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Biomedical subjects

J Furuyama

Publications and source records attributed to J Furuyama.

At least 37 records · Page 2Linked to original sources
PubMed1996

A new alternative splice variant of the mouse Fas antigen with a deletion in the N-terminal portion of the extracellular domain.

The Fas antigen (Fas/APO-1/CD95) has been shown to induce apoptosis when bound to a monoclonal anti-Fas antibody or Fas ligands. Recently, a new soluble human Fas isoform which lacks the transmembrane domain due to alternative splicing has been isolated; however, no mouse Fas isoforms have been reported so far. Analysis of Fas transcripts by RT-PCR detected no Fas transcripts corresponding to the human soluble Fas isoform in mouse thymus, spleen and liver. However, we detected a new isoform with a 117-bp deletion in the second exon in various mouse tissues and cell lines. This isoform, termed truncated Fas (T-Fas), can be generated by alternative splicing and lacks the N-terminal portion of the extracellular domain just after the signal sequence. Since the deletion involves the first cysteine-rich motif believed to be necessary for binding to the Fas ligand, the T-Fas protein may lack the ability to induce apoptosis. The expression of T-Fas relative to that of the normal Fas varies considerably among mouse tissues and cell lines, suggesting preferential transcription of the T-Fas isoform in certain cell types.

Alternative Splicing↗
PubMed1996

Characterization of anti-sperm antibodies and their coding cDNA sequences by Epstein-Barr virus transformed B cell lines from lymphocytes of infertile women possessing anti-sperm antibodies.

Epstein-Barr virus (EBV)-transformed B cell lines that produce human antisperm antibodies were established using peripheral blood lymphocytes (PBLs) from infertile women with sperm immobilizing antibodies in their sera. We obtained three stable cell populations (designated B1, B2, D5) of transformed PBLs originating from three different patients. They produced IgM sperm-reacting antibodies directed against the tail of live, methanol-fixed and NaIO4-treated human spermatozoa. The established antisperm antibodies recognized noncarbohydrate sperm membrane antigens with different specificity and distribution in the male reproductive system. Antisperm antibody-B2 corresponding antigen appears to be specific for the male reproductive system. This antigen is excreted from the epithelial cells of the ductus epididymidis and bound to the spermatozoa in the lumen of the ductus. Antisperm antibodies B1 and D5 corresponding antigens were expressed on the spermatozoa in the seminiferous tubules and were common to the secretions of the ductus epididymidis, prostate and some other somatic organs. The cDNA of the immunoglobulin heavy chain genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using RNA extracted from these clones. The immunoglobulin heavy chain cDNA sequences of these antisperm antibodies showed extremely high homology to previously reported immunoglobulin germline DNA sequences, implying that these antisperm antibodies might be natural autoantibodies rather than antibodies stimulated by external antigen.

Antibodies, Monoclonal↗
PubMed1996

Japanese siblings with missense mutation (717Val --> Ile) in amyloid precursor protein of early-onset Alzheimer's disease.

We report Japanese siblings with the missense mutation 717Val --> Ile in the amyloid precursor protein. The maternal grandmother died of an unknown dementing disorder. The proband's mother had gradually increasing amnesia beginning at age 64, which was diagnosed as Alzheimer's disease (AD). She died in a psychiatric hospital (duration of illness: 16 years). Both the proband and her elder sister were affected at about age 55 years. Disturbances of memory, judgment, and emotion, as well as personality changes, occurred first, with dementia eventually predominating. The elder sister died of pneumonia (duration of illness: 9 years). The amyloid precursor protein (APP) gene was analyzed from each sibling. Genomic DNAs obtained from blood samples were amplified by the polymerase chain reaction (PCR) method. PCR products were digested with the restriction enzyme Bcl I. The resulting restriction fragment length polymorphisms (RFLPs) showed the missense mutation 717Val --> Ile in both patients, but not in a normal control. DNA sequencing showed the presence of the 2149G --> A missense mutation only in the patients. We conclude that this familial AD may originate from the missense mutation 717Val --> Ile in the amyloid precursor protein gene and that the clinical picture is typical of AD, except for normal-pressure hydrocephalus and psychiatric phenomena.

Age of Onset↗
PubMed1995

Localization of Fas antigen mRNA induced in postischemic murine forebrain by in situ hybridization.

The expression of mRNA for the Fas antigen, a membrane-associated protein mediating apoptosis, was localized by in situ hybridization histochemistry in murine brains following 30 min of global cerebral ischemia. Six hours following the ischemia, many labeled cells were detected anew throughout the brain. The hybridization was seen in the small neural cells and in the cells along the walls of the ventricles and vessels, and became undetectable 24 h following the ischemia. These results suggest that the Fas antigen is expressed in the neuron, glia and periventricular cells of the post-ischemic brain.

Animals↗
PubMed1995

A new type of human calcitonin receptor isoform generated by alternative splicing.

There are three isoforms of calcitonin receptors (CTRs). The first type (type 1) has 479 amino acids in rat and 482 amino acids in porcine. The second type (type 2) has a 39 amino acid insertion in the second extracellular domain. The third type (type 3) has a 16 amino acid insertion in the first intracellular domain. The CTR which has been isolated from a human ovarian carcinoma cell line belongs to type 3. Using RT-PCR with primers whose sequences correspond to the human CTR cDNA, we analyzed the expression of the CTR transcripts in seven human tumor cell lines and surgical specimens. The CTR m-RNA were found in all samples. Transcripts which were 48bp shorter than that of the type 3 human CTR were detected, but not transcripts of type 2 and type 3. Since the structure of this CTR is same as that of the type 1 rat and porcine CTRs, we termed it the human type 1 CTR. PCR studies using human genomic DNA as a template revealed that the 48bp sequence constitutes an exon. These results indicate that the type 1 and the type 3 human CTRs are generated by alternative splicing and a majority of human CTR transcripts is type 1.

Alternative Splicing↗
PubMed1995

Molecular characterization of galactosemia (type 1) mutations in Japanese.

We characterized two novel mutations of the galactose-1-phosphate uridyltransferase (GALT) gene in two Japanese patients with GALT deficiency and identified N314D and R333W mutations, previously found in Caucasians. One novel missense mutation was an G-to-A transition in exon 8, resulting in the substitution of arginine by histidine at the codon 231 (R231H). GALT activity of the R231H mutant construct was reduced to 15% of normal controls in a COS cell expression system. The other was a splicing mutation, an A-to-G transition at the 38th nucleotide in exon 3 (318A-->G), resulting in a 38-bp deletion in the GALT cDNA by activating a cryptic splice acceptor site. In seven Japanese families (14 alleles for classic form and one allele for Duarte variant) with GALT deficiency, the R231H and 318A-->G mutations were found only on both alleles of the proband. The N314D and R333W mutations were found on one allele each. The Q188R was prevalent in the United States but not in Japanese patients. The N314D mutation was associated with the Duarte variant in Japanese persons, as well as in the United States. We speculate that classic galactosemia mutations appear to differ between Japanese and Caucasian patients. Our limited data set on galactosemia mutations in Japanese suggests that the N314D GALT mutation encoding the Duarte variant arose before Asian and Caucasian people diverged and that classic galactosemia mutations arose and/or accumulated after the divergence of Asian and Caucasian populations.

Amino Acid Sequence↗
PubMed1995

Are human herpes viruses or measles virus associated with esophageal achalasia?

In order to test the hypothesis that esophageal achalasia may be due to neurotropic viral damage to the esophageal myenteric plexus, esophageal tissue with or without achalasia was analyzed by polymerase chain reaction for the presence of human herpes virus DNA or measles virus RNA. The DNA and RNA were extracted from the esophageal muscle of 12 patients with achalasia and six patients with upper esophageal carcinoma. Peripheral blood mononuclear cells from eight adult volunteers and two samples of umbilical blood mononuclear cells were also used as controls. PCR amplification with a pair of primers specific for herpes simplex type 1 and 2 viruses identified 92-bp fragments in nearly all specimens, including those without achalasia. Each 92-bp fragment was confirmed to be identical to a single herpes simplex virus sequence by automated DNA sequence analysis. No amplification for five other herpes viruses or measles virus was detected. Therefore, a specific viral etiology for achalasia was not identified in this study.

Adult↗
PubMed1995

Multiple alternative splice isoforms of parathyroid hormone-related peptide mRNA in human cell lines.

Parathyroid hormone-related peptide (PTHrP) is thought to be responsible for hypercalcemia in some patients with malignant tumors. The PTHrP gene has seven exons, giving rise to three types of PTHrP isoform through alternative splicing. We studied the expression of mRNAs in 14 human cell lines using the reverse transcription-PCR method, to examine tissue-specific expression. All the cell lines expressed at least two types of PTHrP transcript. Most cell lines expressed all four types of PTHrP mRNA isoform. However, a rhabdomyosarcoma cell line, RD, and a bladder carcinoma cell line, T24, expressed only two types. These results may suggest that PTHrP mRNA is expressed in the majority of tumor and normal tissues and that it shows less tissue- or tumor-specificity.

Base Sequence↗
PubMed1995

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gliomas.

The gene expression of five matrix metalloproteinases (MMPs) and two tissue inhibitors of metalloproteinases (TIMPs) was studied in human gliomas in vivo and in vitro to evaluate their roles in glioma invasion. Simultaneous expression of one to four MMP genes and two TIMP genes was found in 17 surgical glioma specimens, and one MMP (gelatinase A) gene and two TIMP genes were simultaneously expressed in tissue of three brains. The concomitant overexpression of gelatinase A, gelatinase B, and occasional matrilysin genes was associated with the malignancy of gliomas and accompanied by overexpression of the TIMP-1 gene. In five human glioma cell lines, gelatinase A, TIMP-1, and TIMP-2 genes were constitutively expressed in alll cell lines: the matrilysin gene in three cell lines; the stromelysin gene in two cell lines; and the interstitial collagenase gene in one cell line. There was a clear difference in the expression of gelatinase B and stromelysin genes between surgical glioma specimens and glioma cell lines: the gelatinase B gene was not expressed constitutively in vitro but was overexpressed in vivo, whereas the stromelysin gene was not expressed in vivo but was expressed in some cell lines. To find the cause of that difference in vivo and in vitro, the transcriptional regulations of MMP and TIMP genes by tumor promoter, growth factors, or cytokines were studied in vitro. Interstitial collagenase, gelatinase B, stromelysin, and TIMP-1 genes were upregulated in many cell lines by phorbol-12-myristate-13-acetate (PMA) and in some cell lines by epidermal growth factor, tumor necrosis factor-alpha, or interleukin-1 beta. Transforming growth factor-beta 1 (TGF beta 1) upregulated gelatinase A and matrilysin genes in some cell lines, and there were no clear responses from any MMP and TIMP genes to interleukin-6. Thus, the transcriptional modulation of MMP genes by these growth factors and cytokines seemed insufficient to explain the difference in gelatinase B and stromelysin gene expressions in vivo and in vitro and was suggestive of the genetic alteration of glioma cells in vitro, the heterogeneous cell population in glioma tissues, or both. Furthermore, the in vitro invasion of glioma cells through Matrigel in response to PMA, TGF beta 1, or TIMP-1 was assessed by chemoinvasion assay. In most cell lines, invasion was significantly stimulated by PMA or TGF beta 1 but suppressed by TIMP-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1995

Apoptosis of human glioma cells in response to calphostin C, a specific protein kinase C inhibitor.

Calphostin C acts at the regulatory domain as a highly selective inhibitor of protein kinase C (PKC), and staurosporine acts at the catalytic domain as a nonspecific PKC inhibitor. The authors investigated the capacity of calphostin C and staurosporine to promote apoptotic fragmentation of DNA in four human glioma cell lines. The exposure of glioma cell lines to 100 nM calphostin C for 2 to 8 hours induced a decrease in particulate PKC activities and exposure for 16 to 24 hours produced a concentration-dependent increase in internucleosomal DNA cleavage on agarose gel electrophoresis. In addition, the human glioma cells showed the classic morphological features of apoptosis: cell shrinkage, nuclear condensation, and the formation of apoptotic bodies. A 24-hour exposure to staurosporine failed to induce internucleosomal DNA fragmentation at concentrations generally used to achieve maximum inhibition of enzyme activity (50 nM) but promoted fragmentation at considerably higher concentration (more than 200 nM). Deoxyribonucleic acid fragments obtained from cells exposed to 100 nM calphostin C for 16 to 24 hours possessed predominantly 5'-phosphate termini, consistent with the action of a Ca++/Mg(++)-dependent endonuclease. Northern and Western blot analyses revealed that the exposure to 100 nM calphostin C for 4 hours failed to alter bcl-2 transcript and protein, but exposure for more than 8 hours decreased the amount of bcl-2 transcript and protein. Together, these observations suggest that calphostin C is capable of inducing apoptotic DNA fragmentation and cell death in a highly concentration dependent manner in human glioma cells and that the apoptosis is closely associated with the decrease in transcription and translation of bcl-2.

Alkaloids↗
PubMed1995

[Increased expression of gelatinases A and B, matrilysin and TIMP-1 genes in human malignant gliomas ].

Malignant glioma is a local invasive tumor in the central nervous system. The mRNA expression of five matrix metalloproteinases (MMPs) and two tissue inhibitors of metalloproteinases (TIMPs) was examined in surgical specimens of three brain tissues, two astrocytomas, four anaplastic astrocytomas and eleven glioblastomas, including recurrent one anaplastic astrocytoma and two glioblastomas. In the control brain tissues, mRNA expression was high for TIMP-2, low for gelatinase A and TIMP-1, and undetectable for gelatinase B, interstitial collagenase, stromelysin and matrilysin. Gelatinase B and TIMP-1 were concomitantly overexpressed in primary glioblastomas. In addition, the average expression level of gelatinase A increased 3.0 fold in astrocytomas and anaplastic astrocytomas and 6.0 fold in glioblastomas, compared to the brain tissues. Matrilysin was induced variably in more than half of the primary glioblastomas, and interstitial collagenase was slightly induced in some primary and recurrent glioblastomas. Stromelysin was characteristically not expressed in any gliomas, and the expression level of TIMP-2 did not significantly change in the gliomas. These results suggest that the concomitant increased expression of gelatinase A, gelatinase B and occasional matrilysin genes is associated with the malignancy of gliomas and accompanied by the increased expression of TIMP-1 gene.

Astrocytoma↗
PubMed1994

Fas antigen mRNA induction in postischemic murine brain.

Fas antigen mRNA induction in the brain was examined using a transient global cerebral ischemia model in BALB/C mice. Northern blot analysis revealed little Fas antigen mRNA expression in the brains of sham-operated mice. A marked induction of Fas mRNA expression was detected in the brains of mice 6 h after 30 min of cerebral ischemia. These results suggest a possible apoptotic mechanism for cell death, mediated by the Fas antigen, in postischemic brain.

Animals↗
PubMed1994

Characterization of the three genotypes of low Km aldehyde dehydrogenase in a Japanese population.

A deficiency in low Km aldehyde dehydrogenase (ALDH2) is regarded as the main factor responsible for "Oriental flushing" and other symptoms due to alcohol sensitivity. In this study, the relationship of the ALDH2 genotype to alcohol-associated symptoms and drinking behavior was investigated in 524 Japanese workers, using a new, rapid, and nonisotopic polymerase chain reaction (PCR) method. Differences in the frequency of alcohol-associated manifestations between the normal homozygote and the other deficient types were apparent. In addition, among the ALDH2-deficient individuals, the atypical homozygote was obviously more hypersensitive to alcohol than the heterozygote, judging from the frequency of flushing or other drinking-associated manifestations with a small dose of alcohol. Drinking frequency also apparently decreased in the following order: typical homozygote, heterozygote, atypical homozygote. Similarly, mean amounts of alcohol consumption also decreased in the same order, although considerable variation existed within the typical homozygote and the heterozygote group. In contrast, neither the manifestations nor the drinking behavior were, in general, influenced by polymorphism of the alcohol dehydrogenase beta-subunit (ADH2) gene in males. These findings further indicate the important contribution of the ALDH2 genotype to alcohol sensitivity in Orientals.

Adult↗
PubMed1994

Linkage between severe atopy and chromosome 11q13 in Japanese families.

Atopy, characterised by allergic asthma and rhinitis, is due to increased IgE responses to common aeroallergens. An Oxford group has described maternal inheritance of atopy, where there is significant linkage between IgE responsiveness and a VNTR marker D11S97 and a CA microsatellite within a candidate gene, the high affinity IgE receptor beta subunit (Fc epsilon RI beta), on chromosome 11q. Attempts at independent replication have produced conflicting results. We therefore recruited 270 atopic asthmatic probands in a Japanese community population for genetic linkage analysis. Four families, each with more than 15 meioses and a clear phenotype for atopy, were selected for genetic analysis. Atopy was defined as presence of all of raised total IgE, positive RAST and skin tests to three or more aeroallergens; non-atopy, as absence of all these criteria. Linkage analysis showed a maximum two-point lod score of 9.35 for D11S97 and Fc epsilon RI beta under the assumption of unequal rates of maternal and paternal recombination. Two families showed close genetic linkage with Fc epsilon RI beta with a pattern of maternal inheritance. These results from a Japanese population provide further evidence for genetic linkage between severe atopy and chromosome 11q13 and the likelihood of genomic imprinting at the locus.

Base Sequence↗
PubMed1993

Expressions of matrilysin and stromelysin in human glioma cells.

Production of matrilysin and stromelysin by five human glioma cell lines was investigated by Northern blot and immunoblot analyses. Four cell lines constitutively produced matrilysin. Its production was stimulated by phorbol-12-myristate-13-acetate (PMA) in two cell lines and by transforming growth factor-beta 1 (TGF-beta 1) in two other cell lines. Stromelysin transcript was constitutively expressed in only two cell lines, but enhanced or induced by PMA in four cell lines. These results suggest that these enzymes, especially matrilysin, may be involved in the invasive growth of neoplastic glial cells.

Blotting, Northern↗