neuronal damage, postmortem interval, or antipsychotic medication. The dysfunction of GABAergic interneurons observed in psychotic brains in combination with reduced Reln expression and downregulation of Reln-integrin receptor interaction, may provide an explanation for the reported decrease in neuropile expression including dendritic spine density reduction, in neocortex of schizophrenia patients. This downregulation of neuropile plasticity may be a factor to be considered in the etiology of the disintegration of consciousness, which is one of the primary signs of psychosis.