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Biomedical subjects

K Furukawa

Publications and source records attributed to K Furukawa.

At least 91 records · Page 5Linked to original sources
PubMed2000

Expression cloning of human globoside synthase cDNAs. Identification of beta 3Gal-T3 as UDP-N-acetylgalactosamine:globotriaosylceramide beta 1,3-N-acetylgalactosaminyltransferase.

By using a eukaryocytic cell expression cloning system, we have isolated cDNAs of the globoside synthase (beta1, 3-N-acetylgalactosaminyltransferase) gene. Mouse fibroblast L cells transfected with SV40 large T antigen and previously cloned Gb3/CD77 synthase cDNAs were co-transfected with a cDNA library prepared from mRNA from human kidney together with Forssman synthase cDNA, and Forssman antigen-positive cells were panned using an anti-Forssman monoclonal antibody. The isolated cDNAs contained a single open reading frame predicting a type II membrane protein with 351 amino acids. Surprisingly, the cDNA clones turned out to be identical with previously reported beta3Gal-T3, which had been cloned by sequence homology with other galactosyltransferases. Substrate specificity analysis with extracts from cDNA-transfected L cells confirmed that the gene product was actually beta1, 3-N-acetylgalactosaminyltransferase that specifically catalyzes the transfer of N-acetylgalactosamine onto globotriaosylceramide. Results of TLC immunostaining of neutral glycolipids from the cDNA-transfected cells also supported the identity of the newly synthesized component as globoside. The results show that glycosyltransferases apparently belonging to a single glycosyltransferase family do not necessarily catalyze reactions utilizing the same acceptor or even the same sugar donor. The globoside synthase gene was expressed in many tissues, such as heart, brain, testis, etc. We propose the designation beta3GalNAc-T1 for the cloned globoside synthase gene.

Amino Acid Sequence↗
PubMed2000

[Infections].

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Fever↗
PubMed2000

Molecular basis for the p phenotype. Identification of distinct and multiple mutations in the alpha 1,4-galactosyltransferase gene in Swedish and Japanese individuals.

p phenotype individuals lack both P(k) (Gb3) and P (Gb4) glycolipid antigens of the P blood group system. To explore the molecular basis for this phenotype, DNA sequences of Gb3 synthase (alpha1, 4-galactosyltransferase; alpha1,4Gal-T) in six p phenotype individuals from Japan and Sweden were analyzed. A missense mutation P251L and a nonsense mutation W261stop in three and one Japanese indivuiduals, respectively, and missense mutations M183K and G187D in one each of two Swedish p individuals were found, indicating that p individuals from Japan and Sweden have distinct and multiple homozygous point mutations in the coding region. In the function analysis of the mutated alpha1,4Gal-Ts by the transfection of the expression vectors, P251L and M183K mutations showed complete loss of enzyme function, and W261stop and G187D mutations resulted in the marginal activity. BLAST analysis of homologous sequences of alpha1, 4Gal-T revealed that three residues, Met(183), Gly(187), and Pro(251), at which missense mutations were found, were highly conserved among all species examined, suggesting their importance for the function of alpha1,4Gal-T.

Amino Acid Sequence↗
PubMed2000

Glibenclamide acts as an inhibitor of acyl-CoA:cholesterol acyltransferase enzyme.

Sulfonylureas are used in the treatment of non-insulin-dependent diabetes mellitus. Little is known, however, about their effects on cholesterol metabolism. We tested in the present study the effects of glibenclamide (GB) on cholesterol esterification (CE) in macrophage-derived cells. GB inhibited intracellular accumulation of CE induced by acetylated LDL or oxidized LDL in J774 cells, but no such effect on total cholesterol, suggesting that the target of GB was acyl-CoA:cholesterol acyltransferase (ACAT). In the cell-free reconstitution ACAT assay, GB inhibited the ACAT activity with an IC(50) value of 20 microM. Furthermore, GB effectively inhibited the ACAT activity of PMA-stimulated THP-1 cells to the undifferentiated level of THP-1. In the whole-cell ACAT assay using CHO cells overexpressed with ACAT-1 or ACAT-2, GB inhibited the activity of both isozymes with similar potency. Our in vitro data suggest that sulfonylurea could be a potential seed for a new generation of ACAT inhibitors.

Anilides↗
PubMed2000

Versatile transcription of biphenyl catabolic bph operon in Pseudomonas pseudoalcaligenes KF707.

Pseudomonas pseudoalcaligenes KF707 possesses a chromosomally encoded bph gene cluster responsible for the catabolism of biphenyl/polychlorinated biphenyls. The gene cluster consists of (orf0)bphA1A2(orf3)bphA3A4BCX0X1X2X3D. We studied the role of orf0 and transcription in the KF707 bph operon. Primer extension analyses revealed that at least as many as six transcriptional initiation sites exist upstream of orf0, bphA1, bphX0, bphX1, and bphD, including two upstream of bphD. The orf0-disruptant failed to grow on biphenyl but accumulated large amounts of the biphenyl ring meta-cleavage yellow compound (2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate). Western blot analysis revealed that ORF0 protein is inducibly expressed in KF707 in the presence of biphenyl. Gel shift assay revealed that ORF0 directly binds to the orf0 operator region. This binding was greatly enhanced by addition of the biphenyl ring meta-cleavage yellow compound. These results indicated that orf0, bphA1A2(orf3)bphA3A4BC and bphX0X1X2X3D are independently transcribed, and that ORF0 protein belonging to the GntR family is involved in the regulation of the bph operon in KF707 and is absolutely required for the expression of orf0 and bphX0X1X2X3D.

Amino Acid Motifs↗
PubMed2000

Correlated gene expression between beta-1,4-galactosyltransferase V and N-acetylglucosaminyltransferase V in human cancer cell lines.

Since our previous study showed that the gene expression level of beta-1,4-galactosyltransferase (beta-1,4-GalT) V is only increased in mouse NIH3T3 transformant and that beta-1,4-GalT V preferentially galactosylates the GlcNAcbeta1 --> 6Man branch of oligosaccharides [Shirane et al. (1999) Biochem. Biophys. Res. Commun. 265, 434-438], whether its gene expression is correlated with malignant transformation was investigated. Northern blot analysis of beta-1, 4-GalTs I, II, III, IV, V, and VI and N-acetylglucosaminyltransferase (GlcNAcT)V in human cancer cell lines showed that the gene expression levels of beta-1,4-GalT V but not other beta-1,4-GalTs are strongly correlated with those of GlcNAcT V whose activity was shown to increase by malignant transformation. These results indicate that beta-1,4-GalT V is involved in the galactosylation of highly branched oligosaccharides characteristic of malignantly transformed cells.

Blotting, Northern↗
PubMed2000

Degradation of human Aurora2 protein kinase by the anaphase-promoting complex-ubiquitin-proteasome pathway.

Human Aurora2 was originally identified by its close homology to yeast IPL1 and fly aurora, which are key regulators of chromosome segregation and a family of serine/threonine kinases. Here we demonstrate that the Aurora2 protein is degraded rapidly after G2/M phase release in mammalian cells. Aurora2 protein has a rapid turnover rate with a half-life of approximately 2 h. In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of unstable proteins. The treatment of mammalian cells with proteasome inhibitors blocks Aurora2 degradation. Furthermore, Aurora2 is polyubiquitinated in vivo and in vitro using anaphase-promoting complex (APC). These results demonstrate that Aurora2 protein is turned over through the APC-ubiquitin-proteasome pathway. Oncogene (2000) 19, 2812 - 2819

Anaphase-Promoting Complex-Cyclosome↗
PubMed2000

Molecular cloning of globotriaosylceramide/CD77 synthase, a glycosyltransferase that initiates the synthesis of globo series glycosphingolipids.

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (alpha1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed alpha1, 4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.

Amino Acid Sequence↗
PubMed2000

Toward new materials for organic electroluminescent devices: synthesis, structures, and properties of a series of 2,5-diaryl-3,4-diphenylsiloles

A series of 2,5-diaryl-3,4-diphenylsiloles, with various mono-substituted phenyl groups, extended pi-conjugated groups, and heteroaryl groups as aryl groups at the 2,5-positions, has been prepared by a one-pot synthesis from bis(phenylethynyl)silanes based on the intramolecular reductive cyclization followed by the palladium-catalyzed cross-coupling with aryl halides. Crystal structures and chemical reactivities toward the alkaline desilylation reactions have been studied on the 2,5-bis(p-mono-substituted phenyl)silole derivatives to elucidate the effects of the p-substituents. The UV-visible absorption and fluorescence spectra, and cyclic voltammetry of the 2,5-diarylsiloles have been systematically evaluated. Their photophysical properties as well as their electronic structures significantly depend on the nature of the 2,5-aryl groups.

Journal Article↗
PubMed2000

Loss of heterozygosity at 3p24-p25 as a prognostic factor in breast cancer.

Differences in clinical course and biological characteristics among breast cancers will probably be explained ultimately by variations in the pattern of genetic alterations among the many genes that can play roles in carcinogenesis. Loss of heterozygosity (LOH) of a particular chromosomal region in a tumor, which presumably indicates loss of a growth-regulating 'tumor-suppressor' gene in that region, may represent a useful marker for postoperative prognosis. In earlier work we observed LOH at chromosomal regions 3p14-p21 and/or 3p24-p25 in a large proportion of breast cancers. To examine whether allelic losses in either of those regions might correlate with postoperative survival, we tested tumors from a cohort of 504 breast cancer patients for allelic losses of microsatellite markers in the relevant portions of chromosome 3p. Five years postoperatively, patients whose tumors had undergone LOH at 3p24-p25 were found to have borne significantly higher risks of mortality than women whose tumors retained both alleles at that locus; i.e. the 5-year mortality rate was 22% among patients with losses at 3p24-p25 vs. 9% with retentions of heterozygosity at that locus (P=0.0014). These data indicate that LOH at 3p24-p25 is a significant predictive factor for postoperative survival of patients who have undergone surgery for breast cancer.

Adult↗
PubMed2000

A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes.

Protein conjugation, such as ubiquitination, is the process by which the C-terminal glycine of a small modifier protein is covalently attached to target protein(s) through sequential reactions with an activating enzyme and conjugating enzymes. Here we report on a novel protein conjugation system in yeast. A newly identified ubiquitin related modifier, Urm1 is a 99-amino acid protein terminated with glycine-glycine. Urm1 is conjugated to target proteins, which requires the C-terminal glycine of Urm1. At the first step of this reaction, Urm1 forms a thioester with a novel E1-like protein, Uba4. Deltaurm1 and Deltauba4 cells showed a temperature-sensitive growth phenotype. Urm1 and Uba4 show similarity to prokaryotic proteins essential for molybdopterin and thiamin biosynthesis, although the Urm1 system is not involved in these pathways. This is the fifth conjugation system in yeast, following ubiquitin, Smt3, Rub1, and Apg12, but it is unique in respect to relation to prokaryotic enzyme systems. This fact may provide an important clue regarding evolution of protein conjugation systems in eukaryotic cells.

Amino Acid Sequence↗
PubMed2000

Molecular cloning and expression of mouse GD1alpha/GT1aalpha/GQ1balpha synthase (ST6GalNAc VI) gene.

A novel member of the mouse CMP-NeuAc:beta-N-acetylgalactosaminide alpha2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc VI, was identified by BLAST analysis of expressed sequence tags. The sequence of the cDNA clone of ST6GalNAc VI encoded a type II membrane protein with 43 amino acids composing the cytoplasmic domain, 21 amino acids composing the transmembrane region, and 269 amino acids composing the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III, IV, and V, with common amino acid sequences in sialyl motif L and S among these four enzymes. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc VI in an expression vector showed enzyme activity of alpha2,6-sialyltransferase for GM1b, GT1b, and GD1a but not toward glycoproteins. Thin layer chromatography-immunostaining revealed that the products were GD1alpha, GQ1balpha, and GT1aalpha. Northern blotting revealed that this gene was expressed in a wide range of mouse tissues such as colon, liver, heart, spleen, and brain. It is concluded that this enzyme is a novel sialyltransferase involved in the synthesis of alpha-series gangliosides in the nervous tissues and many other tissues.

Amino Acid Sequence↗
PubMed2000

GD3 synthase gene expression in PC12 cells results in the continuous activation of TrkA and ERK1/2 and enhanced proliferation.

A rat pheochromocytoma cell line (PC12) transfected with ganglioside GD3 synthase gene showed a marked change in the ganglioside profile and enhanced proliferation and no response of neurite extension to nerve growth factor (NGF) stimulation. In these transfectant cells, a continuous phosphorylation of TrkA and the activation of ERK1/2 without NGF treatment were observed. Proliferation inhibition experiments with kinase inhibitors such as herbimycin A, K-252a, and PD98059 revealed that the enhanced proliferation was actually due to the activation of the Ras/MEK/ERK pathway. A TrkA dimer was detected in the GD3 synthase transfectant cells regardless of NGF treatment by cross-linking and immunoblotting. The increased expression of GD1b and GT1b in these transfectant cells might induce the conformational change of TrkA to form a dimer and to be activated continuously. These results may indicate regulatory roles of gangliosides in cell proliferation under physiological and malignant processes.

Animals↗
PubMed2000

Inhibition by antisense oligonucleotides of plasma membrane Ca(2+) ATPase in vascular endothelial cells.

Antisense oligodeoxynucleotides were used to knock down plasma membrane Ca(2+) ATPase, and the role of plasma membrane Ca(2+) ATPase was investigated in human aortic endothelial (HAE) cells. The peak of thapsigargin-evoked intracellular Ca(2+) concentration ([Ca(2+)](i)) was higher in antisense-treated than in untreated cells, but the declining time course was unaffected by the antisense treatment. The declining time was prolonged in both antisense-treated and untreated cells by reducing external Na(+), but the prolongation was more marked in the antisense-treated cells. These results provide the evidence of a functional role of plasma membrane Ca(2+) ATPase, although other mechanisms including Na(+)/Ca(2+) exchange may play the primary role in regulating [Ca(2+)](i).

Calcium↗
PubMed2000

Pro-apoptotic effects of tau mutations in chromosome 17 frontotemporal dementia and parkinsonism.

It was recently discovered that mutations of tau cause hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we report that cultured SH-SY5Y human neuroblastoma cells transfected with mutated tau genes are more vulnerable to apoptotic stimulus. Two kinds of mutations of tau causing FTDP-17 were examined in the present study: one was in exon 10 (N279K) and the other was in exon 12 (V337M). SH-SY5Y cells transfected with either mutated tau were more vulnerable to serum withdrawal, whereas cells transfected with the wild-type tau or vector alone showed no significant change in apoptotic vulnerability. The increase in the intracellular calcium concentration by the serum withdrawal was significantly greater in the SH-SY5Y cells transfected with mutated tau genes than in cells transfected with the wild-type tau or vector alone. These results suggest that mutations of tau might cause FTDP-17 by these pro-apoptotic functions by disrupting the intracellular calcium homeostasis.

Apoptosis↗
PubMed2000

Augmentation of somato-sympathetic reflex in the ischemic hindlimb of anesthetized rats.

Activities of the visceral sympathetic nerve increase with noxious mechanical stimulation. This study investigated the effects of noxious mechanical stimulation of hindlimb on arterial blood pressure, heart rate (HR) and renal sympathetic nerve activities (RNA) in anesthetized rats intact or under ischemic conditions. This study utilized two methods of noxious mechanical stimulation. One was pinching of the hindpaw; the other, strangulation of the ankle joint. Twenty-three male Wistar rats were used in the experiment. Pinching and strangulation of the intact hindlimb caused an increase in RNA. The increased RNA was greater during strangulation than during pinch stimulation (p < 0.0001). In the ischemic hindlimb produced by occlusion of the left common iliac artery, pinching and strangulation caused a further increase in RNA (p < 0.0001). Arterial blood pressure and HR significantly increased during strangulation with ischemic condition (p < 0.05). These results suggest that the reflex effects of somatosensory input on RNA were augmented in the ischemic hindlimb.

Analysis of Variance↗