Biomedical subjects

N Ueki

Publications and source records attributed to N Ueki.

At least 19 recordsLinked to original sources

PubMed2000

Identification and characterization of human ZNF274 cDNA, which encodes a novel kruppel-type zinc-finger protein having nucleolar targeting ability.

A human cDNA encoding a novel zinc-finger protein, ZNF274, was identified by the "nuclear transportation trap" method (Ueki, N., Oda, T., Kondo, M., Yano, K., Noguchi, T., and Muramatsu, M., 1998, Nat. Biotechnol. 16: 1338-1342). Based on sequence analysis of the full-length cDNA, this novel gene has two alternative splicing forms, ZNF274a and ZNF274b, which encode putative proteins of 621 and 584 amino acids, respectively. ZNF274a contains five C2H2-type zinc-finger motifs, two KRAB-A (Kruppel-associated box) domains, and one leucine-rich domain. ZNF274b lacks the first KRAB-A domain at the N-terminus. ZNF274 mRNA is detected in various human tissues by Northern analysis. The ZNF274 gene is mapped distal to marker RP S28 1 in the human chromosome 19qter region, by RH mapping. The KRAB domains of ZNF274 exhibited transcription repressor activity when tested in GAL4 fusion protein assays. EGFP-ZNF274 fusion protein expressed in COS7 cells predominantly localized to the nucleoli. A series of deletion constructs revealed that a minimal domain consisting of the third and fourth zinc-fingers possesses nucleolar targeting ability. These results suggest that ZNF274 is a ubiquitous transcription repressor that plays a role in the nucleoli.

Alternative Splicing↗

PubMed2000

Inhibition of hyaluronan synthesis by vesnarinone in cultured human myofibroblasts.

Hyaluronan (HA), which is a major component of the extracellular matrix (ECM), is regulated during myofibroproliferative responses to numerous forms of inflammatory stimuli. It is a key factor involved in cellular migration and adherence. The development of a potent and non-toxic inhibitor of HA synthesis would open up a new avenue for the treatment of fibrocontractive diseases such as pulmonary fibrosis and liver cirrhosis. In this study, the effects of vesnarinone (OPC-8212: 3,4-dihydro-6-[4-(3, 4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone) on the secretion of HA in human myofibroblast cell lines (MRC-5 and LI90 cells, referred to as pulmonary and hepatic myofibroblasts, respectively) were examined. Vesnarinone specifically and dose-dependently inhibited HA secretion by myofibroblasts up-regulated by fetal calf serum (FCS). The treatment of vesnarinone did not modify the phenotype of myofibroblast cells in culture. Vesnarinone also potently inhibited the HA secretion by the two myofibroblast cell lines up-regulated by transforming growth factor-beta1 (TGF-beta1) or tumor necrosis factor-alpha (TNF-alpha). The addition of vesnarinone to myofibroblasts resulted in a significant decrease of HA synthase (HAS) activity, with or without the addition of FCS or either cytokine. These findings suggest that vesnarinone inhibits the secretion of HA in myofibroblasts by specifically suppressing HAS activity, and may therefore prove useful for the treatment of chronic inflammation and tissue fibrosis.

Cell Line↗

PubMed2000

cDNA cloning of a novel human gene NAKAP95, neighbor of A-kinase anchoring protein 95 (AKAP95) on chromosome 19p13.11-p13.12 region.

A-kinase anchoring protein 95 (AKAP95) is a nuclear protein which binds to the regulatory subunit (RII) of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and to DNA. A novel nuclear human gene which shares sequence homology with the human AKAP95 gene was identified by a nuclear transportation trap method. By polymerase chain reaction (PCR)-based analysis with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, the gene was mapped to the chromosome 19p13.11-p13.12 region between markers WI-4669 and CHLC.GATA27C12. Furthermore, alignment with genomic sequences revealed that the gene and human AKAP95 resided tandemly only approximately 250 bp apart from each other. We designated this gene as neighbor of AKAP95 (NAKAP95). The exon-intron structure of NAKAP95 and AKAP95 was conserved, indicating that they may have evolved by gene duplication. The predicted protein product of the NAKAP95 gene consists of 646 amino acid residues, and NAKAP95 and AKAP95 had an overall 40% similarity, both having a potential nuclear localizing signal and two C2H2 type zinc finger motifs. The putative RII binding motif in AKAP95 was not conserved in NAKAP95. A reverse transcription coupled (RT)-PCR experiment revealed that the NAKAP95 gene was transcribed ubiquitously in various human tissues.

Amino Acid Sequence↗

PubMed1999

Isolation and characterization of a novel human gene (HFB30) which encodes a protein with a RING finger motif.

A human cDNA, HFB30, encoding a novel protein that contains a RING finger (C3HC4-type zinc finger) motif was isolated. This cDNA clone consists of 3056 nucleotides and encodes an open reading frame of a 474 amino acid protein. From RT-PCR analysis, the messenger RNA was ubiquitously expressed in various human tissues. The gene was located to the chromosome 5q23.3-q31.1 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. Furthermore, the gene consists of nine exons that span about 20 kb of genome DNA.

Amino Acid Sequence↗

PubMed1999

Stimulation of hyaluronan synthesis by tumor necrosis factor-alpha is mediated by the p50/p65 NF-kappa B complex in MRC-5 myofibroblasts.

The lesions of fibrocontractive diseases result from an excessive myofibroproliferative response to numerous forms of inflammatory stimuli, which elicit the net deposition of extracellular matrix (ECM) in the interstitium of the affected tissue. Hyaluronan (HA), reported to be a key player supporting cellular migration and adherence, is a major component of ECM that undergoes dynamic regulation during inflammation. The molecular regulation of HA biosynthesis by inflammatory cytokines on myofibroblasts is not yet completely understood. Here we report the biochemical characteristics of the lung myofibroblast cell line MRC-5, and we demonstrate that the production of HA by this cell line is inducible by the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), at the message level of HA synthase (HAS). In TNF-alpha-stimulated MRC-5 cells, DNA-binding and competition experiments indicated that the predominant NF-kappa B binding activity detected with nuclear extract-stimulated cells is mediated by the p50/p65 complex. Using antisense oligonucleotides, we confirmed that the TNF-alpha-stimulation of HA synthesis by MRC-5 cells is dependent on the activation of the p50/p65 NF-kappa B complex. These findings indicate that TNF-alpha production within inflamed tissues may enhance the HA synthesis via the transcriptional induction of HAS on myofibroblasts, thereby providing a provisional matrix for supporting cellular migration and adhesion, and that the p50/p65 NF-kappa B complex that plays an important role in the regulation of HA production by TNF-alpha might be an appropriate target for therapeutic compounds to treat tissue fibrosis accompanied by inflammation.

Actins↗

PubMed1999

Isolation and chromosomal assignment of human genes encoding cofactor of LIM homeodomain proteins, CLIM1 and CLIM2.

Cofactors of LIM homeodomain proteins (CLIM) are transcriptional activators that associate with the LIM homeoproteins and coordinate transcription. LIM homeoproteins and CLIMs are involved in a variety of developmental processes. Two CLIMs, CLIM1 and CLIM2, have been identified in the mouse. Here we report the isolation of human CLIM1 and CLIM2 cDNAs and the determination of their chromosome locations by using a human-rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. The proteins deduced from human CLIM1 and CLIM2 cDNAs were composed of 373 and 375 amino acids, respectively, and had 97.3% and 98.7% amino acid identity, respectively, to their mouse counterparts. Human CLIM1 and CLIM2 proteins were 75.5% identical. Human CLIM1 and CLIM2 genes were mapped to the chromosome on 4p15.3 and 10q24-q25 regions, respectively. Mapping of a pair of developmentally important genes may provide new clues to the understanding of genetic disorders caused by these chromosome regions.

Amino Acid Sequence↗

PubMed1999

Isolation and chromosomal assignment of a human gene encoding protein inhibitor of activated STAT3 (PIAS3).

Mouse PIAS3 (protein inhibitor of activated STAT3) is a specific inhibitor of STAT3 that downregulates its signaling pathway. Here we report the isolation and chromosome mapping of the human PIAS3 gene. Human PIAS3 cDNA encoded a predicted protein of 619 aa which has 83% overall amino acid identity to the mouse counterpart. Based on polymerase chain reaction assisted analysis of a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid mapping panel, the human PIAS3 gene was mapped to the chromosome 1q21 region. Mapping of a crucial gene in modulating the STAT3 signaling pathway may provide new clues to the understanding of malignancies or genetic disorders caused by this chromosome region.

Amino Acid Sequence↗

PubMed1999

Isolation, tissue expression, and chromosomal assignment of a human LIM protein gene, showing homology to rat enigma homologue (ENH).

Rat ENH (Enigma homolog) is a LIM domain protein that associates with protein kinase C in an isoform-specific manner. We have identified a human cDNA which shares a significant sequence homology with rat ENH. The isolated cDNA clone, designated human ENH (hENH), was 3287 bp in length and encoded a predicted protein of 596 amino acids which had 88% overall identity to rat ENH protein. Northern blot analysis revealed that 1.9 kb of the hENH messenger RNA was predominantly expressed in heart and skeletal muscle, while 5.6 kb of the hENH messenger RNA was ubiquitously expressed in various human tissues. The chromosomal location of the gene was determined on chromosome 4q22 region, between markers WI-2900 and WI-3273, by polymerase chain reaction (PCR)-based analyses using both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

Adaptor Proteins, Signal Transducing↗

PubMed1998

NOLP: identification of a novel human nucleolar protein and determination of sequence requirements for its nucleolar localization.

This study reports cDNA isolation and partial characterization of a novel human nucleolar protein isolated by "nuclear transportation trap" described previously. The cDNA encodes a putative polypeptide of 524 amino acids with a short Escherichia coli DNA helicase homologous region, an acid-rich domain, three potential base-rich nuclear localization signals (NLSs), a serine-rich domain, and a deduced coiled-coil domain. The protein has no known prominent similarities with any other protein in the protein databases. Tissue distribution analysis demonstrated a predominant expression in brain and testis. To determine the sequence requirements for nucleolar targeting, a set of deletion constructs with a fluorescent tag were transiently expressed in COS-7 cells. We revealed that a region of 30 amino acids (position 342-371), which overlaps the first and second NLS, is sufficient for nucleolar localization. Furthermore, the adjacent region of 30 amino acids (position 372-401), which contains the third NLS, is sufficient for nuclear localization. These results suggest that this novel nucleolar protein has at least two distinct domains for directing to different subnuclear destinations.

Amino Acid Sequence↗

PubMed1998

Induction of calponin-h1 by transforming growth factor-beta1 in cultured human ito cells, LI90.

We investigated the effect of transforming growth factor-beta1 (TGF-beta1) on the expression of calponin-h1, alpha-smooth muscle actin (alpha-SMA), and extracellular matrix (ECM) components in a cultured human Ito cell line, LI90. The TGF-beta1 treatment stimulated productions of hyaluronic acid and laminin, and significantly decreased the secretion of hepatocyte growth factor in LI90 cells. The functional characteristics of LI90 cells were compatible with those of human-activated Ito cells that are known as pericyte-like mesenchymal liver cells. TGF-beta1 induced a slight growth-inhibition of LI90 cells. TGF-beta1 enhanced the expressions of both alpha-SMA and calponin-h1 at the protein level, while tumor necrosis factor-alpha and interleukin-1alpha did not affect the expressions of these cytoskeletal proteins on LI90 cells. The addition of TGF-beta1 to LI90 cells resulted in a significant increase of calponin-h1 mRNA levels, but not calponin-h2. These data suggest that the expression of calponin-h1 is controlled at the level of mRNA under the coordinate regulation together with alpha-SMA as the process of perpetuation of activated Ito cells promoted by TGF-beta1. The identification of smooth muscle features promoted by TGF-beta1 support the hypothesis that the activation of Ito cells coincides with their contractile behavior, indicating that these cells may be important in vasoregulation during liver injury and fibrosis.

Actins↗

PubMed1998

Selection system for genes encoding nuclear-targeted proteins.

Nuclear proteins have essential roles in cell proliferation and differentiation. We have developed a yeast selection system-the nuclear transportation trap (NTT)-to identify genes encoding nuclear transport signals. Both unknown and previously identified nuclear localization signals were identified from a human fetal brain cDNA library. The majority (75%) of the unknown proteins examined were exclusively localized to the nucleus in COS-7 cells. We propose that NTT is an efficient method for isolating cDNAs that encode nuclear targeted proteins that can be applied to the retrieval of novel nuclear proteins and to annotate gene function.

Amino Acid Sequence↗

PubMed1997

Recombinant thermostable cycloinulo-oligosaccharide fructanotransferase produced by Saccharomyces cerevisiae.

A truncated fragment of the cycloinulo-oligosaccharide fructanotransferase (CFTase) gene of Bacillus circulans MCI-2554 was fused to the prepro secretion sequence of the alpha-factor and expressed in Saccharomyces cerevisiae under the control of the 5' upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL). Efficiently secreted recombinant CFTase protein (yeast CFTase) was purified. Yeast CFTase consisted of three protein molecules, each of which had CFTase activity (yeast CFTase 1 [116 kDa], yeast CFTase 2 [117 kDa], and yeast CFTase 3 [116 kDa]). Yeast CFTase 2 was the major product of the expression system employed and was shown to be N glycosylated by endoglycosidase H treatment. Yeast CFTase 1 was N glycosylated but had a short truncation at its N terminus, while yeast CFTase 3 did not contain an N-glycosylated carbohydrate chain(s). Yeast CFTase 2 showed an optimum pH, an optimum temperature, and a pH stability similar to those of CFTase purified from B. circulans but exhibited a significant increase in thermostability. Production of yeast CFTase by the strain which had two copies of the CFTase gene integrated into its chromosomes reached 391 U per liter of culture at 120 h, which corresponded to 8.40 mg of protein per liter, by shake-flask cultivation.

Amino Acid Sequence↗

PubMed1995

Tertiary structure of [2Fe-2S] ferredoxin from Spirulina platensis refined at 2.5 A resolution: structural comparisons of plant-type ferredoxins and an electrostatic potential analysis.

The structure of plant-type [2Fe-2S] ferredoxin isolated from Spirulina platensis has been refined using diffraction data to 2.5 A resolution by alternate cycles of simulated annealing and manual revision of the model. The final R factor is 19.9% for 2,912 reflections with F > 2 sigma F between 8.0 and 2.5 A resolution. S. platensis ferredoxin, like other plant-type [2Fe-2S] ferredoxins, has a major alpha-helix flanking a sheet consisting of four beta strands. The present refinement revises the conformation of residues 56-71, in which a one-turn helix was identified. Superposition of the Spirulina ferredoxin structure on the structures of other ferredoxins that have been well refined showed structural perturbation at a few residues on the amino and carboxyl termini and the turn between the first and second beta-strands. The root-mean-square deviations of the corresponding C alpha atoms of the pairs of ferredoxins range from 0.90 to 1.17 A for all the residues, but from 0.64 to 0.70 A if the few perturbed residues are excluded. Therefore, it may be concluded that the main-chain foldings of all the plant-type [2Fe-2S] ferredoxins are essentially the same. Electrostatic potential analysis showed that the molecular surface around the cluster is negatively charged, whereas that of the beta-sheet of the other side is positively charged. The interaction between ferredoxin and ferredoxin-NADP+ reductase is discussed on the basis of the charge distributions of these molecules and biochemical data.

Amino Acid Sequence↗

PubMed1995

Elevated serum hepatocyte growth factor/scatter factor levels in inflammatory lung disease.

Hepatocyte growth factor/scatter factor (HGF/SF) plays an important role in tissue repair in liver and renal damage. The clinical significance of this growth factor in these diseases has also been reported. The lung is one of the major sources of HGF/SF; because of this, we investigated serum HGF/SF levels in 26 patients with inflammatory lung disease (15 with interstitial pneumonitis [IP], 11 with bacterial pneumonia [BP]) by enzyme-linked immunosorbent assay. As controls, we measured HGF/SF in the serum of 13 stable outpatients with chronic respiratory failure. All patients had no significant liver or renal dysfunction. Serum HGF/SF levels were significantly elevated in patients with IP (1.16 +/- 0.22 ng/ml) or BP (0.96 +/- 0.27 ng/ml) compared with those in the control subjects (0.29 +/- 0.03 ng/ml, both p < 0.01). Serum HGF/SF levels in 14 healthy subjects were also studied, and the results (0.30 +/- 0.02 ng/ml) were not remarkably different from those of the control subjects. There were no significant correlations between serum HGF/SF levels and C-reactive protein and lactate dehydrogenase. Serum HGF/SF levels in the surviving patients rapidly decreased with treatment, but they did not change in the patients who ultimately died. Our results demonstrate the clinical significance of serum HGF/SF level as a useful indicator of prognosis in inflammatory lung disease.

Adolescent↗

PubMed1994

Aspartate aminotransferase-linked immunoglobulin complexes in serum of a patient with primary biliary cirrhosis.

A 51-year-old woman who had been treated for primary biliary cirrhosis (PBC) was admitted to our hospital for evaluation of unexplained, isolated, persistently increased aspartate aminotransferase (AST) activity. Results of laboratory tests on admission showed: AST 171 KU, alanine aminotransferase 28 KU, and anti-mitochondrial titer 1/1280. Results of hepatitis B surface antigen (HBs Ag) and hepatitis C virus antibody (HCV Ab; C100-3) assays were negative. Histology of a liver biopsy specimen was compatible with a diagnosis of PBC (stage III of Scheuer's classification). The molecular size of serum AST was estimated to be more than 500,000 by high-performance size-exclusion liquid chromatography. Electrophoretic analysis showed an abnormal band of AST between supernatant AST (sAST) and mitochondrial AST (mAST), which band was characteristic of AST-immunoglobulin complexes (AST-Ig). Ouchterlony double-diffusion and immunoprecipitation tests identified the immunoglobulin component as IgM. The presence of AST-Ig appeared to be responsible for the elevated serum AST.

Aspartate Aminotransferases↗

PubMed1994

Transforming growth factor-beta 1 (TGF-beta 1)- and beta 2-like activities in malignant pleural effusions caused by malignant mesothelioma or primary lung cancer.

We investigated the levels of TGF-beta in malignant pleural effusions (MPE) caused by malignant mesothelioma (MESO) or primary lung cancer. TGF-beta levels in MPE caused by MESO were 283.9 +/- 219.2 pm (mean +/- s.d.) and were three to six times higher than those due to primary lung cancers (P < 0.01 or P < 0.05). We also evaluated TGF-beta 1- and beta 2-like activities in MPE using specific polyclonal antibodies. Although TGF-beta 1-like activity could be detected in all cases, TGF-beta 2-like activities were detected in five of seven in MESO and in a few cases with primary lung cancer. These results demonstrate that the levels of total TGF-beta and TGF-beta 2-like activity may be clinically useful to differentiate MESO from primary lung cancer. Our data also suggest that TGF-beta may help further characterize the clinical features of MESO.

Carcinoma, Small Cell↗

PubMed1993

Distribution of bent DNA structures in the fission yeast centromere.

To gain a clue as to the functional significance of DNA curvature, we experimentally characterized the distribution of bent DNA structures throughout the 35-kb cen1 sequence, one of the isolated functional centromeric DNA of the fission yeast, Schizosaccharomyces pombe. It was revealed that a relatively large central portion of cen1, covering a 2.2-kb DNA sequence, displays a remarkable DNA curvature.

Centromere↗

PubMed1993

Potentiation of metastatic capacity by transforming growth factor-beta 1 gene transfection.

This study was designed to assess whether the excessive secretion of transforming growth factor-beta 1 (TGF-beta 1) by Chinese hamster ovary (CHO) cells transfected with TGF-beta 1 gene may be linked to the development of a metastatic phenotype. We observed large numbers of metastatic colonies in the lungs of nude mice inoculated with the transfected CHO cells. The tumors derived from these transfected cells demonstrated marked angiogenesis. We postulate that the overproduction of TGF-beta 1 by these tumors may participate in the metastatic progression following establishment of angiogenesis at the primary tumor site.

Animals↗