Hiroyoshi Komatsu

Expression of Stress-related Genes in a Cadmium-resistant A549 Human Cell Line

This study was designed to explain the basis for Cd-acquired tolerance of A549 cells cultured in the presence of Cd. Thirty-day exposure of cultured human pneumocytes (A549 cell line) to 10 μ M Cd was previously found to induce an acquired resistance persisting over several weeks of culture. Moreover, these Cd-resistant cells (R-cells) were found to proliferate faster than controls. No difference was found between R-cells and control cells (S-cells) concerning the basal and Cd-induced level of metallothioneins expression. However, after exposure to Cd, cell glutathione levels were unchanged in R-cells while they were either increased (at 10 μM Cd) or decreased (at 25 μM Cd) in S-cells. cDNA array analysis showed that genes encoding for (GPx1) glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase were similarly expressed in R- and S-cells, whereas the gene of (GPx2) glutathione peroxidase was overexpressed in R-cells. Most genes encoding stress proteins were similarly expressed, except for HSP27 and GRP94 genes, which were respectively under- (ratio 0.5 ± 0.1) and over- (1.8 ± 0.5) expressed in R-cells. Acute exposure to Cd was found to trigger the upregulation of genes encoding the chaperone proteins HSP90A, HSP27, HSP40, GRP78, HSP72, and HO-1 in S-cells. In R-cells, only HO-1 and HSP72 were overexpressed but at a lower level. This suggests that the Cd-related adverse conditions, leading to protein misfolding, are lowered in R-cells. It is likely that the upregulation of GPx2 in R-cells leads to a higher antioxidant defense in these cells. This research was supported by ASUPS from the University Paul Sabatier Toulouse III.

Immunohistochemical Detection of Human Gastrointestinal Glutathione Peroxidase in Normal Tissues and Cultured Cells with Novel Mouse Monoclonal Antibodies

This is the first report to describe the successful detection of human gastrointestinal glutathione peroxidase in normal tissues by Western blotting and immunohistochemical staining techniques. Four hybridoma clones producing monoclonal antibodies (MAbs) against the human gastrointestinal glutathione peroxidase were established from mice immunized with a gastrointestinal glutathione peroxidase-derived peptide. The MAbs did not crossreact with other members of the glutathione peroxidase family, be it cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, or extracellular glutathione peroxidase. Although the MAbs were found to react with a 24-kD protein in a Western blotting assay using gastric carcinoma cell extracts as antigen, they did not react with a B-lymphoblastoid cell extract. Immunohistochemical staining showed gastrointestinal glutathione peroxidase localized in the cytoplasm and in the nucleus of gastric carcinoma cells. Moreover, gastrointestinal glutathione peroxidase was detected in tissue extracts of human stomach, small intestine, large intestine, liver, and gallbladder by Western blotting, and its localization was immunohistochemically confirmed in the mucosal epithelia of the basal area of gastric pits and intestinal crypts.

Oxidative Modulation of the Glutathione-redox Couple Enhances Lipopolysaccharide-induced Interleukin 12 P40 Production by a Mouse Macrophage Cell Line, J774A.1

Interleukin (IL)-12 plays a key role in determining the immune response pattern that results in maturation of Th0 to Th1 and Th2. To investigate the correlation between intracellular redox state and IL-12 production in macrophages, cells from the mouse cell line J774A.1 were treated with reagents modulating the glutathione-redox couple before stimulation with lipopolysaccharide (LPS). It was found that the glutathione reductase inhibitor, 1,3-bis (2-chloroethyl)-1-nitrosourea, markedly augmented LPS induced IL-12p40 production particularly when it was added for 24 h before LPS stimulation, whereas the glutathione-synthesis inhibitor, L-buthionine-(S,R)-sulfoximine, suppressed IL-12p40 production. The profile of IL-12p40 augmentation correlated well with the profile of intracellular glutathione oxidation (GSSG) and the activation profile of nuclear transcription factor kappaB (NF-kappaB), suggesting that GSSG is important in NF-kappaB activation which leads to IL-12p40 production. Our results indicate that the glutathione-redox couple plays an important role in the augmented production of IL-12p40 and thus in influencing immune response patterns.

The RNA Recognition Mechanism of Human Immunodeficiency Virus (HIV) Type 2 NCp8 Is Different from That of HIV-1 NCp7

The nucleocapsid (NC) protein of HIV, which contains two CCHC-type zinc fingers connected by a linker, is a multifunctional protein involved in many of the critical steps of the HIV life cycle. HIV-1 and HIV-2 contain NC proteins NCp7 and NCp8, respectively. The amino acid sequences of both NC proteins are 67% identical. For NCp7, the important elements for RNA binding were found to be the first zinc finger flanked by the linker, as the minimal active domain, and the 3(10) helix in the N-terminus, as the secondary active domain. However, for the NCp8 counterpart in HIV-2, the mechanism for binding to viral RNA has not yet been clarified. In this study, we determined NCp8s three-dimensional structure for the first time and examined the dynamic behavior and chemical shift perturbation as a function of the concentration of viral RNA SL3. Moreover, the specific binding activities of NCp8 and the NCp8-derived peptides with SL3 were examined by a native polyacrylamide gel electrophoresis assay. These results indicate that the RNA recognition mechanism for NCp8 is different from that of NCp7 and that the hydrophobic cleft in the second zinc finger acts as a secondary active domain instead of the 3(10) helix in NCp7. Furthermore, the flexibility of the linker is limited by the hydrogen bond between the first zinc finger (Asn11) and the linker (Arg27), which makes it possible for the sites around Trp10 in the minimal active domain and the secondary active domain to form the binding surface.

Binding properties of human immunodeficiency virus type‐2 (HIV‐2) rna corresponding to the packaging signal to its nucleocapsid protein

The nucleocapsid (NC) protein of human immunodeficiency virus type‐1 (HIV‐1) contains two zinc finger motifs (ZFMs), and binds specifically to the packaging signal which is located in the 5′ leader sequence of the viral genomic RNA between the first splice donor and the gag initiator codon (AUG). In this study, we analyzed the specificity of the binding of the corresponding region of HIV‐2 (Region 3) to its NC protein (NCp8), by performing a competitive ultraviolet (UV) cross‐linking assay using in vitro‐synthesized 32P‐labeled and unlabeled RNAs corresponding to a sequence between the primer binding site and the gag AUG (Region 1). Binding of 32P‐labeled Region 1 RNA to NCp8 was inhibited specifically by adding unlabeled Region 1 and 3 RNAs and no specific binding was detected using deletion mutant peptides of NCp8. These findings suggest that the region(s) which bind(s) specifically to HIV‐2 NCp8 lie(s) between the first splice donor and the gag AUG in the 5′ leader sequence and that NCp8 is the minimum binding region responsible for the specific binding of the region downstream of the first splice donor site of HIV‐2 RNA.

Viral RNA-binding activity of HIV-2 nucleocapsid protein is inhibited by a synthetic peptide containing the first zinc finger motif of HIV-2.

Objective:To analyze the antigenic structure and viral RNA-binding activity of HIV-2GH-1 nucleocapsid protein (NC). Methods:Five synthetic peptides corresponding to hydrophilic regions of HIV-2GH-1 NC were prepared. The reactivity of rabbit antisera directed against these synthetic peptides was examined by Western blot (WB) assay, using lysates of purified HIV-2GH-1 virus and HIV-2GH-1-infected cells as antigen. The binding activity of NC to viral RNA synthesized in vitro, was assessed by Northwestern blot (NWB) assay using 32P-labeled RNA as a probe. Results:One of five antisera against the peptides reacted with a protein of 8 kD (p8) of HIV-2GH-1 in WB. p8 was analyzed for the amino-terminal amino-acid sequence and identified as a portion of NC including two zinc finger motifs (ZFM), comprising the DNA-binding region in the molecule. The binding of the RNA probe to p8 was observed by NWB and did not always depend on zinc. In competition experiments, the reactivity of p8 with the RNA probe ceased to be evident following preincubation of the probe with a peptide containing the first ZFM in the molecule. Conclusion:p8 of HIV-2GH-1 NC binds to viral RNA in vitro. The first ZFM in the p8 molecule appears to be essential to binding activity. Functional analysis of synthetic peptides corresponding to ZFM of HIV p8 may provide important information on the development of antiviral agents.

Adsorption of LLCMK2 Cell‐Grown Sendai Virus onto Human Red Blood Cells and Its Release from the Virus Adsorbed Cells

An early stage of virus adsorption was studied in a system of Sendai virus metabolically labeled with [3H] leucine in LLCMK2 cells and of human red blood cells (RBCs). The efficiency of viral release from the virus‐bound RBCs by incubation at 37 C depended on the number of virus particles which had been used for adsorption onto the RBC at 4 C. When 7.8 × 102 virus particles were previously adsorbed onto the RBC at 4 C, most of the viruses were dissociated from the RBC at 37 C. In the case of adsorption of 3 to 12 virus particles per RBC, however, most of the viruses were not dissociated from the RBC by incubation at 37 C. Such RBC‐bound viruses were released by incubation with various bacterial neuraminidases (Clostridium perfringens, etc.) or with a large number of LLCMK2 cell‐grown Sendai virus (LLCMK2‐Sendai) particles, but not released by treatment with hemagglutinin‐neuraminidase protein (Sendai‐gp) isolated from egg‐grown Sendai virus.

Application of anti-human IgG monoclonal antibodies to antiglobulin reagent. Production and characterization of anti-human IgG monoclonal antibodies.

Ten mouse monoclonal antibodies (mAbs) against human IgG were produced to apply to antiglobulin reagent. Of these 10mAbs, 5 reacted with heavy (H) chains and 4 recognized light (L) chains of human IgG in Western blot analysis. The specificity of a remained mAb, which gave strong positive reaction in enzyme-linked immunosorbent assay using human IgG as antigen, was not defined by Western blot analysis. All mAbs reacted with human IgG sensitized red blood cells on membrane immunofluorescence assay, but anti-H chain mAbs only exhibited strong positivity on antiglobulin tests using human IgG sensitized red blood cells. These results suggest that the anti-H chain mAbs are important as an antiglobulin reagent.