Potential of classification of cancer by multiple discriminant analysis for relationship between cancer and expression of human cellular phosphoprotein.

This is the first report to describe the potential for classification of cancer using anti-phosphoprotein monoclonal antibodies (PPmAbs) and multiple discriminant analysis. Over 150 hybridoma clones producing monoclonal antibodies were generated against a human phosphoprotein mixture derived from a human leukemia cell line. The expression profiles of 22 cell lines from 9 different types of cancer using PPmAbs were examined. The relationship between cancer cells and the expression of human phosphoprotein in the cells was analyzed by multiple discriminant analysis and was used to construct a diagnostic system for cancers. Multiple discriminant analysis was able to successfully classify the cell lines into the correct cancer group by using the diagnostic system for cancers. These results show that multiple discriminant analysis based on phosphoprotein expression in cells or tissues may be a potentially valuable method for assisting in the classification of several cancers.

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 NCp8's 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.

Structural role of the secondary active domain of HIV-2 NCp8 in multi-functionality.

Nucleocapsid protein of HIV, containing two CCHC-type zinc fingers connected by a linker, is a multi-functional protein involved in many critical steps of the HIV life cycle. Several in vitro investigations demonstrated that the reactivities of the first zinc finger flanked by the linker of HIV-1 NCp7 and HIV-2 NCp8 were essential for binding to viral RNA, however, that of the second zinc finger flanked by the linker of NCp7 was very weak and non-specific, whereas the part of NCp8 called NCp8-f2, interacted strongly and specifically with viral RNA. In this study, the three-dimensional structure of NCp8-f2 was determined for the first time. Furthermore, we established that NCp8-f2 specifically binds to the stem-loop SD in viral RNA, and that the hydrophobic cleft and the basic residues close to the cleft were essential for specific binding to SD. We discuss the functional significance of NCp8-f2 for NCp8 being a multi-functional protein.

RNA recognition mechanism of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein.

NCp8 of HIV-2 contains two CCHC-type zinc fingers connected by a linker, and is involved in many critical steps of the virus life cycle. It was previously shown that the first zinc finger flanked by the linker is the minimal active domain for specific binding to viral RNA. In our previous study, we determined the three-dimensional structure of NCp8-f1, including the minimal active domain, and found that a hydrogen bond between Asn(11) N(delta)H and Arg(27) O stabilized the conformation of the linker in the vicinity of the zinc finger [Kodera et al. (1998) Biochemistry 37, 17704-17713]. In this study, RNA binding activities of NCp8-f1 and three types of its mutant peptides were analysed by native PAGE assay. The activity and three-dimensional structure of NCp8-f1/N11A, in which alanine is substituted for Asn(11) thereby affecting the conformation of the linker, was analyzed and compared with those of NCp8-f1. We demonstrated that the existence of Arg(4) and/or Lys(5) and Arg(26) and/or Arg(27) were necessary for binding RNA. Furthermore, the linker's flexible orientation, which is controlled by the hydrogen bond between Asn(11) N(delta)H and Arg(27) O, appears to be a structural basis for NCp8 existing as a multi-functional protein.

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 microM 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 microM Cd) or decreased (at 25 microM 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.

An optimized method for determination of intracellular glutathione in mouse macrophage cultures by fluorimetric high-performance liquid chromatography.

We have optimized a method for the determination of intracellular glutathione by high-performance liquid chromatography, using fluorimetric detection. To minimize artifacts and provide an accurate determination of intracellular glutathione, cell extracts were prepared using extraction conditions specifically designed to inhibit autoxidation and enzymatic degradation of glutathione. The sensitivity of the method was enhanced by adjusting the dansyl chloride derivatization reaction with regard to parameters such as pH, reaction time and dansyl chloride concentration. Both oxidized and reduced forms of glutathione were quantified using the refined method in extracts of oxidatively stressed J774A.1 mouse macrophage cells and reflected an expected shift in cellular redox status.

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.