Biomedical subjects

H Yang

Publications and source records attributed to H Yang.

At least 145 records · Page 8Linked to original sources

PubMed2001

HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.

HIF (hypoxia-inducible factor) is a transcription factor that plays a pivotal role in cellular adaptation to changes in oxygen availability. In the presence of oxygen, HIF is targeted for destruction by an E3 ubiquitin ligase containing the von Hippel-Lindau tumor suppressor protein (pVHL). We found that human pVHL binds to a short HIF-derived peptide when a conserved proline residue at the core of this peptide is hydroxylated. Because proline hydroxylation requires molecular oxygen and Fe(2+), this protein modification may play a key role in mammalian oxygen sensing.

Amino Acid Sequence↗

PubMed2001

The neural recognition molecule L1 is a sialic acid-binding lectin for CD24, which induces promotion and inhibition of neurite outgrowth.

Among the recognition molecules that determine a neuron's interaction with other cells, L1 and CD24 have been suggested to cooperate with each other in neurite outgrowth and signal transduction. Here we report that binding of CD24 to L1 depends on alpha2,3-sialic acid on CD24, which determines the CD24 induced and cell type-specific promotion or inhibition of neurite outgrowth. Using knockout mutants, we could show that the CD24-induced effects on neurite outgrowth are mediated via L1, and not GPI-linked CD24, by trans-interaction of L1 with sialylated CD24. This glycoform is excluded together with L1 from raft microdomains, suggesting that molecular compartmentation in the surface membrane could play a role in signal transduction.

Amino Acid Sequence↗

PubMed2001

Immunoprophylactic strategies against respiratory influenza virus infection.

The objective of this report is to evaluate the prophylactic efficacy of liposome-mediated immunotherapy for prevention of respiratory influenza virus infection in mice. Antiviral antibody, interferon-gamma and poly (ICLC) were encapsulated in liposomes and they were evaluated for their ability to induce protective immunity against lethal influenza infection. Passive immunization using liposome-encapsulated antiviral antibody was found to offer complete protection against the virus challenge. However, this pretreatment must be administered within 24 h prior to virus challenge to be protective. Pretreatment with liposome-encapsulated interferon-gamma was found to stimulate cellular immune responses, but the protection is partial. Immunoprophylaxis using liposome-encapsulated double-stranded (ds) RNA poly (ICLC) provided complete and longer-lasting protection against influenza infection. These results suggest liposome-mediated immunoprophylactic approaches are effective in the prevention of respiratory influenza virus infection.

Adjuvants, Immunologic↗

PubMed2001

Vesicle-associated membrane protein isoforms in the tiger salamander retina.

Vesicle associated membrane protein (VAMP; also known as synaptobrevin) is a key component of the core complex needed for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Recent work indicates that the precise complement of presynaptic proteins associated with transmitter release and their isoforms vary among synapses, presumably conferring specific functional release properties. The retina contains two types of vesicular synapses with distinct morphologic, functional, and biochemical characteristics: ribbon and conventional synapses. Although the precise complement of presynaptic proteins is known to differ between conventional and ribbon synapses and among conventional synapses, the distribution of VAMP isoforms among retinal synapses has not been determined. The expression and localization of VAMP isoforms in the salamander retina, a major model system for studies of retinal circuitry, was examined by using immunocytochemical and immunoblotting methods. Both methods indicated that at least two VAMP isoforms were expressed in salamander retina. One isoform, recognized by an immunoglobulin M antibody that recognizes both mammalian VAMP-1 and VAMP-2, was associated with photoreceptor and bipolar cell terminals as well as many conventional synapses, and probably corresponds to mammalian VAMP-2. A different VAMP isoform associated with a subset of amacrine cells, was recognized only by antibodies directed against the N-terminus of mammalian VAMP-2. An antiserum directed against the N-terminus of mammalian VAMP-1 did not specifically recognize any salamander VAMPs in either immunocytochemical or immunoblotting experiments. Heterogeneous distribution of VAMP isoforms among conventional retinal synapses was confirmed by double labeling for synapsin I, a marker for conventional synapses. These studies indicate that VAMP isoforms are expressed heterogeneously among retinal synapses but cannot account for the differences in transmitter release characteristics at ribbon and conventional synapses. These results also corroborate previous studies in Xenopus indicating that the N-terminus of nonmammalian VAMP isoforms differs from their mammalian counterparts.

Ambystoma↗

PubMed2001

Triplet organometallic reactivity under ambient conditions: an ultrafast UV pump/IR probe study.

The reactivity of triplet 16-electron organometallic species has been studied in room-temperature solution using femtosecond UV pump IR probe spectroscopy. Specifically, the Si-H bond-activation reaction of photogenerated triplet Fe(CO)(4) and triplet CpCo(CO) with triethylsilane has been characterized and compared to the known singlet species CpRh(CO). The intermediates observed were studied using density functional theory (DFT) as well as ab initio quantum chemical calculations. The triplet organometallics have a greater overall reactivity than singlet species due to a change in the Si-H activation mechanism, which is due to the fact that triplet intermediates coordinate weakly at best with the ethyl groups of triethylsilane. Consequently, the triplet species do not become trapped in alkyl-solvated intermediate states. The experimental results are compared to the theoretical calculations, which qualitatively reproduce the trends in the data.

Journal Article↗

PubMed2001

Isolation and characterization of mouse MUC18 cDNA gene, and correlation of MUC18 expression in mouse melanoma cell lines with metastatic ability.

The cell surface adhesion molecule human MUC18 (huMUC18 or Mel-CAM) has been postulated to play a key pathogenic role in metastatic melanoma progression. To establish an immunocompetent syngeneic mouse model that would greatly facilitate our understanding of the role of MUC18 in the metastatic behavior of melanoma, we cloned and characterized the mouse MUC18 (muMUC18) cDNA gene. The gene was amplified by RT-PCR and RACE of the poly(A)+RNA isolated from the mouse melanoma cell line B16F10/Queens. The cloned muMUC18 cDNA gene contained 28 nucleotides of 5'-UTR, 908 nucleotides of 3'-UTR, and an open reading frame (ORF) of 1947 nucleotides encoding a protein of 648 amino acids, which is two amino acids longer than huMUC18. The size of the muMUC18 mRNA is about 3 kb with a shorter 3'-UTR than the huMUC18 mRNA (about 3.3 kb). Besides, the sequence in the 3' UTR of the two mRNAs is diverse with only 31% identity. The 5'-UTR and coding sequences of the muMUC18 cDNA are 72.4 and 80.6% identical to those of huMUC18, respectively. The deduced amino acid sequence of the muMUC18 cDNA is 76.2% identical to that of huMUC18. The amino acid sequences deduced from MUC18 cDNA sequences from six other mouse melanoma cell lines are identical except one to three residues, suggesting that the muMUC18 cDNA sequence determined in this report is correct. The muMUC18 protein is predicted to be slightly more acidic than the human protein. The levels of muMUC18 mRNA and protein in nine mouse melanoma cell lines were directly proportional to their ability to establish metastatic colonies in lungs of syngeneic mice. Most biological functions of the muMUC18 may be similar to the huMUC18.

Amino Acid Sequence↗

PubMed2001

Dynamic recruitment of human CD2 into lipid rafts. Linkage to T cell signal transduction.

CD2 mediates T cell adhesion via its ectodomain and signal transduction utilizing its 117-amino acid cytoplasmic tail. Here we show that a significant fraction of human CD2 molecules is inducibly recruited into lipid rafts upon CD2 cross-linking by a specific pair of mitogenic anti-CD2 monoclonal antibodies (anti-T11(2) + anti-T11(3)) or during cellular conjugate formation by CD58, the physiologic ligand expressed on antigen-presenting cells. Translocation to lipid microdomains is independent of the T cell receptor (TCR) and, unlike inducible TCR-raft association, requires no tyrosine phosphorylation. Structural integrity of rafts is necessary for CD2-stimulated elevation of intracellular free calcium and tyrosine phosphorylation of cellular substrates. Whereas murine CD2 contains two membrane-proximal intracellular cysteines, partitioning CD2 into cholesterol-rich lipid rafts constitutively, human CD2 has no cytoplasmic cysteines. Mapping studies using CD2 point mutation, deletion, and chimeric molecules suggest that conformational change in the CD2 ectodomain participates in inducible raft association and excludes the membrane-proximal N-linked glycans, the transmembrane segment, and the CD2 cytoplasmic region (residues 8-117) as necessary for translocation. Translocation of CD2 into lipid rafts may reorganize the membrane into an activation-ready state prior to TCR engagement by a peptide associated with a major histocompatibility complex molecule, accounting for synergistic T cell stimulation by CD2 and the TCR.

Amino Acid Sequence↗

PubMed2001

A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum.

Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.

Amino Acid Sequence↗

PubMed2001

Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch.

Adenine-DNA glycosylase MutY of Escherichia coli catalyzes the cleavage of adenine when mismatched with 7,8-dihydro-8-oxoguanine (GO), an oxidatively damaged base. The biological outcome is the prevention of C/G-->A/T transversions. The molecular mechanism of base excision repair (BER) of A/GO in mammals is not well understood. In this study we report stimulation of mammalian adenine-DNA glycosylase activity by apurinic/apyrimidinic (AP) endonuclease using murine homolog of MutY (Myh) and human AP endonuclease (Ape1), which shares 94% amino acid identity with its murine homolog Apex. After removal of adenine by the Myh glycosylase activity, intact AP DNA remains due to lack of an efficient Myh AP lyase activity. The study of wild-type Ape1 and its catalytic mutant H309N demonstrates that Ape1 catalytic activity is required for formation of cleaved AP DNA. It also appears that Ape1 stimulates Myh glycosylase activity by increasing formation of the Myh-DNA complex. This stimulation is independent of the catalytic activity of Ape1. Consequently, Ape1 preserves the Myh preference for A/GO over A/G and improves overall glycosylase efficiency. Our study suggests that protein-protein interactions may occur in vivo to achieve efficient BER of A/GO.

Amino Acid Sequence↗

PubMed2001

Sulfanyl- and selanyldifluoromethylphosphonates as a source of phosphonodifluoromethyl radicals and their addition onto alkenes.

[figure: see text] Two different strategies are shown to produce sulfanyl and selanyldifluoromethylphosphonates. Thus, treatment of sulfanyldichloromethylphosphonates by 3HF.NEt3 in the presence of zinc bromide produces the corresponding sulfanyldifluoromethylphosphonates. In addition, lithiation of difluoromethylphosphonates followed by quenching with phenylsulfanyl chloride, phenylselanyl chloride, or diphenyl diselenide yields the corresponding sulfanyl- and selanyldifluorophosphonates. Generation of phosphonodifluoromethyl radicals from such precursors in the presence of alkenes produces the expected adducts.

Journal Article↗

PubMed2001

Crystal structure and catalytic mechanism of the MJ0109 gene product: a bifunctional enzyme with inositol monophosphatase and fructose 1,6-bisphosphatase activities.

Inositol monophosphatase (EC 3.1.3.25) in hyperthermophilic archaea is thought to play a role in the biosynthesis of di-myo-inositol-1,1'-phosphate (DIP), an osmolyte unique to hyperthermophiles. The Methanococcus jannaschii MJ109 gene product, the sequence of which is substantially homologous to that of human inositol monophosphatase, exhibits inositol monophosphatase activity but with substrate specificity that is broader than those of bacterial and eukaryotic inositol monophosphatases (it can also act as a fructose bisphosphatase). To understand its substrate specificity as well as the poor inhibition by Li(+) (a potent inhibitor of the mammalian enzyme), we have crystallized the enzyme and determined its three-dimensional structure. The overall fold, as expected, is similar to that of the mammalian enzyme, but the details suggest a closer relationship to fructose 1,6-bisphosphatases. Three complexes of the MJ0109 protein with substrate and/or product and inhibitory as well as activating metal ions suggest that the phosphatase mechanism is a three-metal ion assisted catalysis which is in variance with that proposed previously for the human inositol monophosphatase.

Amino Acid Sequence↗

PubMed2001

Model-based estimation of myeloid hematopoietic progenitor cells in ex vivo cultures for cell and gene therapies.

Ex vivo production of hematopoietic progenitor cells has potential applications for cell therapy to alleviate cytopenias associated with chemotherapy and for gene therapy. In both therapies, progenitor and stem cells are considered crucial factors for therapeutic success. Assays for progenitor cells, however, take 2 weeks to complete, which is similar to the length of a typical culture. Therefore, a real-time estimation of the percentage or number of progenitor cells, based on rapid measurements, would be useful for optimization of feeding and harvest decisions. In this study, metabolic activity assays and flow cytometric analysis were used to estimate the content of progenitor cells. The measured metabolic activities are a collective contribution from all types of cells. Cells in granulomonocytic cultures have been lumped into six cell types and metabolic rates have been modeled as a linear function of cell composition and growth rate and as a nonlinear function of cell density. Data from 24 experiments were utilized to determine the model parameters in a calibration step. These data include flow cytometric analysis of more mature hematopoietic cells, progenitor cell colony assays, total cell content, and metabolite concentrations, and cover a wide range of cell composition, cell density, and growth rate. After calibration, the model is able to deliver good predictions of progenitor cell content for cultures with higher percentages of progenitor cells, as well as the peak progenitor cell content, based only on parameters that can be rapidly measured. With the aid of those predictions a harvest strategy was developed that will allow optimizing the harvest time based on the culture kinetics of each patient or donor inoculum, rather than using retrospective analysis to determine a uniform harvest time.

Calibration↗

PubMed2001

Identification of two proteins, S14 and UIP1, that interact with UCH37.

By the use of the yeast two-hybrid screen we have identified two proteins that interacted with UCH37: S14, which is a subunit of PA700 and a novel protein, UIP1 (UCH37 interacting protein 1). The interaction of UCH37 with S14 or UIP1 was confirmed by in vitro binding assay and in vivo co-immunoprecipitation analysis. The C-terminal extension of UCH37 is essential for interaction with S14 or UIP1 as shown by the yeast two-hybrid assay and the in vitro binding assay. Furthermore, UIP1 blocked the interaction between UCH37 and S14 in vitro.

Amino Acid Sequence↗

PubMed2001

Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain.

Reporter mouse strains are important tools for monitoring Cre recombinase-mediated excision in vivo. In practice, excision may be incomplete in a given population due to threshold level or variegated expression of Cre. Hence, it is desirable in many experimental contexts to isolate cells that have undergone excision to assess the consequences of gene ablation. To generate alternative reporter mice, an enhanced green fluorescent protein (EGFP) gene was targeted to the retroviral-trapped ROSA26 locus. Upon Cre-mediated excision of "Stop" sequences, EGFP was expressed ubiquitously during embryogenesis and in adult tissues (including T cells, B cells, and myeloid cells). Using this new reporter strain, separation of excised from nonexcised cells in vitro was achieved in thymocytes in a noninvasive manner based on activated EGFP expression. This new EGFP reporter strain should facilitate a variety of conditional gene-targeting experiments, including the functional studies of hematopoietic cells in lineage-specific knockout mice.

Animals↗

PubMed2001

The role of c-Myb in the up-regulation of methionine adenosyltransferase 2A expression in activated Jurkat cells.

Methionine adenosyltransferase (MAT) is a critical cellular enzyme which catalyses the formation of S-adenosylmethionine (SAM), the principal methyl donor. In mammals, two different genes, MAT1A and MAT2A, encode liver-specific and non-liver-specific MATs, respectively. SAM level increases during T-lymphocyte activation and is required for proliferation. A major mechanism for the increase in SAM level is increased MAT2A transcription. In the current work we examined the molecular mechanism of increased MAT2A expression in activated Jurkat cells. Treatment of Jurkat cells with interleukin-2 (IL-2), PMA or PMA plus phytohaemagglutinin (PHA) resulted in a 2-fold increase in MAT2A mRNA levels and a 2-fold increase in luciferase activity driven by the transfected human MAT2A promoter construct -571/+60 but not -270/+60. The region -571 to -270 of the human MAT2A contains a c-Myb consensus binding site. c-Myb is known to be induced during T-lymphocyte activation and its mRNA level was increased after treatment of Jurkat cells with IL-2, PMA or PMA plus PHA. Increased nuclear binding to the MAT2A c-Myb site was confirmed on electrophoretic mobility-shift and supershift analyses. Mutation of the MAT2A c-Myb site abolished the stimulatory effect of these agents on c-Myb nuclear binding and MAT2A promoter activities. Overexpression of c-Myb increased MAT2A promoter activity by 2-fold. Dexamethasone, a known inhibitor of lymphocyte activation, blocked the effect of these agents on MAT2A expression by preventing the increase in c-Myb expression.

Cell Extracts↗

PubMed2001

Epitope specificity of clonally expanded populations of CD8+ T cells found within the joints of patients with inflammatory arthritis.

OBJECTIVE: To investigate the hypothesis that clonality of synovial T cells from patients with rheumatoid arthritis is at least partly due to the presence of virus-specific T cells expressing a restricted repertoire of T cell receptors (TCRs). METHODS: Using fluorescently labeled HLA class I-peptide tetramers, populations of virus-specific CD8+ T cells were identified in samples of peripheral blood and synovial fluid taken from 4 patients with inflammatory arthritis. The TCR repertoire of the virus-specific T cells in the synovial fluid was analyzed using a panel of TCR beta variable region-specific monoclonal antibodies. Where T cells expressing a particular Vbeta chain dominated the response to a viral epitope, the sequences of these Vbeta chains were derived from sorted populations of antigen-specific T cells by reverse transcription-polymerase chain reaction. RESULTS: CD8+ T cells specific for Epstein-Barr virus, cytomegalovirus, and influenza virus were enriched in synovial fluid compared with peripheral blood. Clonal or oligoclonal populations of CD8+ T cells were found to dominate the responses to these viral epitopes in synovial fluid. CONCLUSION: The results support the hypothesis that restricted T cell receptor usage by large populations of virus-specific T cells provides one explanation for the presence of clonally expanded CD8+ T cells within the joints of patients with inflammatory arthritis. Thus, T cell clonality at a site of inflammation may reflect enrichment for memory T cells specific for foreign antigens, rather than proliferation of autoreactive T cells specific for self antigens.

Adult↗

PubMed2001

Autosomal dominant glut-1 deficiency syndrome and familial epilepsy.

Glut-1 deficiency syndrome was first described in 1991 as a sporadic clinical condition, later shown to be the result of haploinsufficiency. We now report a family with Glut-1 deficiency syndrome affecting 5 members over 3 generations. The syndrome behaves as an autosomal dominant condition. Affected family members manifested mild to severe seizures, developmental delay, ataxia, hypoglycorrhachia, and decreased erythrocyte 3-O-methyl-D-glucose uptake. Seizure frequency and severity were aggravated by fasting, and responded to a carbohydrate load. Glut-1 immunoreactivity in erythrocyte membranes was normal. A heterozygous R126H missense mutation was identified in the 3 patients available for testing, 2 brothers (Generation 3) and their mother (Generation 2). The sister and her father were clinically and genotypically normal. In vitro mutagenesis studies in Xenopus laevis oocytes demonstrated significant decreases in the transport of 3-O-methyl-D-glucose and dehydroascorbic acid. Xenopus oocyte membranes expressed high amounts of the R126H mutant Glut-1. Kinetic analysis indicated that replacement of arginine-126 by histidine in the mutant Glut-1 resulted in a lower Vmax. These studies demonstrate the pathogenicity of the R126H missense mutation and transmission of Glut-1 deficiency syndrome as an autosomal dominant trait.

3-O-Methylglucose↗

PubMed2001

Quantitative determination of nitrendipine and its metabolite dehydronitrendipine in human plasma using liquid chromatography-tandem mass spectrometry.

A sensitive and high-throughput LC-MS-MS method was developed for simultaneous determination of nitrendipine (NIT) and its major metabolite, dehydronitrendipine (DNIT) in human plasma using nifedipine as the internal standard. Plasma samples were prepared based on a simple liquid-liquid extraction. The extracted samples were analyzed on a Zorbax SB C(18) column interfaced with a triple quadrupole tandem mass spectrometer. Positive atmospheric pressure chemical ionization was employed as the ionization source. The analytes were detected by use of selected reaction monitoring mode. Standard curves were linear (r > or = 0.995) over the concentration range of 0.4-40 ng/mL for NIT and 0.2-20 ng/mL for DNIT. The intra- and inter-run precision was measured to be below 8.5% for NIT and DNIT. The inter-run accuracy was less than 4% for the analytes. The overall extraction recoveries of NIT and DNIT were determined to be about 75% and 78% on average, respectively. The chromatographic run time was approximately 3 min. More than 120 samples could be assayed daily with this method, including sample preparation, data acquisition and processing. The method developed was successfully used to investigate plasma concentrations of NIT and DNIT in a pharmacokinetic study of volunteers who received NIT orally.

Antihypertensive Agents↗