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Biomedical subjects

Y Xiao

Publications and source records attributed to Y Xiao.

At least 109 records · Page 6Linked to original sources

Epitope-vaccines: a new strategy to induce high levels of neutralizing antibodies against HIV-1.

Based on the experimental evidence that gp120 subunit vaccine did not protect individuals from HIV-1 infection, we suggested that epitope-vaccines of HIV-1 gp41 may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, and characterised immunogenicity of epitope-vaccines. Two epitopes, RILAVERYLKD-epitope (aa586-596) on the N-domain and ELDKWA-epitope (aa669-674) on the C-domain of gp41, were demonstrated by us and others to induce protective activity. After vaccination course, the RILAVERYLKD-dimer epitope-vaccine [C(RILAVERYLKDG)2-BSA] induced strong epitope-specific antibody response by about 1:25,600 dilution, and the ELDKWA-tetramer epitope-vaccine [C-(ELDKWAG)4-BSA] could yet induce strong antibody response to ELDKWA-epitope by 1:12,800-25,600 dilution of antisera in mice, while rgp41 subunit vaccine induced very weak antibody response to both epitopes (1:400). In rabbit experiments, the titres of ELDKWA-epitope-specific antibody induced by ELDKWA-epitope-vaccine [C-(ELDKWAG)4-BSA] reached to 1:6,400, while rgp41 subunit vaccine induced very weak antibody response to this epitope and to P1 and P2 peptides (1:400). Moreover, the ELDKWA-epitope-specific antibodies in mice and rabbit antisera induced by epitope-vaccine could very strongly interact with P2 peptide sequence-corresponding to the C-domain of gp41 (dilution by 1:25,600), and the RILAVERYLKD-epitope-specific antibodies in mice antisera induced by epitope-vaccine could also very strongly interact with P1 peptide sequence-corresponding to the N-domain of gp41 (dilution by 1:102,400). All these results provided experimental evidence that epitope-vaccine may be a new general strategy to induce high levels of neutralizing antibodies against HIV-1 or other viruses.

AIDS Vaccines↗

Induction of high level of specific antibody response to the neutralizing epitope ELDKWA on HIV-1 gp41 by peptide-vaccine.

The monoclonal antibody 2F5 recognizing the neutralizing epitope ELDKWA on the C-domain could neutralize 90% of the investigated HIV-1 isolates. Low levels of ELDKWA-epitope-specific antibodies were observed in HIV-1-infected individuals. To induce high levels of antibodies to ELDKW-epitope, C-domain peptide (P2) was conjugated with a carrier peptide (KGGG)(7)-K (K/G). P2-K/G-conjugate induced high level of antibodies in mice by titer 1:25,600 to ELDKWA-epitope. P2-K/G-BSA-conjugate induced antibody response to ELDKWA-epitope (1:320-6400) in mice. The ELDKWA-epitope-specific antibodies of 19.8 and 34.6 microg/per milliliter serum were isolated from two rabbit antiserums (1:25,600). The levels of ELDKWA-epitope-specific antibodies induced in rabbits were greater than 1 microg/ml, a level considered to confer long-term protection. These results demonstrate the potential role of the C-domain peptide of gp41 to develop an effective ELDKWA-based epitope/peptide-vaccine against HIV-1.

AIDS Vaccines↗

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts.

Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOS-II) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-gamma and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-gamma alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.

Cells, Cultured↗

Expression and distribution of cell-surface proteoglycans in the normal Lewis rat molar periodontium.

Cell-surface proteoglycans participate in several biological functions such as cell cell and cell-matrix interactions, cell adhesion, the binding to various growth factors as co-receptors and repair. To understand better the expression and distribution of cell-surface proteoglycans in the periodontal tissues, an immunohistochemical evaluation of the normal Lewis rat molar periodontium using panels of antibodies for syndecan-1, -2, -4, glypican and betaglycan was carried out. Our results demonstrated the expression and distribution of all proteoglycans in the suprabasal gingival epithelium, soft and hard connective tissues. Both cellular and matrix localization was evident within the various periodontal compartments. The presence of these cell-surface proteoglycans indicates the potential for roles in the process of tissue homeostasis, repair or regeneration in periodontium of which each function requires further study.

Animals↗

Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1.

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that acts as an intracellular and extracellular signalling molecule in numerous biological processes. Many of the cellular actions of SPC are believed to be mediated by the activation of unidentified G-protein-coupled receptors. Here we show that SPC is a high-affinity ligand for an orphan receptor, ovarian cancer G-protein-coupled receptor 1 (OGR1). In OGR1-transfected cells, SPC binds to OGR1 with high affinity (Kd = 33.3 nM) and high specificity and transiently increases intracellular calcium. The specific binding of SPC to OGR1 also activates p42/44 mitogen-activated protein kinases (MAP kinases) and inhibits cell proliferation. In addition, SPC causes internalization of OGR1 in a structurally specific manner.

Calcium↗

Multiepitope vaccines intensively increased levels of antibodies recognizing three neutralizing epitopes on human immunodeficiency virus-1 envelope protein.

A few neutralizing antibodies against human immunodeficiency virus-1 (HIV-1) envelope proteins have been shown to be highly effective at neutralizing different strains in vitro, and exist at very low levels in the sera of HIV-1-infected individuals. Based on our hypothesis that epitope vaccination may be a novel strategy for inducing high levels of antibodies against HIV-1, we prepared multiepitope vaccines using three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope proteins. The PI [C-G-(ELDKWA-GPGRAFY)2-K] and PII (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) peptides were synthesized and conjugated to a carrier protein, bovine serum albumin (BSA). After vaccination, both the PI-BSA and PII-BSA multiepitope vaccines induced high levels of epitope-specific antibodies to the three neutralizing epitopes (antibody titre: 1 : 12,800-102,400). The recombinant glycoprotein 160 (rgp160) subunit vaccine induced strong antibody responses to rgp160, but only very weak epitope-specific antibody responses to the three epitopes. The epitope-specific antibodies were isolated from rabbit sera by single epitope-peptide-conjugated sepharose columns. A yield of 51 microg of epitope-specific antibodies/ml of serum (mean value) was obtained and identified to recognize these epitopes, while 0.35 microg of protein was isolated from 1 ml of pooled preserum by C-(ELDKWAG)4- or C-(RILAVERYLKD-G)2-K- and C-(GPGRAFY)4-sepharose columns. The levels of these epitope-specific antibodies induced in rabbits were much greater than 1 microg/ml, a level that is considered to confer long-term protection against some viruses. Moreover, these antibodies recognized the neutralizing epitopes on peptides and rgp41. Based on the fact that a very low level of ELDKWA epitope-specific antibodies exist in HIV-1-infected individuals, these results suggesting that synthetic epitope vaccines could induce high levels of multiepitope-specific neutralizing antibodies indicate a new strategy for developing an effective neutralizing antibody-based epitope/peptide vaccine against HIV-1.

AIDS Vaccines↗

Ethanol enhances TNF-alpha-inducible NFkappaB activation and HIV-1-LTR transcription in CD4+ Jurkat T lymphocytes.

During the latent phase of human immunodeficiency virus type 1 (HIV-1) infection, CD4+ T cells carrying replication-competent proviral HIV-1 DNA play an important role in persistence of the virus. Several cofactors can induce and or amplify HIV-1 replication and negatively affect disease progression and pathogenesis. Ethanol consumption is an important risk factor for HIV-1 infection, and it has been implicated in increased HIV-1 replication and progression of infection. Because tumor necrosis factor-alpha (TNF-alpha) is an important modulator of HIV-1 replication, in the present study we examined the possible effects of ethanol on TNF-alpha-inducible signaling associated with HIV-1 replication in human CD4+ T cells (Jurkat E6-1). We demonstrate that clinically relevant ethanol concentrations significantly potentiate TNF-alpha-inducible NFkappaB. Although ethanol effectively collaborated with TNF-alpha, by itself it did not have a direct effect on NFkappaB activation. The ethanol-dependent potentiation of TNF-alpha-inducible NFkappaB nuclear translocation was observed to involve the enhanced degradation of IkappaBalpha. Additionally, the ethanol-mediated potentiation of TNF-alpha-inducible NFkappaB activation was abrogated by the known antioxidant pyrrolidinedithiocarbamate, suggesting an important mechanistic role for reactive oxygen species in this process. In correspondence with its effect on NFkappaB, ethanol was also observed to significantly enhance HIV-1 long terminal repeat-dependent transcription induced by TNF-alpha. Overall, the data provide a molecular basis for the possible role of ethanol as a cofactor that can adversely affect HIV-1 infection and pathogenesis.

Cycloheximide↗

Pollination and embryo development in Brassica rapa L. in microgravity.

Plant reproduction under spaceflight conditions has been problematic in the past. In order to determine what aspect of reproductive development is affected by microgravity, we studied pollination and embryo development in Brassica rapa L. during 16 d in microgravity on the space shuttle (STS-87). Brassica is self-incompatible and requires mechanical transfer of pollen. Short-duration access to microgravity during parabolic flights on the KC-135A aircraft was used initially to confirm that equal numbers of pollen grains could be collected and transferred in the absence of gravity. Brassica was grown in the Plant Growth Facility flight hardware as follows. Three chambers each contained six plants that were 13 d old at launch. As these plants flowered, thin colored tape was used to indicate the date of hand pollination, resulting in silique populations aged 8-15 d postpollination at the end of the 16-d mission. The remaining three chambers contained dry seeds that germinated on orbit to produce 14-d-old plants just beginning to flower at the time of landing. Pollen produced by these plants had comparable viability (93%) with that produced in the 2-d-delayed ground control. Matched-age siliques yielded embryos of equivalent developmental stage in the spaceflight and ground control treatments. Carbohydrate and protein storage reserves in the embryos, assessed by cytochemical localization, were also comparable. In the spaceflight material, growth and development by embryos rescued from siliques 15 d after pollination lagged behind the ground controls by 12 d; however, in the subsequent generation, no differences between the two treatments were found. The results demonstrate that while no stage of reproductive development in Brassica is absolutely dependent upon gravity, lower embryo quality may result following development in microgravity.

Brassica↗

Induction of monoclonal antibody with predefined ELNKWA epitope specificity by epitope vaccine.

Since the hybridoma technique to produce monoclonal antibodies (MAbs) was discovered, thousands of MAbs with predefined protein specificity have been produced, and a natural or recombinant protein as antigen is necessary for inducing MAbs in the conventional hybridoma technique. To induce epitope-specific MAbs, we suggest an epitope vaccine as a new technique to induce MAbs with predefined epitope specificity. ELDKWA was identified as an important neutralizing epitope on HIV-1 gp41. The MAb 2F5, recognizing ELDKWA epitope, has shown broad neutralizing activity to many HIV strains, including primary isolates, but the mutant in ELNKWA epitope results in escape 2F5-based neutralization. To produce MAbs recognizing this mutated epitope for consideration of passive immunotherapy against the mutant bearing the ELNKWA epitope, MAbs with predefined ELNKWA epitope specificity were induced by synthetic epitope-peptide instead of a natural or recombinant gp41 bearing this epitope. Three MAbs were identified to recognize ELNKWA epitope on the synthetic epitope-peptide, and interestingly could bind the recombinant gp41 with ELDKWA epitope in an ELISA assay and immunoblotting analysis.

AIDS Vaccines↗

Cocaine induces apoptosis in human coronary artery endothelial cells.

This study was designed to determine the direct cytotoxic effect of cocaine on human coronary artery endothelial cells (HCAECs). Cocaine treatment of cultured HCAECs induced a time- and dose-dependent increase in apoptotic cell death in HCAECs. Cocaine-induced surface exposure of phosphatidylserine in HCAECs was seen as early as at 6 h. With prolonged treatment < or =72 h, cocaine (10-500 microM) produced a dose-dependent increase in apoptosis in the cells. Corresponding DNA fragmentation induced by cocaine was demonstrated in situ by terminal deoxynucleotidyl transferase (Tdt) UTP nick end-labeling TUNEL assay and by electrophoresis of labeled DNA fragments, showing the characteristic apoptotic ladders. Both caspase-9 (Z-LEHD-FMK) and caspase-3 (Ac-DEVD-CHO) inhibitors blocked cocaine-induced apoptosis. In addition, cyclosporin A inhibited cocaine-induced apoptosis in a concentration-dependent manner with a median inhibitory concentration (IC50) of 0.3 microM. The maximum of 62% inhibition was obtained with 3 microM cyclosporin A. Cocaine-induced apoptosis also was blocked by naloxone and nifedipine in a dose-dependent manner. These findings suggest that cocaine induces apoptosis in cultured HCAECs, which may be mediated by opioid receptors. The release of cytochrome c from the mitochondria and its subsequent activation of caspase-9 and caspase-3 may play a key role in cocaine-induced apoptosis.

Apoptosis↗

Identification of bone morphogenetic proteins 2 and 4 in commercial demineralized freeze-dried bone allograft preparations: pilot study.

BACKGROUND: Demineralized freeze-dried bone allografts (DFDBAs) have been proposed as a useful adjunct in periodontal therapy to induce periodontal regeneration through the induction of new bone formation. The presence of bone morphogenetic proteins (BMPs) within the demineralized matrix has been proposed as a possible mechanism through which DFDBA may exert its biologic effect. However, in recent years, the predictability of results using DFDBA has been variable and has led to its use being questioned. One reason for the variability in tissue response may be attributed to differences in the processing of DFDBA, which may lead to loss of activity of any bioactive substances within the DFDBA matrix. Therefore, the purpose of this investigation was to determine whether there are detectable levels of bone morphogenetic proteins in commercial DFDBA preparations. METHODS: A single preparation of DFDBA was obtained from three commercial sources. Each preparation was studied in triplicate. Proteins within the DFDBA samples were first extracted with 4M guanidinium HCI for seven days at 40 degrees celsius and the residue was further extracted with 4M guanidinium HCL/EDTA for seven days at 40 degrees celsius. Two anti-human BMP-2 and -4 antibodies were used for the detection of the presence of BMP's in the extracts. RESULTS: Neither BMP-2 nor BMP-4 was detected in any of the extracts. When recombinant human BMP-2 and -4 were added throughout the extraction process of DFDBA extraction, not only were intact proteins detected but smaller molecular weight fragments were also noted in the extract. CONCLUSIONS: These results indicate that all of the DFDBA samples tested had no detectable amounts of BMP-2 and -4. In addition, an unknown substance present in the DFDBA may be responsible for degradation of whatever BMPs might be present.

Antibodies, Monoclonal↗

Evaluation of plasma lysophospholipids for diagnostic significance using electrospray ionization mass spectrometry (ESI-MS) analyses.

We previously reported that lysophosphatidic acid (LPA) represents a potential biomarker for ovarian and other gynecologic cancers. To further improve the accuracy and potentially increase the sensitivity and specificity of the assay, we developed an electrospray ionization mass spectrometry (ESI-MS)-based method to analyze LPA and related lysophospholipids. LPA, lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS), and lysophosphatidylcholine (LPC) could be detected with high sensitivity (in low pmol range) using this method. Standard curves were established for quantitative analysis. LPA and closely related lysophospholipids isolated from thin-layer chromatography (TLC) plates were analyzed directly by ESI-MS. This ESI-MS-based assay allows simultaneous detection and quantitation of all different species of LPAs and LPIs in a sample over a range of at least 5-300 pmol. Moreover, this test was at least 50 times more sensitive when a multiple reaction monitoring (MRM) mode was used. Using these protocols in a limited set of analysis, we found that both LPA and LPI were elevated in patients with ovarian cancer.

Adolescent↗

An optimized forward-planning technique for intensity modulated radiation therapy.

Intensity modulated radiation therapy (IMRT) has stirred considerable excitement in the radiation oncology community. Its objective is to make the dose conform to the tumor and spare other organs. Instead of resorting to the rather complex inverse-planning, the technique described here is an extension of the conventional treatment planning technique. The beam orientation and wedge angles are chosen in the conventional rule-based manner. However, within each conformal beam's eye view (BEV) field including margin, a number of sub-field openings are added. The smaller field openings are designed to irradiate the tumor, while sparing the normal tissue of the organs at risk (OARs) that intrude into the target region in the BEV. As the number of intrusions into the target BEV increases, the number of sub-fields for each beam increases. The Cimmino simultaneous projection method was employed to obtain the optimized weighting for each field of each beam. In cases where the dose constraints for the tumor and for the OARs are reasonable, it is possible to obtain a plan with a fairly small number of beams that satisfies the specified dose objectives. This is illustrated for the treatment of prostate cancer, where the rectum creates a concavity in the planning target volume. An advantage of this technique is that the quality assurance for the delivery of these plans does not require extensive special efforts.

Algorithms↗

Shared roles of yeast glycogen synthase kinase 3 family members in nitrogen-responsive phosphorylation of meiotic regulator Ume6p.

Nitrogen limitation activates meiosis and meiotic gene expression in yeast, but nitrogen-responsive signal transduction mechanisms that govern meiotic gene expression are poorly understood. We show here that Ume6p, a subunit of the Ume6p-Ime1p meiotic transcriptional activator, undergoes increased phosphorylation in vivo in response to nitrogen limitation. Phosphorylation depends on an N-terminal glycogen synthase kinase 3 (GSK3) target site in which substitutions cause reduced Ume6p-Ime1p interaction and meiotic gene expression, thus arguing that phosphorylation promotes functional Ume6p-Ime1p interaction. Phosphorylation of this site depends on two GSK3 homologs, Rim11p and Mck1p. Prior studies indicate that Rim11p phosphorylates both Ume6p and Ime1p in vitro and is required for Ume6p-Ime1p interaction, but no evidence has linked Mck1p function to Ume6p activity. Here we find that Mck1p-Ume6p interaction is detectable by two-hybrid assays and that meiosis in a partially defective rim11-K68R mutant is completely dependent on Mck1p. These findings argue that nitrogen limitation governs Rim11p/Mck1p-dependent phosphorylation of Ume6p, which in turn is required for Ume6p-Ime1p interaction and meiotic gene activation.

Base Sequence↗

Immunogenicity of neutralizing epitopes on multiple-epitope vaccines against HIV-1.

Based on our hypothesis that epitope vaccine may be a new strategy to induce high levels of neutralization antibodies against HIV-1, we prepared multiple-epitope vaccines using three neutralizing epitopes (GPGRAFY, RILAVERYLKD and ELDKWA) of HIV-1 gp160, and characterized their immunogenicity. Peptide 1 [C-G-(ELDKWA-GPGRAFY)(2)-K] and peptide 2 (CG-GPGRAFY-ELDKWA-G-RILAVERYLKD) were synthesized and conjugated with carrier protein bovine serum albumin (BSA). After vaccination antibody responses to these immunogens were induced and evaluated by ELISA. The C-G-(ELDKWA-GPGRAFY)(2)-K-BSA (BSA: carrier protein) multiple-epitope vaccine induced a strong antibody response to the C-G-(ELDKWA-GPGRAFY)(2)-K peptide (antibody titer: 1:25,600) and C-(ELDKWAG)(4) peptide (antibody titer: 1:12,800), but a weak antibody response to the C-(GPCGRAFY)(4) peptide. The CG-GPGRAFY-ELDKWA-G-RILAVERYLKD-K-BSA (BSA: carrier protein) multiple-epitope vaccine also induced strong antibody response to the CG-GPGRAFY-ELDKWA-G-RILAVERYLKD-K peptide (antibody titer: 1:25, 600) and C-(ELLDKWAG)(4) peptide (antibody titer: 1:6,400), a very strong response to C-(RIVALVERYLKD-G)(2)-K peptide (dilution: 1:102, 400), and a very weak response to the C-(GPGRAFY)(4) peptide (dilution: 1:400) in mice. Both antisera induced by both multiple-epitope vaccines interacted with the recombinant soluble gp41 (rgp41), but did not bind two control peptides. In comparison with both epitope vaccines, the rgp160 subunit vaccine could induce weak epitope-specific antibody response to these three epitopes on the three epitope peptides and V3, N-domain and C-domain peptides (dilution: 1:400-1:1,600). These results indicate that both multiple-epitope vaccines could induce high levels of antibodies to both neutralizing epitopes RILAVERYLKD and ELDKWA, while the GPGRAFY epitope on both vaccines appeared to have weak immunogenicity. Both multiple-epitope vaccines showed significant potency on inducing high levels of epitope-specific neutralization antibodies in comparison with rgp160 subunit vaccine.

AIDS Vaccines↗

HIV-1 gp41 by N-domain binds the potential receptor protein P45.

Recent crystal structure analysis of HIV-1 gp41 revealed that two domains (N- and C-domains) on gp41 play an important role in mediating membrane fusion and HIV-1 entry. The experimental evidence that gp41 by N-domain bound the potential receptor protein P45 could help to understand the mechanism of HIV entry. A recombinant soluble gp41 (rsgp41: Env aa539-684) could bind to P45 in the affinity capillary electrophoresis analysis and the surface plasmon resonance assay. In a blockade assay, peptide P1 (Env aa583-599) could inhibit interaction between rsgp41 and P45, while a control peptide could not. Direct binding of rsgp41, rgp41DP (aa567-648), P1 peptide and (P1)(2) peptide [(aa586-596)(2)] to P45 was examined in an ELISA assay. Rsgp41 bound the potential receptor protein P45 strongly, while rgp41DP and P1 as well as (P1)(2) could all weakly bind to P45, indicating that gp41 by N-domain weakly binds P45 and the region RILAVERYLKD located in the N-domain is defined as the binding site for P45 binding.

Amino Acid Motifs↗

Induction of high levels of antibodies recognizing the neutralizing epitope ELDKWA and the D- or K-position-mutated epitopes by candidate epitope vaccines against HIV-1.

Monoclonal antibody 2F5 recognizing the ELDKWA epitope on HIV-1 gp41 has a significant neutralization potency against 90% of the investigated viruses of African, Asian, American, and European strains, but the antibody responses to the epitope 2F5 in HIV-1-infected individuals were very low. We attempted to induce high levels of epitope-specific antibodies to ELDKWA and its three mutated epitopes by candidate epitope vaccines. The four candidate epitope vaccines all induced strong antibody responses at dilutions from about 1:6,400 to 1:25,600. We tested the cross-reactions between these antisera and four epitope peptides. The ELDKWA-specific antisera showed strong cross-reactivity with three neutralizing-resistant mutated epitopes which contain changes in the D or K positions of the epitope sequence. Virus variants containing these changes could escape neutralization by monoclonal antibody 2F5. In immunoblotting analysis, the ELDKWA, ELDEWA, and ELEKWA epitope specific antibodies all recognized rsgp41 which confirms that the antibodies against both mutated epitopes, ELDEWA and ELEKWA, could cross-react with the native epitope on rsgp41. Although it is not clear whether the polyclonal antibodies induced by the ELDKWA epitope vaccine could neutralize the mutated viruses containing these mutated epitopes, it is conceivable that epitope vaccines based on mutated epitopes could induce strong antibody responses with predefined epitope specificity to neutralize mutated viruse containing the mutated epitope. An epitope vaccine, using different epitopes including mutated epitopes, could provide a new concept for developing a new vaccine against HIV-1.

AIDS Vaccines↗