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Biomedical subjects

Y Furuichi

Publications and source records attributed to Y Furuichi.

At least 91 records · Page 5Linked to original sources
PubMed1997

Human endothelin receptors ET(A) and ET(B) expressed in baculovirus-infected insect cells--direct application for signal transduction analysis.

We expressed human endothelin receptors, ET(A) and ET(B), in insect Sf9 cells infected by recombinant baculoviruses that contained the respective cDNAs. Ligand-binding experiments showed that the two expressed receptors have the same affinities as observed for the receptors in mammalian cells, i.e. the ET(A) receptor showed an affinity order of ET-1 > or = ET-2 >> ET-3, and the ET(B) receptor remained nonselective for three isopeptide ligands. The ET(B) receptor was purified by affinity chromatography with K9-biotinyl-ET-1 without losing the ligand-binding activity from the membrane of infected Sf9 cells. Protein chemical analysis of the purified ET(B) receptor showed that it is glycosylated, and that the N-terminal 38-amino-acid peptide is susceptible to proteolytic digestion, resulting in a small 35-kDa receptor like that found in the human placenta. Surprisingly, the infected and unlysed cells showed a strong intracellular Ca2+ concentration increase ([Ca2+]i), which was generated by a unique signal-transduction pathway consisting of the insect GTP-binding protein and human endothelin receptors expressed in the late phase of virus infection. Due mainly to an efficient expression (over 200,000 receptors/cell), to a low background owing to no endogenous homolog receptor in insect Sf9 cells, and to a sensitive fluorescent reagent Fura-2, this insect Sf9 cell system can detect the [Ca2+]i induced by picomolar levels of endothelin-receptor. We propose that this highly sensitive system be used to screen for potential antagonists/agonists of endothelin receptors.

Amino Acid Sequence↗
PubMed1997

Abnormal telomere dynamics of B-lymphoblastoid cell strains from Werner's syndrome patients transformed by Epstein-Barr virus.

The characteristics of B-lymphoblastoid cell strains transformed by Epstein-Barr virus (EBV) from normal individuals and Werner's syndrome (WRN) patients were compared. We continuously passaged cell strains from 28 WRN patients and 20 normal individuals for about 2 years corresponding to over 160 population doubling levels (PDLs). First, the WRN mutation significantly suppressed the immortalization: all the 28 cell strains from WRN patients, as well as 15 out of 20 cell strains from normal individuals, died out before 160 PDLs mostly without developing a significant telomerase activity. The remaining five cell strains from normal individuals became moderately/strongly telomerase-positive and, three of them were apparently immortalized with an infinitively proliferating activity. Second, the monitoring of the telomere length of both normal and WRN cell strains during the culture period suggests that the WRN gene mutation causes abnormal dynamics of the telomere: (1) a significant proportion of WRN cell strains showed drastic shortening or lengthening of telomere lengths during cell passages compared with normal cell strains, and (2) WRN cell strains terminated their life-span at a wide range of telomere length (between 3.5 and 18.5 Kbp), whereas normal cell strains terminated within a narrow telomere length range (between 5.5 and 9 Kbp). The chromosomal aberration characteristic of WRN cells, including translocation was confirmed in our experiment. We discussed the correlation between the chromosomal instability, abnormal telomere dynamics and inability of immortalization of the WRN B-lymphobloastoid cell strains.

B-Lymphocytes↗
PubMed1997

Characterization of human endothelin B receptor and mutant receptors expressed in insect cells.

Endothelin type-B receptor (ET(B)R) forms a stable complex with its ligand, endothelin-1. To facilitate biochemical and biophysical studies of human ET(B)R, several ET(B)R mutants carrying a hexahistidine tag sequence at the N or C terminus were expressed in Sf9 cells and were purified by a combination of biotinylated endothelin-1-ligand-affinity and nickel-affinity chromatographies. The ligand-free receptor was purified by dissociating the ligand x receptor complex with 2 M NaSCN, whereas the ligand-bound ET(B)R was purified by the use of thiol-sensitive biotinylated endothelin-1. While the wild-type ET(B)R was expressed at about 100 pmol 125I-endothelin-1-binding activity/mg membrane protein, the deletion of 36 residues from the N-terminus reduced the expressed activity to about 30%. On the other hand, the lack of glycosylation and the replacement of 2-9 residues in the N-terminal tail resulted in a 20-40% reduction in the expressed activity. Among the mutant proteins, [H57-H62, G63-G65]ET(B)R, carrying six His residues in the N-terminal tail, was studied extensively because it was purified most effectively. Ligand-free [H57-H62, G63-G65]ET(B)R, purified in digitonin, retained full ligand-binding activity, while other detergents led to partial denaturation of the receptor after solubilization or after elution with NaSCN. On the other hand, ligand-bound [H57-H62, G63-G65]ET(B)R could be purified in various detergents, such as n-octyl-beta-D-glucopyranoside or n-decyl-beta-D-maltopyranoside. Ligand-free [H57-H62, G63-G65]ET(B)R reconstituted in phospholipid vesicles stimulated the binding of guanosine 5'-3-O-(thio)triphosphate by Gq in the presence of endothelin-1. Ligand-bound [H57-H62, G63-G65]ET(B)R showed similar catalytic activity in nucleotide exchange by Gq. These results indicate that the ligand x receptor complex in a detergent-micellar solution retained the biologically active structure, and that the presence of ligand, endothelin-1, in the receptor molecule reinforces the stable assembly of a helical bundle and therefore the active structure.

Amino Acid Sequence↗
PubMed1997

DNA helicase activity in Werner's syndrome gene product synthesized in a baculovirus system.

The gene responsible for Werner's syndrome (WRN) contains a region homologous to the Escherichia coli RecQ type DNA helicase and was thought to code for a DNA helicase belonging to this helicase family. However, no evidence has been shown before to substantiate this prediction. Here, we show data that the product of the WRN gene is indeed a DNA helicase. The gene product, a polypeptide with a relative molecular mass of 170 kDa, expressed in the insect Spodoptera frugiperda (Sf21) cell and purified by affinity column chromatography contained both the ATPase and DNA unwinding activities characteristic of DNA helicase. Expressions in Sf21, as well as in HeLa cells, showed that the WRN DNA helicase is exclusively transported to the nucleoplasm, which is consistent with its function in DNA metabolism. Our studies on strand displacement suggest that WRN helicase can unwind not only a duplex DNA, but also an RNA-DNA heteroduplex, while the latter reaction seems less efficient. Enzymological features learned from the purified WRN helicase are discussed with respect to the biological function, which remains to be clarified.

Adenosine Triphosphatases↗
PubMed1997

Down-regulation of the defective transcripts of the Werner's syndrome gene in the cells of patients.

Werner's syndrome (WS), an adult progeria, is a recessive genetic disorder caused by the mutations in the DNA helicase gene (WRN). In this study, a comparative northern blot analysis was made for poly(A)+ RNAs extracted from fibroblasts and B-lymphoblastoid cells of WS patients, relatives of patients, and normal individuals. The levels of mutant WRN mRNA from patient cells were significantly lower than those of intact mRNA from the cells of normal individuals by an average of 70%. Furthermore, an extremely low level of WRN mRNA(s), presumably a mixture of mutant and intact mRNAs, was observed for the patient's family members who carry one mutated allele. These results strongly suggest that a relatively low level of helicase mRNA is sufficient to prevent the onset of Werner's syndrome.

Adult↗
PubMed1997

Cloning and characterization of a novel gene, WS-3, in human chromosome 8p11-p12.

A novel human gene referred to as the WS-3 gene, in the short arm of human chromosome 8, was cloned by a combination of exon trapping, thermal asymmetric interlaced-PCR (TAIL-PCR) and the Marathon-Ready cDNA amplification method. The gene consists of 7 exons separated by 6 introns, and is at the telomere side of the STS marker, D8S1055. The full-length WS-3 gene contains 1052 nucleotides and codes for a protein of 190 amino acids with a calculated mol. wt. of 20,747. Southern blot experiments showed that the WS-3 gene exists as a single copy in the human genome. A protein encoded by the WS-3 gene has an R-G-D (Arg-Gly-Asp) motif in the N-terminal region, which seems to confer adhesive properties to macromolecular proteins like fibronectin. Although WS-3 is a small gene with unknown biological function, its ubiquitous expression in various tissues and organs suggests that the encoded protein is one of the essential components of all organs and tissues.

Amino Acid Sequence↗
PubMed1997

A novel yeast gene, RHK1, is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits beta-1,3-glucan synthesis.

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall beta-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of beta-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of Rep-8 (D8S2298E) in the human chromosome 8p11.2-p12.

A novel human gene referred to as the Rep-8 gene (D8S2298E) was cloned by a combination of exon trapping, thermal asymmetric interlaced-PCR, and screening of a cDNA library. It is located in human chromosome 8p11.2-p12. The gene consists of eight exons and spans about 20 kb between the glutathione S-reductase and the protein phosphatase 2A beta subunit genes. The full-length Rep-8 gene contains 1483 nucleotides and codes for a protein of 270 amino acids. Southern blot experiments showed that the Rep-8 gene exists as a single copy per haploid. With a zoo blot analysis, human Rep-8 DNA hybridized strongly with the monkey DNA, but only weakly with the DNAs of species other than Homo sapiens. Northern blot analysis showed that it is expressed abundantly in the testis and ovary, suggesting that the Rep-8 gene product may play a role in reproduction.

Amino Acid Sequence↗
PubMed1997

Gene expressions of transferred human chromosome 8 in mouse cell lines.

Gene expressions were studied for the human chromosome 8 which were introduced by microcell fusion to three mouse tumor cell lines: A9, mouse melanoma B16F10 and SCVA2 derived from SV40-transformed scid fibroblasts. Nineteen genes in the human chromosome 8 were chosen, and the presence of their transcripts in these cells was examined by RT-PCR. The data showed that most of the human genes were expressed, with a few exceptions: the indoleamine 2, 3-dioxygenase gene was expressed in none of these cell lines, while two other genes, calbindin and neuronal nicotinic acetylcholine receptor subunit (Ach Rbeta3) genes, were not expressed in the A9 and SCVA2 cell lines, respectively, suggesting some cell- or species-specific transcriptional regulations exist. These results show that the mouse cell lines carrying a human chromosome are powerful tools for chromosome-specific cDNA cloning.

Animals↗
PubMed1997

Analysis of helicase gene mutations in Japanese Werner's syndrome patients.

The profile of helicase gene mutations was studied in 89 Japanese Werner's syndrome (WRN) patients by examining the previously described mutations 1-4 as well as a new mutation found during this study, designated mutation 5. Of 178 chromosomes (89 patients), 89 chromosomes (50%) had mutation 4, 11 (6.2%) chromosomes had mutation 1, and two chromosomes (1.1%) contained mutation 5. Mutations 2 and 3 were not observed in this patient population. The remaining 76 (42.7%) chromosomes had none of these mutations. A significant fraction of all patients (22 total patients, 24.7%) appear to be compound heterozygotes, including those carrying mutations of both types 1 and 4. The genotypes analysis of the markers surrounding the. WRN helicase gene strongly suggests that most of the chromosomes carrying either mutation 1 or 4 were derived from two single founders.

DNA Helicases↗
PubMed1997

Mutation and haplotype analyses of the Werner's syndrome gene based on its genomic structure: genetic epidemiology in the Japanese population.

The correlation between mutations in the Werner's syndrome (WRN) gene and the haplotypes of surrounding markers was studied in Japanese patients. We have elucidated the genomic structure of WRN helicase, and found five additional mutations, designated mutations 6-10. Mutations 4 and 6 were found to be the two major mutations in this population; these mutations comprised 50.8% and 17.5%, respectively, of the total in a sample of 126 apparently unrelated chromosomes. Almost all the patients homozygous for mutation 4 shared a haplotype around the WRN gene, consistent with the view that they are derived from a single ancestor. This important advantage demonstrated in the identification of the WRN gene suggests that the Japanese present a unique population for the cloning of other disease genes. The conserved haplotype was observed across 19 loci, extending a distance estimated to be more than 1.4 Mbp around the WRN gene. This haplotype is rare among random Japanese individuals. Unexpectedly, all the nine patients homozygous for mutation 6 shared a haplotype that was identical to this haplotype at 18 of these 19 markers. These results suggest that mutations 4 and 6 arose independently in almost identical rare haplotypes. The remaining mutations (1, 5, 7, 8, 9, and 10) occurred rarely, and were each associated with different haplotypes.

DNA Helicases↗
PubMed1997

Immortalization of immunologically committed Epstein-Barr virus-transformed human B-lymphoblastoid cell lines accompanied by a strong telomerase activity.

The immunological characteristics and immortalization processes of three EBV-transformed human B-lymphoblastoid cell lines, N0003, N0005 and N6803, with strong telomerase and infinitively proliferating activities are described. The three cell lines were apparently immortalized: they developed a strong telomerase activity at the population doubling levels (PDLs) between 11 and 135, and continued proliferation over 250 PDLs. All the cell lines expressed CD22, CD19 and CD20 antigens. They were uniformly stained with IgM (N0005), IgG (N6803) or IgA (N0003) at early PDLs between 17 and 20, and they secreted the corresponding class of Ig into the medium; the N6803 and N0003 cell lines continued to secrete each class of Ig at decreased levels while the N0005 cell line expressed or secreted virtually no Ig after immortalization. Karyotype analysis of the immortalized cell lines showed that they were derived from a single cell because they shared a set of abnormal chromosomes within each cell population, and two of the cell lines attained clonal characteristics before they developed a strong telomerase activity. These results indicate that the three immortalized cell lines with a strong telomerase activity correspond to the intermediate stages of B-cell differentiation naturally committed to a specific Ig class, and suggest that they were derived from a B-lymphoblastoid cell committed to a specific class of Ig with poor telomerase activity, rather than from a strongly telomerase-positive B-lymphoblastoid cell either committed or multipotential.

B-Lymphocytes↗
PubMed1997

Short-term effects of triclosan on healing following subgingival scaling.

The present clinical trial was performed to evaluate short-term effects of a triclosan-containing dentifrice/gel combination on soft tissue healing, when applied supra-/sub-gingivally at periodontal sites treated with scaling and root planing. 16 subjects with moderate periodontitis participated in a 2x 2-week, split-mouth designed clinical trial. 2 combinations of gel/dentifrice (the test combination containing triclosan) were used. 2 pairs of contralateral sites with probing pocket depth (PPD) > or 5 mm, and which bled on probing (BoP +) were selected in each patient as experimental units. A baseline examination included assessments of PPD, BoP, gingival index scores, plaque index scores, and the composition of the subgingival microbiota (dark-field microscopy). The assigned quadrant was anaesthetized and the teeth exposed to meticulous scaling and root planing. Immediately after the completion of mechanical therapy, either the test or control gel was applied subgingivally at the experimental sites. The volunteer was instructed to brush his/her teeth with an assigned dentifrice and to apply the gel (via a custom-made stent) supra-gingivally 2x daily for the following 2 weeks. He/she was recalled on day 7 for a second professional subgingival gel application. Re-examinations were carried out on days 2, 7 and 14 after treatment. 1-week wash-out periods separated the 2 experimental periods. The mean PPD reductions (between days 0 and 14) were 1.8 mm and 1.9 mm for the test and control gel/dentifrice sites. The reduction in BoP and gingival index scores was significantly greater during the test than during the control regimen. No significant differences were observed between the 2 regimens regarding plaque scores and composition of the subgingival microbiota. The findings from the present investigation demonstrated that triclosan, applied both sub- and supra-gingivally reduced soft tissue inflammation following scaling and root planing.

Anti-Infective Agents, Local↗
PubMed1997

The use of a triclosan/copolymer dentifrice may retard the progression of periodontitis.

The present investigation was performed to examine if triclosan and a copolymer, incorporated in a dentifrice and used by periodontitis-susceptible subjects could influence clinical symptoms characteristic of recurrent periodontitis. 60 subjects, 34 to 67 years of age, were recruited for the study. They were randomly selected from a group of patients previously treated for advanced periodontal disease. This treatment had included oral hygiene instruction, subgingival debridement, but no surgical therapy. The patients had, during a 3-5 year period following active therapy, been enrolled in a maintenance care program but had, at various intervals, exhibited signs of recurrent periodontitis. The patients were stratified into 2 balanced groups with respect to mean probing pocket depth. The test group, included 30 individuals who used a dentifrice containing triclosan/copolymer/fluoride, i.e. 0.3% triclosan, 2% copolymer and 1100 ppm F from 0.243% sodium fluoride (Colgate Total). The control group also included 30 subjects who used a dentifrice identical to the one used in the test group but without the triclosan/copolymer content. Following the baseline examination, including clinical and radiographical assessments, all volunteers received detailed information on how to brush their teeth in a proper way. This information was repeated on an individual need basis during the course of the subsequent 36 months. No professional subgingival therapy was delivered between the baseline and the 36-month examinations, but the subjects were recalled every 3 months. Re-examinations were performed after 6, 12, 24, and 36 months of the trial. A 2nd set of radiographs was obtained at the final examination, i.e., at 36 months. The results demonstrated that in subjects susceptible to periodontal disease, meticulous, self-performed, supragingival plaque control maintained over a 3-year period failed to prevent recurrent periodontitis. In a similar group of subjects and plaque control program, however, the daily use of a triclosan-containing dentifrice reduced (i) the frequency of deep periodontal pockets, and (ii) the number of sites that exhibited additional probing attachment and bone loss.

Adult↗
PubMed1997

Effect of triclosan on the subgingival microbiota of periodontitis-susceptible subjects.

The present study evaluated the long-term effect of (i) meticulous self-performed, supragingival plaque control and (ii) the use of a triclosan/copolymer containing dentifrice in adult subjects susceptible to destructive periodontitis. 40 individuals were recruited into the trial. 3-5 years prior to the baseline examination, they had all been treated by nonsurgical means- for advanced periodontal disease. During the subsequent maintenance phase, all subjects had at different time intervals exhibited sites with recurrent periodontitis. At a baseline examination, 6 surfaces per tooth were examined regarding bleeding on probing, probing pocket depth, and probing attachment level. The deepest pocket site in each quadrant (i.e. 4 sites per subject) was selected and samples of the subgingival bacteria were taken. At baseline, all volunteers received detailed information on proper oral hygiene techniques. This information was repeated on an individual need basis during the course of the subsequent 36-months. No professional subgingival therapy was delivered between the baseline and the 36-month examinations. The subjects were randomly distributed into 2 equal groups of 20 individuals each, 1 test and 1 control group. The members of the test group were supplied with a fluoridated dentifrice containing triclosan/copolymer (Total, Colgate), while the controls received a corresponding dentifrice but without triclosan/copolymer. The findings demonstrated that in subjects with advanced and recurrent periodontitis, carefully practiced supragingival plaque control had some effects on the subgingival microbiota, but also that this was insufficient to prevent disease progression. In a corresponding group of subjects, however, who used a triclosan/copolymer dentifrice, the subgingival microbiota was reduced in both quantitative and qualitative terms and recurrent periodontitis was almost entirely prevented.

Actinomyces↗
PubMed1997

A unique downregulation of h2-calponin gene expression in Down syndrome: a possible attenuation mechanism for fetal survival by methylation at the CpG island in the trisomic chromosome 21.

To understand the effect of trisomic chromosome 21 on the cause of Down syndrome (DS), DNA methylation in the CpG island, which regulates the expression of adjacent genes, was investigated with the DNAs of chromosome 21 isolated from DS patients and their parents. A methylation-sensitive enzyme, BssHII, was used to digest DNAs of chromosome 21, and the resulting DNA fragments were subjected to RLGS (restriction landmark genomic scanning). Surprisingly, the CpG island of the h2-calponin gene was shown to be specifically methylated by comparative studies with RLGS and Southern blot analysis. In association with this methylation, h2-calponin gene expression was attenuated to the normal level, although other genes in the DS region of chromosome 21 were expressed dose dependently at 1.5 times the normal level. These results and the high miscarriage rate associated with trisomy 21 embryos imply that the altered in vivo methylation that attenuates downstream gene expression, which is otherwise lethal, permits the generation of DS neonates. The h2-calponin gene detected by the RLGS procedure may be one such gene that is attenuated.

Base Sequence↗