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Biomedical subjects

Y Furuichi

Publications and source records attributed to Y Furuichi.

At least 73 records · Page 4Linked to original sources
PubMed1998

Bloom's and Werner's syndrome genes suppress hyperrecombination in yeast sgs1 mutant: implication for genomic instability in human diseases.

Bloom's syndrome (BS) and Werner's syndrome (WS) are genetic disorders in which an increased rate of chromosomal aberration is detected. The genes responsible for these diseases, BLM and WRN, have been found to be homologs of Escherichia coli recQ and Saccharomyces cerevisiae SGS1 genes. Here we show that yeast Sgs1 helicase acts as a suppressor of illegitimate recombination through homologous recombination and that human BLM and WRN helicases can suppress the increased homologous and illegitimate recombinations in the S. cerevisiae sgs1 mutant. The results imply a role of BLM and WRN helicases to control genomic stability in human cells. Similar to Sgs1 helicase, BLM helicase suppressed the cell growth in the top3 sgs1 mutation background and restored the increased sensitivity of the sgs1 mutant to hydroxyurea, but the WRN helicase did not. We discussed differential roles of BLM and WRN helicases in human cells. BLM- and WRN-bearing yeasts provide new useful models to investigate human BS and WS diseases.

Base Sequence↗
PubMed1998

Extra-chromosomal telomere repeat DNA in telomerase-negative immortalized cell lines.

We found novel extra-chromosomal telomere repeat (ECTR) DNAs in telomerase-negative immortalized KMST-6 cells, by staining these cells with a (TTAGGG)n probe using both cycling oligonucleotide-primed in situ synthesis and by fluorescence in situ hybridization. Relatively small amounts of ECTR DNAs were also observed in telomerase-negative VA13 and SUSM-1 cells, but not observed in telomerase-positive immortalized HeLa cells. The ECTR DNAs existed mainly in the nucleoplasm with a small amount in the cytoplasm. The nucleoplasm ECTR DNAs were co-stained with an antibody directed to the telomeric-repeat binding factor 1 (TRF1), suggesting that they exist as a complex with TRF1. In consistent with these cytological studies, Southern blot analysis showed the existence of small telomere repeat DNAs. The ECTR DNA may provide an insight into the elucidation of the mechanisms responsible for the maintenance of telomeres in telomerase-negative immortalized cells.

Cell Line↗
PubMed1998

Cloning and characterization of a human DEAH-box RNA helicase, a functional homolog of fission yeast Cdc28/Prp8.

During the splicing process, spliceosomal snRNAs undergo numerous conformational rearrangements that appear to be catalyzed by proteins belonging to the DEAD/H-box superfamily of RNA helicases. We have cloned a new RNA helicase gene, designated DBP2 (DEAH-boxprotein), homologous to the Schizosaccaromyces pombe cdc28(+)/prp8(+) gene involved in pre-mRNA splicing and cell cycle progression. The full-length DBP2 contains 3400 nucleotides and codes for a protein of 1041 amino acids with a calculated mol. wt of 119 037 Da. Transfection experiments demonstrated that the GFP-DBP2 gene product, transiently expressed in HeLa cells, was localized in the nucleus. The DBP2 gene was mapped by FISH to the MHC region on human chromosome 6p21.3, a region where many malignant, genetic and autoimmune disease genes are linked. Because the expression of DBP2 gene in S.pombe prp8 mutant cells partially rescued the temperature-sensitive phenotype, we conclude that DBP2 is a functional human homolog of the fission yeast Cdc28/Prp8 protein.

Amino Acid Sequence↗
PubMed1998

Physical map of the human chromosome 8p12-p21 encompassing tumor suppressor and Werner's syndrome gene loci.

Detailed physical maps of the human genome are important resources for identification and isolation of genes responsible for diseases and for the study of their structure and function. We constructed a 2.0-Mb high-resolution physical map within the human chromosome 8p12-p21 region extending from marker D8S131 to D8S283. The map comprises a series of contigs mostly P1/PAC clones, which span the loci of potential tumor suppressor genes and the Werner's syndrome gene. Each P1/PAC DNA was defined by its size, restriction sites, terminal sequences, intermarker distances and location relative to major genes and markers. The genes on these P1/PAC DNAs were analyzed by an exon amplification method to determine their locations. The genes newly found by the exon amplification method together with other known genes, including those of glutathion reductase, a general transcription factor, protein phosphatase 2A beta subunit and Werner's syndrome, were precisely mapped within the contigs. These P1/PAC DNAs are useful reagents for the generation of new microsatellite markers to narrow the candidate region of the tumor suppressor gene(s) and/or genes responsible for other diseases, which are believed to exist in this region by linkage analysis.

Animals↗
PubMed1998

Human stanniocalcin (STC): genomic structure, chromosomal localization, and the presence of CAG trinucleotide repeats.

Stanniocalcin (STC) is a glycoprotein hormone that is secreted by the corpuscle of Stannius, an endocrine gland of bony fish. It prevents hypercalcemia via mechanisms including inhibition of calcium uptake across the gills. Mammalian homologues have recently been reported but their function is unknown. Here we report the genomic organization and the transcription start site of the human STC gene and the existence of a polymorphic CAG trinucleotide repeat complex within the 5' untranslated region (UTR) of the mRNA and a smaller [CAG]6 repeat in the 3' UTR. As CAG repeats are associated with various human diseases, we used dual-color fluorescence in situ hybridization to localize the STC gene near markers D8S131 and D8S339 on chromosome 8p11.2-p21. STC should be considered a candidate gene for hereditary diseases mapped to this region.

Base Sequence↗
PubMed1998

A comparative analysis of the proteins between the fibroblasts from Werner's syndrome patients and age-matched normal individuals using two-dimensional gel electrophoresis.

Werner's syndrome (WS) is an autosomal recessive disorder causing symptoms of premature aging. The fibroblasts of WS patients have a shorter life-span than normal fibroblasts. We analyzed the fibroblast proteins from three WS patients and from three age-matched normal individuals using two-dimensional gel electrophoresis and image processing. The expressions of 12 proteins were shown to be augmented or suppressed in WS fibroblasts compared with normal fibroblasts: 11 of 12 spots on the electrophoresis gel of WS fibroblasts were denser than the corresponding spots of normal individual fibroblasts, while the remaining one spot was fainter in WS fibroblasts than in normal fibroblasts. The abundance of these proteins were compared to those of the corresponding proteins from normal fibroblasts at various cell passages in vitro reported in the TMIG-2DPAGE database. The result shows that the change in the protein patterns in in vitro aging did not necessarily correspond to the change in WS fibroblasts, except for three proteins abundant in WS fibroblasts, which increased their abundance during in vitro aging. These results suggest that the premature aging process of WS fibroblasts shares only part of the in vitro aging process of normal fibroblasts.

Adult↗
PubMed1998

Comparative effects of short- and long-term feeding of safflower oil and perilla oil on lipid metabolism in rats.

Diets high in linoleic acid (20% safflower oil contained 77.3% linoleic acid, SO-diet) and alpha-linolenic acid (20% perilla oil contained 58.4% alpha-linolenic acid, PO-diet) were fed to rats for 3, 7, 20, and 50 days, and effects of the diets on lipid metabolism were compared. Levels of serum total cholesterol and phospholipids in the rats fed the PO-diet were markedly lower than those fed the SO-diet after the seventh day. In serum and hepatic phosphatidylcholine and phosphatidylethanolamine, the proportion of n-3 fatty acids showed a greater increase in the PO group than in the SO group in the respective feeding-term. At the third and seventh days after the commencement of feeding the experimental diets, expressions of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA were significantly higher in the SO group than those in the PO group, although the difference was not observed in the longer term. There were no significant differences in the LDL receptor mRNA levels between the two groups through the experimental term, except 3-days feeding. These results indicate that alpha-linolenic acid has a more potent serum cholesterol-lowering ability than linoleic acid both in short and long feeding-terms.

Animals↗
PubMed1998

Severe growth defect in mouse cells lacking the telomerase RNA component.

The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA onto chromosome ends. Telomere length is maintained, by the presence of telomerase activity, in the vast majority of primary tumours and stem cells, suggesting that telomere maintenance is essential for cellular immortalization. Recently, the telomerase RNA component in human and mouse (TERC and Terc, respectively), a telomerase-associated protein TEP1/TLP1 (refs 6,7) and the human catalytic subunit protein TERT (refs 8,9) have been identified. To examine the role of telomerase in telomere maintenance and cellular viability, we established Terc-deficient embryonic stem (ES) cells. It is known that telomerase activity is absent in cells from Terc-knockout mice. Although the study showed that telomere shortening was observed in the Terc-deficient cells from first to six generation animals, whether telomerase-dependent telomere maintenance was essential for cellular viability remained to be elucidated. To address this issue, we examined Terc-deficient ES cells under long-term culture conditions. Accompanying the continual telomere shortening, the growth rate of Terc-deficient ES cells was gradually reduced after more than 300 divisions. An impaired growth rate was maintained to approximately 450 divisions, and then cell growth virtually stopped. These data clearly show that telomerase-dependent telomere maintenance is critical for the growth of mammalian cells.

Animals↗
PubMed1998

A surface plasmon resonance assay for the binding of Amaranthus hypochondriacus var. Mexico lectin to glycoprotein.

We have used the analytical system based on surface plasmon resonance to monitor the interaction between Amaranthus hypochondriacus var. Mexico lectin and four different fetuins; fetuin, asialofetuin, agalactofetuin, and agalactosaminofetuin. Agalactofetuin and agalactosaminofetuin were prepared by enzymic digestion of asialofetuin using jack bean beta-galactosidase or endo-alpha-N-acetylgalactosaminidase from Diplococcus pneumoniae. Ligands were immobilized onto a sensor surface via amide linkages. The lectin interacted most strongly with asialofetuin, but not with agalactosaminofetuin. The binding of the lectin to asialofetuin was inhibited by N-acetylgalactosamine or Gal beta 1-->3GalNAc in a dose-dependent manner.

Asialoglycoproteins↗
PubMed1998

Sp1-mediated transcription of the Werner helicase gene is modulated by Rb and p53.

The regulation of Werner's syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5' upstream region (2.8 kb) of WRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides -67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRN promoter-luciferase reporter (WRN-Luc) plasmids that contained the 5'-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2. 5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

Base Sequence↗
PubMed1998

Action properties of HYI killer toxin from Williopsis saturnus var. saturnus, and antibiotics, aculeacin A and papulacandin B.

The mechanism of the killing and cytocidal effects produced by HYI toxin from Williopsis saturnus var. saturnus, and by the amphiphilic antibiotics aculeacin A and papulacandin B on yeast Saccharomyces bayanus cells was studied. When the yeast cells were treated with these molecules, a discharge of cell materials at the budding position was observed by phase-contrast and scanning electron microscopy. The cytocidal effect of these molecules was most pronounced when the cells were in the logarithmic growth phase. Washing the HYI toxin incubation mixture completely eliminated the killing activity, but in the case of the antibiotics, it only partially reduced the cytocidal activity. Full recovery of the killing activity in the supernatant of the washing solution was observed after HYI toxin incubation, but in the case of the antibiotics, the recovery of cytocidal activity was time-dependent. The activity of membrane beta-1,3-glucan synthase was potently inhibited by HYI toxin, and the concentration of this enzyme in the budding tip was observed. These results suggest that HYI toxin exerts a cytocidal effect on the budding of sensitive yeast cells by inhibiting cell wall synthesis. This mechanism is similar to that of the HM-1 toxin of W. saturnus var. mrakii, and to aculeacin A and papulacandin B, although there are some differences from that of HYI toxin.

Aminoglycosides↗
PubMed1998

Differential effects of cytotoxic drugs on mortal and immortalized B-lymphoblastoid cell lines from normal and Werner's syndrome patients.

We studied the effects of nine cytotoxic drugs on three groups of B-lymphoblastoid cell lines transformed by Epstein-Barr virus (EBV): group 1, mortal cell lines from normal individuals; group 2, immortalized cell lines from normal individuals with strong telomerase activity; group 3, mortal cell lines from Werner's syndrome (WS) patients. Aminoglycoside antibiotics and alkylating drugs showed significantly stronger cytotoxic effects on immortalized cell lines than on mortal cell lines or the cell lines before immortalization. In contrast, topoisomerase II inhibitors showed no difference or they tended to be less cytotoxic to immortalized cell lines. Mortal cell lines from normal individuals and WS patients showed no difference in sensitivity against all the drugs examined except for the topoisomerase I inhibitor, camptothecin, which had a stronger cytotoxic effect on WS cell lines than other cell lines. We discuss the mechanisms underlying these cytotoxic effects.

Aminoglycosides↗
PubMed1998

Characterization of the nuclear localization signal in the DNA helicase involved in Werner's syndrome.

The nuclear localization signal (NLS) of the DNA helicase involved in Werner's syndrome (WS) was studied. Previously, we noted that the C-terminal region of WS helicase contains the NLS. In this study, we generated in HeLa cells various chimeric proteins consisting of the N-terminal tagged with an enhanced green fluorescent protein and the C-terminal fragments of the WS helicase that were truncated either from N- or C-termini, and we examined the ability of fragments to transfer the fusion proteins to the nucleoplasm by fluorescence microscopy. A small C-proximal region containing 34 amino acid residues (residues 1369-1402) was found to contain full nuclear migration activities. Subsequent amino acid substitution experiments showed that a sequence of three positively charged amino acids (Lys1371-Arg1372-Arg1373) in this region are particularly important. Similar sequence has previously been defined as the nuclear localization signal of SV-40 large T antigen that also acts as a viral DNA helicase. Conservation of this motif was found in the C-terminal regions of the other RecQ type DNA helicases, including murine WS helicase, yeast sgs1 and rqh+1 and human Bloom syndrome DNA helicases.

Adenosine Triphosphatases↗
PubMed1998

Characterization of recombinant precursor proteins of the human seminal plasma sperm motility inhibitor synthesized in insect cells.

Human seminal plasma sperm motility inhibitor (SPMI) proteins which are exclusively secreted from seminal vesicles, inhibit sperm motility. It is secreted as biologically active 52 kDa and a mixture of 71 and 76 kDa precursor forms, which are identical to semenogelin-I and II (Sg-I and Sg-II), respectively. To understand the molecular mechanism underlying the inhibition of sperm motility by SPMI proteins, we expressed human Sg-I and Sg-II genes in insect cells using a baculovirus system. The baculoviruses expressing full-size Sg-I and Sg-II proteins that were N-terminally-tagged with a hexahistidine were selected, and were infected with Sf 21 cells. The Sg-I and Sg-II proteins were purified from infected cells by column chromatography using Ni-NTA resin 48 h after infection. The full-size Sg-I and Sg-II proteins were obtained in soluble forms. However, they tended to aggregate to form a gel, as expected from naturally occurring semenogelin. Both the purified recombinant Sg-I and Sg-II proteins showed strong SPMI activities with a complete inhibition of sperm motility at 60 units/mg, equivalent to the natural proteins. This production system that permits the generation of purified Sg-I and Sg-II proteins, as well as mutant derivatives, will be helpful for further study on male infertility.

Animals↗
PubMed1997

Cloning and characterization of a putative human RNA helicase gene of the DEAH-box protein family.

A human RNA helicase gene, DBP1, was cloned by PCR methodsusing degenerate oligonucleotide primers corresponding to highly conserved motifs among known members of the DEAH-box protein family. The full-length DBP1 contains 3028 nucleotides and codes for a protein of 813 amino acids with a calculated mol. wt. of 92723 daltons. The predicted amino acid sequence shares extensive homology with Prp2, Prp16, and Prp22 proteins, which are required to splice mRNA precursors in budding yeast. The protein encoded by DBP1 has RGD, RD, and HS(A/T) repeat motifs close to the N-terminus. Southern blot analysis suggested the presence of a homologue of the DBP1 genes in other species, and Northern blot analysis showed that DBP1 is expressed ubiquitously in various human organs investigated. The DBP1 gene was found to be on chromosome 4p15.3 and encodes a putative nuclear ATP-dependent RNA helicase.

Adenosine Triphosphatases↗
PubMed1997

BLM (the causative gene of Bloom syndrome) protein translocation into the nucleus by a nuclear localization signal.

Bloom syndrome (BS) is a rare genetic disorder characterized by small body size, sun sensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS, and BLM protein is homologous to DNA helicase. There are two putative nuclear localization signals (NLSs) within amino acid residues 1334-1349 in the C-terminus of the BLM protein, which has the distinctive structure of two basic residue arms separated by a spacer. The entire coding or deleted BLM sequences of various sizes were ligated into an enhanced green fluorescent protein (EGFP) vector and transfected into HeLa cells. The EGFP vector harboring the entire BLM coding sequence was transported to the nucleus. The BLM protein truncated at 1341 amino acid, containing an intact helicase domain and only one proximal arm, was not transported to the nucleus. The BLM protein truncated at 1357 amino acid, containing an intact helicase domain and two arms, was transported to the nucleus. The EGFP vector harboring DNA fragments encoding a protein having only the distal arms of basic amino acids in the C-terminus was also transported to the nucleus. The truncated BLM proteins corresponding to previously reported mutated BLM proteins were retained in the cytoplasm or both the cytoplasm and the nucleus as was the EGFP vector with no insert. These results show that the BLM protein translocates into the nucleus and that the distal arm of the bipartite basic residues in the C-terminus of the BLM protein is essential for targeting the nucleus.

Adenosine Triphosphatases↗