Identification of immunoglobulin E (IgE)-binding epitopes and recombinant IgE reactivities of a latex cross-reacting Indian jujube Ziz m 1 allergen.

Ziz m 1 is a major Indian jujube (Zizyphus mauritiana) allergen involved in latex-fruit syndrome, and cDNA of the allergen has been cloned, sequenced and expressed in yeast by our laboratory previously. In this study, we performed an immunoglobulin E (IgE)-binding epitope analysis of Ziz m 1 using overlapping recombinant fragments. Eight overlapping recombinant fragments were generated from the recombinant Ziz m 1 allergen. The fragments were expressed in Escherichia coli and IgE-binding activities were evaluated by sera of latex-Indian jujube-allergic subjects and normal subjects using immunoblotting. Human allergic sera are not able to recognize fragments consisting of amino acid sequences 26-71, 119-280 and 119-291. However, residues at positions 26-199, 26-105, 26-86, 119-320 and 238-330 were found relevant in the IgE-binding. Our results indicate that (72)NISGHCSDCTFLGEE(86) and (292)VWNRYYDLKTNYSSSIILEYVNSGTKYLP(320) of Ziz m 1 are the sequences required for human IgE binding. Four corresponding peptides, (72)NISGHCSDCTE(86), (292)VWNRYYDLKT(301), (300)KTNYSSSIILEY(311) and (309)LEYVNSGTKYLP(320), were synthesized, and these peptides reacted with 70%, 100%, 70% and 70% of 10 allergic sera tested, as revealed by enzyme-linked immunosorbent assay. Sensitization to (292)VWNRYYDLKT(301) correlated significantly with the presence of allergic symptoms (P < 0.001). These findings will be useful in designing diagnostic and therapeutic approaches, thereby contributing to the development of specific immunotherapy for subjects with latex-fruit syndrome.

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.

Hypersensitivity to Forcipomyia taiwana (biting midge): clinical analysis and identification of major For t 1, For t 2 and For t 3 allergens.

BACKGROUND: Forcipomyia taiwana is a tiny, blood-sucking midge that cause intense pruritus and swelling in sensitive individuals. It is distributed island-wide in rural Taiwan and Southern China. OBJECTIVE: This study aimed to study the allergic immune responses and identify F. taiwana allergens. METHODS: Crude whole body F. taiwana extracts were prepared with phosphate-buffered saline. The specific IgE antibody was determined by enzyme-linked immunoassay and immunoblotting. Protein was analyzed by electrospray ionization tandem mass spectrometry. RESULTS: Among the 372 subjects that were exposed to F. taiwana bites, 179 (48%) reported an immediate skin reaction with/without delay reaction and 41(11.1%) reported a solely delay reaction. The skin of 21 subjects was tested with F. taiwana extract. Of these 21 subjects, 12 (57.1%) produced immediate skin reactions and contained high levels of specific IgE antibody against F. taiwana. Immunoblotting revealed that 11 allergenic components are able to bind specific IgE. Allergens of 22, 24, 35, 36, and 64 kDa bound 50, 50, 75, 66.7, and 75% of IgE-containing sera tested, respectively. Tryptic fragments of the 24, 35, 36, and 64 kDa allergens were analyzed by ESI-MS/MS. Selected tryptic peptides of 24, 35, and 36, and 64 kDa allergens exhibited significant sequence identity with triosephosphate isomerase of Anopheles merus,Tenebrio molitor,Ochlerotatus togoi, and Chrysops vittatus, fructose 1,6-bisphosphate aldolase of Antheraea yamamai and Homalodisca coagulata, and a slow muscle myosin S1 heavy chain of Homarusamericanus and a protein with unknown function from A. gambiae, respectively. The 35 and 36 kDa proteins may represent different isoforms of the fructose 1,6-bisphosphate aldolase. CONCLUSION: We conclude that immediate reaction to F. taiwana bites is IgE mediated and the 24 (For t 1), 35 (For t 2), and 64 kDa (For t 3) proteins are candidates for major F. taiwana allergens. Further studies are needed to confirm these allergens.

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.

Emerging erythromycin resistance among group B streptococci in Korea.

In order to determine possible trends in the susceptibility and distribution of group B streptococci (GBS) serotypes in a Korean population and to elucidate any relationship between the serotypes and the antimicrobial susceptibility patterns found, 185 clinical isolates of GBS were investigated between 1990 and 1998. The rate of erythromycin resistance increased from 0% during the period 1990-1995 to 26% in 1996 and 40% in 1998. The overall rates of resistance to erythromycin and clindamycin were 20% and 22.2%, respectively. GBS serotype V was not detected until 1995, but it was isolated in 1996 and ranked third in frequency (18.8%) in 1997. Among the 37 erythromycin-resistant strains detected, 54.1% and 29.7% were of serotype III and V, respectively. The emerging erythromycin resistance detected among these GBS isolates was mainly due to a sudden increase in the incidence of GBS serotypes with multidrug-resistant phenotypes.

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.

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.

Immunolocalization of high-density lipoproteins in arterial walls of rats.

The inverse correlation between serum high-density lipoprotein (HDL) levels and coronary heart disease in humans suggests that HDL has a protective effect against the development of atherosclerosis. However, there is a lack of data concerning its distribution across the arterial wall. In order to detect this lipoprotein, we performed immunogold labeling on ultrathin sections of L.R. White embedded rat arterial tissue. Electron microscopic examination revealed that HDL was localized in the cytoplasm of the endothelial cells and the smooth muscle cells, but not in the nucleus or other organelles. The HDL was also present in the subendothelial space, the extracellular matrix as well as the intercellular clefts between the endothelial cells. Quantitative study revealed that rats on a high cholesterol diet for one month have more immunogold labeling (P < 0.05) in the subendothelial space, the smooth muscle cells and the extracellular matrix as compared to rats on a normal diet. After 12 months of normal diet, the intracellular labeling was significantly increased (P < 0.05) in the endothelial cells and the smooth muscle cells as compared to 1 month on the normal diet. The increase was greater (P < 0.05) for the high-cholesterol diet than for the normal diet treatment.

A PDGF-regulated immediate early gene response initiates neuronal differentiation in ventricular zone progenitor cells.

When exposed to platelet-derived growth factor (PDGF), uncommitted neuroepithelial cells from the developing cortex of embryonic day 14 (E14) rats develop into neurons. Outward signs of the neuronal phenotype are not observed for 4 days following exposure to PDGF. However, only a brief (2-3 hr) period of PDGF receptor activation is required to initiate neuronal development. During the window of receptor activation, RNA synthesis is essential, but protein synthesis is not. These observations indicate that specification of neuronal fate is mediated by an immediate early gene response.

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.

Sequence analyses and antigenic epitope mapping of the putative RNA-directed RNA polymerase of five U.S. bluetongue viruses.

We determined the complete nucleotide sequences of the cognate L1 double-stranded RNA segments of bluetongue virus (BTV) serotypes 2, 11, 13, and 17, which encode the putative RNA-directed RNA polymerase VP1. Each L1 gene contained 3944 nucleotides and was 10 bases shorter than the previously reported L1 gene of BTV 10. A single open reading frame which could encode the reported VP1 protein, 1302 amino acids in size, began with an initiation codon at nucleotides 12-14 and a termination codon at nucleotides 3918-3920. Analyses of the nucleotides of L1 genes and the deduced amino acid sequences of VP1 proteins of the five U.S. BTV serotypes indicated that the most recently isolated BTV-2 serotype from Florida was more distantly related than BTV-10, 11, 13, and 17, which were isolated primarily in the western U.S.A. The results are consistent with our hypothesis that BTVs-10, -11, -13, and -17 are derived from a single and common gene pool, and that BTV-2 belongs to a second, distinct gene pool. These genetic distinctions also reflected well with the known geographic distribution of the five U.S. BTV serotypes in North America. This putative RNA-directed RNA polymerase (149 KDa) was a basic protein, and the deduced amino acid sequences of the VP1 proteins contained seven highly conserved hydrophobic domains and many other sequence motifs which were also found in other known RNA polymerases. Four immunodominant but linear antigenic epitopes conserved among the VP1 of five U.S. BTVs were also been identified and mapped using monospecific oligoclonal antibodies.

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.

Identification and localization of a serotypic neutralization determinant on the VP2 protein of bluetongue virus 13.

We have used a serotype-specific monoclonal antibody to locate a neutralization epitope on the outer capsid protein, VP2, of the bluetongue virus 13. This surface-accessible region of the virion was recognized by a monoclonal antibody, D24.15, which exhibited serotype-specific neutralizing activity as determined by plaque reduction assay. In Western blots, this monoclonal antibody reacted only with the VP2 of bluetongue virus 13, but not with any of the other US BTV serotypes. Competition with sequence-specific synthetic peptides identified only one linear synthetic peptide (EMDD-DETEYE), corresponding to amino acids 642-651 of VP2 of bluetongue virus 13 that could block both the neutralizing activity of MAb D24.15 and its specific binding to VP2 of BTV-13. However, oligoclonal antibody against this synthetic peptide did not exhibit any neutralizing activity. These data suggest that the serotype-specific neutralization determinant of the outer capsid protein VP2 is located on the surface of the BTV-13 virion and represents the major component of a conformational epitope.

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.

Evolutionary analyses of five US bluetongue viruses using the cognate S2 genes.

Full-length cDNA copies of S2 genes (segment 8), coding for non-structural protein 2 (NS2) of bluetongue virus serotypes 2, 11, 13 and 17 were selectively synthesized by a modified polymerase chain reaction (Clamp-R) and cloned into the PstI site of the pUC19 plasmid. Each of these S2 cognate genes was 1125 nucleotides in length with an initiation and a termination codon at nucleotides 20-22 and 1082-1084, respectively, resulting in a long open reading frame capable of coding for a protein of 354 amino acids. The deduced amino acid sequence of NS2 protein had a high concentration of lysine and contained a relatively low number of tryptophan and histidine residues. There was a highly conserved hydrophilic region at the carboxyl termini of predicted NS2 proteins in all five BTV serotypes, even though the amino acid sequence in this region in BTV-2 was more variable than in the other four serotypes. There was significant sequence homology of the cognate S2 genes at both the nucleotide and the amino acid levels. Phylogenetic analyses using the S2 gene sequences indicated that BTV-10, -11, -13 and -17 were more closely related and BTV-2 was the most distantly related serotype among the five US bluetongue viruses.

Comparative sequence analyses of the cognate structural protein VP6 genes of five US bluetongue viruses.

The S3 segment (the small segment 3), encoding the structural protein, VP6, from the five United States (US) prototype bluetongue virus (BTV) serotypes were amplified by the Clamp-R method and cloned as full-length entities. The complete nucleotide sequence of each cognate gene segment was determined. Each cognate S3 segment of BTV-10, 11, 13 and 17 was 1049 nucleotides long and contained an open reading frame (ORF) capable of encoding a 325-amino acid protein. However, the S3 segment of BTV-2, which also contained 1049 nucleotides, had a longer 5'-non-coding region of 99-nucleotide and contained an ORF capable only of encoding a 301-amino acid protein. Comparative analyses of the predicted amino acid sequences of S3 segments of BTV-2, 10, 11, 13 and 17 revealed that VP6 was unusually high in glycine and contained few aromatic amino acids, but a high concentration of charged amino acids, which is a characteristic of a hydrophilic protein. Phylogenetic analyses indicated that BTV-11, 13 and 17 were more closely related than the other two US BTV serotypes. BTV-2 was the most distantly related.

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.