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Biomedical subjects

N Seki

Publications and source records attributed to N Seki.

At least 55 records · Page 3Linked to original sources
PubMed2001

Thrombospondin-1 and -2 messenger RNA expression in invasive cervical cancer: correlation with angiogenesis and prognosis.

PURPOSE: TSP association with clinicopathological features, including microvessel count, regarding prognostic significance was examined in patients presenting with invasive cervical cancer. EXPERIMENTAL DESIGN: Gene expression of TSP-1 and TSP-2 was assessed by reverse transcription-PCR in 10 normal cervix and 78 invasive cervical cancer samples. RESULTS: TSP-1 and TSP-2 mRNA expression was detected in seven (70.0%) of the normal cervical specimens. TSP-2 mRNA expression in normal cervix was significantly higher than that in cases involving cervical cancer (P = 0.032). TSP-1 mRNA expression was significantly lower in tumors characterized by advanced stage (P = 0.047). Fifty-three patients displaying stage Ib-IIb cervical cancer underwent radical hysterectomy and pelvic lymphadenectomy. Expression of TSP-1 and TSP-2 mRNA was significantly lower in tumors exhibiting parametrial invasion (P = 0.016 and P = 0.049, respectively). Microvessel counts were significantly higher when decreased TSP-1 expression was evident (P = 0.029). The microvessel count in patients lacking TSP-2 mRNA expression was higher than that observed in patients displaying TSP-2 mRNA expression, although it was not statistically significant (P = 0.062). Subjects demonstrating TSP-1 mRNA expression exhibited significantly better prognosis than those lacking TSP-1 mRNA expression (P = 0.0038). Furthermore, TSP-1 mRNA expression was an independent prognostic factor in the multivariate analysis. CONCLUSIONS: These findings provide evidence that TSP-1 expression is of value as a prognostic factor in cervical cancer. The inverse correlation between TSP expression and microvessel count also indicates that decreased TSP expression may be associated with an angiogenic phenotype in this class of neoplasm.

Adult↗
PubMed2000

Isolation and characterization of murine orthologue of PTP-BK.

We isolated and characterized a murine orthologue of PTP-BK, a receptor-type protein tyrosine phosphatase expressed in brain and kidney. The deduced amino acid sequences showed 89, 95, and 92% identities with human, rabbit, and chicken homologues of PTP-BK. Mouse PTP-BK encodes a polypeptide of 1,226 amino acids with calculated molecular weight of 138,598 Da. The mature form of PTP-BK constitutes of 3 domain structures including extracellular, transmembrane, and a single intracellular PTPase domain. Western blot analysis indicated that anti-PTP-BK antibody specifically immunoreacted to 2 distinct molecules of 200 kDa and 220 kDa in COS cells, possibly due to differential glycosylation. The recombinant PTP-BK showed the phosphatase activity specific for the phosphotyrosine, but not for the phosphoserine residue of phosphopeptides in vitro. Radiation hybrid panel assigned mouse PTP-BK gene to 5.02cR distal to from the marker D6Mit339 on chromosome 6. Mouse PTP-BK was classified in PTPRO family in novel nomenclature. We discuss here the diversity and physiological functions of PTPRO family of PTP.

Amino Acid Sequence↗
PubMed2000

Isolation of a cDNA for a novel human RING finger protein gene, RNF18, by the virtual transcribed sequence (VTS) approach(1).

We have recently developed a novel database system, designated as the virtual transcribed sequence (VTS) which efficiently extracts many genes from public human genome databases, and tested the feasibility of this novel computational approach (N. Miyajima, C. Burge, T. Saito, Biochem. Biophys. Res. Commun. 272 (2000) 801; http://host45.maze.co.jp/vts/). In this study, using the VTS approach, we isolated a cDNA for a novel human gene with RING finger motif (C(3)HC(4)), which is not deposited in public EST databases. The isolated cDNA clone is 2163 bp in length, and contains an open reading frame of 452 amino acids. We designated the novel gene as RNF18. A database search showed that the RNF18 gene had the moderate similarity to SS-A/Ro52 protein, which is a ribonucleoprotein reactive with autoantibodies in patients with Sjögren's syndrome and systemic lupus erythematosus. Tissue distribution analyses by Northern blot and RT-PCR methods demonstrated that the RNF18 messenger RNA was preferentially expressed in testis. The exon-intron boundaries of RNF18 gene were determined by aligning the cDNA sequence with the corresponding genome sequence. The isolated cDNA consists of eight exons that span about 11 kb of the genome DNA. The precise chromosomal location of the RNF18 gene was determined by PCR-based radiation hybrid mapping, and the gene was located to centromere region of chromosome 11 between markers NIB1900 and D11S1350. Taken together, the VTS approach should provide a novel cDNA cloning strategy for isolating unidentified genes, which are not found even in EST databases but are detectable computationally.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line.

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Oncogene (2000) 19, 4302 - 4307

Carrier Proteins↗
PubMed2000

Isolation of novel mouse genes differentially expressed in brain using cDNA microarray.

The major issue in the post-genome sequencing era is determination of gene expression patterns in variety of biological systems. A microarray system is a powerful technology for analyzing the expression profile of thousands of genes at one experiment. In this study, we identified highly expressed genes in mouse brain using the cDNA microarray carrying 2304 cDNAs derived from oligo-capped mouse cDNA library. Nine genes were highly expressed in adult mouse brain compared to kidney, liver, and skeletal muscle. Tissue distribution analysis by reverse transcription-coupled polymerase chain reaction (RT-PCR) revealed that consistent with the microarray data, all of the selected 9 genes were predominantly expressed in the brain. A database search showed that 5 of the 9 genes, MBP, SC1, HiAT3, S100 protein-beta, and SNAP25, were previously known to be expressed at high level in the brain and in the nervous system. One gene was highly sequenced similar to rat S-Rex-s/human NSP-C, suggesting that the gene is a mouse homologue. The remaining three genes did not match to known genes in the GenBank/EMBL database, indicating that these are novel genes highly expressed in the brain. Taken together, our cDNA microarray system can be an excellent tool for identifying differentially expressed genes in mouse brain.

Animals↗
PubMed2000

T cell receptor-induced calcineurin activation regulates T helper type 2 cell development by modifying the interleukin 4 receptor signaling complex.

The activation of downstream signaling pathways of both T cell receptor (TCR) and interleukin 4 receptor (IL-4R) is essential for T helper type 2 (Th2) cell development, which is central to understanding immune responses against helminthic parasites and in allergic and autoimmune diseases. However, little is known about how these two distinct signaling pathways cooperate with each other to induce Th2 cells. Here, we show that successful Th2 cell development depends on the effectiveness of TCR-induced activation of calcineurin. An inhibitor of calcineurin activation, FK506, inhibited the in vitro anti-TCR-induced Th2 cell generation in a dose-dependent manner. Furthermore, the development of Th2 cells was significantly impaired in naive T cells from dominant-negative calcineurin Aalpha transgenic mice, whereas that of Th1 cells was less affected. Efficient calcineurin activation in naive T cells upregulated Janus kinase (Jak)3 transcription and the amount of protein. The generation of Th2 cells induced in vitro by anti-TCR stimulation was inhibited significantly by the presence of Jak3 antisense oligonucleotides, suggesting that the Jak3 upregulation is an important event for the Th2 cell development. Interestingly, signal transducer and activator of transcription (STAT)5 became physically and functionally associated with the IL-4R in the anti-TCR-activated developing Th2 cells that received efficient calcineurin activation, and also in established cloned Th2 cells. In either cell population, the inhibition of STAT5 activation resulted in a diminished IL-4-induced proliferation. Moreover, our results suggest that IL-4-induced STAT5 activation is required for the expansion process of developing Th2 cells. Thus, Th2 cell development is controlled by TCR-mediated activation of the Ca(2+)/calcineurin pathway, at least in part, by modifying the functional structure of the IL-4R signaling complex.

Amino Acid Sequence↗
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

Identification of the homozygously deleted region at chromosome 1p36.2 in human neuroblastoma.

BACKGROUND: We have identified for the first time a homozygously deleted region within the smallest region of overlap at 1p36.2-3 in two neuroblastoma cell lines. PROCEDURE: The 800-kb PAC contig covering the entire homozygously deleted region was made and sequenced. To date, approximately 70% of sequencing has been accomplished, and the estimated length of the deleted region was 500 kb. RESULTS: Currently, we have found six genes within the region, which include three known genes as well as three other genes that have been reported during processing of our present project for the last 3(1/2) years. We report here the results of expression and mutation analyses of those genes. CONCLUSIONS: Full sequencing for the region of homozygous deletion as well as further analyses of the genes mapped within the region may reveal whether or not there is a neuroblastoma suppressor gene as proposed by the Knudson's two-hit hypothesis.

Child, Preschool↗
PubMed2000

cDNA cloning, expression profile, and genomic structure of human and mouse RNF10/Rnf 10 genes, encoding a novel RING finger protein.

RING finger (C3HC4-type zinc finger) is a variant zinc finger motif present in a new family of proteins including transcription regulators. A new member of the RING finger protein family was identified through a mouse expressed sequence tag (EST) database search, and its full-length cDNA was isolated from a mouse brain full length-enriched cDNA library. The gene was designated as Rnf10, for RING finger protein 10. The cDNA clone consists of 3110 nucleotides and encodes an open reading frame (ORF) of an 804-amino acid protein. A database search revealed that human KIAA0262 protein (accession number, D87451) has strong homology to mouse Rnf10. To confirm that mouse Rnf10 is the homolog or an isolog of human KIAA0262, a human RNF10 cDNA was cloned in our hands from a fetal brain cDNA pool. The newly isolated cDNA contained an ORF for 811 amino acids which had almost identical structure to mouse Rnf10 protein, indicating that the human ORF codes for RNF10 protein. This finding was also supported by comparative chromosome mapping in which both genes were localized in a conserved linkage homology region between mouse and human. Comparison of the RNF10 and KIAA0262 proteins revealed that both were transcribed from the same gene and that the longer RNF10 ORF would be the authentic form. The complete genomic organization of RNF10 was determined to consist of 17 exons spanning at least 40kb in the genome.

Amino Acid Sequence↗
PubMed2000

cDNA cloning, tissue expression, and chromosome mapping of human homolog of SOX18.

The SRY (sex-determining region Y) gene encodes a transcription factor characterized by a DNA-binding motif termed the HMG (high mobility group) domain. The SOX (Sry-box) genes comprise a large family related by homology to the HMG-box region. We isolated a cDNA clone with an open reading frame encoding a putative protein of 384 amino acids, which shared 83% identity to the mouse Sox18 protein. Northern blot analysis revealed that a 1.9-kb band of human SOX18 messenger RNAs was predominantly expressed in heart, although weak signals were seen in brain, liver, testis, and leukocyte. By polymerase chain reaction (PCR)-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, the gene was mapped to the chromosome 20q13.33 region.

Amino Acid Sequence↗
PubMed2000

cDNA cloning of a human RAB26-related gene encoding a Ras-like GTP-binding protein on chromosome 16p13.3 region.

Members of the RAB protein family are important regulators of vesicular fusion and trafficking. A putative new member of the RAB family of genes was identified through a public database search, and its full-length cDNA was isolated from a human fetal brain cDNA library. The predicted protein product of the gene consists of 190 amino acid residues and has 87% identity with rat Rab26. Thus, we designated this gene as the human RAB26-related gene. Reverse transcription-coupled polymerase chain reaction (RT-PCR) demonstrated that the RAB26-related messenger RNA was predominantly expressed in adult and fetal brain. Furthermore, an RT-PCR experiment for brain subregions showed that the mRNA was highly expressed in the amygdala, cerebellum, caudate nucleus, and hippocampus. By PCR-based analysis with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, the gene was mapped to the chromosome 16p13.3 region between markers WI-7742 and WI-3061. The RAB26-related gene consists of eight exons that span about 44kb of the genome DNA.

Amino Acid Sequence↗
PubMed2000

cDNA cloning of a new member of the Ras superfamily, RAB9-like, on the human chromosome Xq22.1-q22.3 region.

Members of the RAB protein family regulate vesicular trafficking and reside in specific intercellular compartments. A new member of the RAB family was identified through a public database search, and its full-length cDNA was isolated from a human fetal brain cDNA library. The predicted protein product of the gene consists of 201 amino acid residues, and the protein has 86% similarity to human RAB9 at the amino acid level. We designated the new gene RAB9-like. Northern blot analysis showed that the gene was transcribed ubiquitously in various human tissues. A database search revealed that the gene is divided into three exons and spans approximately 7.2kb of the genome DNA of chromosome Xq22.1-q22.3 region.

Amino Acid Sequence↗
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↗
PubMed2000

Vascular endothelial growth factor and platelet-derived endothelial cell growth factor expression are implicated in the angiogenesis of endometrial cancer.

Although many angiogenic factors have been described, it is not well defined which factors are expressed in endometrial cancer. The object of this study was to examine mRNA levels of the two angiogenic factors, vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) in endometrial cancer tissues and their association with clinicopathological features including microvessel density. The level of VEGF and PD-ECGF mRNAs was assessed by semi-quantitative reverse transcription-polymerase chain reaction using beta-actin as an internal standard in 38 patients with endometrial cancer. Microvessel counts were also assessed by immunostaining for factor VIII-related antigen in the most vascularised area of the specimen. VEGF/beta-actin ratios of non-endometrioid tumours were significantly higher than those of endometrioid tumours (P = 0.013). VEGF/beta-actin ratios of cases with lymph-vascular space involvement were significantly higher than those of cases without lymph-vascular space involvement (P = 0.021). Although it was not statistically significant, PD-ECGF/beta-actin ratios in grade 3 tumours were higher than those in grade 1 and 2 tumours (P = 0.066). The microvessel density was significantly correlated with the level of VEGF and PD-ECGF mRNA expression (P = 0.041 and P < 0.0001, respectively). Our findings provide evidence that the expression of both VEGF and PD-ECGF is involved in the promotion of angiogenesis in endometrial cancer. In addition, VEGF and PD-ECGF might contribute to the aggressive potential of high grade tumours or certain histological subtypes with unfavourable prognosis through the induction of angiogenesis.

Endometrial Neoplasms↗
PubMed2000

Analysis of tumor necrosis factor-alpha promoter polymorphism in type 1 diabetes: HLA-B and -DRB1 alleles are primarily associated with the disease in Japanese.

Polymorphisms in the 5'-flanking region of the tumor necrosis factor (TNF)-alpha gene were examined to study the genetic background of type 1 diabetes in Japanese. Five different biallelic polymorphisms were examined in 136 type 1 diabetic patients and 300 control subjects. The frequencies of individuals carrying TNF-alpha-857T allele (designated as TNFP-D allele) or -863A/-1,031C allele (designated as TNFP-B allele) were significantly increased in the patients as compared with the controls. Since these TNF-alpha alleles are in linkage disequilibria with certain DRB1 and HLA-B alleles, two-locus analyses were carried out. The TNFP-D allele did not increase the risk in either the presence or absence of the DRB1*0405 or HLA-B54 allele, while the DRB1*0405 and HLA-B54 alleles per se could confer susceptibility in both the TNFP-D allele-positive and -negative populations. Moreover, an odds ratio was remarkably elevated in the population carrying both DRB1*0405 and HLA-B54. Similarly, the TNFP-B allele did not show significant association with the disease in either the HLA-B61-positive or -negative population, while the HLA-B61 allele could significantly increase the risk in the TNFP-B allele-positive population. These data suggest that the associations of TNFP-D and -B alleles may be secondary to their linkage disequilibria with the susceptible HLA class I and class II alleles. Because HLA-B and DRB1 genes were independently associated, both of these genes may be contributed primarily to the pathogenesis of type 1 diabetes in Japanese.

Adolescent↗
PubMed2000

Susceptible locus for obese type 2 diabetes mellitus in the 5'-flanking region of the tumor necrosis factor-alpha gene.

We have identified three novel polymorphisms in the promoter/enhancer region of the tumor necrosis factor (TNF)-alpha gene. In this study we investigated the role of the polymorphisms in predisposition to Type 2 diabetes. The allele frequency of C-->T substitution at -857 tended to be higher in obese patients with diabetes than in lean subjects with normal glucose tolerance (0280 vs. 0.190). This tendency was due to a significantly higher ratio of -857T homozygotes in the obese patients with diabetes (15%) than in lean subjects with normal glucose tolerance (4%). There was no significant association between polymorphisms at positions -863 or -1,013, and diabetes. The homozygous -857T allele may be associated with genetic susceptibility to type 2 diabetes in obese subjects.

5' Untranslated Regions↗
PubMed2000

Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulin-dependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.

Aged↗