Induction by beta-bungarotoxin of apoptosis in cultured hippocampal neurons is mediated by Ca(2+)-dependent formation of reactive oxygen species

The component of the venom of the Taiwanese banded krait Bungarus multicinctus, beta-bungarotoxin (beta-BuTx), acts as an extremely potent inducer of neuronal apoptosis when applied to rat hippocampal cultures. While induction of cell death is dependent on toxin binding to voltage-activated K+ channels and subsequent internalization, the pro-apoptotic signals triggered by picomolar concentrations of beta-BuTx are not understood. Following toxin binding, a dramatic increase in intracellular Ca2+ became detectable after 30 min, and in reactive oxygen species (ROS) after 3-4 h. Conversely, Ca2+ chelators, radical quenchers and antioxidants efficiently antagonized beta-BuTx induced apoptosis. As shown for the antioxidant 2,3-dihydroxybenzoic acid, analysis by matrix assisted laser desorbtion-time of flight (MALDI-TOF) mass spectrometry excluded the protective effects to be due to reductive cleavage of the toxic beta-BuTx dimer. Inhibitors of the intracellular antioxidant defence system enhanced neuronal susceptibility to beta-BuTx, supporting the essential role of ROS in beta-BuTx-initiated apoptosis. Cell damage was accompanied by an accumulation of markers of oxidative cell stress, phospholipid hydroxyperoxides and the lipid peroxidation product, malonyl dialdehyde. These observations indicate that beta-BuTx-induced cell death resulted from an intracellular signalling cascade involving subsequent stages of a dramatic rise in free Ca2+, the accumulation of ROS, membrane lipid peroxidation and, finally, apoptosis.     

An umbraviral protein,involved in long-distance RNA movement,binds viral RNA and forms unique,protective ribonucleoprotein complexes

Umbraviruses are different from most other viruses in that they do not encode a conventional capsid protein (CP); therefore, no recognizable virus particles are formed in infected plants. Their lack of a CP is compensated for by the ORF3 protein, which fulfils functions that are provided by the CPs of other viruses, such as protection and long-distance movement of viral RNA. When the Groundnut rosette virus (GRV) ORF3 protein was expressed from Tobacco mosaic virus (TMV) in place of the TMV CP [TMV(ORF3)], in infected cells it interacted with the TMV RNA to form filamentous ribonucleoprotein (RNP) particles that had elements of helical structure but were not as uniform as classical virions. These RNP particles were observed in amorphous inclusions in the cytoplasm, where they were embedded within an electron-dense matrix material. The inclusions were detected in all types of cells and were abundant in phloem-associated cells, in particular companion cells and immature sieve elements. RNP-containing complexes similar in appearance to the inclusions were isolated from plants infected with TMV(ORF3) or with GRV itself. In vitro, the ORF3 protein formed oligomers and bound RNA in a manner consistent with its role in the formation of RNP complexes. It is suggested that the cytoplasmic RNP complexes formed by the ORF3 protein serve to protect viral RNA and may be the form in which it moves through the phloem. Thus, the RNP particles detected here represent a novel structure which may be used by umbraviruses as an alternative to classical virions.     

Subcellular localization and regulation of coenzyme A synthase

CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.     

Identification of three genes encoding P(II)-like proteins in Gluconacetobacter diazotrophicus: studies of their role(s) in the control of nitrogen fixation

In our studies on the regulation of nitrogen metabolism in Gluconacetobacter diazotrophicus, an endophytic diazotroph of sugarcane, three glnB-like genes were identified and their role(s) in the control of nitrogen fixation was studied. Sequence analysis revealed that one P(II) protein-encoding gene, glnB, was adjacent to a glnA gene (encoding glutamine synthetase) and that two other P(II) protein-encoding genes, identified as glnK1 and glnK2, were located upstream of amtB1 and amtB2, respectively, genes which in other organisms encode ammonium (or methylammonium) transporters. Single and double mutants and a triple mutant with respect to the three P(II) protein-encoding genes were constructed, and the effects of the mutations on nitrogenase expression and activity in the presence of either ammonium starvation or ammonium sufficiency were studied. Based on the results presented here, it is suggested that none of the three P(II) homologs is required for nif gene expression, that the GlnK2 protein acts primarily as an inhibitor of nif gene expression, and that GlnB and GlnK1 control the expression of nif genes in response to ammonium availability, both directly and by relieving the inhibition by GlnK2. This model includes novel regulatory features of P(II) proteins.     

Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking

Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT-20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C-terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C-terminal region functions in TGN-retention; removal of 10 amino acids from the C-terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half-life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10-residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half-life than either the wild type sequence or the EDE mutant. Adaptor protein-1 and -2 bind to most of the deletion mutants, the EDE point mutant, and the CK2-phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C-terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins.     

A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg

Hepatitis C virus (HCV) evolution is thought to proceed by mutations within the six genotypes. Here, we report on a viable spontaneous HCV recombinant and we show that recombination may play a role in the evolution of this virus. Previously, 149 HCV strains from St. Petersburg had been subtyped by limited sequencing within the NS5B region. In the present study, the core regions of 41 of these strains were sequenced to investigate the concordance of HCV genotyping for these two genomic regions. Two phylogenetically related HCV strains were found to belong to different subtypes, 2k and 1b, according to sequence analysis of the 5' untranslated region (5'UTR)-core and the NS5B regions, respectively. By sequencing of the E2-p7-NS2 region, the crossover point was mapped within the NS2 region, probably between positions 3175 and 3176 (according to the numbering system for strain pj6CF). Sequencing of the 5'UTR-core regions of four other HCV strains, phylogenetically related to the above-mentioned two strains (based on analysis within the NS5B region), revealed that these four strains were also recombinants. Since a nonrecombinant 2k strain was found in St. Petersburg, the recombination may have taken place there around a decade ago. Since the frequency of this recombinant is now high enough to allow the detection of the recombinant in a fraction of the city's population, it seems to be actively spreading there. The reported recombinant is tentatively designated RF1-2k/1b, in agreement with the nomenclature used for HIV recombinants. Recombination between HCV genotypes must now be considered in the classification, laboratory diagnosis, and treatment of HCV infection.     

Family of hemorphins: co-relations between amino acid sequences and effects in cell cultures

Hemorphins, i.e. endogenous fragments of beta-globin chain segment (32-41) LVVYPWTQRY(F) suppress the growth of transformed murine fibroblasts L929 cell culture, the effect is due to cytotoxicity and inhibition of cell proliferation. The contribution of cytotoxicity depends on the presence of Leu(32): VV-hemorphins, except VV-hemorphin-4, exhibit cytotoxicity significantly higher than respective LVV-hemorphins. Decrease of cell number induced by hemorphins depend on the extent of N- and C-terminal degradation of hemorphins: VV-hemorphins in most cases are more active than LVV-, V-hemorphins, and hemorphins. In the group of VV-hemorphins the activity of VV-hemorphin-5 (valorphin) is significantly higher than of VV-hemorphin-7, VV-hemorphin-6, and VV-hemorphin-4, meaning that the presence of C-terminal Gln is important for suppressing of cell number. The amino acid sequence VVYPWTQ corresponding to valorphin was identified as important for manifestation of the both cytotoxic and antiproliferative effects.     

The origin of neointimal cells in the rat carotid artery after balloon angioplasty

At present the issue of a possible role of circulating stem cells and precursors in pathological vascular wall remodeling after angioplasty remains unsolved. Therefore the origin of neointimal cells was examined in the rat carotid artery after balloon angioplasty using morphological and immunocytochemical approaches. It is shown that at the early stages (1-7 days) after vessel injury acute inflammatory response arises in the arterial wall recruiting neutrophils, monocytes, macrophages as well as large amounts of low-differentiated blood-derived cells. At the late stages (10-28 days), at the area of injured intima, a new hyperplastic intima (neointima) is formed, which consists of cells carrying specific smooth muscle markers--alpha-actin and smoothelin. The study on cell proliferative behaviour in the injured vessel wall by bromodeoxyuridine showed that in the process of neointima formation blood-born rather than resident cells are involved. Probably, early smooth muscle and endothelial precursor cells penetrate into injured area with blood stream, where they proliferative and differentiate into mature cells.