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Biomedical subjects

B Yang

Publications and source records attributed to B Yang.

At least 325 records · Page 18Linked to original sources
PubMed1995

Lateral mobility of Fc gamma RIIa is reduced by protein kinase C activation.

The lateral mobility of membrane proteins can reflect the extent of various protein-protein interactions. Using the fluorescence recovery after photobleaching technique, we have studied the lateral mobility of human Fc gamma RIIa and some Fc gamma RIIa mutants expressed in either P388D1 cells, a mouse macrophage-like cell line, or in Chinese hamster ovary (CHO) cells [1]. After treatment with phorbol myristate acetate (PMA), only the Fc gamma RIIa molecules capable of mediating rapid endocytosis of immune complexes exhibited a reduced lateral diffusion coefficient with respect to untreated controls. Wild type Fc gamma RIIa expressed in CHO cells, and nonfunctional Fc gamma RIIa mutants expressed in P388D1 cells did not show any differences upon PMA treatment. This finding suggests that protein kinase C activation evokes additional protein-protein interactions with the cytoplasmic domain of functional Fc gamma RIIa, which reduced receptor lateral mobility. The identity of these putative interacting proteins and the nature of the interactions remain to be elucidated.

Animals↗
PubMed1995

Identification and characterization of the dystrophin anchoring site on beta-dystroglycan.

Dystrophin, the product of the Duchenne muscular dystrophy gene, is tightly associated with the sarcolemmal membrane to a large glycoprotein complex. One function of the dystrophin-glycoprotein complex is to link the cytoskeleton to the extracellular matrix in skeletal muscle. However, the molecular interactions of dystrophin with the membrane components of the dystrophin-glycoprotein complex are still elusive. Here, we demonstrate and characterize a specific interaction between beta-dystroglycan and dystrophin. We show that skeletal muscle and brain dystrophin as well as brain dystrophin isoforms specifically bind to beta-dystroglycan. To localize and characterize the dystrophin and beta-dystroglycan interaction domains, we reconstituted the interaction in vitro using dystrophin fusion proteins and in vitro translated beta-dystroglycan. We demonstrated that the 15 C-terminal amino acids of beta-dystroglycan constituted a unique binding site for the second half of the hinge 4 and the cysteine-rich domain of dystrophin (amino acids 3054-3271). This dystrophin binding site is located in a proline-rich environment of beta-dystroglycan within amino acids 880-895. The identification of the interaction sites in dystrophin and beta-dystroglycan provides further insight into the structure and the molecular organization of the dystrophin-glycoprotein complex at the sarcolemma membrane and will be helpful for studying the pathogenesis of Duchenne muscular dystrophy.

Amino Acid Sequence↗
PubMed1995

Characteristics of ATP-dependent peptide transport in isolated microsomes.

This report examines the transport properties and specificity of ATP-dependent peptide transport by the murine transporter for Ag presentation (TAP) complex in isolated microsome preparations from H-2d haplotype mice. The murine TAP complex has a Km of 661 nM and a maximum velocity of 2.9 fmol/min.micrograms microsome protein for a modified peptide corresponding to a defined MHC class I binding epitope from influenza nucleoprotein recognized by CD8+ CTL in association with the Kd molecule. This high Km value for peptide transport suggests that the rate and efficiency of peptide transport of the TAP complex are influenced by the concentration of processed peptides derived from self and foreign proteins in the cell cytoplasm. Furthermore, these findings imply that competition among peptides for TAP-dependent transport is unlikely to be an important factor in determining the immunodominance of certain peptide epitopes within a foreign protein recognized by CD8+ T lymphocytes. We also examined the specificity of TAP transport for peptides containing bona fide murine MHC class I binding epitopes and provide evidence that certain flanking residues can affect the efficiency of peptide epitope transport by the TAP complex.

ATP-Binding Cassette Transporters↗
PubMed1995

Characterization of the murine gene encoding the hyaluronan receptor RHAMM.

We describe the isolation and characterization of the murine gene encoding RHAMM, a hyaluronan receptor which regulates focal adhesion turnover, is required for cell locomotion and is a critical downstream regulator of ras transformation. The RHAMM gene spans at least 20 kb and comprises 14 exons ranging in size from 75 to 1099 bp. Primer extension studies indicate that the major transcription start point is in position -31, relative to the start Met. Northern blot analysis of mouse fibroblast RNA identified two hybridizing species of 4.2 and 1.7 kb. Comparison of cDNA clones and RT-PCR products with the genomic clones identified alternately spliced exons in both the coding and 5' noncoding regions of RHAMM. In the coding region exon 4 is alternately spliced. The major RHAMM transcript (RHAMM1) in 3T3 fibroblasts does not contain exon 4 and encodes a protein of 70 kDa. A minor transcript containing exon 4, namely RHAMM v4, encodes a 73-kDa protein, as demonstrated by isoform-specific antibodies. Western analysis demonstrated both a major 70-kDa (RHAMM 1) and minor 73-kDa RHAMM protein (v4) in 3T3 murine fibroblast cell lysates. The functional significance of these two isoforms is currently being investigated.

Amino Acid Sequence↗
PubMed1995

cDNA cloning, gene organization, and chromosomal localization of a human mercurial insensitive water channel. Evidence for distinct transcriptional units.

Two distinct cDNAs encoding a human mercurial insensitive water channel (hMIWC) were cloned from a fetal brain cDNA library. The longest open reading frame of cDNA clone hMIWC1 encoded 301 amino acids with 94% identity to rat MIWC (Hasegawa, H., Ma, T., Skach, W., Matthay, M. M., and Verkman, A. S. (1994) J. Biol. Chem. 269, 5497-5500). A second cDNA (hMIWC2) had a distinct 5'-sequence upstream from base pair (bp) -34 in clone hMIWC1 and contained two additional inframe translation start codons. Expression of hMIWC cRNAs in Xenopus oocytes increased osmotic water permeability by 10-20-fold in a mercurial insensitive manner. Cell-free translation in a reticulocyte lysate/microsome system generated single protein bands at 30 kDa (hMIWC1) and 32-34 kDa (hMIWC2) without glycosylation. Northern blot and polymerase chain reaction/Southern blot analysis showed expression of mRNA encoding hMIWC in human brain - muscle >> heart, kidney, lung, and trachea. Analysis of hMIWC genomic clones indicated two distinct but overlapping transcription units from which multiple hMIWC mRNAs are transcribed. The promoter region of hMIWC1 was identified and contained TATA, CAAT, AP-1, and other regulatory elements. Primer extension revealed hMIWC1 transcription initiation at 46 bp downstream from the TATA box. There were three introns (lengths 0.9, 0.2, and 6 kilobases) in the hMIWC1 coding sequence at bp 381, 546, and 627. A distinct 5'-sequence in clone hMIWC2 suggested an alternative upstream transcription initiation site. Two alternatively spliced, nonfunctional hMIWC transcripts with exon 3 deletion and partial exon 4 deletion were identified. A poly(A)+ signal sequence was identified at 138 bp downstream of the translation stop codon. Genomic Southern blot analysis indicated the presence of a single copy hMIWC gene; chromosome-specific polymerase chain reaction and in situ hybridization localized hMIWC to human chromosome 18q22. The structural organization of the hMIWC gene represents a first step in definition of hMIWC differential expression, regulation, and possible role in human disease.

Amino Acid Sequence↗
PubMed1995

Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation.

Overexpression of the RHAMM gene by transfection into fibroblasts is transforming and causes spontaneous metastases in the lung. H-ras-transformed fibrosarcomas transfected with a dominant suppressor mutant of RHAMM exhibit a so-called revertant phenotype and are completely nontumorigenic and nonmetastatic. Conversely, fibroblasts stably expressing low levels of RHAMM as a result of antisense transfection are resistant to ras transformation. Collectively, these results indicate that RHAMM acts downstream of ras. The loss of functional RHAMM ablates signaling within focal adhesions, in particular changes in focal adhesion kinase phosphorylation, and as a result these focal adhesions are unable to turn over in response to hyaluronan. These results provide evidence of the oncogenic potential of a novel extracellular matrix receptor and establish a functional link between transformation by ras and signaling within focal adhesions that are required for transformation by this oncogene.

Amino Acid Sequence↗
PubMed1995

Biotinylated hyaluronic acid as a probe for identifying hyaluronic acid-binding proteins.

The glycosaminoglycans hyaluronan (HA), heparin, and chondroitin sulfate were biotinylated using biotin-x-hydrazide (biotin-epsilon-aminocaproyl hydrozyde) in conjunction with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, an activating agent for carboxyl groups. The biotin-x-hydrazide was shown to be coupled directly to the glycosaminoglycans in enzymatic digestions and competition experiments. The biotinylated HA was shown to bind to link protein and receptor for hyaluronic acid-mediated motility, two proteins known to bind HA. The labeled HA was used as a probe to detect known HA-binding proteins in chicken cartilage extract and to identify new HA-binding motifs in the G3 domain of the proteoglycan aggrecan. The significance of the biotinylation of HA, heparin, and chondroitin sulfate A are discussed.

Binding, Competitive↗
PubMed1995

T-cell specific avian TdT: characterization of the cDNA and recombinant enzyme.

A cDNA clone coding for avian terminal deoxynucleotidyl transferase (TdT) has been isolated and sequenced. The size of this cDNA was 2545 bp with an open reading frame of 1521 bp and a predicted translation product of 58 kDa. Comparison of this TdT sequence with other known TdT sequences has revealed a very high degree of homology at both the DNA and predicted amino acid levels. The chicken TdT cDNA was expressed in a bacterial system and the protein was purified by affinity chromatography. The purified recombinant enzyme, with a specific activity of approximately 1700 U/mg protein, was significantly less active than TdTs from mammalian species. This finding correlates with the observation that TdT isolated from avian thymus has lower activity than that isolated from any mammalian thymus source. Northern blot hybridization analyses and reverse transcription PCR of RNA preparations were carried out with the chicken cDNA. The data generated from these experiments revealed that the TdT RNA was only expressed in the thymus and not in the bone marrow or the bursa of Fabricius during pre- and post hatching chicken development. These data suggest that while TdT is probably involved in N region addition in chicken T-cell receptor genes, it is unlikely to play a role in diversification of immunoglobulin genes.

Animals↗
PubMed1995

SH3 domain-mediated interaction of dystroglycan and Grb2.

Dystroglycan is a novel laminin receptor that links the extracellular matrix and sarcolemma in skeletal muscle. The dystroglycan complex containing alpha- and beta-dystroglycan also serves as an agrin receptor in muscle, where it may regulate agrin-induced acetylcholine receptor clustering at the neuromuscular junction. beta-Dystroglycan has now been expressed in vitro and shown to directly interact with Grb2, an adapter protein involved in signal transduction and cytoskeletal organization. Protein binding assays with two Grb2 mutants, Grb2/P49L and Grb2/G203R, which correspond to the loss-of-function mutants in the Caenorhabditis elegans sem-5, demonstrated that the dystroglycan-Grb2 association is through beta-dystroglycan C-terminal proline-rich domains and Grb2 Src homology 3 domains. Affinity chromatography has also shown endogenous skeletal muscle Grb2 interacts with beta-dystroglycan. Immunoprecipitation experiments have demonstrated that Grb2 associates with alpha/beta-dystroglycan in vivo in both skeletal muscle and brain. The specific dystroglycan-Grb2 interaction may play an important role in extracellular matrix-mediated signal transduction and/or cytoskeleton organization in skeletal muscle that may be essential for muscle cell viability.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Identification of alpha-syntrophin binding to syntrophin triplet, dystrophin, and utrophin.

Syntrophin represents three cytoplasmic components of the dystrophin-glycoprotein complex that links the cytoskeleton to the extracellular matrix in skeletal muscle. alpha-Syntrophin has now been translated in vitro and shown to associate directly with all three components of the syntrophin triplet and with dystrophin. The in vitro translated 71-kDa non-muscle dystrophin isoform, containing the cystein-rich/C-terminal domain, can also interact with the syntrophin triplet. The syntrophin binding motif in dystrophin was localized to exons 73 and 74 including amino acids 3447-3481 by comparing the interactions of alpha-syntrophin and seven overlapping human dystrophin fusion proteins. More than one syntrophin interaction site in this binding motif was suggested. alpha-Syntrophin also interacts directly with a C-terminal utrophin fusion protein. alpha-Syntrophin is localized to the muscle sarcolemma as well as to the neuromuscular junction in control mouse muscle. However, similar to utrophin, alpha-syntrophin is only present at the neuromuscular junction in mdx mouse muscle in which dystrophin is absent. Our data suggest that alpha-syntrophin binds all syntrophin isoforms, and syntrophin directly interacts with dystrophin through more than one binding site in dystrophin exons 73 and 74 including amino acids 3447-3481.

ATP-Binding Cassette Transporters↗
PubMed1995

Organ culture of benign, aging, and hyperplastic human prostate.

Organ culture of the human prostate began in the 1970s and was modeled after the work of Lasnitzki and her collaborators in the mouse two decades earlier. In organ culture of human prostates, one sees a rapid increase in epithelial cells and decrease in stromal cells during the first 3-5 days of culture. While modulation of many phenotypic properties occurs, these cultures provide a simple and rapid way to achieve large numbers of human prostatic epithelial cells in cultured tissues that are markedly depleted of stromal cells. There is some evidence that organ cultures are maintained in slightly better functional states in the presence of androgens; however, most of this evidence is less than quantitative. Most organ culture of prostates has been accomplished with tissues from unspecified locations within the prostate; interpretation of cultures carried out in this fashion has been less complete than would have been possible if they had been carried out from specific anatomic locations within the prostate. Careful pathological characterization of locations contiguous to the cultured tissue is mandatory if cultures are to be interpreted meaningfully.

Forecasting↗
PubMed1995

A novel form of Epstein-Barr virus latency in normal B cells in vivo.

We have developed a PCR assay that can detect a single Epstein-Barr virus (EBV) genome in the presence of 10(6) uninfected cells. Using this assay, we demonstrate that EBV persists, in the peripheral blood of all seropositive individuals tested, in CD19+, CD23-, and CD80 (B7)- B cells. We further show that the virus in these cells is latent, but readily reactivated to produce infectious immortalizing virus; therefore, these cells represent a true site of latent persistence. EBV was not significantly detected in monocytes or T cells. The frequency of infected cells in nine healthy donors varied from 23 to 625 per 10(7) B cells, but was relatively stable for each individual over the course of 2 years. We conclude that the EBV-infected cells in vivo are B cells with a nonactivated phenotype. This represents a novel form of latency in normal B cells.

Adult↗
PubMed1995

Perceptions of fear in other children and adolescents: the role of gender and friendship status.

Gender differences in fear were examined in 693 Chinese children and adolescents. Subjects were asked to rate their own fears, the fears of their best friends, and the fears of "other" classmates using the Fear Survey Schedule for Children--Revised (Ollendick, 1983). Consistent with previous investigations in Western and Eastern countries, girls rated themselves as more fearful than boys. In addition, both girls and boys rated their best friends as similar in number, content, and intensity of fears. However, girls rated their classmates as less fearful than themselves or their best friends, while boys rated their classmates as more fearful then themselves or their best friends. Findings are discussed in terms of gender role expectations and similarity-attraction hypotheses.

Adolescent↗
PubMed1995

Cytological effects of 50 Hz electromagnetic fields on human lymphocytes in vitro.

Incubation of human peripheral blood cultures in the presence of an electromagnetic field (EMF) of 50 Hz and 5 mT leads to stimulation of the cell cycle of dividing lymphocytes but has no influence on the frequencies of sister-chromatid exchanges. Comparative studies with two different exposure systems and with different culture temperatures indicate that the effect on the cell cycle results from the EMF and is not a thermal effect. These data support the assumption that with respect to their suspected carcinogenic effects EMFs have no initiating but probably promoting effects.

Carcinogens↗
PubMed1995

An ankyrin-related gene (unc-44) is necessary for proper axonal guidance in Caenorhabditis elegans.

Caenorhabditis elegans unc-44 mutations result in aberrant axon guidance and fasciculation with inappropriate partners. The unc-44 gene was cloned by transposon tagging, and verified by genetic and molecular analyses of six transposon-induced alleles and their revertants. Nucleotide sequence analyses demonstrated that unc-44 encodes a series of putative ankyrin-related proteins, including AO49 ankyrin (1815 aa, 198.8 kD), AO66 ankyrin (1867 aa, 204 kD), and AO13 ankyrin (< or = 4700 aa, < or = 517 kD). In addition to the major set of approximately 6 kb alternatively spliced transcripts, minor transcripts were observed at approximately 3, 5, 7, and 14 kb. Evidence is provided that mutations in the approximately 14-kb AO13 ankyrin transcript are responsible for the neuronal defects. These molecular studies provide the first evidence that ankyrin-related molecules are required for axonal guidance.

Amino Acid Sequence↗
PubMed1995

The stability and prediction of fears in Chinese children and adolescents: a one-year follow-up.

The stability in number, level, content, and pattern of fears over a one-year period was examined in Chinese children and adolescents aged 7-17. In addition, perceptions of fear in friends and parents as well as perceptions of the controllability and modifiability of fears were explored as predictors of subsequent fear level. Evidence for moderate stability of fears was obtained. However, stability was qualified by age and sex interaction effects. A developmental-cultural hypothesis was put forth to account for those findings. Hierarchical regression analyses revealed that perceptions of fears in friends and parents, along with perceptions of the controllability and modifiability of fears, were predictive of fear level at follow-up. Limitations of the study are noted.

Adolescent↗
PubMed1995

Stimulation of Gs and inhibition of Gi protein functions by minimally oxidized LDL.

We have previously shown that treatment of aortic endothelial cells with minimally oxidized LDL (MM-LDL) induces their interaction with monocytes but not neutrophils and that these induced responses are associated with increased cAMP levels. Here we studied the mechanism of by which MM-LDL elevates cAMP levels. Treatment of human aortic endothelial cells with MM-LDL resulted in a saturable dose-dependent increase in cAMP levels. Studies using a combination of pertussis toxin and MM-LDL suggested that part of the cAMP increase was due to the stimulation of Gs complexes. Studies with pertussis toxin-treated membranes in which Gi was completely inhibited were used to directly address the effect of MM-LDL on the Gs pathway. MM-LDL and an oxidized lipid (palmitoyl arachidonyl phosphatidylcholine), the effects of which mimic those of MM-LDL, caused a 40% to 100% increase in cAMP levels in these isolated membranes that was augmented by GTP, thus showing Gs stimulation. These results also show that MM-LDL increases cAMP levels by inhibiting Gi. MM-LDL inhibited ADP ribosylation of Gi by about 30% and completely abolished the ability of serotonin to interact with Gi complexes, whereas direct activation of Gi by mastoparan was not inhibited. This observation suggests that MM-LDL interferes with the interaction of Gi molecules with inhibitory receptors. There was no direct effect of MM-LDL on adenylate cyclase. Overall, these studies show that MM-LDL increases cAMP levels both by stimulating Gi and inhibiting Gi complexes.

Adenylyl Cyclases↗