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Biomedical subjects

J Hou

Publications and source records attributed to J Hou.

At least 145 records · Page 8Linked to original sources
PubMed1994

Molecular cloning and expression of the gene for a major leucine-rich protein from human hepatoblastoma cells (HepG2).

The human hepatoblastoma cell line, HepG2, exhibits an array of stable properties in culture that have made it a popular cell culture model for studies on regulation of liver-specific gene expression and properties of hepatoma cells. In contrast to other hepatoma cell lines, HepG2 cells overexpress a characteristic detergent-extractable, wheat germ lectin-binding protein with apparent molecular mass of 130 kDa. Using an antibody to screen a phage expression library of HepG2 complementary DNA (cDNA), we identified and cloned a 4734 base pair cDNA which codes for a 130-kDa leucine-rich protein (lrp 130) when expressed in transfected cells. The deduced sequence of lrp130 exhibits sequences weakly homologous to the consensus sequence for the ATP binding site in ATP-dependent kinases and the protein kinase C phosphorylation site of the epidermal growth factor receptor. Consistent with the higher levels of expression of lrp130 antigen, Northern hybridization analysis indicated that HepG2 cells express high levels of the major 4.8 kilobase lrp130 mRNA relative to other hepatoma cells. Although currently of unknown function, lrp130 may be of utility as a marker for liver cell lineages represented by the HepG2 cell line.

Amino Acid Sequence↗
PubMed1994

Antibodies to glutamic acid decarboxylase and P2-C peptides in sera from coxsackie virus B4-infected mice and IDDM patients.

The possible role of amino acid sequence and epitope homologies between a protein P2-C of Coxsackie virus B4 and human GAD in the development of host-specific immune response in insulin-dependent diabetes mellitus (IDDM) (molecular mimicry) was investigated. Peptide antibodies to the P2-C protein, GAD65, and GAD67 were raised to analyze their immunoreactivity by enzyme-linked immunosorbent assay and immunoblotting with GAD purified from the brain and pancreas of mice that develop hyperglycemia after the infection. Additionally, antibody reactivity to these peptide antigens was assessed in sera from the virus-infected mice and IDDM patients. All three peptide antisera reacted very strongly with homologous peptides; P2-C antiserum cross-reacted with GAD65 as efficiently as GAD65 antiserum with P2-C, but no cross-reaction was detected between P2-C and GAD67 although cross-reaction between the two GADs was quite pronounced. P2-C antiserum immunocomplexed with GAD65 from mouse brain or pancreas, whereas GAD65 and GAD67 antisera both immunocomplexed with the two GADs from these sources. Most of the sera from virus-infected mice were reactive to brain and pancreas GAD65 and also to P2-C peptide, whereas some reacted to GAD65 and a few to GAD67 peptides. A number of IDDM sera reacted with mouse GAD65 and also with P2-C and GAD65 peptides, whereas only a few reacted with GAD67 peptide. The immunoreactivity of the mouse and IDDM sera to P2-C and GAD65 peptides was blocked by pre-adsorption with mouse GAD. The results suggest that molecular mimicry may play a role in the pathogenesis of the disease.

Amino Acid Sequence↗
PubMed1994

[Clinical evaluation of cefpodoxime proxetil on the treatment of bacterial infections].

122 patients with bacterial infections of respiratory tract, ear, nose, and throat, urinary tract and skin and soft tissue were treated with cefpodoxime proxetil. In the treatments of patients with clinical efficacy tates of cefpodoxime proxetil for infections in these four systems were 90.0%, 97.5%, 90.0% and 86.4%, respectively. The bacterial clearance rate of gram-positive bacterial was 96.9%, and that of gram-negative bacteria 96.4%. Adverse drug reaction rate was 18.9%.

Adolescent↗
PubMed1993

An essential heparin-binding domain in the fibroblast growth factor receptor kinase.

Heparin or heparin-like heparan sulfate proteoglycans are obligatory for activity of the heparin-binding fibroblast growth factor (FGF) family. Heparin interacts independently of FGF ligand with a specific sequence (K18K) in one of the immunoglobulin-like loops in the extracellular domain of the FGF receptor tyrosine kinase transmembrane glycoprotein. A synthetic peptide corresponding to K18K inhibited heparin and heparin-dependent FGF binding to the receptor. K18K and an antibody to K18K were antagonists of FGF-stimulated cell growth. Point mutations of lysine residues in the K18K sequence abrogated both heparin- and ligand-binding activities of the receptor kinase. The results indicate that the FGF receptor is a ternary complex of heparan sulfate proteoglycan, tyrosine kinase transmembrane glycoprotein, and ligand.

Amino Acid Sequence↗
PubMed1993

Regulation of the human hsp70 promoter by p53.

The tumor suppressor p53 is a nuclear phosphoprotein with characteristics of a transcription factor. It displays sequence-specific DNA binding, contains a potent transactivation domain, and has been implicated as both a transcriptional activator and a repressor. Transcription of the human hsp70 gene is stimulated by adenovirus E1a protein. This E1a transactivation of the hsp70 promoter is mediated by CCAAT binding factor (CBF). It is demonstrated here that p53 both represses transcription from the human hsp70 promoter and also interacts with CBF. Thus, the repression of the hsp70 promoter by p53 may be mediated by direct protein-protein interaction with CBF. These results suggest that protein-protein interaction between p53 and specific transcription factors may be an additional mechanism by which p53 regulates gene expression.

Adenovirus E1A Proteins↗
PubMed1993

Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.

Amino Acid Sequence↗
PubMed1993

Coxsackievirus B4 alters pancreatic glutamate decarboxylase expression in mice soon after infection.

The 64,000-M(r) (64K) islet autoantigen, which is considered to be a target protein of beta cell destruction in insulin-dependent diabetes mellitus (IDDM), has recently been identified as the enzyme glutamic acid decarboxylase (GAD). We reported a two- to three-fold increased expression of the antigen in islets of diabetes-susceptible mice following infection with a diabetogenic strain of Coxsackievirus B4 (CB4) at 72-h postinfection (p.i.), a time point of active virus replication in the islets. Most of the infected animals subsequently developed 64K autoantibodies and hyperglycemia. Since the infection increases 64K expression, we have analysed immunoreactive GAD expression with a panel of peptide antisera and two widely-used polyclonal antisera against GAD, and measured GAD activity in the brain, pancreas and islets of these mice. Two isoforms, GAD65 and GAD67, are detected in these tissues from non-infected mice. Both GADs are also present in the infected mice brain at 72 h p.i.; however, their islets contain about three-fold more GAD65, and essentially no detectable GAD67. GAD activity is significantly higher in the brain compared with whole pancreas or islets, and islet GAD activity is higher than pancreas GAD activity. The infection significantly reduces islet GAD activity, but not brain GAD activity. CB4-induced abnormalities in islet GAD expression may play a role in virus-induced diabetes.

Animals↗
PubMed1993

Heparin-binding fibroblast growth factors and prostate cancer.

Studies of model rat prostate tissue and derived cells indicate the insulin-like (IGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-beta) and heparin-binding fibroblast growth factor (HBGF) families and their receptors may play important roles in regulation of normal prostate cell growth. Tumor cells at different levels in the progression from slow-growing, hormone-dependence to fast-growing, hormone-independence exhibit distinct alterations in expression of specific growth factors and their receptor phenotype. Distinct IGF-I and HBGF mRNAs are constitutively expressed in the mesenchymal cells of slow-tumors, but alteration in HBGF receptor phenotype occurs in the epithelial cells. Fast-tumors exhibit even higher constitutive expression of multiple HBGFs. Splice variants in cDNA for the HBGF receptor in fast-tumors suggest constitutive expression of an intracellular receptor, that together with intracellular HBGFs, may constitute an intracellular autocrine system that is independent of exogenous hormones and growth factors.

Amino Acid Sequence↗
PubMed1993

Control of fibroblast growth factor receptor kinase signal transduction by heterodimerization of combinatorial splice variants.

A differentiated liver cell (HepG2), which exhibits a dose-dependent growth-stimulatory and growth-inhibitory response to heparin-binding fibroblast growth factor type 1 (FGF-1), displays high- and low-affinity receptor phenotypes and expresses specific combinatorial splice variants alpha 1, beta 1, and alpha 2 of the FGF receptor (FGF-R) gene (flg). The extracellular domains of the alpha and beta variants consist of three and two immunoglobulin loops, respectively, while the intracellular variants consist of a tyrosine kinase (type 1) isoform and a kinase-defective (type 2) isoform. The type 2 isoform is also devoid of the two major intracellular tyrosine autophosphorylation sites (Tyr-653 and Tyr-766) in the type 1 kinase. An analysis of ligand affinity, dimerization, autophosphorylation, and interaction with src homology region 2 (SH2) substrates of the recombinant alpha 1, beta 1, and alpha 2 isoforms was carried out to determine whether dimerization of the combinatorial splice variants might explain the dose-dependent opposite mitogenic effects of FGF. Scatchard analysis indicated that the alpha and beta isoforms exhibit low and high affinity for ligand, respectively. The three combinatorial splice variants dimerized in all combinations. FGF enhanced dimerization and kinase activity, as assessed by receptor autophosphorylation. Phosphopeptide analysis revealed that phosphorylation of Tyr-653 was reduced relative to phosphorylation of Tyr-766 in the type 1 kinase component of heterodimers of the type 1 and type 2 isoforms. The SH2 domain substrate, phospholipase C gamma 1 (PLC gamma 1), associated with the phosphorylated type 1-type 2 heterodimers but was phosphorylated only in preparations containing the type 1 kinase homodimer. The results suggest that phosphorylation of Tyr-653 within the kinase catalytic domain, but not Tyr-766 in the COOH-terminal domain, may be stringently dependent on a trans intermolecular mechanism within FGF-R kinase homodimers. Although phosphotyrosine 766 is sufficient for interaction of PLC gamma 1 and other SH2 substrates with the FGF-R kinase, phosphorylation and presumably activation of substrates require the kinase homodimer and phosphorylation of Tyr-653. We propose that complexes of phosphotyrosine 766 kinase monomers and SH2 domain signal transducers may constitute unactivated presignal complexes whose active or inactive fate depends on homodimerization with a kinase or heterodimerization with a kinase-defective monomer, respectively. The results suggest a mechanism for control of signal transduction by different concentrations of ligand through heterodimerization of combinatorial splice variants from the same receptor gene.

Amino Acid Sequence↗
PubMed1993

[Immunohistochemical study of lung tissues from patients with idiopathic pulmonary fibrosis].

Immunohistochemical analysis using 24 different monoclonal and polyclonal antibodies was applied to lung biopsy and autopsy material from 9 patients with idiopathic pulmonary fibrosis (IPF) and 6 patients without lung disorders as control. Lymphoid, non-lymphoid cells and their subsets as well as extracellular matrix (fibronectin, laminin and collagens type I, III and IV) were observed in lung interstitium. Our results suggest that local B and T lymphoid immune responses are occurring in IPF. Extracellular matrix alterations are closely linked with the local immune responses.

CD4-CD8 Ratio↗
PubMed1993

[Correlation of hepatic necrosis and complement system activation in fulminant viral hepatitis].

The relationship between complement activation and hepatic necrosis was investigated in 110 cases of fulminant, acute or chronic viral hepatitis by examining plasma complement conversion products C3d and C4d and intact C3 and C4 molecules, as well as the local hepatic deposition of C3 IgG globulins in necrotic areas of the liver. The results revealed an excessive activation of plasma C3 in the fluminant hepatitis group. The C3d/C3 ratio was proved to be directly related to the severity of the clinical symptoms. C3 and IgG hepatic depositions in adjacent necrotic areas were found in over 85% of the fulminant hepatitis cases, as compared to 37.5% in cases of chronic active hepatitis. The conclusion was drawn that activation of the complement cascade is responsible for liver tissue damage, while the plasma complement activation represented by increased C3d and C3c fragment production might well reflect hepatic pathology. Therefore, the C3d/C3 ratio could serve as an index to aid clinical management, as well as pointing out a new therapeutic approach.

Complement Activation↗
PubMed1993

Sequence comparison of putative regulatory DNA of the 5' flanking region of the myeloperoxidase gene in normal and leukemic bone marrow cells.

Myeloperoxidase (MPO) is an enzyme which is exclusively expressed in immature myeloid cells with downregulation of gene expression occurring during granulocytic maturation. Levels of MPO RNA, protein, and enzyme activity differ, usually in a concordant fashion, among the various classes of acute leukemia and among different cases within a particular class. One portion of the gene thought to be involved in regulation of MPO expression is the proximal 5' flanking region. To determine if mutations in this putatively regulatory region of the MPO gene might be responsible for some of the differences in level of MPO expression among different cases or classes of acute leukemia, we compared the nucleotide sequence of this part of the gene from 16 patients with acute leukemia, with DNA from normal human bone marrow cells and selected other neoplasms and cell lines. The sequence of this regulatory region was found to be identical in cases of acute myeloid leukemia (AML) with tha of normal DNA except for a dA to dG transition in the Alu region, 463 bases upstream from the transcription start site. This base substitution was seen in almost all cases of AML studied, regardless of the level of MPO which they expressed. It was absent from normal human DNA obtained from various tissues, and cases of acute and chronic lymphocytic leukemia, carcinoma of lung, and most cell lines examined. The base substitution was also absent in a remission blood sample from one of the cases which showed the dA to dG transition in leukemic marrow, suggesting that the base substitution is a mutation rather than a polymorphism. Our results suggest that mutations in promoter or enhancer DNA are not an important cause of the differences in level of MPO gene expression seen among different cases or different classes of AML. However, the base substitution we have detected could potentially serve as a useful marker for detection of residual disease in patients with AML following treatment.

Base Sequence↗
PubMed1992

Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms.

Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.

Amino Acid Sequence↗
PubMed1992

Substitution of putative half-cystine residues in heparin-binding fibroblast growth factor receptors. Loss of binding activity in both two and three loop isoforms.

Alternate use of an exon coding for an 89-residue NH2 terminal immunoglobulin-like disulfide loop results in isoforms of the heparin-binding fibroblast growth factor receptor (FGF-R) with three (FGF-R alpha) and two (FGF-R beta) Ig-like loops in the extracellular domain. Both FGF-R alpha and FGF-R beta isoforms exhibit qualitatively similar ligand-binding activities. In this report, we show by site-directed mutagenesis and analysis of ligand-binding activity in transfected cells that substitution of a cysteine that potentially forms an intra-loop disulfide in either juxtamembrane Loop II or III disrupted maturation and formation of the ligand-binding site in both FGF-R alpha and FGF-R beta isoforms. Neither three loop FGF-R alpha constructions coding for intact Loops I and II adjacent to defective Loop III nor intact Loops I and III separated by defective Loop II exhibited ligand-binding activity. In addition, a two-loop molecule of tandem Loops I and III was inactive. The results suggest that single Loops I, II, or III of FGF-R are insufficient to form a ligand-binding site. Loop I does not form an independent ligand-binding site with either Loop II or III, but interacts with a common ligand-binding site formed by Loops II and III (Xu, J., Nakahara, M., Crabb, J. W., Shi, E., Matuo, Y., Fraser, M., Kan, M., Hou, J., and McKeehan, W. L. (1992) J. Biol. Chem. 267, 17792-17803, 1992).

Amino Acid Sequence↗
PubMed1992

Expression and transforming activity of a variant of the heparin-binding fibroblast growth factor receptor (flg) gene resulting from splicing of the alpha exon at an alternate 3'-acceptor site.

Splicing at an alternate 3'-acceptor site results in deletion of a CCCAG in the 5'-sequence of the exon coding for the NH2-terminal immunoglobulin-like disulfide loop of the heparin-binding fibroblast growth factor receptor (flg) alpha isoform. The result is an in-frame stop codon 138 base pairs after the first flg consensus translational initiation site. The next more favorable site predicts the same two loop intracellular receptor isoform, gamma, which was predicted from two different human cDNAs that arise by alternate use of two exons at the same site. Although expressed in normal tissue, the gamma mRNA is increased in rat prostate tumors and confers ability of anchorage-dependent cells expressing non-secreted heparin-binding fibroblast growth factors to grow in soft agar.

Amino Acid Sequence↗