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Biomedical subjects

R Yang

Publications and source records attributed to R Yang.

At least 19 recordsLinked to original sources
PubMed2003

Thrombin induces angiogenesis and vascular endothelial growth factor expression in human endothelial cells: possible relevance to HIF-1alpha.

The serine protease thrombin present at the site of vascular injury triggers fibrin formation, platelet activation and different cellular responses including angiogenesis. We report a role for thrombin in the human monolayer cultured endothelial cell growth and angiogenesis in 3D collagen gel angiogenesis assay. The angiogenic activity of thrombin is, in part, related to the expression of the vascular endothelial growth factor (VEGF)165 mRNA, assessed by reverse transcriptase-polymerase chain reaction, either in monolayer cultured endothelial cells or in endothelial cells forming capillary-like structures in the 3D collagen gel assay. This expression of VEGF mRNA is associated with a VEGF secretion in the supernatant of thrombin-treated human umbilical vein endothelial cells. The thrombin-induced VEGF165 mRNA expression is associated with the regulation of hypoxia-inducible factor 1alpha, analyzed by Western Blot, in endothelial cells.

Cell Division↗
PubMed2002

Hyaluronic acid and chondroitin sulphate A rapidly promote differentiation of immature DC with upregulation of costimulatory and antigen-presenting molecules, and enhancement of NF-kappaB and protein kinase activity.

Dendritic cells (DCs) have been identified as effective antigen-presenting cells (APCs). We demonstrate that extracellular matrix (ECM), hyaluronic acid (HA) and chondroitin sulphate A (CSA), in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), can rapidly promote the differentiation of monocyte-derived immature DCs, as characterized by the remarkable upregulation of human leucocyte antigen (HLA-DR), CD40, CD54, CD80 and CD86 expression to levels higher than those in the DCs generated by culturing with GM-CSF and interleukin (IL)-4 for 7 days and aggregation of the cells within 48 h. The upregulation of expression of HLA-DR, CD40, CD54, CD80 and CD86 was dose-dependent. Further studies showed that HA and CSA were able to augment nuclear factor (NF)-kappaB activity, as determined by gel mobility shift assay and promote protein phosphorylation. Inhibition of NF-kappaB by pyrolidine dithiocarbamate and sodium salicylate, and serine-threonine and tyrosine kinase by starosporine as well as phosphatidylinositide-3-kinase (PI-3-K) by wortmannin could prevent the effects of HA and CSA on the expression of HLA-DR, CD40, CD80 and CD86 in various degrees. Thus, our data demonstrate that HA or CSA can effectively and rapidly promote the differentiation of immature DC, suggesting that HA and CSA may possess a potential capacity in regulating immune responses.

Antigen Presentation↗
PubMed2001

"Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin.

Taste buds contain a variety of morphological and histochemical types of elongate cells. Serotonin, neuron-specific enolase (NSE), ubiquitin carboxyl terminal hydrolase (PGP 9.5), and neural cell adhesion molecule (N-CAM) all have been described as being present in the morphologically defined Type III taste cells in rats. In order to determine whether these substances coexist in a single cell, we undertook immunohistochemical and ultrastructural analysis of taste buds in rats. Double-label studies show that PGP 9.5 and NSE always colocalize. In contrast, PGP 9.5 and serotonin seldom colocalize. Further, whereas the serotonin-immunoreactive cells are always slender and elongate, the PGP 9.5/NSE population comprise two morphological types--one slender, the other broader and pyriform. Although gustducin-immunoreactive taste cells appear similar in overall shape to the pyriform PGP 9.5/NSE population, gustducin never colocalizes with PGP 9.5 or NSE. The serotonin-immunoreactive taste cells have an invaginated nucleus, synaptic contacts with nerve fibers, and taper apically to a single, large microvillus. These are all characteristics of Type III taste cells described previously in rabbits (Murray [1973] Ultrastructure of Sensory Organs I. Amsterdam: North Holland. p 1-81). PGP 9.5-immunoreactive taste cells exhibit two morphological varieties. One type is similar to the serotonin-immunoreactive population, containing an invaginated nucleus, synapses with nerve fibers, and a single large microvillus. The other type of PGP 9.5-immunoreactive taste cell has a large round nucleus and the apical end of the cell tapers to a tuft of short microvilli, which are characteristics of Type II taste cells. Thus, in rats, some Type III cells accumulate serotonin but do not express PGP 9.5, whereas others express PGP 9.5 but do not accumulate amines. Similarly, Type II taste cells come in at least two varieties: those immunoreactive for gustducin and those immunoreactive for PGP 9.5.

Animals↗
PubMed2001

Immature dendritic cells kill ovarian carcinoma cells by a FAS/FASL pathway, enabling them to sensitize tumor-specific CTLs.

Dendritic cells (DCs) can acquire antigen(s) from apoptotic tumor cells, resulting in an immunogen that can induce class I-restricted cytotoxic T lymphocytes (CTLs) and protective tumor rejection. Here, we investigated whether DCs derived from ascitic monocytes of patients with ovarian carcinoma could kill autologous ovarian tumor cells and if as a result they would acquire antigen(s) enabling them to induce a tumor-specific immunity. We found that the immature DCs could exert a significant cytotoxicity towards autologous and allogeneic ovarian tumor cells. This cytotoxicity was independent of Ca(2+) and could be inhibited by anti-Fas IgG1 monoclonal antibody, indicating the involvement of the Fas/Fas ligand (FasL) pathway in the cytotoxic mechanism. Further supporting this conclusion, the ascitic monocyte-derived DCs expressed high levels of FasL mRNA and intracellular FasL and significant levels of Fas were also revealed on the surface of ovarian tumor cells. Coculture of DCs induced apoptosis in ovarian carcinoma cells, as well as uptake of apoptotic tumor cells into the cytoplasma of the DCs, as visualized by immunofluoresence. Autologous DCs cocultured with apoptotic ovarian tumor cells were able to specifically stimulate tumor-specific CTLs, whereas DCs cocultured with necrotic ovarian cells were unable to do so. Collectively, these results demonstrate that immature DCs can kill autologous ovarian carcinoma cells via the Ca(2+)-independent Fas/FasL pathway and that this may have important consequences for their ability to stimulate tumor-specific CTLs.

Antibodies, Monoclonal↗
PubMed2001

Isolation and characterization of a full-length molecular DNA clone of Ghanaian HIV type 1 intersubtype A/G recombinant CRF02_AG, which is replication competent in a restricted host range.

We have isolated a replication-competent, full-length molecular clone of HIV-1 CRF02_AG, designated p97GH-AG1, by reconstituting two separately amplified genomic regions of an HIV-1 provirus of a 1997 Ghanaian isolate. The phylogenetic and recombination breakpoint analyses revealed that 97GH-AG1 had an A/G recombinant structure similar to that of prototype Nigerian isolate IbNG. The 17-nucleotide insertion downstream of the primer-binding site appeared to be a common sequence signature specific to most CRF02_AG strains, including 97GH-AG1. 97GH-AG1 showed an R5 phenotype and exerted productive infection in both HOS and NP2 cell infectivity assays, whereas it failed to show a detectable level of progeny production in peripheral blood mononuclear cells (PBMCs). The data may suggest the presence of unknown determinant(s) that dictate efficient replication in PBMCs, but that are not required for replication in immortalized cell lines.

Amino Acid Sequence↗
PubMed2001

[26S rDNA D1/D2 domain sequence analysis of the pathogenic strain from the first case of disseminated trichosporonosis in China].

OBJECTIVE: To determine the large-subunit (26S) rDNA D1/D2 domain sequence of AS 2.217 4, the pathogenic strain from the first case of disseminated trichosporosis in China, and study the feasibility of applying DNA sequencing in rapid identification of pathogenic yeast. METHOD: The strain of Trichosporon (AS 2.2174) was collected from the skin lesion of a case of disseminated trichosporosis. Nuclear DNA was extracted from the cells of AS 2.2174 by a simplified protocol and the 26S rDNA D1/D2 domain was amplified by PCR. The PCR product was then directly sequenced with the ABI PRISM BigDye cycle sequencing kit. RESULTS: The 26S rDNA D1/D2 domain of strain AS 2.2174 was determined (GenBank accession number: AF337949). Comparison analysis indicated that the D1/D2 sequence of AS 2.2174 is identical with that of the type strain of Trichosporon asahii, and differs from those T. asteroides, T. faecale and T. coremiiforme in 3, 5 and 8 nucleotides, respectively. The latter three species are phenotypically indistinguishable from T. asashii. CONCLUSION: The strain AS 2.2174 was confirmed to belong to Trichosporon asahii. This species is the first record in China clinically and taxonomically. DNA sequencing shows an advantage in fast and accurate identification of clinically relevant yeast species.

Adult↗
PubMed2001

Molecular cloning of a third member of the potassium-dependent sodium-calcium exchanger gene family, NCKX3.

We describe here the identification and characterization of a novel member of the family of K(+)-dependent Na(+)/Ca(2+) exchangers, NCKX3 (gene SLC24A3). Human NCKX3 encodes a protein of 644 amino acids that displayed a high level of sequence identity to the other family members, rod NCKX1 and cone/neuronal NCKX2, in the hydrophobic regions surrounding the "alpha -repeat" sequences thought to form the ion-binding pocket for transport. Outside of these regions NCKX3 showed no significant identity to other known proteins. As anticipated from this sequence similarity, NCKX3 displayed K(+)-dependent Na(+)/Ca(2+) exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive (45)Ca(2+) uptake. The N-terminal region of NCKX3, although not essential for expression, increased functional activity at least 10-fold and may represent a cleavable signal sequence. NCKX3 transcripts were most abundant in brain, with highest levels found in selected thalamic nuclei, in hippocampal CA1 neurons, and in layer IV of the cerebral cortex. Many other tissues also expressed NCKX3 at lower levels, especially aorta, uterus, and intestine, which are rich in smooth muscle. The discovery of NCKX3 thus expands the K(+)-dependent Na(+)/Ca(2+) exchanger family and suggests this class of transporter has a more widespread role in cellular Ca(2+) handling than previously appreciated.

Animals↗
PubMed2001

Cyclin A1 directly interacts with B-myb and cyclin A1/cdk2 phosphorylate B-myb at functionally important serine and threonine residues: tissue-specific regulation of B-myb function.

Cyclin A1 is tissue-specifically expressed during spermatogenesis, but it is also highly expressed in acute myeloid leukemia (AML). Its pathogenetic role in AML and in the cell cycle of leukemic blasts is unknown. B-myb is essential for G1/S transition and has been shown to be phosphorylated by the cyclin A2/cdk2 complex. Here it is demonstrated that cyclin A1 interacts with the C-terminal portion of B-myb as shown by glutathione S-transferase (GST) precipitation. This interaction is confined to cyclin A1 because binding could not be detected between cyclin A2 and B-myb. Also, cdk2 was not pulled down by GST-B-myb from U937 lysates. In addition, co-immunoprecipitation of cyclin A1 and B-myb in leukemic cells evidenced protein interaction in vivo. Baculovirus-expressed cyclin A1/cdk2 complexes were able to phosphorylate human as well as murine B-myb in vitro. Tryptic phosphopeptide mapping revealed that cyclin A1/cdk2 complexes phosphorylated the C-terminal part of B-myb at several sites including threonine 447, 490, and 497 and serine 581. These phosphorylation sites have been demonstrated to be important for the enhancement of B-myb transcriptional activity. Further studies showed that cyclin A1 cooperated with B-myb to transactivate myb binding site containing promoters including the promoter of the human cyclin A1 gene. Taken together, the data suggest that cyclin A1 is a tissue-specific regulator of B-myb function and activates B-myb in leukemic blasts. (Blood. 2001;97:2091-2097)

Animals↗
PubMed2001

Effects of early angiotensin-converting enzyme inhibition on cardiac gene expression after acute myocardial infarction.

BACKGROUND: ACE inhibition after myocardial infarction (MI) has been shown to have beneficial effects on cardiac anatomy and function. The purpose of this study was to examine the effects of ACE inhibition on cardiac gene expression after MI. METHODS AND RESULTS: Rats were randomized to receive captopril or no treatment 1 day after MI. Eight weeks later, cardiac function and hemodynamics were measured by use of indwelling catheters and perivascular flow probes. Myocardial gene expression was assessed with DNA microarrays and real-time reverse transcription-polymerase chain reaction. The ratios of heart and left ventricular weights to body weight were significantly increased by MI and normalized by captopril. Cardiac index and stroke volume index were lower in the untreated MI group than in sham controls but were normal in the MI+captopril group. Thirty-seven genes were found to be differentially expressed between the untreated MI group and sham controls; 31 were induced and 6 repressed. Captopril partially or completely inhibited changes in 10 of the genes. The 37 genes clustered into 11 functional groups, and 6 had >/=1 genes whose expression was modified by ACE inhibition. CONCLUSIONS: ACE inhibition after MI inhibits cardiac hypertrophy, preserves cardiac function, and attenuates changes in myocardial gene expression. Gene expression profiling reveals, however, that some elements of the pathophysiology may be unaffected by the treatment and be targets for new therapies.

Acute Disease↗
PubMed2001

Closely related HIV-1 CRF01_AE variant among injecting drug users in northern Vietnam: evidence of HIV spread across the Vietnam-China border.

To investigate the nature of recent HIV outbreaks among injecting drug users (IDUs) near the Vietnam-China border, we genetically analyzed 24 HIV-positive blood specimens from 2 northern provinces of Vietnam (Lang Son and quang Ninh) adjacent to the China border, where HIV outbreaks among IDUs were first detected in late 1996. Genetic subtyping based on gag (p17) and env (C2/V3) sequences revealed that CRF01_AE is a principal strain circulating throughout Vietnam, including the provinces near the China border. The majority of CRF01_AE sequences among IDUs in Quang Ninh and Lang Son showed significant clustering with those found in nearby Pingxiang City of China's Guangxi Province, sharing a unique valine substitution 12 amino acids downstream of the V3 loop. This particular subtype E variant, uniquely found among IDUs in northern Vietnam and southeastern China, is designated E(v). The genetic diversity of CRF01_AE distributed in Quang Ninh (1.5 +/- 0.6%) and Pingxiang City (1.9 +/- 1.2%) was remarkably low, indicating the emerging nature of HIV spread in these areas. It is also noted that the genetic diversity of CRF01_AE among IDUs was consistently lower than that in persons infected sexually, suggesting that fewer closely related CRF01_AE variants were introduced into IDUs and, conversely, that multiple strains of CRF01_AE had been introduced via the sexual route. The data in the present study provide additional evidence that HIV outbreaks among IDUs in northern Vietnam were caused by the recent introduction of a highly homogeneous CRF01_AE variant (E(v)) closely related to that prevailing in nearby southern China.

Adolescent↗
PubMed2001

Study on the preparation of recombinant human HSP70 and its presenting-antigen function.

The preparation of recombinant human HSP70 and its presenting-antigen function were investigated. Cultured in glucose-free M9ZB medium and induced with IPTG and lactose at a final concentration of 0.02 mmol/L and 5 mmol/L respectively, the engineered bacteria carrying expression vector of human HSP70 gene expressed rHSP70 at an efficiency fo 60%. After the purification with DEAE ion-exchange chromatography, HSP70 with a purity of higher than 90% was obtained. The purified product could bind tumor-antigen peptide in vitro, and the binding was identified by native PAGE containing 5% glycerol. HSP70-peptide complex could activate lymphocytes to produce specific cytotoxicity to tumor cells, suggesting that the recombinant human HSP70 could be used as an antigen-presenting reagent in tumor therapy.

Animals↗
PubMed2001

Solid state NMR measurements of conformation and conformational distributions in the membrane-bound HIV-1 fusion peptide.

The solid state NMR lineshape of a protein backbone carbonyl nucleus is a general diagnostic of the local conformational distribution in the vicinity of that nucleus. In addition, measurements of carbonyl chemical shifts and 2D exchange spectra provide information about the most probable conformation in the distribution. These types of solid state NMR methodologies have been applied to structural studies of the membrane-bound HIV-1 fusion peptide. This peptide is derived from a domain of the HIV-1 gp41 envelope protein, which is critical for viral-host cell-membrane fusion. Even in the absence of the rest of the envelope protein, the fusion peptide will fuse liposomes or erythrocytes. The solid state NMR measurements demonstrate that the center of the membrane-bound HIV-1 fusion peptide is structured, while the C-terminus is highly disordered. The structural distribution at the peptide center is lipid-dependent, with the greatest degree of structural homogeneity in a lipid environment whose composition reflects that of the target T cells. When bound to the lipid mixture, the peptide center is predominately beta sheet. The beta-sheet structure may be diagnostic of peptide oligomerization, which is thought to be a requirement for membrane fusion activity. Although the peptide partially disrupts bilayer orientational ordering in stacked glass-plate samples, 2H NMR demonstrates that the bilayers remain intact in the presence of the fusion peptide and are not micellized. The retention of the bilayer phase may relate to the biological requirement that the virus should fuse with, but not destroy, the target host cell membrane.

HIV-1↗
PubMed2001

Fluorometric study of the inclusion interaction of beta-cyclodextrin derivatives with tetraphenylporphyrin and its analytical application.

The effects of native beta-cyclodextrin (beta-CD) and four kinds of alkylated beta-cyclodextrin (beta-CDs), i.e. heptakis (2,6-di-O-isobutyl-beta-cyclodextrin) (I), heptakis (2,6-di-O-octyl-beta-cyclodextrin) (II), heptakis (2,6-di-O-dodecyl-beta-cyclodextrin) (III), and heptakis (2,6-di-O-hexadecyl-beta-cyclodextrin) (IV), on the fluorescence behaviors of tetraphenylporphyrin (TPP) are investigated. An obvious fluorescence enhancement is observed from TPP by using alkylated derivatives compared to that obtained in beta-CD aqueous or in water. A 114-N fluorescence emission intensity enhancement is found for the complex with 2,6-di-O-octyl-beta-cyclodextrin relative to the free analyte. The exact stoichiometric ratios and the formation constants of the inclusion complexes have been examined by application of curve fitting method. The linear calibration plots between fluorescence intensity and TPP concentration are determined in the 1.14 x 10(-8)-5.06 x 10(-6) mol l(-1) range.

Alkylation↗
PubMed2001

Transcatheter closure of secundum atrial septal defect with a new self-expanding nitinol double disk device (Amplatzer device): experience in Nanjing.

PURPOSE: Various devices have been developed for the transcatheter closure of secundum atrial septal defect (ASD II) to avoid the morbidity, discomfort, and thoracotomy scar associated with surgical closure. The purpose of this study was to evaluate the safety and efficacy of the Amplatzer septal occluder for transcatheter closure of ASD II. PATIENTS AND METHODS: Only patients who were clinically diagnosed with ASD II were selected. The anatomy of ASD had to meet certain echocardiographic criteria. Under the guidance of echocardiography and fluoroscopy, the implantation of the device was accomplished as recommended by the manufacturer. RESULTS: Thirty patients (median age 18.4 years) with an ASD II underwent transcatheter closure. Procedure time ranged from 30-200 minutes and fluoroscopy time from 10-50 minutes. The diameter of the ASD measured by echocardiography ranged from 13-25 mm, while both the stretched diameters of the ASDs and the sizes of the devices ranged from 18-34 mm. The successful placement rate was 100%. The residual shunt rate was 100% immediately after device implantation and 10% after 24 hours. After 3 months, 3.3% of the patients had a (trivial) residual shunt. The device did not affect the surrounding structures of ASD. No embolization of the device occurred. CONCLUSION: The Amplatzer device designed for the closure of ASD II can be implanted easily and also is retrievable. Due to a low ratio of residual shunt and few complications, this device is a good choice for transcatheter closure of ASD II. Long-term follow-up will be required for widespread clinical use.

Adolescent↗
PubMed2001

Modeling and mutational analysis of a putative sodium-binding pocket on the dopamine D2 receptor.

A homology model of the dopamine D2 receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB ) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.

Amino Acid Sequence↗
PubMed2001

Augmentation of human immunodeficiency virus type 1 subtype E (CRF01_AE) multiple-drug resistance by insertion of a foreign 11-amino-acid fragment into the reverse transcriptase.

A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the beta3-beta4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of the pol sequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3'-azido-3'-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the beta3-beta4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.

Adult↗