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Y Tone

Publications and source records attributed to Y Tone.

At least 37 records · Page 2Linked to original sources
PubMed1999

Phosphotyrosine of macrophage by low-density lipoproteins from hemodialysis patients.

BACKGROUND: Because of the possible importance of tyrosine phosphorylation in the signal transduction process, we investigated whether an interaction of low-density lipoprotein (LDL) from hemodialysis patients (HD-LDL) and human macrophages induces tyrosine-phosphorylated proteins in the macrophages. METHODS: Human monocyte-derived macrophages were incubated with HD-LDL (100 micrograms/ml) or native LDL (100 micrograms/ml) for 15 minutes at 37 degrees C. Whole cells were lyzed with Tris-HCl buffer containing vanadate and Triton X-100. After centrifugation, lyzed proteins were divided into Triton-soluble and -insoluble fractions. Both fractions (soluble and insoluble) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Immunoblotting was performed using an antibody against phosphotyrosine or c-Src. RESULTS: Several proteins in the range 40 to 100 kDa were found to be phosphorylated constitutively in the macrophages and not affected by the addition of HD-LDL. HD-LDL did not induce any tyrosine-phosphorylated proteins either in the soluble or insoluble fractions. Macrophages pretreated with tyrosine kinase inhibitor genestein drastically inhibited tyrosine phosphorylation of these proteins. The nonreceptor tyrosine kinase, c-Src p60, was also strongly tyrosine phosphorylated in the macrophages, and this was not enhanced by the stimulation of HD-LDL. CONCLUSION: These data suggest that tyrosine autophosphorylated proteins may play a role in the early step of signal transduction in the macrophages.

Enzyme Inhibitors↗
PubMed1998

Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans.

We isolated a cDNA encoding a novel glucuronyltransferase from human placenta cDNA with the use of the degenerate reverse transcriptase-polymerase chain reaction method. Degenerate primers were designed based upon the amino acid sequence alignment of rat glucuronyltransferase (GlcAT-P) involved in the biosynthesis of the carbohydrate epitope HNK-1 with putative proteins in Caenorhabditis elegans and Schistosoma mansoni. The new cDNA sequence revealed an open reading frame coding for a protein of 335 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 43% identity to the rat GlcAT-P, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active glucuronyltransferase with marked specificity for a glycoserine Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. In contrast, asialoorosomucoid, which contains the Galbeta1-4GlcNAc sequence and is a good acceptor substrate for the GlcAT-P, did not serve as an acceptor. The reaction product was sensitive to beta-glucuronidase digestion and co-chromatographed with authentic GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser in high-performance liquid chromatography, suggesting that the enzyme is a beta1, 3-glucuronyltransferase. These results indicate that this new member of the glucuronyltransferase gene family is the enzyme previously described as glucuronyltransferase I that forms the glycosaminoglycan-protein linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans.

Amino Acid Sequence↗
PubMed1997

Regulation of IL-18 (IFN-gamma-inducing factor) gene expression.

IL-18 (also known as IFN-gamma-inducing factor), although structurally unrelated to IL-12, shares with it the role of activating NK cells and polarizing T cells toward Th1 cell function. To understand how the IL-18 gene (and consequently Th1 function) is regulated, we have determined the gene structure and investigated the mechanisms of transcriptional control and cell type expression. The mouse IL-18 gene comprises seven exons distributed over 26 kb. Exons 1 and 2 of this gene are 5'-noncoding exons. Promoter activity was detected upstream of these noncoding exons in two distinct regions. Both promoters are TATA-less and not G+C rich. The promoter activity located upstream of exon 2 was shown to act constitutively, while the activity located upstream of exon 1 was up-regulated in activated macrophage and T cell lines. IL-18 gene expression may be regulated in a wide range of cell types by the activities of these two distinct promoters. IL-18 is known to be synthesized as a precursor, pro-IL-18, and its maturation is controlled by IL-1beta-converting enzyme (ICE). We observed concordant expression of IL-18 and ICE mRNAs in a wide range of cell types, unlike the more restricted expression of IL-12 p40 mRNA. The widespread IL-18 mRNA distribution and the special relationship with ICE lead us to the hypothesis that IL-18 expression may be coupled with apoptotic processes involving activation of ICE or ICE-like proteinase.

Animals↗
PubMed1997

Developmental regulation of the sulfation profile of chondroitin sulfate chains in the chicken embryo brain.

Developmentally regulated and cell type-specific expression of distinct sulfated glycosaminoglycan structures on cell surface proteoglycans is increasingly recognized as providing information relevant to cell-cell interactions and differentiation in developing organisms. In this report, developmental regulation of both the sulfation profile of chondroitin sulfate chains and activities of chondroitin 4-sulfotransferase (C4ST) and chondroitin 6-sulfotransferase (C6ST) were evaluated in embryonic chicken brain. The results revealed that the sulfation profile and the sulfotransferase activities changed markedly with development, and these alterations were precisely coordinated. Specifically, the proportions of both chondroitin 6-sulfate to 4-sulfate and C6ST to C4ST activities progressively decreased with development. In addition, the total amounts of both chondroitin sulfate chains and the sulfotransferase activities were highest during early embryonic stages and decreased sharply as the development reached completion. The developmental expression of the C6ST gene was also found to parallel the developmental down-regulation of both the C6ST activity and the chondroitin 6-sulfate structure. These findings suggest that the developmentally regulated expression of the sulfotransferases is a predominant factor for stage-specific regulation of chondroitin sulfate structures.

Animals↗
PubMed1997

Inverse gene expression of prostacyclin and thromboxane synthases in resident and activated peritoneal macrophages.

Prostacyclin and thromboxane A2 produced from prostaglandin H2 are known to be important modulators with opposite biological activities. To examine possible roles of these prostanoids in immune responses, we have studied the gene expression of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) in murine resident macrophages or in macrophages elicited with casein or bacillus Calmette-Guérin (BCG). Northern blot analyses showed that the PGIS mRNA was expressed in a decreasing order in the resident, and casein- and BCG-elicited macrophages. In contrast, the TXS mRNA was expressed in an increasing order in the resident, and casein- and BCG-elicited macrophages. On the other hand, the mRNA for cyclooxygenase-2, which produces PGH2 and participates in the production of prostanoids in inflammation, was expressed in both the resident and BCG-elicited macrophages but barely in the casein-elicited cells. In situ hybridization analysis showed that the expression of mRNAs for PGIS and TXS was ascribable not only to the alteration of the expression levels of both mRNAs in the each macrophage but also to the changes in subpopulations of the cells expressing these mRNAs. These observations suggested that the inverse gene expression of PGIS and TXS in macrophages contributes to immune responses by modulating the relative levels of prostacyclin and thromboxane A2.

Animals↗
PubMed1997

Thyroid hormone-mediated enhancement of heterodimer formation between thyroid hormone receptor beta and retinoid X receptor.

A subset of nuclear receptors, including those for thyroid hormone (TR), retinoic acid, vitamin D3, and eicosanoids, can form heterodimers with the retinoid X receptor (RXR) on DNA regulatory elements in the absence of their cognate ligands. In a mammalian two-hybrid assay, we have found that recruitment of a VP16-RXR chimera by a Gal4-TRbeta ligand-binding domain fusion is enhanced up to 50-fold by thyroid hormone (T3). This was also observed with a mutant fusion, Gal4-TR(L454A), lacking ligand-inducible activation function (AF-2) and unable to interact with putative coactivators, suggesting that the AF-2 activity of TR or intermediary cofactors is not involved in this effect. The wild-type and mutant Gal4-TR fusions also exhibited hormone-dependent recruitment of RXR in yeast. Hormone-dependent recruitment of RXR was also evident with another Gal4-TR mutant, AHTm, which does not interact with the nuclear receptor corepressor N-CoR, suggesting that ligand-enhanced dimerization is not a result of T3-induced corepressor release. Finally, we have shown that the interaction between RXR and TR is augmented by T3 in vitro, arguing against altered expression of either partner in vivo mediating this effect. We propose that ligand-dependent heterodimerization of TR and RXR in solution may provide a further level of control in nuclear receptor signaling.

Cell Nucleus↗
PubMed1997

The regional distribution and cellular localization of mRNA encoding rat prostacyclin synthase.

The cloned cDNA for rat prostacyclin synthase was found to contain a 1503-bp open reading frame which encoded a 501-amino acid protein sharing 84.0% identity with the human enzyme. RNA blot analysis revealed that the rat prostacyclin synthase mRNA, as a single species of 2.1 kb, is expressed abundantly in the aorta and uterus. High levels of expression were also observed in the stomach, lung, heart, testis, liver, and skeletal muscle. Low but significant expression was also seen in the brain and kidney. Furthermore, the regional distribution and cellular localization of prostacyclin synthase mRNA were examined by in situ hybridization analysis of rat tissue sections. The definitive signals for the mRNA were localized in smooth muscle cells of the arteries, bronchi and uterus, and in the cells of the fibrous tunic surrounding the seminiferous tubules, which are characterized as smooth muscle cells. Besides smooth muscle cells, signals were also detected in the fibroblasts of the heart myocardium, lung parenchyma cells and kidney inner medulla tubules and interstitial cells.

Amino Acid Sequence↗
PubMed1996

Human gene encoding prostacyclin synthase (PTGIS): genomic organization, chromosomal localization, and promoter activity.

The prostacyclin synthase gene isolated from human genomic libraries (PTGIS) consists of 10 exons spanning approximately 60 kb. All the splice donor and acceptor sites conform to the GT/AG rule. Genomic Southern blot and fluorescence in situ hybridization analyses revealed that the human prostacyclin synthase gene is present as a single copy per haploid genome and is localized on chromosome 20q13. 11-q13.13. The 1.5-kb sequence of the 5'-upstream of the translational initiation site contained both GC-rich and pyrimidine-rich regions and consensus sequences of the transcription factor recognition sites such as Sp1, AP-2, the interferon-gamma response element, GATA, NF-kappaB, the CACCC box, and the glucocorticoid response element. The core binding sequence (GAGACC) of the shear stress responsive element was also found in the 5'-flanking region of the gene. The major product of the primer extension analysis suggested that the transcription of the gene started from the positions around 49 bp upstream of the translational initiation codon. Transient transfection experiments using human aortic and bovine arterial endothelial cells demonstrated that the GC-rich region (positions -145 to -10) possessed a significant promoter activity. The 6-kb downstream sequence of the translational termination codon contained multiple polyadenylation signals, Alu repeat sequences, and the consensus sequence of the primate-repetitive DNA element, MER1. Two sizes of the prostacyclin synthase mRNAs (approximately 6 and 3.3 kb) were detected with the human aorta and lung. RNA blot hybridization analysis using the 3'-untranslated region as probe indicated that the sizes of the 3'-flanking regions were different in the major 6-kb and minor 3.3-kb mRNAs.

Animals↗
PubMed1996

Structure and chromosomal location of the mouse interleukin-12 p35 and p40 subunit genes.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of p35 and p40 subunits and is required for induction of T helper 1 (Th1) responses. Knowledge of how the IL-12 gene is regulated will permit an understanding of susceptibility and resistance to pathogenic microbes and to autoiummune diseases. In this report, we provide the gene structures, nucleotide sequences and chromosomal assignment for the p35 and p40 subunits of mouse IL-12. The p35 and p40 subunit genes are distributed over 8 kb and 14 kb, and map to chromosomes 3 and 11, respectively. The p35 subunit gene consists of eight exons, including a 5'-noncoding exon that was defined by sequence comparison of genomic DNA with the 5'ends of novel cDNA molecules. Transcription of p35 mRNA can start from the first exon but can also initiate further downstream. Potential transcription regulatory elements, AP1, AP2, AP3, NF-kB and GATA recognition sequences, are located within 523 bp upstream of the p35 gene; however, no TATA box was identified. The p40 subunit gene consists of eight exons. A TATA box is located 30 bp upstream from the transcription start site, and AP1, AP3, GATA, and Pu.1 recognition sequences are located within 690 bp upstream of the p40 gene. An AGTTTCTACTTT sequence, which acts as an interferon-gamma response element in the promoter of the major histocompatibility complex class I gene, was also found upstream of the p40 gene.

Animals↗
PubMed1995

Overexpression of prostacyclin synthase inhibits growth of vascular smooth muscle cells.

To define the local effects of prostacyclin (PGI2) on the growth of vascular smooth muscle cells (VSMC), we transfected VSMC with an expression vector harboring the cDNA for PGI2 synthase (PGIS), which catalyzes the rearrangement of prostaglandin H2 to PGI2. Transfection of the human PGIS cDNA into rat VSMC did not affect DNA synthesis under serum-free basal conditions, but it increased PGI2 synthesis and decreased DNA synthesis under serum-stimulated conditions (in the presence of 1 or 5% fetal calf serum). These results demonstrated that locally synthesized PGI2 can exert autocrine and/or paracrine inhibitory effects on VSMC growth. It was also suggested that in vivo transfer of PGIS gene may be useful for the gene therapy for vascular disease such as neointimal hyperplasia.

Animals↗
PubMed1995

Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element.

There exist two distinct isozymes of prostaglandin-endoperoxide synthase (PES). PES-2 mRNA is synergistically induced by lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol-13-acetate (TPA) in bovine arterial endothelial cells. On the other hand, PES-1 mRNA is constitutively expressed under these conditions. Therefore, the promoter activities of the human genes for PES-1 and -2 in bovine arterial endothelial cells were examined. The 5'-flanking region of the human PES-2 gene (nucleotides -327 to +59) showed promoter activity inducible by LPS and TPA using transient transfection analysis, whereas that of the PES-1 gene (nucleotides -1010 to +69) showed constitutive promoter activity. Destruction of both consensus sequences for the nuclear factor responsible for the interleukin-6 expression (NF-IL6) site (nucleotides -132 to -124) and the cyclic AMP response element (CRE) (nucleotides -59 to -53) of the human PES-2 gene markedly reduced the promoter activity (25%) of the PES-2 gene after combined treatment with LPS and TPA, although single destruction of the NF-IL6 site or the CRE slightly reduced the promoter activity (60 or 90%, respectively). Moreover, cotransfection experiments showed that a trans-acting factor, CCAAT enhancer binding protein delta (C/EBP delta), which binds to both the NF-IL6 site and the CRE, increased the promoter activity of the PES-2 gene mainly through the CRE. C/EBP delta mRNA was rapidly induced by LPS. Collectively, these results suggest that transcription of the PES-2 gene in vascular endothelial cells is regulated through combination of the NF-IL6 site and the CRE and that C/EBP delta functions as one of the trans-acting factors.

Animals↗
PubMed1994

Functional analysis of a transactivation domain in the thyroid hormone beta receptor.

Hormone-dependent transcriptional activation (AF-2) by the thyroid hormone beta receptor (TR beta) localizes to its carboxyl-terminal domain. A putative transactivation sequence within this domain was analyzed by mutating individual residues to alanine. Mutant receptor carboxyl-terminal domains were tested coupled to the heterologous DNA binding domain of Gal4. A single mutant receptor (E460A) showed normal hormone binding and activation, whereas several others (P453A, F455A, L456A, F459A) exhibited impaired transactivation which correlated with their reduced ligand binding. Two mutations (L454A, E457A) were able to dissociate these properties, generating transcriptionally defective mutant proteins with preserved hormone binding. A further conservative substitution (E457D) was also nonfunctional, and these three mutations were equally deleterious when tested in the context of full-length TR beta with a natural thyroid hormone response element containing promoter. This loss of activity was not due to altered DNA binding or expression of mutant receptors in cultured cells. They also retained the ability to recruit VP16-tagged retinoid X receptor in vivo as well as bind the basal transcription factors TFIIB and TBP in vitro. Our observations indicate that conserved hydrophobic (Leu454) and charged (Glu457) residues mediate AF-2 activity of TR beta, possibly via a co-activator that has yet to be identified.

Amino Acid Sequence↗
PubMed1994

Abundant expression of thromboxane synthase in rat macrophages.

The cloned cDNA for rat thromboxane (TX) synthase with a size of 1851 bp contained a 1599-bp open reading frame which encoded a 533-amino acid protein sharing 79.7% identity with human TX synthase. RNA blot analysis was carried out with rat cells and tissues. Rat peritoneal macrophages most abundantly expressed mRNA for TX synthase, and its level was not changed by in vivo stimulation of casein. Bone marrow, spleen, lung and thymus also expressed the TX synthase gene. These findings suggest the possibility that TXA2 plays a role in the immune system.

Amino Acid Sequence↗
PubMed1994

A human monoclonal antibody to high-frequency red cell antigen Jra.

A human-mouse heterohybridoma (HMR0921) secreting human monoclonal IgG3, lambda antibody was produced from peripheral blood lymphocytes of a healthy blood donor with serum antibody to Jra, by EBV transformation and hybridization with mouse myeloma cell line P3X63Ag8.653. The reactivity of HMR0921 antibody was assessed by antiglobulin test with a panel of red cells including 14 different rare blood types. Only Jr(a-) red cells were negative. The strict specificity of this antibody to Jra antigen was further confirmed by absorption test with fluorescence flow cytometry. On screening of 28,744 blood donor samples by HMR0921 antibody, we detected 19 agglutination-negative samples, which were confirmed as Jr(a-) by conventional anti-Jra antisera. Therefore, our HMR0921 antibody is extremely useful for detecting rare Jr(a-) blood.

Animals↗
PubMed1994

Genetic analysis of 29 kindreds with generalized and pituitary resistance to thyroid hormone. Identification of thirteen novel mutations in the thyroid hormone receptor beta gene.

Resistance to thyroid hormone (RTH), with elevated serum free thyroid hormones and nonsuppressed thyrotropin levels, is either relatively asymptomatic, suggesting a generalized disorder (GRTH) or associated with thyrotoxic features, indicating possible selective pituitary resistance (PRTH). 20 GRTH and 9 PRTH cases, sporadic or dominantly inherited, were analyzed. Affected individuals were heterozygous for single nucleotide substitutions in the thyroid hormone receptor beta gene, except for a single case of a seven nucleotide insertion. With one exception, the corresponding 13 novel and 7 known codon changes localized to and extended the boundaries of two mutation clusters in the hormone-binding domain of the receptor. 15 kindreds shared 6 different mutations, and haplotype analyses of the mutant allele showed that they occurred independently. The majority (14 out of 19) of the recurrent but a minority (1 out of 10) of unique mutations were transitions of CpG dinucleotides. Mutant receptor binding to ligand was moderately or severely impaired and did not correlate with the magnitude of thyroid dysfunction. There was no association between clinical features and the nature or location of a receptor mutation. These observations suggest that GRTH and PRTH are phenotypic variants of the same genetic disorder, whose clinical expression may be modulated by other non-mutation-related factors.

Adolescent↗
PubMed1994

Spectrum of transcriptional, dimerization, and dominant negative properties of twenty different mutant thyroid hormone beta-receptors in thyroid hormone resistance syndrome.

Resistance to thyroid hormone (RTH) is usually dominantly inherited and characterized by elevated thyroid hormone levels, impaired feedback inhibition of pituitary TSH production, and variable hormonal responsiveness in peripheral tissues. We have identified 20 different mutations in the thyroid hormone beta-receptor (TR beta) gene in RTH and assayed mutant receptor properties using the TSH alpha subunit gene promoter or promoters containing three different types of positive thyroid response element (TRE). Dominant negative inhibition of wild type TR beta action by mutant receptors was also tested. The mutant receptors exhibited differing transcriptional inhibitory properties and dominant negative potential with the TSH alpha promoter that correlated with their impaired hormone binding, whereas transactivation and dominant negative effects with promoters containing positive TREs varied depending on their configuration. Heterodimeric mutant receptor-retinoid X receptor (RXR) interactions, either in cultured cells or as TRE-bound complexes in gel retardation assays, were uniformly preserved, whereas homodimeric receptor interactions could not be detected in vivo, and in vitro homodimer formation on TREs was variably reduced or absent for some mutant proteins. We correlate these findings with the distribution of receptor mutations that cluster in two areas within the hormone binding domain outside putative dimerization regions and show that artificial mutations that impaired heterodimerization abrogated dominant negative activity. Therefore, we suggest that the dominant negative effect of mutant receptors in the pituitary-thyroid axis generates the characteristic biochemical abnormality of RTH and that variable resistance in other tissues may be due to response element-dependent differences in their dominant negative potential.

Amino Acid Sequence↗
PubMed1993

Primary amenorrhoea and infertility due to a mutation in the beta-subunit of follicle-stimulating hormone.

We report a woman with primary amenorrhoea and infertility associated with an isolated deficiency of pituitary follicle-stimulating hormone (FSH), but normal luteinizing hormone (LH) secretion. Ovulation was induced by administration of exogenous FSH and resulted in a successful pregnancy. Sequence analysis of the FSH beta-subunit gene indicated that she is homozygous for a two nucleotide frameshift deletion in the coding sequence. Her mother and son are heterozygous for this mutation. This deletion results in an alteration of amino acid codons 61-86 followed by a premature termination codon. The predicted truncated beta-subunit peptide lacks regions which are important for association with the alpha subunit and for binding to and activation of the FSH receptor. Abnormalities of FSH structure or function might be an under recognised but treatable cause of infertility.

Adult↗