Biomedical subjects

M Shibuya

Publications and source records attributed to M Shibuya.

At least 73 records · Page 4Linked to original sources

PubMed2000

A new one-Pot method for the synthesis of alpha-siloxyamides from aldehydes or ketones and its application to the synthesis of (-)-bestatin

A new one-pot method for the synthesis of alpha-siloxyamides is described. The three substrates, H-C(CN)(2)O-SiMe(2)t-Bu, aldehydes or ketones, and primary or secondary amines, are simply mixed in one portion in acetonitrile or ether; the alpha-siloxyamides are obtained within short peroids in excellent yields in many cases. As a demonstration of our method, the synthesis of (-)-bestatin was carried out.

Journal Article↗

PubMed2000

Hemorheological abnormalities in experimental cerebral ischemia and effects of protein kinase inhibitor on blood fluidity.

The aim of this study was to investigate the mechanisms of the pathogenesis of hyperviscosity following cerebral ischemia. Focal ischemia was produced by embolic occlusion of the right middle cerebral artery (MCA) in rats for 1 hour, followed by recirculation. Twenty-four hours after MCA occlusion, fasudil, a protein kinase inhibitor, was administered intraperitoneally. Blood samples were taken from the abdominal aorta, and viscosity was measured using a cone-plate viscometer. The viscosity of whole blood in the ischemic attack group was significantly increased compared with the sham operated group 24 hours after MCA occlusion. Fasudil dose-dependently and significantly decreased the blood viscosity, and reduced to the normal range after administration of 10 mg/kg of fasudil (sham-operated rats, 5.17+/-0.05 cP; pre dose/ischemic rats, 6.05+/-0.08 cP; post dose/ischemic rats, 5.23+/-0.14 cP; 37.5 sec(-1)). Our findings suggest that cerebral ischemia induces a potent, systemic and long-lasting hyperviscosity, and that the inhibition of protein kinases, especially rho kinase, is efficacious in preventing this hyperviscosity.

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

PubMed2000

Identification of amino acid residues important in the cyclization reactions of chalcone and stilbene synthases.

Chalcone synthase (CHS) and stilbene synthase (STS) catalyse condensation reactions of p-coumaroyl-CoA and three C(2) units from malonyl-CoA up to a common tetraketide intermediate but then catalyse different cyclization reactions to produce naringenin chalcone and resveratrol respectively. On the basis of sequence alignment with other condensing enzymes including 3-ketoacyl-(acyl carrier protein) synthases of polyketide and fatty-acid synthases, site-directed mutagenesis was performed on the active-site G(372)FGPG loops in CHS and STS. The CHS-P375G mutant showed a 6-fold decrease in overall condensing activity with selectively increased production of p-coumaroyltriacetic acid lactone (CTAL, the derailment product of the tetraketide intermediate). Meanwhile, resveratrol production by STS-P(375)G strongly decreased to give various products in the order CTAL> resveratrol approximately bisnoryangonin>naringenin. As a result, naringenin production (cross-reaction) by STS-P(375)G was close to 30% of resveratrol production. Both G(374)L mutants of CHS and STS showed no condensing activity with residual malonyl-CoA decarboxylase activity. These results suggested that the G(372)FGPG loop in CHS and STS contribute to a determination of the outcome during cyclization reactions by serving as a part of the active-site scaffold on which the stereochemistry of cyclization is performed. These observations provide the first biochemical indication that cyclization reactions are modulated by active-site geometry. The implications for the evolutionary relationship of these enzymes are also discussed.

Acyltransferases↗

PubMed2000

In vitro processing of human immunodeficiency virus type 1 Gag virus-like particles.

Human immunodeficiency virus (HIV) Gag proteins are assembled into virus particles and then cleaved by the virion-associated HIV protease. Concomitant with Gag processing, doughnut-like HIV particles (the immature form) are converted to particles containing condensed cores (the mature form). Here we describe the in vitro processing of immature HIV Gag virus-like particles (VLP) by exogenously added HIV protease. Following delipidization, sequential processing of immature VLP showed that the matrix (MA)/capsid (CA) junction was cleaved faster than the CA/nucleocapsid (NC) junction, an altered order of processing when compared with authentic processing. When the in vitro processed VLP were analyzed on density gradients, most of the MA, CA-p15 intermediate, and NC were detected as a highly multimeric form, equivalent to the unprocessed VLP. In contrast, CA was found as a monomer dissociated from the multimeric CA-p15 following cleavage of the CA/NC junction. Electron microscopy revealed that the in vitro processing was accompanied by conversion of the doughnut-like particles to particles containing condensed cores and spherical outer shells. The cores, however, lacked core shells, which are normally observed for authentic HIV, suggesting that the in vitro processing of immature VLP failed to produce core shells.

Animals↗

PubMed2000

A conformationally reliable spacer for molecular tweezers

[figure: see text] A reliable and rigid spacer, trans,trans,trans-perhydronaphthacene, for molecular tweezers was designed. A synthesis and molecular recognition study of its 1,14-disubstituted derivatives was carried out.

Journal Article↗

PubMed2000

Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a principal regulator of vasculogenesis and angiogenesis. VEGF expresses its effects by binding to two VEGF receptors, Flt-1 and KDR. However, properties of Flt-1 and KDR in the signal transduction of VEGF-mediated effects in endothelial cells (ECs) were not entirely clarified. We investigated this issue by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF elicits DNA synthesis and cell migration of human umbilical vein endothelial cells (HUVECs). The pattern of inhibition of these effects by two mAbs indicates that DNA synthesis is preferentially mediated by KDR. In contrast, the regulation of cell migration by VEGF appears to be more complicated. Flt-1 regulates cell migration through modulating actin reorganization, which is essential for cell motility. A distinct signal is generated by KDR, which influences cell migration by regulating cell adhesion via the assembly of vinculin in focal adhesion plaque and tyrosine-phosphorylation of focal adhesion kinase (FAK) and paxillin.

3T3 Cells↗

PubMed2000

Somatic mutation of the hBUB1 mitotic checkpoint gene in primary lung cancer.

Mutations in mitotic checkpoint genes have been detected in several human cancers, and these cancers exhibit chromosomal instability. Aneuploid stem cells seem to result from chromosomal instability and have been reported in many lung cancers. To determine whether alteration of mitotic checkpoint regulators is involved in carcinogenesis and tumor progression in primary lung cancer, we screened the genomic DNA sequence of 30 human lung cancer cell lines and 30 primary lung cancer tumors for a mutation in the hBUB1 mitotic checkpoint gene. First, we designed 26 sets of intron-based primers to amplify each of the 25 exons of the hBUB1 gene to examine the entire coding region of the hBUB1 gene. Using these primers, we performed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis as well as direct sequencing in the mutation analysis of the hBUB1 gene. Three different nucleotide substitutions were detected in the coding region of the hBUB1 gene in some of the cancer cell lines and primary tumors as follows. The hBUB1 gene of one adenocarcinoma tumor contained a somatic missense mutation, a cytosine-to-guanine substitution in codon 51 of exon 5 that resulted in a histidine-to-aspartic acid amino acid substitution. The hBUB1 gene of three lung cancer cell lines contained a thymine-to-cytosine substitution in codon 430 of exon 12, which did not result in an amino-acid substitution. We were unable to determine whether the nucleotide substitution in exon 12 was a polymorphism or a silent mutation because matched normal tissue was not available. A polymorphism in codon 93 of exon 4, a guanine-to-thymine substitution, in hBUB1 was found in one lung cancer cell line and one primary lung tumor. This is the first report of a somatic missense mutation of a gene involved in a mitotic checkpoint in primary lung cancer. The presence of a point mutation in the hBUB1 gene is consistent with the hypothesis that alteration of mitotic checkpoint genes is involved in the development of primary lung cancers. Because the frequency of hBUB1 gene mutations was low, future studies should focus on other mechanisms of inactivation of the hBUB1 gene as well as mutation analysis of other mitotic checkpoint genes in lung cancers.

Adenocarcinoma↗

PubMed2000

Clinical safety and performance of Sugita titanium aneurysm clips.

BACKGROUND: In spite of advantages for neuro-imaging, titanium aneurysm clips are not yet chosen for routine use for clipping of intracranial aneurysms, probably because their reliability has not been demonstrated in large numbers of cases. We assess reliability and safety of Sugita titanium aneurysm clips for routine clinical use in a large number of operated cases. METHOD: Intra-operative performance and safety of Sugita titanium aneurysm clips were evaluated in 347 patients in our institutions. Aneurysms in 261 patients had ruptured: 86 patients had unruptured aneurysms. RESULTS: A total of 441 clips of 52 different types were used. No early or delayed complications occurred in relation to the titianium clips, such as deformity or slippage of a clip. Patient outcome according to the Glasgow Outcome Scale was similar to that when conventional cobalt-based aneurysm clips were used. INTERPRETATION: Reliability and safety of Sugita aneurysm clips were demonstrated in a large number of patients. The clips are suitable for routine use in aneurysm surgery.

Adult↗

PubMed2000

Mutation analysis of the gene encoding the human mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) in human cell lines resistant to growth inhibition by transforming growth factor beta(1) (TGF-beta(1)).

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is involved in activating the transforming growth factor beta(1) (TGF-beta(1)), an inhibitor of the cell proliferation, and limiting the insulin-like growth factor 2 mediated-growth stimulation. The M6P/IGF2R gene has been reported to be mutated and deleted in various cancers, and is a candidate tumor suppressor gene. We studied the genomic structure of the M6P/IGF2R gene and designed the intron primers to detect mutations in the M6P/IGF2R gene of genomic DNA samples. The M6P/IGF2R gene consists of 48 exons. The previously reported 23 mutations of the M6P/IGF2R gene in human cancers, liver, breast, and gastrointestinal tumors, are located in five exons, exon 27, 28, 31, 40, 48. Using the intron primers designed in this study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, and direct sequencing, we performed an initial analysis of the complete coding sequences of the M6P/IGF2R gene in 21 human cell lines resistant to growth inhibition by TGF-beta(1). An adenine-to-guanine transition, resulting in an asparagine-to-serine amino acid substitution, was found in one lung adenocarcinoma cell line at exon 40 where the mutation has been previously reported in human cancers. This is the first report of a mutation of the M6P/IGF2R gene in lung tumor. These results indicated that the mutation in M6P/IGF2R may be involved in human lung cancinogenesis.

Cell Line↗

PubMed2000

Molecular cloning and functional expression of triterpene synthases from pea (Pisum sativum) new alpha-amyrin-producing enzyme is a multifunctional triterpene synthase.

Ursane type triterpene is one of the most widespread triterpene aglycones found in plants, together with oleanane type, and these two types often occur together in the same plant. Pisum sativum is known to produce both types of triterpenes. Homology based PCRs with degenerate primers designed from the conserved sequences found in the known beta-amyrin synthases have resulted in cloning of two triterpene synthase cDNAs from immature seeds of P. sativum. They show high sequence identities to each other (78%) and also to the known beta-amyrin synthases (70-90%). ORFs of the full-length clones named as PSY (2277 bp, codes for 759 amino acids) and PSM (2295 bp, codes for 765 amino acids) were ligated into the yeast expression vector pYES2 under the control of GAL1 promoter. Heterologous expression in yeast revealed PSY to be a P. sativum beta-amyrin synthase. Surprisingly, however, PSM turned out to be a novel mixed amyrin synthase producing both alpha- and beta-amyrin. Several minor triterpenes were also identified as the PSM byproducts. The presence of such multifunctional triterpene synthase would account for the co-occurence of ursane and oleanane type triterpenes in plants.

Amino Acid Sequence↗

PubMed2000

An oncogenic form of the Flt-1 kinase has a tubulogenic potential in a sinusoidal endothelial cell line.

We have previously reported a constitutively activated form of the Flt-1 kinase (BCR-FLTm) molecularly engineered based on the structural backbone of the activated tyrosine kinase BCR-ABL. Here we show that it can induce not only growth stimulation but also tubulogenic differentiation of non-tubulogenic NP31 (non parenchymal) sinusoidal endothelial cells of rat liver in basement membrane matrix. Tubules formed in vitro were accompanied by fenestration structures and allowed circulation when transplanted into syngeneic animals. This biological response was not observed in other activated forms of kinases constructed in a similar fashion, which include Trk (BCR-TRK), KDR (BCR-KDR), and the parental BCR-ABL. Interestingly, formation of fine tubules was accomplished with lower but not higher expression levels of BCR-FLTm. Compared to NP cells in primary culture NP31 is deficient in expression of alpha1 integrin subunit, which was restored by expression of BCR-FLTm that had tubulogenic ability. Matrix-induced tyrosine phosphorylation of an adaptor protein Shc with recruitment of Grb-2 was observed even when tubulogenesis was nearly completed at G1 stage of the cell cycle in 2-3 weeks. Activation of matrix metalloproteinase 2 (MMP-2) and expression of urokinase type plasminogen activator (uPA) was observed with cellular invasion into matrix at the depth of 200-300 microm. Inhibitors for MAP kinase activator MEK1 and for serine proteases showed deleterious effects on the tubulogenesis. We suppose that matrix ligand-induced integrin signals cooperate with a low level of Flt-1 kinase activity to promote tubulogenic behaviors of endothelial cells in this system.

3T3 Cells↗

PubMed2000

Adeno-associated virus-mediated vascular endothelial growth factor gene transfer into cardiac myocytes.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that stimulates endothelial cell proliferation, increases endothelial permeability, and promotes collateral vessel formation. We transferred human VEGF gene into rat cardiac myocytes using adeno-associated virus (AAV) vectors and investigated whether VEGF secreted from the transduced cardiac myocytes promoted proliferation of endothelial cells. We produced VEGF-expressing AAV vectors (AAV-VEGF) by the adenovirus-free method. Immunoblotting revealed VEGF protein expression in AAV-VEGF-transduced rat cardiac myocytes. More than 60% of cardiac myocytes were stained positively on immunohistochemical staining using anti-VEGF antibody. Concentration of VEGF in the culture medium of AAV-VEGF-transduced myocytes was increased in a vector dose-dependent manner, and VEGF secretion from the transduced myocytes persisted for > or = 14 days. Thymidine incorporation into human vascular endothelial cells was significantly increased by incubation with the conditioned medium from AAV-VEGF-transduced myocytes. This increased thymidine uptake was significantly inhibited by anti-VEGF antibody. We demonstrated here that AAV-mediated VEGF gene transfer into cardiac myocytes induces the secretion of functional VEGF.

Animals↗

PubMed2000

Inhibition of vascular endothelial growth factor activity by transfection with the soluble FLT-1 gene.

Vascular endothelial growth factor (VEGF)-induced angiogenesis is involved in the etiology of some cardiovascular diseases. The soluble form of VEGF receptor, FLT-1 (sFLT-1), is a potent antagonist of VEGF. Therefore, we investigated whether transfection with the sFLT-1 gene could inhibit VEGF-induced angiogenesis. Human embryonic kidney (HEK)-293 cells were transfected with plasmids containing VEGF and sFLT-1 (pCMV-VEGF and pCMV-sFLT-1) by the calcium-phosphate co-precipitation method. VEGF- and/or sFLT-1-transfected HEK-293 cells were incubated for 24 h, and then conditioned medium was collected. The effects of conditioned medium on angiogenesis were tested by incorporation of [3H]thymidine into human umbilical vein endothelial cells (HUVECs). Expression of VEGF protein was determined by Western blotting. The conditioned medium from sFLT-1 gene-transfected HEK-293 cells significantly inhibited recombinant VEGF-induced increase in [3H]thymidine incorporation by HUVECs. VEGF gene-transfected HEK-293 cells secreted VEGF protein into conditioned medium. This conditioned medium increased [3H]thymidine incorporation by HUVECs, which was significantly inhibited by co-transfection of sFLT-1 gene with VEGF gene. These observations suggested that sFLT-1 gene transfer could inhibit VEGF-induced DNA synthesis of vascular endothelial cells.

Blotting, Western↗

PubMed2000

Properties of two VEGF receptors, Flt-1 and KDR, in signal transduction.

The properties of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF in human umbilical vein endothelial cells (HUVECs) were investigated by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF stimulated the expression of transcription factor Ets-1 as well as matrix metalloproteinase-1 (MMP-1) and Flt-1 in HUVECs. The KDR/Flt-1 heterodimer and the KDR homodimer mediate the expression of Ets-1, MMP-1, and Flt-1. VEGF also stimulated DNA synthesis and migration of HUVECs. DNA synthesis is mediated by the same signaling system as the expression of Ets-1. In contrast, cell migration is regulated by two distinct signaling systems. The Flt-1 homodimer is required for actin reorganization. The KDR/Flt-1 heterodimer and the KDR homodimer are required for the assembly of vinculin in focal adhesion plaque by regulating the phosphorylation of focal adhesion kinase (FAK) and paxillin.

Endothelial Growth Factors↗

PubMed2000

Molecular cloning and characterization of a cDNA for Glycyrrhiza glabra cycloartenol synthase.

A cDNA clone (GgCAS1) encoding cycloartenol synthase (CAS) has been isolated from Glycyrrhiza glabra (licorice) by cross-hybridization with that of Pisum sativum CAS as a probe. The deduced amino acid sequence of GgCAS1 exhibits 89%, 83% and 81% identity to those of Pisum sativum, Panax ginseng and Arabidopsis thaliana CASs, respectively. CAS activity has been detected in the homogenate of the yeast transformed with the expression vector containing the open reading frame of GgCAS1. Southern blot analysis suggested that at least two CAS genes exist in the licorice genome. In Northern blot analysis, the strong signal for CAS mRNA is detected in the cultured licorice cells of all growth phases, but no significant increase of CAS mRNA expression was observed in the cells treated with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, pravastatin.

Amino Acid Sequence↗

PubMed2000

Chalcone and stilbene synthases expressed in eucaryotes exhibit reduced cross-reactivity in vitro.

Chalcone synthase (CHS) and stilbene synthase (STS) catalyze different cyclization reactions of the common tetraketide to give different products, naringenin chalcone and resveratrol, respectively. We have previously observed in vitro cross-reaction of CHS and STS overexpressed in Escherichia coli, resveratrol production by CHS and chalcone production by STS. When expressed in eucaryotic cells, or in E. coli as thioredoxin-fusion proteins, CHS and STS exhibited reduced cross-reaction. STS refolded from inclusion bodies also showed reduced cross-reaction. While addition of bovine serum albumin and pH in the reaction were without noticeable effect, addition of glycerol decreased the cross-reaction of CHS likely due to its stabilizing effect on enzyme conformation. These results were interpreted to provide supporting evidence to our earlier proposition (Yamaguchi T. et al., FEBS Lett., 460, 457-4 61 (1999)) that the in vitro cross-reaction of CHS and STS is due to intrinsic capability of these enzymes to catalyze different types of cyclization, which, in turn, is endowed by conformational flexibility of their active sites.

Acyltransferases↗

PubMed2000

Structural features for fluorescing present in methoxycoumarin derivatives.

Structural features of fluorescent methoxycoumarins were examined from the viewpoint of substituent effect and ring structure in connection with intramolecular charge-transfer (ICT). The fluorescence of methoxycoumarins depended primarily upon the ICT from a C6-electron-donating group to the substituents at the C3-position of the coumarin ring. Furthermore, the presence of a lactone ring itself, including a carbonyl group, cyclic ether oxygen and ethylenic bond as partial ring structures, was found to be essential for fluorescing in methoxycoumarins according to the fluorescent behaviors of chemically deformed model compounds.

Chemical Phenomena↗

PubMed2000

Altered expression and function of beta1 integrins in a highly metastatic human lung adenocarcinoma cell line.

In order to investigate the relationship between beta1 integrins and the metastatic ability of cancer cells, we established a novel and highly metastatic cell line designated PC9-f9 from a poorly metastatic human lung adenocarcinoma cell line (PC9) in nude mice. PC9-f9 cells showed higher invasive activity in the Matrigel invasion assay than PC9 cells. Additionally, in cell adhesion assays, PC9-f9 cells adhered to laminin more strongly than PC9 cells and, unlike PC9 cells, adhered to collagen type IV and fibronectin. FACS analysis showed expression of the integrins alpha2beta1, alpha3beta1 and alpha6beta1 on both of the cell lines but alpha4beta1 and alpha5beta1 were neo-expressed on PC9-f9 cells. In cell adhesion inhibition assays, alpha3beta1 was the major laminin receptor for PC9 cells but not for PC9-f9 cells. Alternatively, PC9-f9 cells adhered to collagen type IV via alpha2beta1 and adhered to fibronectin mainly via alpha5beta1 but also moderately via alpha4beta1. The pretreatment of PC9-f9 cells with anti-beta1 monoclonal antibodies suppressed lung metastases by more than 50%. These data suggest that the altered expression and function of beta1 integrins allow PC9-f9 cells to become more adhesive and invasive, and lead to increased metastatic potential.

Adenocarcinoma↗