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Biomedical subjects

Y Huang

Publications and source records attributed to Y Huang.

At least 145 records · Page 8Linked to original sources
PubMed2001

A new focal adhesion protein that interacts with integrin-linked kinase and regulates cell adhesion and spreading.

Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH(2)-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti-CH-ILKBP antibodies, and green fluorescent protein-CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP-ILK-PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.

Actinin↗
PubMed2001

Action of hormone responsive sequence in 2.3 kb promoter of CYP11A1.

The CYP11A1 gene encodes cytochrome P450scc, the enzyme catalyzing the first step of steroid biosynthesis in the adrenal and gonad. We generated transgenic mice containing 2.3 kb of the 5'-flanking region of CYP11A1 driving LacZ reporter gene expression, in order to study hormonal control of CYP11A1 gene expression in different tissues. This 2.3 kb fragment contains information for hormonal control; by ACTH and hCG which increased reporter gene expression, in the adrenal and testis of transgenic mice respectively, while dexamethasone administration decreased reporter activity in the adrenal. The 5'-fragment of CYP11A1 has appreciable promoter activities in mouse adrenal Y1 cells but not in non-steroidogenic COS-1 cells, showing cell-type specificity. Transcription factor SF-1 activates the 2.3 kb promoter, which can be potentiated by cotransfection with c-Jun in steroidogenic JEG3 cells but not in COS-1 cells. We conclude that the 2.3 kb region of CYP11A1 contains elements controlling hormonal-dependent, cell-type-specific expression. In addition, c-Jun and SF-1 could act synergistically to activate CYP11A1 gene expression.

Adrenal Glands↗
PubMed2001

Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors.

Microbial pathogens have been selected for the capacity to evade or manipulate host responses in order to survive after infection. Chlamydia, an obligate intracellular pathogen and the causative agent for many human diseases, can escape T lymphocyte immune recognition by degrading host transcription factors required for major histocompatibility complex (MHC) antigen expression. We have now identified a chlamydial protease- or proteasome-like activity factor (CPAF) that is secreted into the host cell cytosol and that is both necessary and sufficient for the degradation of host transcription factors RFX5 and upstream stimulation factor 1 (USF-1). The CPAF gene is highly conserved among chlamydial strains, but has no significant overall homology with other known genes. Thus, CPAF represents a unique secreted protein produced by an obligate intracellular bacterial pathogen to interfere with effective host adaptive immunity.

Cell-Free System↗
PubMed2001

[Family study of genomic imprinting in tic disorder].

OBJECTIVE: To investigate whether genomic imprinting is involved in the etiology of tic disorder. METHODS: International standard structural schedule for the genetic research of tic disorder and standard process of phenotype evaluation, in addition to both family study method and family history method were used in the family study of 171 probands with tic disorder. RESULTS: Maternal transmission was associated with the symptom of complex motor tics in the probands (beta ratio = 6.6, P = 0.01); Maternal transmission was more likely to present earlier-onset of the disease (Log Rank = 4.71, P = 0.029); However, paternally transmitted tic disorder was characterized by increased attention problem score in CBCL behavioral scale among the proband (t = 2.78, chi(2) = 0.01). CONCLUSION: Parental specific expression exists in the transmission of tic disorder, which gives evidence that genomic imprinting may be involved in the genetic mechanism of tic disorder.

Adolescent↗
PubMed2001

Stretch-sensitive switching among different channel sublevels of an endothelial cation channel.

A mechanosensitive Ca(2+)-permeable cation channel was recorded by patch clamp in isolated rat aortic endothelial cells. A low level of channel activity could be observed after seal formation. The channel displayed some inward rectification and had a conductance for inward current of approx. 32 pS in Ca(2+)-free pipette and bath solutions. Negative suction of -10 to -20 mmHg increased the probability of the channel being open. When the negative pressure in the pipette was raised to -35 to -45 mmHg, the channel underwent an abrupt transition to a large conductance substate that was interrupted occasionally by two other low conductance levels. Under this condition, the overwhelming majority of openings and closings were between a main level of 83 pS and the closed level. Compared to the 32 pS substate, the 83 pS large conductance substate had shorter mean open and closed times. The two channel substates had similar ionic selectivity and both were sensitive to the inhibition of cGMP and protein kinase G. This is the first demonstration showing that mechanostress can change the single channel conductance level of an ion channel in eukaryotic cells.

Alkaloids↗
PubMed2001

Co-existence of alternative forms of 8q gain in cytogenetic clones of three patients with acute myeloid leukemia, pointing to 8q22 approximately 8qter as a region of biologic significance.

Clonal trisomy 8 chromosome abnormalities can be detected in 15% of patients with acute myeloid leukemia (AML). The most common form of change is complete gain of the whole chromosome 8, followed by partial gains in unbalanced forms. The biologic consequences of trisomy 8 remain unclear, but a gene dosage effect is suspected. We report on three patients with AML who had alternative forms of chromosome 8 gain in their bone marrow cells. The partial gains resulted from a breakpoint in the chromosome band 8q22. This indicates that the region 8q22 to 8qter may be of particular pathogenetic importance.

Acute Disease↗
PubMed2001

Electric manipulation of bioparticles and macromolecules on microfabricated electrodes.

Bioparticle separation, bioparticle enrichment, and electric field-mediated immune detection were carried out on microfabricated semiconductor chips utilizing ac and dc electric fields. Microscale separation on a chip surface having an active area of approximately 16 mm2 was demonstrated for a mixture of Bacillus globigii spores and Escherichia coli bacteria. Dielectrophoretic enrichment was performed by collecting target bioparticles from a flow stream in flow cells of 47.5 microL, achieving a 20-fold increase in the concentration of E. coli bacteria from a diluted sample, a 28-fold enrichment for peripheral blood mononuclear cells from red blood cells, and a 30-fold increase in white blood cells from diluted whole blood. The ability to manipulate and collect bioparticles and macromolecules at microfabricated electrodes with ac and dc fields was further illustrated in electric field-mediated immunoassays for analyzing the biological identities of E. coli bacteria and B. globigii spores. According to these results, the electric methods for manipulating bioparticles present themselves as viable techniques for novel biomedical applications in sample preparations and biochemical assays on microelectrode arrays.

Bacillus↗
PubMed2001

Construction of FLAG and histidine tagging vectors for Schizosaccharomyces pombe.

Schizosaccharomyces pombe is becoming an increasingly popular model system for investigating important cellular processes. To facilitate detection, purification and functional studies of Sz. pombe gene products, we constructed two tagging expression vectors for use in Sz. pombe. These vectors allow proteins to be expressed ectopically as fusion proteins with a FLAG epitope and six histidine residue tags attached to their N-terminus or C-terminus. The function and applicability of these vectors were examined and the results are shown using the N-terminal tagging vector encoding Sfc6p, a subunit of the Sz. pombe RNA polymerase III general transcription factor, TFIIIC.

Amino Acid Sequence↗
PubMed2001

Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex.

Apoptosis is a highly regulated process that is crucial for normal development and homeostasis of multicellular organisms. The p35 protein from baculoviruses effectively prevents apoptosis by its broad-spectrum caspase inhibition. Here we report the crystal structure of p35 in complex with human caspase-8 at 3.0 A resolution, and biochemical and mutagenesis studies based on the structural information. The structure reveals that the caspase is inhibited in the active site through a covalent thioester linkage to p35, which we confirmed by gel electrophoresis, hydroxylamine treatment and mass spectrometry experiments. The p35 protein undergoes dramatic conformational changes on cleavage by the caspase. The repositioning of the amino terminus of p35 into the active site of the caspase eliminates solvent accessibility of the catalytic dyad. This may be crucial for preventing hydrolysis of the thioester intermediate, which is supported by the abrogation of inhibitory activity through mutations at the N terminus of p35. The p35 protein also makes conserved contacts with the caspase outside the active-site region, providing the molecular basis for the broad-spectrum inhibitory activity of this protein. We demonstrate a new molecular mechanism of caspase inhibition, as well as protease inhibition in general.

Binding Sites↗
PubMed2001

Molecular cloning, mapping and characterization of the human neurocalcin delta gene (NCALD).

We identified a new human gene that encodes a cognate of the bovine neurocalcin delta from a human fetal brain cDNA library; hence we named it human neurocalcin delta (NCALD) gene. The deduced polypeptide product of the cDNA is 22 kDa in size, and its amino acid sequence is 100% and 99% identical to that of the bovine and chicken neurocalcin, respectively. Northern blots showed that the NCALD gene is more abundantly expressed in brain, testis, ovary and small intestine. Tissue in situ hybridization confirmed the existence of the NCALD mRNA in the adult human testis. Radiation hybrid panel mapping localized the gene to chromosome 8 between molecular markers D8S270 and D8S257.

Adult↗
PubMed2001

Structural basis of caspase inhibition by XIAP: differential roles of the linker versus the BIR domain.

The inhibitor of apoptosis proteins (IAPs) represent the only endogenous caspase inhibitors and are characterized by the presence of baculoviral IAP repeats (BIRs). Here, we report the crystal structure of the complex between human caspase-7 and XIAP (BIR2 and the proceeding linker). The structure surprisingly reveals that the linker is the only contacting element for the caspase, while the BIR2 domain is invisible in the crystal. The linker interacts with and blocks the substrate groove of the caspase in a backward fashion, distinct from substrate recognition. Structural analyses suggest that the linker is the energetic and specificity determinant of the interaction. Further biochemical characterizations clearly establish that the linker harbors the major energetic determinant, while the BIR2 domain serves as a regulatory element for caspase binding and Smac neutralization.

Apoptosis Regulatory Proteins↗
PubMed2001

A human hepatoma cell line expressing hepatitis c virus nonstructural proteins tightly regulated by tetracycline.

Nonstructural (NS) proteins of hepatitis C virus (HCV) play major roles in viral replication and the pathogenesis of liver diseases. Current studies on antiviral strategies targeting these proteins have been hampered by the lack of efficient cell culture systems. Combining tetracycline-regulated gene expressing system and enhanced green fluorescent protein (EGFP), we generated a human hepatoma cell line inducibly expressing the HCV NS proteins. This cell line exhibited high induction of a full NS transcript ( approximately 7 kb). In the absence of tetracycline, NS proteins 3, 4A, and 5A of mature sizes were detected by immunoblot analysis and the induction of NS proteins 3 to 5B are confirmed by immunofluorescent staining. Using DNA microarray analysis, we characterized the changes in mRNA expression profile of 6416 genes and identified several genes, whose mRNAs are specifically upregulated by the induction of NS proteins. This cell line provides a unique in vitro hepatoma cell system for the investigation of structural and functional properties of HCV NS proteins.

Carcinoma, Hepatocellular↗
PubMed2001

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

Carrier Proteins↗
PubMed2001

N-terminal domains of the class ia phosphoinositide 3-kinase regulatory subunit play a role in cytoskeletal but not mitogenic signaling.

Phosphoinositide (PI) 3-kinases are required for the acute regulation of the cytoskeleton by growth factors. We have shown previously that in the MTLn3 rat adenocarcinoma cells line, the p85/p110alpha PI 3-kinase is required for epidermal growth factor (EGF)-stimulated lamellipod extension and formation of new actin barbed ends at the leading edge of the cell. We have now examined the role of the p85alpha regulatory subunit in greater detail. Microinjection of recombinant p85alpha into MTLn3 cells blocked both EGF-stimulated mitogenic signaling and lamellipod extension. In contrast, a truncated p85(1-333), which lacks the SH2 and iSH2 domains and does not bind p110, had no effect on EGF-stimulated mitogenesis but still blocked EGF-stimulated lamellipod extension. Additional deletional analysis showed that the SH3 domain was not required for inhibition of lamellipod extension, as a construct containing only the proline-rich and breakpoint cluster region (BCR) homology domains was sufficient for inhibition. Although the BCR domain of p85 binds Rac, the effects of the p85 constructs were not because of a general inhibition of Rac signaling, because sorbitol-induced JNK activation in MTLn3 cells was not inhibited. These data show that the proline-rich and BCR homology domains of p85 are involved in the coupling of p85/p110 PI 3-kinases to regulation of the actin cytoskeleton. These data provide evidence of a distinct cellular function for the N-terminal domains of p85.

Adenocarcinoma↗
PubMed2001

Cloning and characterization of a novel human leptin receptor overlapping transcript-like 1 gene (LEPROTL1).

A new full-length cDNA encoding a novel protein was isolated from our human fetal brain cDNA library. The cDNA consists of 2701 bp and has a putative open reading frame encoding 131 amino acids which possesses a JAK binding site (Pro(46)-Ile-Pro(48) which is preceded by a cluster of hydrophobic residues) and is highly homologous to the leptin receptor gene-related protein (OB-RGRP). Northern blot analysis showed that this new gene is widely expressed in human tissues and radiation hybrid mapping placed the gene to human chromosome 8p21.1-8p21.2.

Amino Acid Sequence↗
PubMed2001

A family of AP-2 proteins down-regulate manganese superoxide dismutase expression.

Manganese superoxide dismutase (Mn-SOD) is a primary antioxidant enzyme whose expression is essential for life in oxygen. Mn-SOD has tumor suppressor activity in a wide variety of tumors and transformed cell systems. Our initial observations revealed that Mn-SOD expression was inversely correlated with expression of AP-2 transcription factors in normal human fibroblasts and their SV-40 transformed counterparts. Thus we hypothesized that AP-2 may down-regulate Mn-SOD expression. To examine the functional role of AP-2 on Mn-SOD promoter transactivation we cotransfected AP-2-deficient HepG2 cells with a human Mn-SOD promoter-reporter construct and expression vectors encoding each of the three known AP-2 family members. Our results indicated that AP-2 could significantly repress Mn-SOD promoter activity, and that this repression was both Mn-SOD promoter and AP-2-specific. The three AP-2 proteins appeared to play distinct roles in Mn-SOD gene regulation. Moreover, although all three AP-2 proteins could repress the Mn-SOD promoter, AP-2alpha and AP-2gamma were more active in this regard than AP-2beta. Transcriptional repression by AP-2 was not a general effect in this system, because another AP-2-responsive gene, c-erbB-3, was transactivated by AP-2. Repression of Mn-SOD by AP-2 was dependent on DNA binding, and expression of AP-2B, a dominant negative incapable of DNA binding, relieved the repression on Mn-SOD promoter and reactivated Mn-SOD expression in the AP-2 abundant SV40-transformed fibroblast cell line MRC-5VA. These results indicate that AP-2-mediated transcriptional repression contributes to the constitutively low expression of Mn-SOD in SV40-transformed fibroblasts and suggest a mechanism for Mn-SOD down-regulation in cancer.

Amino Acid Motifs↗
PubMed2001

Directed assembly of one-dimensional nanostructures into functional networks.

One-dimensional nanostructures, such as nanowires and nanotubes, represent the smallest dimension for efficient transport of electrons and excitons and thus are ideal building blocks for hierarchical assembly of functional nanoscale electronic and photonic structures. We report an approach for the hierarchical assembly of one-dimensional nanostructures into well-defined functional networks. We show that nanowires can be assembled into parallel arrays with control of the average separation and, by combining fluidic alignment with surface-patterning techniques, that it is also possible to control periodicity. In addition, complex crossed nanowire arrays can be prepared with layer-by-layer assembly with different flow directions for sequential steps. Transport studies show that the crossed nanowire arrays form electrically conducting networks, with individually addressable device function at each cross point.

Journal Article↗