Biomedical subjects

Y Furuichi

Publications and source records attributed to Y Furuichi.

At least 109 records · Page 6Linked to original sources

PubMed1996

A unique human gene that spans over 230 kb in the human chromosome 8p11-12 and codes multiple family proteins sharing RNA-binding motifs.

A unique gene, RBP-MS, spanning over 230 kb in the human chromosome 8p11-12 near the Werner syndrome gene locus is described. The single-copy RBP-MS gene is alternatively spliced, resulting in a family of at least 12 transcripts (average length of 1.5 kb). Nine different types of cDNAs that encode an RNa-binding motif at the N terminus and helix-rich sequences at the C terminus have been identified thus far. Among the 16 exons identified, four 5'-proximal exons contained sequences homologous to the RNA-binding domain of Drosophila couch potato gene. Northern blot analysis showed that the RBP-MS gene was expressed strongly in the heart, prostate, intestine, and ovary, and poorly in the skeletal muscle, spleen, thymus, brain, and peripheral leukocytes. The possible role of this gene in RNA metabolism is discussed.

Alternative Splicing↗

PubMed1996

Purification and characterization of a pod lectin from Great Northern bean, Phaseolus vulgaris L.

The pods of the Great Northern bean plant contain a lectin (GNpL) that highly resembles seed lectins (GNLs) of the same plant. Purification of GNpL from pod extracts was achieved by ion-exchange chromatographies on CM- and DEAE-celluloses and gel filtration chromatography on Sephacryl S-300 HR. GNpL has a similar SDS-PAGE pattern to that of GNLs. GNpL and GNLs yield three subunits though each GNpL subunit is 0.5 kDa smaller than the corresponding GNLs subunit (GNpL; pod-alpha-subunit of 34.0 kDa, pod-beta-subunit of 36.5 kDa, and pod-gamma-subunit of 38.5 kDa). GNpL and GNLs display indistinguishable carbohydrate specificities and have similar amino acid compositions. Pod-alpha-subunit cross-reacts with antibodies against GNLs on western blotting. On the other hand, the N-terminal amino acid sequence of pod-alpha-subunit suggests that GNpL is a distinct gene product from those of GNLs genes although they are shown to be homologous proteins.

Amino Acid Sequence↗

PubMed1996

Estimation of the physical distance between major genomic markers in the Werner syndrome locus (8p11.2-12) by dual-color fish analysis.

The gene responsible for Werner syndrome (WS) is considered to be located between D8S131 and D8S87 in the 8p11.2-12 region that includes the closest marker D8S339 (Goto et al. (1992) Nature 355: 735-738). In this experiment, the order of major markers in this region was determined and the physical distances between them were estimated by dual-color fluorescence in situ hybridization (FISH) using P1, PAC and cosmid clone DNAs as probes. The fine overall order of telomere-D8S131-D8S339-GSR-PP2A beta-D8S283-D8S87-centromere was determined for the first time. The distance from D8S131 to D8S87 was estimated to be 1,634 kb. To our surprise, the distance between D8S131 and D8S87 is much shorter than previously estimated by recombination analysis, 8.3 cM equivalent to 8.3 Mb in physical distance. These information provide the basis for the positional cloning of WS gene and the identification of its mutation.

DNA↗

PubMed1996

Pore formation on proliferating yeast Saccharomyces cerevisiae cell buds by HM-1 killer toxin.

The cytocidal effect of HM-1 produced by Hansenula mrakii on yeast Saccharomyces cerevisiae cells was studied. The HM-1 strongly inhibited the growth of S. cerevisiae cells at a low concentration (IC50: 2.1 x 10(-8) M) by reducing the number of viable cells. The killer action of HM-1 was most efficient when cells were actively proliferating. Cells in a resting state were resistant, but they became HM-1-sensitive after about 90 min of culturing at 30 degrees C, concomitantly with the increment of budding index. In association with the reduction of viable cell number, ultraviolet light-absorbing cellular components were discharged from sensitive cells. HM-1 molecules appear to bind to susceptible cells rather loosely since cells incubated with HM-1 were able to proliferate after having been washed. By phase-contrast light microscopy and scanning electron microscopy, discharge of cell material was observed at the budding portions of HM-1-treated cells. Addition of sorbitol to make the culture medium isotonic partially reduced the cell death induced by HM-1. These results suggest that HM-1 acts on the budding region of proliferating yeast cells, resulting in pore formation, leakage of cell material and eventual cell death.

Cell Division↗

PubMed1996

The effects of antimicrobial mouthrinses on de novo plaque formation at sites with healthy and inflamed gingivae.

The objective of the present investigation was to evaluate to what extent mouthrinses containing triclosan and chlorhexidine may modify the amount of de novo plaque that forms on tooth surfaces adjacent to healthy and inflamed gingival units. 10 volunteers were recruited. On day 0, gingival crevicular fluid (GCF) was obtained at predetermined sites and gingivitis (GI) was assessed. A careful oral prophylaxis was given to each of the volunteers who subsequently abstained from all mechanical plaque control measures for the following 18 days. During the first 4 days (rinse phase I), they rinsed with either 0.12% chlorhexidine, 0.06% triclosan or placebo solution. Clinical examinations (GCF, GI) were repeated and the amount of plaque formed determined on days 4, 7 and 14. On day 14, the participants received a new professional tooth cleaning after which rinse phase II was initiated. During this 2nd phase, the participants rinsed for 4 days with the same mouthwash preparation and in the same manner as during rinse phase I. The examinations were repeated on day 18. Each participant received a comprehensive oral prophylaxisis and was instructed to perform meticulous mechanical plaque control during the following 4 weeks. A 2nd experimental period was then initiated. A total of 3 experimental periods were repeated until all subjects had rinsed with the 3 different mouthwash preparations. The results demonstrated (i) that significantly more plaque formed at sites with gingivitis than at surfaces adjacent to healthy gingival units and (ii) pre-existing gingivitis significantly increased the amount of de novo plaque that formed in subjects who rinsed with mouthwash preparations containing chlorhexidine and triclosan.

Adult↗

PubMed1996

Some effects of mouthrinses containing salifluor on de novo plaque formation and developing gingivitis.

Three clinical trials were carried out to evaluate the effects of mouthrinses containing 5n-octanoyl-3'-trifluormethylsalicylanilide (salifluor) on plaque and gingivitis. Each trial was performed as a double-blind, randomised and cross-over designed study (studies 1, 2 and 3). In each study, 10 young individuals with healthy gingiva abolished all means of mechanical plaque control during the course of the experimental period including 6 x 4 days (study 1), 3 x 18 days (study 2) and 3 x 14 days (study 3). They rinsed, 2 x daily, with various mouthwash preparations for 4 days (study 1), for the last 4 days of a 18 day period (study 2) or for 14 days (study 3). 6 (control, vehicle control, 0.08%, 0.12% and 0.2% salifluor and 0.12% chlorhexidine), 3 (control, 0.12% salifluor and 0.12% chlorhexidine) and 3 (control, 0.12% salifluor and 0.12% chlorhexidine) mouthwash preparations were tested in studies 1, 2 and 3, respectively. The findings of study 1 indicated that (i) mouthrinses containing salifluor were significantly more effective than control rinses and that (ii) the salifluor mouthrinses were equally effective as the 0.12% chlorhexidine mouthrinse, in retarding 4-day de novo plaque formation. The findings of study 2 indicated that (i) the mouthrinse containing 0.12% salifluor retarded de novo plaque formation to the same extent as the 0.12% chlorhexidine mouthrinse at healthy as well as at inflamed sites but that (ii) the anti-plaque effects of the salifluor and chlorhexidine mouthrinses were significantly smaller at sites with inflamed than with healthy gingiva. In study 3, it was observed that there was no significant difference between the 0.12% salifluor and 0.12% chlorhexidine mouthrinses in retarding de novo plaque formation and the development of gingivitis during a 14-day period of no mechanical plaque control. Thus, the results of the 3 clinical trials demonstrated the potential of salifluor as an effective anti-plaque and anti-inflammatory agent.

Adult↗

PubMed1996

In vitro and in vivo studies on salifluor/PVM/MA copolymer/NaF combination as an antiplaque agent.

Salifluor (5-n-octanoyl-3'-trifluoromethyl-salicylanilide), a broad spectrum antimicrobial agent, was investigated for its ability to inhibit dental plaque formation. A combination of salifluor with PVM/MA copolymer and NaF was optimized for its antiplaque effect in mouthrinse and dentifrice formulations based on a series of both laboratory and clinical studies. It was found that salifluor, a highly hydrophobic compound, could not be adequately solubilized with the conventional amount of sodium lauryl sulfate (SLS), the most commonly used anionic surfactant in oral hygiene products. However, it was possible to prepare stable mouthrinse formulations using a mixed surfactant system containing both anionic and nonionic surfactants. The most suitable mixture was found to be a combination of SLS, Pluronic and Tauranol in a proportion of 1:1:1. This combination provided adequate stability and high antimicrobial activity as determined by in vitro microbiological tests. Addition of a PVM/MA copolymer to the formulation improved the adsorption and retention of salifluor on stimulated tooth surfaces in vitro (saliva coated hydroxyapatite disks) by almost two-fold and also increased the antiplaque efficacy in both laboratory and human clinical studies. It was also found that a non fluoride dentifrice containing a combination of salifluor and PVM/MA copolymer with a dicalcium phosphate dihydrate abrasive, was highly effective in reducing smooth surface and fissure caries in rats. The results of the present studies demonstrated that salifluor is an effective antiplaque agent in mouthrinse and dentifrice when carefully formulated to maximize its delivery and bioavailability on oral surfaces. They also illustrated the difficulties encountered in exploiting the antimicrobial efficacy of highly hydrophobic, nonionic antimicrobial agents such as salifluor in commonly used oral hygiene vehicles.

Adsorption↗

PubMed1996

HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae.

We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae.

Bacterial Outer Membrane Proteins↗

PubMed1996

Purification and characterization of a lectin from Amaranthus hypochondriacus var. Mexico seeds.

A lectin from Amaranthus hypochondriacus var. Mexico (AHML) was purified by affinity chromatography using asialofetuin-Sepharose 4B. AHML is specific for N-acetyl-D-galactosamine as are the other Amaranthus lectins. AHML has no carbohydrate moiety and requires no metal ion for the hemagglutination activity. The pI of AHML is 6.8. AHML has a native molecular mass of 45.0 kDa and is composed of homo-subunits having molecular masses of 36.8 kDa.

Amino Acids↗

PubMed1996

Characterization of a lectin from the leaves of great northern bean, Phaseolus vulgaris L.

A novel lectin (GNLL) was isolated from the leaves of the Great Northern bean, Phaseolus vulgaris. GNLL was purified by affinity chromatography on ovomucoid-Sepharose 4B. GNLL had a molecular mass of 135 kDa on gel filtration and gave two bands on SDS-polyacrylamide gel electrophoresis (PAGE)(band A of 34.0 kDa and band B of 34.2 kDa). Binding assay of horseradish peroxidase (HRP)-glycoproteins to the bands electroblotted onto polyvinylidene difluoride (PVDF) membrane showed that both bands could bind to complex-type N-linked oligosaccharide chains in glycoproteins. The N-terminal amino acid sequences of both bands were identical through the 10 residues and identical to that of alpha-subunit of a pod lectin (pod-alpha-subunit) from the same bean. On the other hand, band B cross-reacted with monoclonal antibody against a seed lectin from the same bean, but band A did not.

Amino Acid Sequence↗

PubMed1995

Reproducible alterations of DNA methylation at a specific population of CpG islands during blast formation of peripheral blood lymphocytes.

We investigated the changes in the methylation patterns of CpG islands associated with blast formation of human peripheral blood lymphocytes activated by anti-CD3 and interleukin-2 (IL-2), using restriction landmark genomic scanning with a methylation-sensitive restriction enzyme (RLGS-M) system. Of about 2,100 NotI spot/loci which were analyzed, only 10 showed changes, whereas drastic changes have been observed in cases of malignant and SV40 transformation. These changes were highly reproducible for samples from both the same and different individuals. Even the timing of the changes after cultivation was the same. Thus, we concluded that at least the genomic DNA methylation state in vivo was essentially retained in T blast cells activated in vitro by induction with IL-2 and anti-CD3, which are commonly used in biological experiments as well as clinical diagnosis and therapy.

CD3 Complex↗

PubMed1995

Characterization and gene cloning of 1,3-beta-D-glucan synthase from Saccharomyces cerevisiae.

1,3-beta-D-Glucan synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment. The specific activity of the partially purified enzyme was around 4 mumol glucose incorporated.min-1.mg protein-1. In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity. mAbs that could immunoprecipitate the 1,3-beta-D-glucan synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot. Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion. With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (glucan synthase of S. cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains. Disruption of each gene was not lethal, but disruption of both genes was lethal. The 1,3-beta-D-glucan synthase activities of membrane and partially purified enzyme of gsc1::URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.

Amino Acid Sequence↗

PubMed1995

Cloning of boar SPMI gene which is expressed specifically in seminal vesicle and codes for a sperm motility inhibitor protein.

Boar semen contains a seminal plasma motility inhibitor (SPMI) that blocks the motility of demembranated-reactivated spermatozoa as well as of intact spermatozoa. In this paper, we describe the primary structure of SPMI, the coding of boar SPMI cDNA gene and its expression in various porcine tissues. Nucleotide sequence analysis of the 645-bp SPMI cDNA predicts a coded polypeptide of 137 amino acid residues which includes a 21-residue signal peptide and a 116-residue secreted protein. The amino acid sequence of SPMI was found to be highly homologous to AQN-3, a member of spermadhesin family proteins of boar that bind to spermatozoa. Expression of the boar SPMI gene detected by Northern blot analysis revealed that its expression is very abundant in seminal vesicles and specific to this tissue.

Amino Acid Sequence↗

PubMed1995

Separable binding sites for the natural agonist endothelin-1 and the non-peptide antagonist bosentan on human endothelin-A receptors.

A three-dimensional model for the transmembrane domains of human endothelin-A receptor was built using structural information from bacteriorhodopsin and sequence alignment to other guanine-nucleotide-binding regulatory(G) protein-coupled receptors. Based on this model, 18 amino acids located at the inside of the receptor were mutated and analyzed for binding of the natural ligand endothelin-1 and bosentan, a recently described potent orally active endothelin antagonist [Clozel, M., Breu, V., Gray, G., Kalina, B., Löffler, B.-M., Burri, K., Cassal, J.-M., Hirth, G., Müller, M., Neidhart, W. & Ramuz, H. (1994) Pharmacological characterization of bosentan, a new potent orally active nonpeptide endothelin receptor antagonist, J. Pharmacol. Exp. Ther. 270, 228-235]. Mutation of Gly97, Lys140, Lys159, Gln165 and Phe315, located in transmembrane region 1, 2, 3, 3, and 6, respectively, caused reduced specific binding of 125I-labelled endothelin-1, despite an expression level similar to wild-type endothelin-A receptor. Mutation of Tyr263, Arg326 and Asp351 preserved endothelin-1 binding but caused reduced binding of bosentan. These amino acids, located on transmembrane regions 5, 6 and 7, respectively, are conserved among endothelin-A and endothelin-B receptors but not in other G-protein-coupled receptors. These observations demonstrate a dissociation of the binding site for the peptidic natural agonist endothelin-1 and the synthetic non-peptide antagonist bosentan. They provide the molecular basis for bosentan being a specific antagonist for both, endothelin-A as well as endothelin-B receptors and may in combination with studies on structure/activity relationship support the design of novel and more potent endothelin receptor antagonists.

Amino Acid Sequence↗

PubMed1995

Ligand binding domain of the human endothelin-B subtype receptor.

We have employed both protein chemical and molecular biological approaches to determine the ligand binding domain of the endothelin-B subtype (ETB) receptor. The human ETB receptor purified from human placenta by using affinity chromatography was cross-linked with 125I-labeled endothelin-1 (ET-1) and then incubated in the presence of trypsin or thermolysin under nondenaturing conditions. The N-terminal amino acid sequence of the radiolabeled polypeptide encompassed approximately 115 amino acid residues starting from Ile85 of the human ETB receptor. This was confirmed by experiments in which the binding activity of endothelin-1 to various chimeric endothelin receptors was monitored in the presence and absence of competitive endothelin receptor antagonists such as BQ-123 and bosentan. The region from Ile138 to Ile197 (60 amino acid residues) of the ETB receptor was found to interact with both antagonists. Therefore, this sequence was determined to be the ligand binding domain. In addition, we found that part of the N-terminal domain in close proximity to the first transmembrane region was required for the ligand binding activity of the ETB receptor, and the 12 amino acid residues from Ser390 to Leu401 at the proximal cytoplasmic tail are perhaps necessary to maintain the ligand binding site in active form. The cysteine rich region from residue 400 to residue 403 in the C-terminus of the ETB receptor is involved in coupling of the guanine nucleotide-binding regulatory protein for ET-1-induced signal transduction.

Amino Acid Sequence↗

PubMed1995

The effect of triclosan on developing gingivitis.

The aim of the study was to examine whether triclosan has an effect on developing gingival inflammation. 10 volunteers, with clinically healthy gingivae were enrolled. The study was performed as a 2-week, double-blind, cross-over, experimental gingivitis trial. Between each plaque accumulation period, there was a wash-out phase of 4 weeks. A baseline examination was performed which included assessment of plaque and gingivitis. The volunteers were asked to refrain from mechanical oral hygiene measures for 2 weeks. During this period, they rinsed 2x daily with one of the randomly assigned mouthrinse preparations. Solution A (period A): 0.06% triclosan+ 2%tween 80. Solution B (period B): 0.06% triclosan+ 0.25% sodium lauryl sulphate. Re-examinations were performed on days 4, 7, 11 and 14. The mean plaque score increased during period A to 2.2 (day 4), 2.8 (day 7), 3.1 (day 11) and 3.1 (day 14). The corresponding scores for period B were significantly lower; 1.2 (day 4), 1.8 (day 7), 2.0 (day 11) and 2.2 (day 14). The mean gingivitis scores at baseline were 0.17 (periods A and B). The mean gingivitis scores increased to 0.45 (day 4), 0.69 (day 7), 0.83 (day 11) and 0.96 (day 14) when the subjects rinsed with solution A and 0.42 (day 4), 0.64 (day 7), 0.78 (day 11) and 0.92 (day 14) in period B. There were no statistically significant differences between periods A and B with respect to gingivitis. Thus, although significantly more plaque formed during period A than period B, no differences could be found between the gingivitis scores in the 2 periods.

Adult↗

PubMed1995

[Prior enrichment of HIV-DNA with probe-DNA particles for an efficient PCR diagnosis].

PCR mediated detection of HIV-DNA has been widely used. However, compared with traditional immunological diagnoses, the extraction of DNA is a laborious and time consuming step. We have developed a procedure for the efficient isolation and concentration of HIV-DNA from cell lysates. We report here a novel method by one can recover HIV-DNA in a small volume (approximately 50 microliters) of solution from a large volume of crude cell lysate which contains as few as several copies. The method uses the specific hybridization of HIV-DNA to HIV probe-DNA particles. This prior enrichment augmented the sensitivity in the detection of HIV-DNA by PCR, and allows us to make a diagnosis even if the specimen contained an extremely low copy number of HIV-DNA molecules in a large volume, which would have otherwise resulted in false-negative data with the conventional extraction method. The method also enables the examination of 100 individual blood specimens in a combined form. Thus, the application of the present enrichment procedure with HIV probe-DNA particles should reduce the labor and cost of HIV diagnosis, since the HIV positive samples represent a very minor group of people among specimens subjected to clinical laboratory tests, and particularly, among blood samples voluntarily donated to be used for transfusions.

Base Sequence↗

PubMed1995

Prior enrichment of human immunodeficiency virus DNA with probe DNA particles for efficient PCR diagnosis.

We report here a novel method by which one can recover human immunodeficiency virus (HIV) DNA in a small volume (approximately 50 microliters) of solution from 8 ml of crude cell lysate which contains as few as several HIV DNA copies. The method uses the specific binding of HIV DNA to HIV probe DNA particles. The HIV DNA thus concentrated on the particles can be subjected to the PCR assay. The method also enables the examination of 100 individual blood specimens in a combined form.

Base Sequence↗