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T Koike

Publications and source records attributed to T Koike.

At least 181 records · Page 10Linked to original sources
PubMed1999

Ras pathway is required for the activation of MMP-2 secretion and for the invasion of src-transformed 3Y1.

To search for the signaling pathway critical for tumor invasion, we examined the effects of dominant negative ras (S17N ras) expression on the activation of matrix metalloproteinase-2 (MMP-2) in src-transformed 3Y1, SR3Y1, under the control of conditionally inducible promoter. In SR3Y1 clones transfected with S17N ras, augmented secretion and proteolytic activation of MMP-2 were dramatically suppressed by S17N Ras expression, while tyrosine phosphorylation of cellular proteins was not suppressed. We found that invasiveness of SR3Y1 cells assayed by the modified Boyden Chamber method was strongly suppressed by S17N Ras expression. In contrast, cell morphology reverted partially and glucose uptake remained unchanged by S17N Ras expression. In addition, treatment of SR3Y1 with manumycin A, a potent inhibitor of Ras farnesyltransferase, strongly suppressed both augmented secretion and proteolytic activation of MMP-2. Contrary, treatment of SR3Y1 with wortmannin or TPA showed no clear effect on MMP-2 activation. Thus, these results strongly suggest that Ras-signaling, but neither P13 kinase- nor protein kinase C-signalings, plays a critical role in activation of MMP-2 and, subsequently, in the invasiveness of src-transformed cells.

Androstadienes↗
PubMed1999

Up-regulation of cyclin D1 occurs in apoptosis of immature but not mature cerebellar granule neurons in culture.

Cerebellar granule neurons isolated from 7-day-old rats and cultured in normal medium undergo apoptosis, but remain healthy under depolarizing conditions with elevated K(+) (>==25 mM) or in the presence of brain-derived neurotrophic factor. Northern blot analysis showed that cyclin D1 mRNA was up-regulated in this apoptotic process. Both granule neurons and microglia were immunostained with anti-cyclin D1 antibodies, which is consistent with our previous finding that microglia become activated in response to neuronal cell death under these conditions. Only granule neurons, however, showed an enhanced expression of both mRNA and protein levels of cyclin D1 in the presence of aphidicolin that completely eliminated non-neuronal cells. The entire cell body of granule neurons became immunostained prior to cell shrinkage or nuclear condensation. Moreover, cell cycle blockers and an inhibitor of cyclin-dependent kinases suppressed both increased immunoreactivity and cell death, further substantiating the involvement of an abortive cell cycle in this process. In contrast, both levels of cyclin D1 remained unaltered in mature granule neurons undergoing apoptosis following combined serum withdrawal and low K(+) shift, suggesting developmental stage dependence of granule neuron apoptosis in vitro. This culture system is suitable for further analysis of the role of cyclin D1 in cell death.

Animals↗
PubMed1999

The cadherin-like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin.

The Bacillus thuringiensis CryIAa toxin binds a cadherin-like protein (BtR175) on the brush-border membranes of the Bombyx mori midgut columnar cells, which are the targets. By introducing the BtR175 gene with a baculovirus, Spodoptera frugiperda Sf9 cells expressed BtR175 protein on the cell membrane and became susceptible to the CryIAa toxin. The toxin bound the cadherin repeat adjacent to the membrane and made a pore that passed inorganic ions, causing the cell to swell and burst. This was not observed with a BtR175 variant lacking the toxin-binding site. This in vitro experiment mimicked the specific insecticidal action of the toxin in vivo well.

Bacillus thuringiensis↗
PubMed1999

Phosphatidylinositol 3-kinase is involved in the protection of primary cultured human erythroid precursor cells from apoptosis.

Little is known about the physiologic role of phosphatidylinositol 3-kinase (PI-3K) in the development of erythrocytes. Previous studies have shown that the effects of the PI-3K inhibitor wortmannin on erythropoietin (EPO)-dependent cell lines differed depending on the cell type used. Wortmannin inhibited EPO-induced differentiation of some cell lines without affecting their proliferation; however, the EPO-induced proliferation of other cell lines was inhibited by wortmannin. In neither case were signs of apoptosis observed. We have previously reported that signaling in highly purified human colony forming units-erythroid (CFU-E), generated in vitro from CD34(+) cells, differed from that in EPO-dependent cell lines. In the current study, we examined the effects of a more specific PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002 dose-dependently inhibits the proliferation of erythroid progenitor cells with a half-maximal effect at 10 micromol/L LY294002. LY294002 at similar concentrations also induces apoptosis of these cells, as evidenced by the appearance of annexin V-binding cells and DNA fragmentation. The steady-state phosphorylation of AKT at Ser-473 that occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are specific. Interestingly, the acceleration of apoptosis by LY294002 was observed in the presence or absence of EPO. Further, deprivation of EPO resulted in accelerated apoptosis irrespective of the presence of LY294002. Our study confirms and extends the finding that signaling in human primary cultured erythroid cells is significantly different from that in EPO-dependent cell lines. These data suggest that PI-3K has an antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive and one that is EPO-dependent and at least partly insensitive to LY294002.

Androstadienes↗
PubMed1999

Enhancement of G2 checkpoint function by gelsolin transfection in human cancer cells.

We have previously reported that human gastric (TMK1) and urinary bladder (UMUC2) cancer cell lines show markedly reduced expression of an actin-regulatory protein, gelsolin [S. Moriya et al., (1994), Int. J. Oncol. 5, 1347-1351, M. Tanaka et al. (1995), Cancer Res. 55, 3228-3232]. When gelsolin expression is restored by transfection, cancer cells lost tumorigenicity in vivo [M. Tanaka et al. (1995), Cancer Res. 55, 3228-3232]. Here, we show that gelsolin-overexpressing TMK1 and UMUC2 cells are more resistant to UVC irradiation. Increased resistance is associated with increases in the proportion of cells in the G2 phase of the cell cycle compared to similarly treated control neotransfectants. After UVC irradiation, synchronized gelsolin-overexpressing UMUC2 cells had a prolonged S phase followed by delayed G2 accumulation compared to neotransfected UMUC2 cells as determined by cell cycle analysis. The levels of cyclin B1 and cdk1 histone H1 kinase activity in gelsolin transfectants remained low during S and early G2 phase and the production of diacylglycerol induced by UVC was reduced in gelsolin transfectants compared to neotransfectants. These observations suggest that gelsolin enhances G2 checkpoint function of cells through lipid metabolism, leading to UVC resistance. Considered together with recent evidence that radiation clastogenesis and chemical carcinogenesis are cell-cycle-dependent, down regulation of gelsolin may lead to the malignant transformation of human gastric or urinary bladder cancers by attenuating G2 checkpoint function.

Aphidicolin↗
PubMed1999

Induction of apoptotic cell death in vascular endothelial cells cultured in three-dimensional collagen lattice.

Endothelial cells derived from fetal bovine aorta (BAECs) undergo apoptosis in three-dimensional (3-D) type I collagen lattice in the absence of specific angiogenic factor. In the presence of angiogenic factor, BAECs survive and form a capillary-like tube structure in 3-D culture. In the present study we elucidate the mechanisms of BAECs apoptosis or survival and tube formation in 3-D culture. When BAECs embedded in collagen lattice were cultured with angiogenic factor (fibroblast growth factor-2 (FGF-2) or 4beta-phorbol 12-myristate 13-acetate (PMA)) in the presence of PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, BAECs did not form tube structures and underwent apoptosis in collagen lattice. Function-blocking antibody against alphavbeta3 integrin also inhibited tube formation and induced apoptosis in 3-D culture in the presence of angiogenic factors. Exposure of BAECs to FGF-2 and PMA had no effect on the alphavbeta3 integrin expression but induced the activation of alphavbeta3 integrin. PD98059 attenuated alphavbeta3 integrin activation in response to angiogenic factor. KB-R8301, a hydroxamic acid-based matrix metalloproteinase (MMP) inhibitor, prevented apoptotic cell death in the absence of angiogenic factor in 3-D culture and enhanced capillary-like tube formation in the presence of angiogenic factor, which was not inhibited by the anti-alphavbeta3 integrin antibody. The results suggest that angiogenic factor-induced alphavbeta3 integrin activation through the MEK-ERK pathway regulates the BAEC fate between apoptosis and angiogenesis in collagen lattice. MMP derived from BAECs seems to play a key role in the release of cryptic ligands for alphavbeta3 integrin from intact collagen.

Angiogenesis Inducing Agents↗
PubMed1999

A novel putative transporter maps to the osteosclerosis (oc) mutation and is not expressed in the oc mutant mouse.

The phenotype of mice homozygous for the osteosclerosis (oc) mutation includes osteopetrosis, and a variety of studies demonstrate that osteoclasts in these mice are present but nonfunctional. We have identified a novel gene that has homology to a family of 12-transmembrane domain proteins with transport functions and maps to proximal mouse chromosome 19, in a region to which the oc mutation has been previously assigned. The putative transporter is abundant in normal kidney, but its expression is markedly reduced in kidneys from oc/oc mice when tested using Northern and Western analyses. Southern analysis of this gene, which we call Roct (reduced in oc transporter), demonstrates that it is intact and unrearranged in oc/oc mice. In situ studies show that Roct is expressed in developing bone. We propose that the absence of Roct expression results in an osteopetrosis phenotype in mice.

Amino Acid Sequence↗
PubMed1999

Novel technique for the direct flow cytofluorometric analysis of human basophils in unseparated blood and bone marrow, and the characterization of phenotype and peroxidase of human basophils.

BACKGROUND: No technique has been reported to analyze directly the antigen expression on basophil leukocytes when using a flow cytometer; therefore, the exact phenotype of human basophils and the character of the peroxidase in basophils are not well understood. METHODS: Human blood basophils were purified by using an antibody against high-affinity Fc epsilon receptor (hFcepsilonR) and a MACS magnetic cell sorting system and then cytochemically stained. The phenotype and peroxidase of the human basophils were flow cytofluorometrically analyzed directly in unseparated blood and bone marrow samples as hFcepsilonR+/MBP+ (major basic protein)/Hist+ (histamine) light-density cells distributed in the high sidescatter area of lymphocytes on light scattergrams. RESULTS: The peroxidase granules of human basophils were stained by an anti-eosinophil peroxidase (EPO) antibody. The human blood basophils had common granulocyte markers plus CD25, i.e., they were CD11a/ CD11b/CD11c/CD25/CD38/CD13/CD33/hFcepsi lonR/MBP/Hist/ EPO positive, CD71 dim positive, CD14/CD15 partially positive, and CD2/CD3/CD7/CD122/CD16/CD56/CD57/ CD10/CD19/CD20/CD22/HLA-DR/MPO (myeloperoxidase)/CD23 negative. Further examination was done to analyze the expression of colony-stimulating factor receptors on three lineages of granulocytes, i.e., basophils, eosinophils, and neutrophils. The neutrophils were CD114 (G-CSFR)/CD116 (GM-CSFR)/CD124 [interleukin (IL)-4R]/CD126 (IL-6R) positive and CD123 (IL-3R)/CD125 (IL-5R) negative. In contrast, the eosinophils and basophils were CD116/CD123/CD125/CD126 positive and CD114/CD124 negative. CONCLUSIONS: This novel technique for directly characterizing human basophil leukocytes with flow cytometry may be a convenient way to screen the expression of surface antigens and the cytoplasmic expression of CD antigens and other proteins in human blood basophils and to analyze alterations of the character of basophils by cytokines and other biological substances in vivo and in vitro.

Adult↗
PubMed1999

Molecular cloning of cDNA encoding human Rab3D whose expression is upregulated with myeloid differentiation.

To identify genes expressed in myeloid differentiation, we isolated a cDNA fragment by differential display using RNA prepared from HT93A cells, a human cell line capable of differentiating into neutrophil and eosinophil lineages in response to retinoic acid (RA). Evaluation of the full-length clone isolated from an HT93A cDNA library showed that it encoded a 24 kDa protein comprised of several domains conserved in the Ras superfamily. Comparison of the deduced amino acid sequence of this clone with Rab proteins revealed that it had highest homology to a small GTP-binding protein, murine Rab3D. The mRNA expression of human Rab3D was upregulated in the course of myeloid differentiation, and it was preferentially expressed in granulocytes. These results suggest that human Rab3D may play a specific role in granulocytes, for example in exocytosis of neutrophil-specific granules or in degranulation of both eosinophils and basophils.

Amino Acid Sequence↗
PubMed1999

Thapsigargin induces microglial transformation from amoeboid to ramified type in vitro.

Microglia generally display amoeboid morphology under prevalent culture conditions. We found that cultured microglia derived from rat cerebral cortex undergo a morphological transformation from amoeboid to process-bearing microglia upon treatment with thapsigargin (TG), a specific Ca2+-ATPase inhibitor of endoplasmic reticulum. Microglial transformation was further enhanced by exposure of amoeboid microglia to serum-free (N2) medium containing TG (TG/N2 treatment). TG/N2-treated microglia showed a marked reduction in the activity of phagocytosis and showed down-regulated expression of MRF-1 or F4/80, which are markers for activated microglia. Thus, both morphological and physiological criteria suggest that TG promotes the ramification of amoeboid microglia in vitro. This method would be helpful in characterization of ramified microglia in vitro.

Animals↗
PubMed1999

Regulation of the expression of MHC class I and II by class II transactivator (CIITA) in hematopoietic cells.

In order to develop an effective immunotherapy for hematological malignancies, we investigated the applicability of class II transactivator (CIITA), which had been demonstrated to regulate the expression of MHC class II (MHC-II) by assembling the transcription factors of MHC-II molecules, for immunotherapy by potentiating the antigenicity of tumour cells by inducing MHC expression. First, 32 hematopoietic cell lines were analysed for the expression of HLA-DR, CIITA, RFX5 or HLA-ABC. Fourteen cell lines were positive and 18 were negative for HLA-DR. All the 14 HLA-DR positive cell lines were demonstrated to express CIITA mRNA by RT-PCR. On the other hand, in all the 18 HLA-DR negative cell lines, the expression of CIITA was not demonstrated. RFX5, which is one of the transcription factors of MHC-II, was expressed ubiquitously in all 32 cell lines. Three cell lines out of 23 hematopoietic cell lines examined were negative for HLA-ABC, and all three of these cell lines were negative for both HLA-DR and CIITA expression. Furthermore, CIITA cDNA was transfected into K562 cells, which were negative for HLA-ABC, -DR and -DQ, but positive for HLA-DP. The transfection rendered HLA-DR negative to positive and increased the expression level of HLA-DP, but HLA-DQ remained negative. In addition to HLA-DR, HLA-ABC was also induced to express by the transfection of CIITA gene. The present study demonstrated that the expression of HLA-DR in hematopoietic cells is regulated in subordination to CIITA and the expression of HLA-DR (and HLA-ABC in K562) is induced by transfection with the CIITA gene. These findings revealed the applicability of CIITA in potentiating anti-tumour immunity of HLA-DR negative tumour cells for immunotherapy of hematological malignancies.

Acute Disease↗
PubMed1999

Beta2-glycoprotein I is necessary to inhibit protein C activity by monoclonal anticardiolipin antibodies.

OBJECTIVE: To clarify mechanisms of the thrombosis associated with anticardiolipin antibodies (aCL), we examined the effects on activated protein C (APC) of monoclonal aCL and beta2-glycoprotein I (beta2GPI), which is required for formation of the epitopes of aCL. METHODS: We developed the chromogenic assay, in which the degradation of coagulation factor Va by APC is reflected in the reduced generation of thrombin from prothrombin, using soybean trypsin inhibitor to inhibit APC. APC activities were measured in the presence and absence of 3.4 microM beta2GPI and/or 2.5 microg/ml of IgM monoclonal aCL (EY2C9 and EY1C8) established from peripheral blood lymphocytes obtained from a patient with aCL. RESULTS: Without APC, the formed thrombin activity decreased by the addition of 3.4 microM beta2GPI. When 12.8 nM APC was added, beta2GPI partially reversed the APC-induced inhibition of thrombin generation in a concentration-dependent manner. With 3.4 microM beta2GPI, the thrombin generation in monoclonal aCL (2.5 microg/ml) decreased to 77.1-80.2% by the addition of 12.8 nM APC, but the values were above that in the control IgM (72.7%). Without beta2GPI, the APC activity was unaffected by the addition of monoclonal aCL. CONCLUSION: Beta2-glycoprotein I exhibits procoagulant activity by inhibiting APC activity and anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL and only in the presence of beta2GPI.

Adenomatous Polyposis Coli Protein↗
PubMed1999

A chimeric antibody with the human gamma1 constant region as a putative standard for assays to detect IgG beta2-glycoprotein I-dependent anticardiolipin and anti-beta2-glycoprotein I antibodies.

OBJECTIVE: Thromboembolic manifestations or thrombocytopenia in association with anticardiolipin antibodies (aCL) or lupus anticoagulant are known as the antiphospholipid syndrome (APS). Efforts have been made to elucidate precise clinical features and adequate therapeutic options for treating patients with APS. However, the lack of a proper international standard for measurement of aCL makes it difficult to compare data derived from different laboratories. We attempted to design a chimeric antibody with human gamma constant regions and variable regions of WBCAL-1, a monoclonal antibody established from an APS-prone mouse which has a specificity similar to that of aCL in sera from humans with APS. METHODS: Variable-region genes of WBCAL-1, which were cloned using reverse transcription-polymerase chain reaction, were inserted into plasmids containing human gamma1 and kappa constant-region genes. The construct was transfected to a mouse myeloma cell line. Stable transfectants that secreted a chimeric antibody, HCAL, into the culture supernatant were obtained. The reactivity of HCAL to cardiolipin and to beta2-glycoprotein I (beta2GPI) was studied using a solid-phase enzyme immunoassay. The binding of HCAL was compared with the binding of standards for IgG aCL and anti-beta2GPI antibody assays done in 18 independent laboratories. RESULTS: In the presence of beta2GPI, HCAL bound to the wells of cardiolipin-coated microtiter plates in a dose-dependent manner and reacted with beta2GPI on oxygenated polystyrene plates. The aCL activity of HCAL can be converted into GPL units (IgG phospholipid units), which is widely used to quantify IgG aCL activity, using the following formula: 1 GPL unit = 32.9 x (concentration of HCAL [in microg/ml])(0.503). The reactivity of HCAL to cardiolipin or beta2GPI was similar to the reactivity of standards for IgG aCL or anti-beta2GPI antibody assays done in collaborative laboratories. CONCLUSION: Because the reactivity of HCAL is similar to that of aCL in sera from humans with APS, HCAL will be useful as a standard for human IgG aCL and anti-beta2GPI antibody assays.

Amino Acid Sequence↗
PubMed1999

Cytokine regulation of env gene expression of human endogenous retrovirus-R in human vascular endothelial cells.

To determine whether human endogenous retroviruses are implicated in the pathogenesis of inflammatory vascular diseases of unknown etiology, we examined mRNA expression of a human endogenous retrovirus, HERV-R, which has a long open reading frame in the env region, in cultured human vascular endothelial and smooth muscle cells stimulated in the presence of various cytokines. mRNA of HERV-R was always evident in these cells but not in fibroblastic cells. Levels of expression in vascular endothelial cells were significantly regulated by treatment with tumor necrosis factor-alpha, interleukin (IL)-1alpha, and IL-1beta as up-regulators and interferon-gamma as a down-regulator. These observations are interpreted to mean that HERV-R expression may be up- or down-regulated at sites of inflammation in vessels in vivo and hence may play a pathogenetic role in inflammatory vascular diseases in humans, perhaps similar to endogenous retroviruses in mouse models of polyarteritis nodosa in humans.

Cells, Cultured↗
PubMed1999

Kynurenine concentration of serum was increased by exercise.

Kynurenine pathway of tryptophan makes a lot of physiological active substances, such as quinolinate, NAD and so on, suggesting that kynurenine itself may play a very important role physiologically. Therefore, we examined the influence of exercise on serum kynurenine concentration. At first, we assayed kynurenine concentration of students (n = 13) who took part in a rugby camp for three days. The mean value of kynurenine concentration of before and after training were 1.362 +/- 0.306 microM and 1.725 +/- 0.511 microM respectively. These data means that severe exercise rise the serum kynurenine concentration. Then we tried to examine the relationship between the level of exercise and serum kynurenine concentration. Serum kynurenine concentration had significantly increased immediately after the exercise from 1.869 +/- 0.285 microM to 2.138 +/- 0.248 microM of 24 hours later by loading of 65% heart rate max exercise for each subject. These results suggested that at least the severe exercise affect on the tryptophan metabolism. We will discuss the change of serum kynurenine concentration by another sports such as soccer game and 20 km run.

Chromatography, High Pressure Liquid↗