Oxidative stress in nonallergic nasal polyps associated with bronchial hyperresponsiveness.

BACKGROUND: Nasal polyposis (NP) is a chronic inflammatory disease of upper airway with unknown etiology. NP is frequently associated with asthma; the interaction between these comorbidities remains interesting. Oxidative stress has been implicated in the pathophysiology of NP and asthma. The aim of this study is to investigate the significance of oxidative stress in sinonasal microenvironments by evaluating its association with clinopathological parameters and its impacts on the pathogenesis of bronchial hyperresponsiveness (BHR) in NP. METHODS: Polyp biopsy specimens were obtained from 20 nonallergic patients; control mucosas were obtained from 20 volunteers. The levels of free radicals in the tissues and in blood were determined by a sensitive chemiluminescence (CL) method. NP patients were substratified into three subgroups, NP without BHR, NP with asymptomatic BHR, and NP with BHR and asthma by the results of provocative testing. Four histological characteristics of NP, inflammatory cells, eosinophil infiltration, edema and fibrosis were estimated and applied to correlate with the tissue-CL. RESULTS: The mean CL level in polyp-tissues, but not in blood, was higher than in the control specimens. In NP patients, tissue-CL was associated with endoscopy score; high tissue-CL levels were positively correlated with the abundance of inflammatory cells and eosinophils. Tissue-CL and endoscopy score were associated with BHR/asthma phenotype. CONCLUSION: These results suggest an important role for oxidative stress in the pathophysiology of NP and a causal relation between oxidative stress and inflammatory cells, especially the eosinophils. Free radical levels in polyp-tissues associated with NP severity and with BHR/asthma phenotype in nonallergic NP patients.

Identification and expression of scavenger receptor SR-BI in endothelial cells and smooth muscle cells of rat aorta in vitro and in vivo.

In this study, we used immunoelectron microscopy to investigate the subcellular localization of scavenger receptor class B type I (SR-BI) in the arterial walls of rats. The expression of SR-BI in cultured endothelial and smooth muscle cells of rat aorta after exposure to high-density lipoprotein (HDL) was also investigated by immunofluorescence microscopy and immunoblotting analysis. A peptide containing residues 495-509 from mouse SR-BI (mSR-BI) plus an NH2-terminal cysteine was coupled to hemocyanin to generate mSR-BI antiserum in rabbits. Reactivity of antiserum against the synthetic peptides was confirmed with an enzyme-linked immunosorbent assay (ELISA). The results showed that SR-BI was specifically localized on the surface of the endothelial cells and smooth muscle cells. SR-BI was also observed in the cytoplasm of smooth muscle cells. Immunoblotting analysis indicated that SR-BI was expressed in the cell membrane. The levels of SR-BI increased gradually from 1 to 3 h and decreased at 24 and 48 h after cholesterol-loaded cells were incubated in the culture medium containing HDL. We conclude that SR-BI, a functional receptor for HDL, is expressed in the aortic endothelial cells as well as in smooth muscle cells. This receptor also responds to the presence of HDL in the culture medium.

Immunological characterization of two major secreted forms of recombinant hepatitis B virus e antigens.

Plasmids containing PCR-amplified hepatitis B virus e antigen (HBeAg) genes (HBeAg-MV and HBeAg-SV) were constructed and expressed in E. coli strain DH5alpha. The induced intracellular glutathione S-transferase (GST) fusion proteins of HBeAg-MV and HBeAg-SV were recovered and purified from bacterial lysates by affinity chromatography with glutathione-sepharose beads. The HBeAg-MV protein contained an additional 19 amino acids at its amino terminus. These two proteins were specifically cleaved from GST by the protease factor Xa and recognized by a monoclonal antibody against HBeAg. HBeAg-MV and HBeAg-SV were found to be the two major components of the post-modified HBcAg during viral infection. The antigenic specificities of the fusion and purified HBeAgs (factor Xa-digested) were confirmed by the Abbott HBe enzyme immunoassay (EIA) detection system. Sera from patients with confirmed hepatocellular carcinoma (HCC) specifically reacted only with HBeAg moiety of fusion proteins. HCC sera bound more strongly to the HBeAg-SV protein than to the HBeAg-MV one. This indicates that HBeAg-SV is either more antigenic than -MV or is the major target protein for the elicitation of antibody production after HBV infection. Thus, the two recombinant HBeAgs expressed and obtained in this study are appropriate immunological agents for the diagnostic detection of hepatitis B virus infection in humans.

Induction of p21(CIP1/Waf1) and activation of p34(cdc2) involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells.

The biological activity of retinoic acid (RA) was examined in human hepatoma Hep3B cells. Under serum-deprived conditions, RA induced S/M-phase elevation and mitotic index increase within 24 h, followed by apoptosis. This RA-induced apoptosis was accompanied by p53-independent up-regulation of endogenous p21(CIPI/Waf1) and Bax proteins, as well as activation of p34(cdc2) kinase, and increase of Rb2 protein level and phosphorylation pattern. In addition, RA had no effect on the levels of Bcl-XL; Bcl-XS; cyclins A, B, D1, D3, or E; or Rb1 expression but markedly down-modulated Cdk2 kinase activity and reduced Cdk4 expression. RA also slightly delayed p27(Kip1) expression. Olomoucine, a potent p34(cdc2) and Cdk2 inhibitor, effectively blocked RA-mediated p34(cdc2) kinase activation and prevented RA-induced apoptosis. Furthermore, antisense oligonucleotide complementary to p21(CIP2/Waf1) and p34(cdc2) mRNA significantly rescued RA-induced apoptosis. Our data indicate that p21(CIP2/Waf1) overexpression may not be the only regulatory factor necessary for RA-induced apoptosis in human hepatoma Hep3B cells. RA treatment leads to Rb2 hyperphosphorylation, and p34(cdc2) kinase activation is coincident with an aberrant mitotic progression, followed by appearance of abnormal nucleus. This aberrant cell cycle progression appeared requisite for RA-induced cell death. These findings suggest that inappropriate regulation of the cell cycle regulators p21(CIP2/Waf1) and p34(cdc2) is coupled with induction of Bax and involved in cell death with apoptosis when Hep3B cells are exposed to RA.

Platelet-derived growth factor induction of the immediate-early gene MCP-1 is mediated by NF-kappaB and a 90-kDa phosphoprotein coactivator.

A broad panel of agents including serum, interleukin-1, double-stranded RNA, and platelet-derived growth factor (PDGF) stimulate transcription of the "slow" immediate-early gene MCP-1. These disparate inducers act through a tight cluster of regulatory elements in the distal 5'-flanking sequences of the MCP-1 gene. We describe a 22-base element in this cluster which, in single copy, confers PDGF-inducibility to a tagged MCP-1 reporter gene. In mobility shift assays, the element binds a PDGF-activated form of NF-kappaB, and a 90-kDa protein (p90) which binds constitutively. Antibody supershift and UV cross-linking experiments indicate that the PDGF-activated NF-kappaB species is a Rel A homodimer. The DNA binding form of p90 is a nuclear-restricted serine/threonine phosphoprotein. Mutagenesis of the 22-base element shows that the NF-kappaB and p90 binding sites overlap, but binding of the two species is mutually independent. Both sites, however, are required for optimum PDGF induction of MCP-1. Therefore, p90 appears to be a coactivator with NF-kappaB in PDGF-mediated induction of MCP-1.

Analyses and conservation of sequences among the cognate L3 segments of the five United States bluetongue viruses.

We determined the complete nucleotide sequences of the cognate L3 double-stranded RNA (ds-RNA) segments of bluetongue virus (BTV) serotypes 2, 11, and 13 encoding the major viral inner capsid protein, VP3. Each cognate L3 segment was 2772 nucleotides long and contained a single open reading frame (ORF) with an initiation codon at nucleotides #18-20 and a termination codon at nucleotides #2721-2723. This ORF can encode the 901-amino acid VP3 protein (103 kDa) with a calculated isoelectric point of 6. Phylogenetic analyses using both the nucleotide and the deduced amino acid sequences of the L3 cognate gene of the five US BTV serotypes indicated that the BTV-2 serotype recently isolated in Florida was more distantly related than BTV-10, 11, 13 or 17. The five US BTV serotypes were derived apparently from two distinct gene pools, findings consistent with their current geographic distribution in North America.

High-sequence conservation among the United States bluetongue viruses cognate M2 genes which encode the nonstructural NS1 tubule protein.

Full-length cDNA copies of the M2 gene of BTV-2, -11, and -13 serotypes obtained by a modified polymerase chain reaction (Clamp-R) were cloned into pUC19 plasmid. The entire nucleotide sequences of each M2 gene were determined and compared with BTV-10 and BTV-17, thus completing the sequencing of these cognate M2 gene segments from all five U.S. BTV serotypes. Each M2 segment contained 1769 nucleotides, a single open reading frame (ORF) with an initiation codon at nucleotides 35-37, and a termination codon at nucleotides 1691-1693. This ORF can encode the 552-amino-acid NS1 protein (64 KDa) which has an isoelectric point of 7. Analyses of the nucleotide and deduced amino acid sequences of the five U.S. BTV serotypes indicated that the most recently isolated BTV-2 serotype was more distantly related than BTV-10, -11, -13, or -17. Analyses of the evolutionary relatedness of the cognate M2 genes by codon positions indicate that the rate of mismatch accumulations in the first and second base codon positions are less than 4%. However, the mismatch accumulations in the third base codon position are quite evident (23%) when BTV-2 serotype was compared with the other U.S. BTV serotypes. This suggests that BTV-2 has separated from the other four U.S. serotypes long before they themselves diverged. These data also indicate that the five U.S. BTV serotypes were apparently derived from two distinct gene pools that reflected geographic distribution in North America.

Sequence conservation among the cognate nonstructural NS3/3A protein genes of six bluetongue viruses.

Full-length cDNA copies of segment 10 genes of bluetongue virus serotypes 2, 11, 13 and 17 were synthesized by the Clamp-R method and inserted into the plasmid pUC19. The complete nucleotide sequences of these four cognate genes were sequenced and determined to be 822 nucleotides in length, smallest of the 10 genes in the bluetongue virion. These four cognate gene segments contained two in-phase and overlapping open reading frames capable of coding for two non-structural proteins of 229 and 216 amino acids with net charges of +4.5 and +5.5, respectively, at neutral pH. Comparative analyses of the predicted amino acid sequences of bluetongue virus serotypes 1, 2, 10, 11, 13 and 17 revealed (i) a high degree of sequence homology and conservation, (ii) a single conserved tryptophan located at residue 159, (iii) the presence of two conserved cysteines at residues 137 and 181 and two potential N-linked glycosylation sites at residues 63-65 and 150-152, (iv) a cluster of six prolines within a 15-amino acid region near the amino terminus, and (v) the longest 3' noncoding sequence of 113 bases among the 10 bluetongue viral genes. Phylogenetic analyses indicated that BTV-10 and -11 are very closely related and BTV-2 is the distantly related serotype of the five US bluetongue virus serotypes.

Chromatographic purification and characterization of EBV DNase from chemically induced lymphoid cells.

Epstein-Barr virus-associated deoxyribonuclease (EBV-DNase) was purified to homogeneity, as determined by silver staining, sequential column chromatography, and FPLC from Raji and P3HR-1 cells treated with 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. This viral protein was immunogenic and elicited high neutralization titer sera in rabbits. By silver staining of SDS-PAGE, Western immunoblot, and radioimmunoprecipitation using NPC patient sera and both polyclonal and monoclonal antibodies, the EBV DNase was identified as a 58K protein. The potential presence of two EBV DNases was also discussed.