Biomedical subjects

Y Furuichi

Publications and source records attributed to Y Furuichi.

At least 127 records · Page 7Linked to original sources

PubMed1995

Structure and activity of HYI killer toxin from Hansenula saturnus.

The primary structure of HYI killer toxin produced by Hansenula saturnus was determined in its reduced and pyridylethylated form, as well as the peptides resulting from protease digestion. It was found that the HYI killer toxin consisted of 87 amino acid residues and the molecular weight was calculated to be 9543 Da. It showed 87% homology with HM-1 killer toxin produced by H. mrakii, while there were multiple mutations including one amino acid deletion which, nevertheless, did not alter the strong cytocidal effect on sensitive yeasts.

Amino Acid Sequence↗

PubMed1995

Modulation of glomerular endothelin and endothelin receptor gene expression in aminonucleoside-induced nephrosis.

This study assessed glomerular endothelin (ET)-1, ET-3, and ET-receptor A and B mRNA levels in puromycin aminonucleoside (PAN)-induced nephrosis. During the nephrotic stage, 8 days after PAN injection, ET-1 and ETB receptor mRNA were elevated by 2.8 +/- 0.8-fold (P < 0.01) and 2.4 +/- 0.9-fold (P < 0.01), respectively, as compared with controls. These mRNA levels decreased to control levels by Day 20, when the nephrosis was in remission. In contrast, glomerular ETA receptor mRNA levels did not change in PAN nephrosis or control rats during the experimental period. ET-3 mRNA was not detected in the glomeruli of PAN nephrosis or control rats. Additionally, plasma ET concentration and glomerular ET production were measured in PAN nephrosis and control rats by radio-immunoassay. Eight days after PAN injection, ET-1 levels in plasma and glomeruli were not significantly altered in rats with PAN-induced nephrosis (glomeruli, 104.68 +/- 16.46 pg/mg of protein versus 98.24 +/- 13.68 pg/mg of protein; plasma, 2.68 +/- 1.10 versus 2.52 +/- 0.98 pg/mL). The administration of methylprednisolone to PAN rats resulted in the rapid disappearance of proteinuria and partially attenuated the increased ET-1 and ETB receptor gene expression in the glomeruli. These data indicate that glomerular ET-1 and ETB receptor expression in PAN nephrosis in increased at the mRNA level and that methylprednisolone treatment results in an attenuated increase.

Animals↗

PubMed1995

Cell wall synthesis specific cytocidal effect of Hansenula mrakii toxin-1 on Saccharomyces cerevisiae.

HM-1 toxin produced by Hansenula mrakii kills sensitive Saccharomyces cerevisiae. We found that the budding cells and the cells that responded to mating factor were sensitive to HM-1 toxin. These findings indicate that the target sites of HM-1 toxin are developing buds and conjugating tubes. The in vitro activity of beta-1,3-glucan synthase solubilized and partially purified from S. cerevisiae membranes was inhibited by HM-1 toxin at a concentration (around 50 nM, 0.5 micrograms/ml) that coincided well with its minimum inhibitory concentration for the growth of yeast cells. These data indicate that the HM-1 toxin perturbs the synthesis of yeast cell walls by inhibiting the glucan synthesis occurring at a budding site or a conjugating tube, which results in cell lysis.

Cell Cycle↗

PubMed1994

Identification of a region of the human endothelin ETA receptor required for interaction with bosentan.

Bosentan (Ro 47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2- methoxy-phenoxy)-2,2'-bipyrimidin-4-yl]-benzenesulfonamide) is a new non-peptidic mixed antagonist of endothelin receptors whose binding activity was two orders higher for the endothelin ETA receptor than that for the endothelin ETB receptor. To identify which region of the human endothelin ETA receptor interacts with bosentan, we created various chimeric endothelin receptors containing domains from the endothelin ETA and ETB receptors in Chinese hamster ovary cells and studied the effect of bosentan on the binding of endothelin-1 to the chimeric receptors. We found that the chimeric endothelin ETB receptor containing domains from the endothelin ETA receptor, the second extracellular region including the proximal transmembrane region (B-region) revealed an affinity toward bosentan which was similar to that of the endothelin ETA receptor. In contrast, the chimeric endothelin ETA receptor, containing the B-region of the endothelin ETB receptor, reduced the binding affinity to the level of the endothelin ETB receptor. Since bosentan competes with endothelin-1 for binding to the endothelin ETA receptor, this receptor antagonist seems to interact with the (140-144) KLLAG sequence located at the carboxylterminus of the second transmembrane region of the endothelin ETA receptor, required for the natural ligand binding.

Amino Acid Sequence↗

PubMed1994

Down-regulation of mitochondrial gene expression by the anti-tumor arotinoid mofarotene (Ro 40-8757).

To understand the mechanism of action of the antitumor arotinoid mofarotene (Ro 40-8757), differential screening of cDNA libraries with cDNA probes prepared from treated or untreated breast-cancer cells was performed. Several genes were identified that appeared to be regulated by mofarotene, including a mitochondrial gene encoding a subunit of NADH dehydrogenase (NDI). This gene was down-regulated in the breast-cancer cell line MDA-MB-231 after treatment with the arotinoid for 3 to 6 hr. Down-regulation of NDI was detected in 2 other breast-carcinoma cell lines (ZR-75-I and MCF-7) and a pancreatic cancer cell line (BxPC3), but not in the normal fibroblast cell line Wi-38 or several other tumor cell lines. This effect was blocked by addition of cycloheximide to the medium. The retinoids, all-trans and 9-cis retinoic acids, did not affect the expression of NDI in MDA-MB-231 cells, demonstrating that mofarotene was not acting through the nuclear retinoic-acid receptors. In the estrogen-receptor-expressing breast-cancer line ZR-75-I, tamoxifen had no effect on NDI expression. The cytotoxic drugs doxorubicin, 5-FU and vincristine also had no effect on regulation of this gene. Two mitochondrial proteins encoded in the nucleus, ATPase beta subunit and mitochondrial transcription factor I, were not down-regulated by mofarotene. Addition of mofarotene to cells incubated in glucose-free medium led to their death. These results indicate that down-regulation of mitochondrial gene transcription is specific to mofarotene and may explain, in part, the anti-proliferative effects of this compound.

Antineoplastic Agents↗

PubMed1994

Identification of specific regions of the human endothelin-B receptor required for high affinity binding with endothelin-3.

To investigate the endothelin-3 (ET-3) binding region of the endothelin-B (ETB) receptor, we have transiently produced various chimeric endothelin receptors in transfected Chinese hamster ovary cells. Using 125I-ET-1 as the radioactive ligand in the displacement experiment, the replacement of both the second and third extracellular regions including the flanking transmembranes of the ETB receptor with the corresponding domains of the endothelin-A (ETA) receptor, increased the apparent Ki value for ET-3 from 5 x 10(-11) M to 10(-8) M. The affinity of this chimeric receptor, ETB-BC, for ET-3 was about two orders lower than ETB yet one order higher than ETA. Previously we have reported the involvement of Lys-140 located in the C-terminus of the second transmembrane region of the ETA receptor for ET-1 binding (Eur. J. Biochem., 220, 37-43, 1994). To assess the importance of the corresponding Lys-161 of the ETB receptor in ET-3 binding, we have replaced it with Ile in the ETB receptor. The mutant receptor had a 5.6-fold reduction in its affinity for ET-3, but its affinity for ET-1 remained similar. These results demonstrate that Lys-161 of the receptor is important for high affinity binding with ET-3 which, in part, confers the non-selective binding characteristics of the ETB receptor for ET isopeptides.

Amino Acid Sequence↗

PubMed1994

Involvement of cell wall beta-glucan in the action of HM-1 killer toxin.

HM-1 killer toxin secreted from Hansenula mrakii inhibits the growth of Saccharomyces cerevisiae cells by interfering with beta-1,3-glucan synthesis. We found that HM-1 killer toxin killed intact cells but not protoplasts. In addition, cells lacking the functional KRE6 allele (kre6 delta) became resistant to higher concentration of HM-1 killer toxin. As reported by Roemer and Bussey [(1991) Proc. Natl. Acad. Sci. 88 11295-11299], cells lacking functional KRE6 had a reduced level of the cell wall beta-1,6-glucan compared to that in cells harboring the normal KRE6. These results suggest that the cell wall beta-glucan is involved in the action of HM-1 killer toxin. Addition of HM-1 killer toxin with several kinds of oligosaccharides revealed that either beta-1,3- or beta-1,6-glucan blocked the cytocidal action of HM-1 killer toxin whereas alpha-1,4-glucan and chitin did not. Mannan also interfered with HM-1 killer toxin action, but this inhibitory effect was much weaker than that observed with beta-1,3- or beta-1,6-glucans. Thus, it appears that the cell wall beta-glucan interacts with HM-1 killer toxin, and that this toxin-beta-glucan commitment is required for the action of HM-1 killer toxin.

Cell Wall↗

PubMed1994

Human endothelin receptor ETB. Amino acid sequence requirements for super stable complex formation with its ligand.

ETB type endothelin receptor (ETBR) forms a stable complex with its ligand endothelin-1 (ET-1). The ligand-receptor complex can survive in the presence of 2% SDS and migrate in SDS-polyacrylamide gel electrophoresis at reduced temperature. This is not the case for the ETA type endothelin receptor (ETAR). Most of ETBR from various animals, except rat, can form such a stable complex. Examination of stability in the ligand-receptor complex which was formed by chimeric receptors constructed from human ETBR and rat ETBR demonstrated that the region containing 29 amino acids in the N-terminal extracellular domain is responsible for the formation of such a stable complex. Site-directed mutagenesis focused on this particular region revealed that Asp75 and Pro93 are involved in the formation of the stable complex. This unique observation contributes to the elucidation of the structure of the stable ETBR.ET-1 complex at the molecular level.

Amino Acid Sequence↗

PubMed1994

Stimulation of calf thymus DNA polymerase alpha activity by nucleolar protein B23.

Protein B23 is a major RNA-associated nucleolar protein and putative ribosome assembly factor which exists in at least two isoforms designated B23.1 and B23.2. Recently, it has been reported that B23 is copurified with DNA polymerase alpha-primase complex. To examine its possible role in DNA replication, the effects of B23 on DNA polymerase activities were investigated. B23.1 purified from rat Novikoff hepatoma ascites cell nucleoli stimulated the activity of DNA polymerase alpha by as much as 3-to 4-fold in a dose-dependent manner, while it showed little effect on the activities of DNA polymerase beta, gamma, and primase. Rat recombinant B23.1 showed the same stimulation as that of B23.1 from Novikoff cells. In contrast, isoform B23.2 showed no effect on the activity of DNA polymerase alpha, suggesting that C-terminal region of B23.1 is important in its activity in the stimulation of DNA polymerase alpha.

Animals↗

PubMed1994

Identification of a ligand-binding site of the human endothelin-A receptor and specific regions required for ligand selectivity.

To investigate the ligand-binding site of the human endothelin-A-receptor subtype (ETA), we have produced various chimeric and mutated receptors in chinese hamster ovary cells. The substitution of Lys140 with Ile located in the C-terminus of the second transmembrane region caused a 13-fold reduction in affinity for endothelin-1 (ET-1) and 3.6-fold lower Bmax than those values for the original receptor. Correspondingly, the mutated ETA receptor with the Lys140-->Ile substitution failed to induce an increase in the intracellular calcium concentration in the presence of 1 nM ET-1. Thus, the Lys140 in the ETA receptor is important in ligand binding. ETA and ETB receptors possess the ET isopeptides selective and non-selective binding activities, respectively. Displacement experiments and the binding of 125I-ET-3 to various chimera receptors demonstrated that both the third and fourth extracellular regions, including the flanking transmembrane regions, are responsible for the ligand-binding selectivity of the ETA receptor.

Amino Acid Sequence↗

PubMed1994

Spot mapping on the standard profile of restriction landmark genomic scanning (RLGS) of sorted chromosome 20 using methylation-insensitive enzyme.

We established the spot mapping system on a restriction landmark genomic scanning (RLGS) profile using sorted chromosome as RLGS material. In this mapping system, we can mapped RLGS spots physically, regardless of their polymorphism, using methylation-insensitive enzymes in all RLGS steps. Here, we report that we identified 28 spots derived from human chromosome 20 on an RLGS profile, and that number was in good agreement with the number predicted from the length of the chromosome 20.

Cell Line↗

PubMed1994

Cloning of the Saccharomyces cerevisiae gene whose overexpression overcomes the effects of HM-1 killer toxin, which inhibits beta-glucan synthesis.

A gene whose overexpression can endow Saccharomyces cerevisiae cells with resistance to HM-1 killer toxin was cloned from an S. cerevisiae genomic library. This gene, designated HKR1 (Hansenula mrakii killer toxin-resistant gene 1), contains a 5.4-kb open reading frame. The predicted amino acid sequence of the protein specified by HKR1 indicates that the protein consists of 1,802 amino acids and is very rich in serine and threonine, which could serve as O-glycosylation sites. The protein also contains two hydrophobic domains at the N-terminal end and in the C-terminal half, which could function as a signal peptide and transmembrane domain, respectively. Hkr1p is found to contain an EF hand motif of the calcium-binding consensus sequence in the C-terminal cytoplasmic domain. Thus, Hkr1p is expected to be a calcium-binding, glycosylated type I membrane protein. Southern and Northern (RNA) analyses demonstrated that there is a single copy of the HKR1 gene in the S. cerevisiae genome, and the transcriptional level of HKR1 is extremely low. Gene disruption followed by tetrad analysis showed that HKR1 is an essential gene. Overexpression of the truncated HKR1 encoding the C-terminal half of Hkr1p made the cells more resistant to HM-1 killer toxin than the full-length HKR1 did, demonstrating that the C-terminal half of Hkr1p is essential for overcoming the effect of HM-1 killer toxin. Furthermore, overexpression of HKR1 increased the beta-glucan content in the cell wall without affecting in vitro beta-glucan synthase activity, suggesting that HKR1 regulates beta-glucan synthesis in vivo.

Amino Acid Sequence↗

PubMed1994

Effect of nilvadipine on the development of neurological deficits in stroke-prone spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: Several types of antihypertensive drugs have been reported to protect stroke-prone spontaneously hypertensive rats from stroke. However, the clinical relevance remains unclear. This study was performed to investigate the effect of nilvadipine, a calcium channel blocker, on the development of neurological deficits in stroke-prone spontaneously hypertensive rats. In addition, plasma levels of nilvadipine were measured to determine the clinical relevance. METHODS: Salt-loaded stroke-prone spontaneously hypertensive rats were orally administered nilvadipine mixed with a powder diet (0.01% and 0.03%, wt/wt). Non-salt-loaded rats were maintained on tap water. Chronological changes in neurological deficit scores and systolic blood pressure were recorded. After 6 weeks of medication, measurement of plasma levels of nilvadipine, serum biochemical analysis, and pathological observation of both the brain and the kidney were performed. RESULTS: In the salt-loaded control group, both severe hypertension and neurological deficit developed, and the final survival rate was 30%. Systolic blood pressure decreased significantly in the high-dose nilvadipine-treated group but not in the low-dose nilvadipine-treated group. However, the development of neurological deficit was almost completely inhibited in both nilvadipine-treated groups that had no deaths (P < .01). The mean plasma levels of nilvadipine in the low-dose group and in the high-dose group at the time of death were 0.21 ng/mL and 0.61 ng/mL, respectively. CONCLUSIONS: Nilvadipine inhibited the development of neurological deficit in stroke-prone spontaneously hypertensive rats at plasma concentrations lower than that in clinical use. Thus, nilvadipine might prevent cerebral vascular disorders at doses routinely used for essential hypertension.

Animals↗

PubMed1994

Differential regulation of c-fos gene expression by two types of human endothelin receptor in Chinese hamster ovary cells.

To investigate the nuclear signalling pathway induced by endothelin (ET) isopeptides, we have established permanent Chinese hamster ovary (CHO) cell lines, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ, that produce both a c-fos-beta-galactosidase fusion protein and either the type A or the type B human ET receptor. These cell lines permitted a colorimetric measurement of c-fos expression, which was induced by the signal transduction system with ET receptors and ET isopeptides. We found that the ET-1-dependent c-fos expression was so efficient that it could respond to low concentrations (even a physiological concentration) of ET-1. For example, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ responded to ET concentrations of 5 x 10(-9) M and 5 x 10(-13) M respectively. Using this highly sensitive system, the H-7 sensitive protein kinase was found to be involved in signal transduction mediated by ETA, and also partly in the ETB-mediated pathway. These lines of evidence suggest that c-fos expression occurs through at least two different pathways, depending on the concentration of ET in plasma.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1993

Purification and characterization of novel lectins from Great Northern bean, Phaseolus vulgaris L.

Two lectins, GNL-1 and 2, were isolated from extracts of Great Northern bean powder through fractionation with ammonium sulfate, ion-exchange chromatographies on CM- and DEAE-celluloses, and gel filtration chromatography on Sephacryl S-200 HR. These lectins were shown to be homogenous by gel electrophoresis, gel filtration, and isoelectric focusing. The lectins (GNL-1 and 2) have molecular masses of 175 and 145 kDa on gel filtration, respectively. They yield three bands having the respective same molecular masses on SDS-PAGE (GNL-1; alpha-subunit of 34.5 kDa, beta of 37.0, and gamma of 39.0: GNL-2; alpha' of 34.5 kDa, beta' of 37.0, and gamma' of 39.0). Two lectins are shown to be glycoproteins and the carbohydrate contents of GNL-1 and 2 are 5.1 and 4.5%, respectively. The isoelectric points are 5.5 and 5.1 and the extinction coefficients (A 1cm 1%) at 280 nm are 11.37 and 11.45, respectively. These lectins are nonspecific in agglutination for rabbit and any types of human erythrocytes. Inhibition study shows no specificity against mono and disaccharides. On the other hand, binding assay of horseradish peroxidase-glycoproteins to the bands electroblotted onto PVDF membrane reveals that all of the subunits can bind to sugar moieties in fetuin, asialofetuin, and porcine thyroglobulin specifically. Moreover, assay of mitogenic activity shows that GNL-1 is a strong mitogen, but GNL-2 is lack of the activity.

Amino Acid Sequence↗

PubMed1993

Application of oligo(dT)30-latex for rapid purification of poly(A)+ mRNA and for hybrid subtraction with the in situ reverse transcribed cDNA.

The carboxyl groups on the surface of latex beads were linked to amino moiety of cytidine residue of oligo(dC)10(dT)30. The resultant latex beads-(dC)10(dT)30 showed a very stable suspension and yet is precipitable to a small pellet by centrifugation. These properties merits the oligomer-linked beads to be applied for experiments in which poly(A)+ mRNAs are involved. An efficient (> 95%) hybridization to poly(A)+ mRNA occurred in a short reaction period (10 min), and more than 95% of bound mRNAs were recovered from the beads by heating (65 degrees C, 5 min) followed by centrifugation. Interestingly, the poly(A)+ mRNAs could be transcribed to cDNAs in situ by reverse transcriptase, with the covalently linked oligo(dT)30 as primers. These properties allowed the oligo(dT)30-latex to prepare the cDNA covalently bound to latex which was used for mRNA hybrid subtraction. In a model experiment with the mixture of vaccinia virus and HeLa mRNAs, about 200-fold enrichment of vaccinia mRNA species was obtained after four cycles of hybrid subtraction with HeLa cDNA-latex.

DNA↗

PubMed1993

Molecular characterization of the 5'-flanking region of human genomic ETA gene.

A clone containing the promoter, the exon 1 and exon 2 of human genomic ETA gene was isolated and sequenced. The transcription initiation site and TATA box were identified at 528 bp upstream of the initiation codon and 24 bp upstream of the transcription initiation site, respectively. The 534 bp fragment containing 5'-flanking sequence and most of the exon 1 of the human ETA gene showed a promoter activity corresponding to 55% of SV40 early promoter activity when placed upstream of the luciferase gene and transfected into CHO cells. Thus, the TATA box which consists of the TAAAAA sequence is functional for transcription of the human ETA gene. The coding region of the human ETA receptor, therefore, starts from exon 2 which contained the first and the second transmembrane regions. The human ETA gene was demonstrated to be a single pair of copy by Southern blot analysis, and it was localized in chromosome 4 by analysis with a panel of human-rodent somatic cell hybrid lines.

Amino Acid Sequence↗

PubMed1993

Expression in Escherichia coli of the cloned polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus.

Cloned cDNA of genomic segment 10 of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was placed downstream from the lambda PL promoter in expression plasmid pRC23 and expressed in Escherichia coli cells. A polypeptide of the same molecular weight (28 kDa) as natural polyhedrin was synthesized at the level of approximately 10% of total host cell protein. This polypeptide was identified as CPV polyhedrin (r-polyhedrin) after comparative studies. The r-polyhedrin did not form any crystalline structure in E. coli cells but instead accumulated in the form of an insoluble inclusion body, even though natural polyhedrin is known to form a crystalline matrix (polyhedra) in infected insect cells. The purified r-polyhedrin complex, like natural polyhedra, was not soluble in neutral or acidic buffer but soluble in alkaline buffer. Upon solubilization, the r-polyhedrin complex did not undergo proteolytic degradation, while natural polyhedra were digested into small peptides by the associated protease. Incubation of r-polyhedrin with natural polyhedra in alkaline buffer, however, degraded the r-polyhedrin, resulting in an identical profile of peptide products to that of natural polyhedra. These results indicate that even though r-polyhedrin molecules produced in E. coli cells are not in the natural conformation, the molecules can present the identical cleavage sites to the polyhedra-associated alkaline protease. Experiments showed that the alkaline protease was associated with the matrix of polyhedra and not with virus particles.

Alkalies↗