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Biomedical subjects

T Kurata

Publications and source records attributed to T Kurata.

At least 109 records · Page 6Linked to original sources
PubMed1999

Epidermal growth factor-dependent dissociation of CrkII proto-oncogene product from the epidermal growth factor receptor in human glioma cells.

Human glioma cells frequently overexpress epidermal growth factor receptor (EGFR). We found that the CrkII proto-oncogene product was associated with the EGFR in human glioma cells in the absence of epidermal growth factor (EGF). EGF stimulation of glioma cells induced the phosphorylation of tyrosine 221 of the CrkII protein, which correlates with its dissociation from the EGFR. By contrast, Shc and Grb2 were inducibly associated with the EGFR in response to EGF stimulation of glioma cells. In A431 cells, epidermoid carcinoma cells which overexpress EGFR, CrkII was tyrosine-phosphorylated and associated with the EGFR in an EGF-dependent manner. Therefore, the dissociation of CrkII from the EGFR upon stimulation with EGF appears to be specific to glioma cells. The Cbl oncogene product was also tyrosine-phosphorylated in U87MG glioma cells upon EGF stimulation. However, unlike in other cell lines, CrkII was not inducibly bound to Cbl in U87MG glioma cells. Thus, EGF-dependent binding of CrkII to phosphotyrosine-containing proteins appears to be suppressed in glioma cells. To evaluate the physiological role of dissociation of CrkII from EGFR, we expressed the CrkII-23 mutant in glioma cells. CrkII-23 mutant, which was isolated as a suppressor gene of the EGF-dependent transformation of NRK cells, binds constitutively to EGFR. We found that expression of CrkII-23 inhibited the anchorage-independent growth of the glioma cells in the presence of EGF. Taken together, these data implicate EGF-dependent dissociation of CrkII from EGFR in the oncogenicity of human glioma cells.

Cell Adhesion↗
PubMed1999

Differential expression of facilitative glucose transporter (GLUT) genes in primary lung cancers and their liver metastases.

Glucose uptake is mediated by members of the facilitative glucose transporter (GLUT) family. Malignant cells take up more glucose than their normal counterparts. The aim of this study was to investigate the gene expression levels of the GLUT family, especially GLUT1, GLUT3, and GLUT5 in primary lung cancer, metastatic liver tumors, and normal lung tissues, and to compare the expression levels of primary and metastatic tumors with those of normal tissues. We analyzed 105 autopsy samples (35 primary lung tumors, 35 corresponding normal lung tissues, 25 normal liver tissues, and 10 metastatic liver tumors) from 35 patients using the quantitative reverse transcription polymerase chain reaction. The GLUT1 gene expression levels in primary lung tumors were significantly higher than those in normal lung tissues. In liver metastatic lesions, the GLUT3 and GLUT5 gene expression levels were significantly higher than those in primary lung tumors, but there were no differences in GLUT1 expression levels between primary and metastatic liver tumors. Our results show that the gene expression pattern of the GLUT family is different between primary and metastatic liver tumors and suggest that the energy transporters in metastatic tumors may be different from those in primary tumors.

Actins↗
PubMed1999

Increased levels of epsilon and gamma isoforms of 14-3-3 proteins in cerebrospinal fluid in patients with Creutzfeldt-Jakob disease.

We established four hybridoma cell lines producing monoclonal antibodies (MAbs) against 14-3-3 proteins. Immunoblot analysis revealed that epsilon and gamma isoforms were specifically increased in premortem cerebrospinal fluid samples from patients with sporadic Creutzfeldt-Jakob disease. Furthermore, dot immunoblot analysis showed that MAbs were more specific for native antigen than polyclonal antibodies were.

14-3-3 Proteins↗
PubMed1999

Human herpesvirus 6 latently infects mononuclear cells but not liver tissue.

AIM: To investigate whether human herpesvirus 6 (HHV-6) can cause latent infection of liver tissue. METHODS: Peripheral blood and liver tissue were collected from 25 living related liver transplant recipients at the time of transplantation. An avidin-biotin complex peroxidase method was used to identify HHV-6 antigen in the liver tissue. A nested polymerase chain reaction (PCR) was used to detect HHV-6 DNA in the liver tissue and mononuclear cells. Variant of HHV-6 was determined by the presence of the Hind III site in a second PCR product. RESULTS: Immunohistochemical analysis for HHV-6 antigen was negative in all the liver specimens. HHV-6 DNA was not detected in liver tissue. Virus DNA was detected in peripheral blood mononuclear cells in nine of 25 recipients. All nine HHV-6 identified in the mononuclear cells were variant B. CONCLUSIONS: HHV-6 variant B latently infects mononuclear cells but not liver tissue.

Adolescent↗
PubMed1999

Stabilization of L-ascorbic acid by superoxide dismutase and catalase.

The effects of superoxide dismutase (SOD) and catalase on the autoxidation rate of L-ascorbic acid (ASA) in the absence of metal ion catalysts were examined. The stabilization of ASA by SOD was confirmed, and the enzyme activity of SOD, which scavenges the superoxide anion formed during the autoxidation of ASA, contributed strongly to this stabilization. The stabilization of ASA by catalase was observed for the first time; however, the specific enzyme ability of catalase would not have been involved in the stabilization of ASA. Such proteins as bovine serum albumin (BSA) and ovalbumin also inhibited the autoxidation of ASA, therefore it seems that non-specific interaction between ASA and such proteins as catalase and BSA might stabilize ASA and that the non-enzymatic superoxide anion scavenging ability of proteins might be involved.

Animals↗
PubMed1999

Characterization of antibodies raised against bovine-PrP-peptides.

To analyze the antigenicity of peptides derived from bovine prion protein (PrP) cDNA, we immunized rabbits with four synthetic peptides and compared the immunoreactivity of antibodies to PrPs from various species by immunoblotting and immunohistochemistry. Two of the antibodies reacted strongly with all PrPs. The other antibodies, raised against overlapping peptides close to two glycosylation sites, did not recognize PrPSc-mouse but did recognize PrPSc-sheep which contains two sugar residues and PrPCJD with or without a sugar residue. Our results suggest that these antibodies may have species-specificity for both glycosylation status and amino acid sequences of the protein. In conclusion, we identified two regions in bovine-PrP which appear suitable for raising antibodies that detect various kinds of PrPs, and one region (Ab103-121) which appears suitable for raising antibodies that detect several species of PrPs. These antibodies may be useful for diagnosing prion diseases and for researching their pathogenesis.

Amino Acid Sequence↗
PubMed1999

[Interventional radiology for the peripheral vascular occlusive disease].

Recently interventional radiology (IVR) has been playing an important role as a treatment for the vascular occlusive disease. We describe the recent advances in the transcatheter techniques. Thrombolysis is widely used for the treatment of acute thrombotic occlusion. And balloon angioplasty (PTA) is still accepted as the gold standard for chronic arterial occlusive disease. Some other procedures, such as mechanical thrombectomy, atherectomy and metallic stent, are now available. More clinical experiences are necessary to improve the initial and long-term success rate of these techniques.

Arterial Occlusive Diseases↗
PubMed1999

[Association between chlamydial infection and coronary artery disease].

Recent epidemiological studies have demonstrated the association between Chlamydia pneumoniae infection and coronary atherosclerosis. However, the relationship is less clear in the Japanese population. Serum IgA and IgG antibodies to Chlamydia-specific lipopolysaccharide were measured by enzyme-linked immunosorbent assay in 152 consecutive patients(112 males, 40 females, mean age 57 years)who underwent coronary angiography. Patients(n = 123)with coronary artery disease(CAD)were defined as having more than 50% diameter stenosis in at least one major coronary artery. The control group(n = 29) had normal coronary angiograms. In the CAD group, there was a high tendency of prevalence of IgA(20% vs 7%, p = 0.08)and IgG(54% vs 34%, p = 0.052). Prevalence of either IgA or IgG was significantly higher (59% vs 38%, p = 0.045) compared with the control group. Although the index of IgA antibody was not significantly different between the CAD and control groups(median 0.52 vs 0.36, p = 0.19), the index of IgG antibody was significantly higher in the CAD group than in the control group(median 1.29 vs 0.82, p = 0.026). The odds ratios for CAD were 3.4[95% confidence interval(CI)0.6-18.7]for the prevalence of IgA, 2.3(95% CI 0.9-5.2)for the prevalence of IgG, and 2.3(95% CI 1.0-5.2)for the prevalence of either IgA or IgG. Patients with CAD tended to have high prevalence of antibodies to Chlamydia spp, and these findings suggest an association between chlamydial infection and coronary atherosclerosis in the Japanese population.

Antibodies, Bacterial↗
PubMed1999

Prevalences of human herpesvirus 6 and human herpesvirus 7 in normal Thai population.

Prevalences of human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) DNA were investigated in normal Thai population. Peripheral blood mononuclear cells (PBMC) and saliva were collected from 238 healthy adults in five provinces which might be a representative of each part of the country, and 120 normal children in one province. Prevalences of HHV-6 DNA PBMC were 45.5-74.3% in adults and 78.3% in children, and in saliva, very low prevalences were detected; 5.7-8.6% in adults and 15.0% in children, respectively. Additionally, all HHV-6 DNA detected in this study were variant B. Comparingly to those of HHV-7 DNA, the prevalences were significantly higher than those of HHV-6, ie, 82.9-91.4% in PBMC of adults, 85% in PBMC of children, 84.8-89.0% in saliva of adults and 92.5% in saliva of children. HHV-6 and HHV-7 isolation from saliva specimens were also performed. No HHV-6 could be isolated from any samples, whereas, in the present study, HHV-7 could be isolated as 90.0% from children and as 20.0-54.5% from adults.

Adolescent↗
PubMed1998

Activation of Rac1 by a Crk SH3-binding protein, DOCK180.

DOCK180 is involved in integrin signaling through CrkII-p130(Cas) complexes. We have studied the involvement of DOCK180 in Rac1 signaling cascades. DOCK180 activated JNK in a manner dependent on Rac1, Cdc42Hs, and SEK, and overexpression of DOCK180 increased the amount of GTP-bound Rac1 in 293T cells. Coexpression of CrkII and p130(Cas) enhanced this DOCK180-dependent activation of Rac1. Furthermore, we observed direct binding of DOCK180 to Rac1, but not to RhoA or Cdc42Hs. Dominant-negative Rac1 suppressed DOCK180-induced membrane spreading. These results strongly suggest that DOCK180 is a novel activator of Rac1 and involved in integrin signaling.

3T3 Cells↗
PubMed1998

Producer cell-dependent requirement of the Nef protein for efficient entry of HIV-1 into cells.

A proviral nef gene mutant of human immunodeficiency virus type 1 (HIV-1) was evaluated for its defective early replication step. Virus stocks were prepared from six CD4-positive and -negative cell lines transfected with wild-type (wt) or the nef mutant clone and inoculated into two target CD4-positive cell lines to monitor the efficiency of viral entry process. The nef mutant virions produced in one cell line exhibited a severe defect in the entry process, although those produced in the other five cell lines were only slightly less efficient than the wt virions at entering into cells. These results have demonstrated that the HIV-1 Nef is critical for efficient viral entry in a producer cell-dependent manner.

CD4-Positive T-Lymphocytes↗
PubMed1998

Evidence that DOCK180 up-regulates signals from the CrkII-p130(Cas) complex.

DOCK180 is one of the two principal proteins bound to the SH3 domain of the adaptor protein CrkII. Here, we have studied the involvement of DOCK180 in integrin signaling. DOCK180 was neither phosphorylated nor bound to CrkII in quiescent NIH 3T3 cells and 3Y1 cells. We found that DOCK180 was phosphorylated and bound to CrkII in NIH 3T3 cells stimulated with integrin and also in 3Y1 cells transformed by v-src or v-crk. The binding of DOCK180 to CrkII correlated with the binding of CrkII to p130(Cas), which is a major CrkII SH2 domain-binding protein at focal adhesions. In a reconstitution experiment, expression of DOCK180 induced hyperphosphorylation of p130(Cas) and a concomitant increase in the amount of CrkII bound to p130(Cas). Similarly, binding of DOCK180 to CrkII was also enhanced by the coexpression of p130(Cas). Finally, we found that coexpression of p130(Cas) and CrkII with DOCK180 induced local membrane spreading and accumulation of DOCK180-CrkII-p130(Cas) complexes at focal adhesions. These findings suggest that DOCK180 positively regulates signaling from integrins to CrkII-p130(Cas) complexes at focal adhesions.

3T3 Cells↗
PubMed1998

Productive infection of Epstein-Barr virus (EBV) in EBV-genome-positive epithelial cell lines (GT38 and GT39) derived from gastric tissues.

We characterized the expression of Epstein-Barr virus (EBV) on two epithelial cell lines, GT38 and GT39, derived from human gastric tissues. The EBV nuclear antigen (EBNA) was detected in all cells of both cell lines. The EBV immediate-early BZLF 1 protein (ZEBRA), the early antigen diffuse component (EA-D), and one of the EBV envelope proteins (gp350/220) were expressed spontaneously in small proportions in the cells. EBNA 1, EBNA2, latent membrane protein 1, ZEBRA, and EA-D molecules were then observed by Western blotting in the cells. The lytic cycle was enhanced with treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or n-butyrate. The virus particles were observed in the TPA treated GT38 cells by electron microscopy. Infectious EBV was detected with the transformation of cord blood lymphocytes and also with the induction of early antigen to Raji cells by the supernatants of both cells lines. A major single and minor multiple fused terminal fragments and a ladder of smaller fragments of the EBV genome were detected with a Xhol probe in both cell lines. These epithelial cells lines and viruses will be useful in studying their association with EBV in gastric epithelial cells.

Animals↗
PubMed1998

Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth factor receptor.

CrkII adaptor protein becomes tyrosine-phosphorylated upon various types of stimulation. We examined whether tyrosine 221, which has been shown to be phosphorylated by c-Abl, was phosphorylated also by other tyrosine kinases, such as epidermal growth factor (EGF) receptor. For this purpose, we developed an antibody that specifically recognizes Tyr221-phosphorylated CrkII, and we demonstrated that CrkII was phosphorylated on Tyr221 upon EGF stimulation. When NRK cells were stimulated with EGF, the tyrosine-phosphorylated CrkII was detected at the periphery of the cells, where ruffling is prominent, suggesting that signaling to CrkII may be involved in EGF-dependent cytoskeletal reorganization. The EGF-dependent phosphorylation of CrkII was also detected in a c-Abl-deficient cell line. Moreover, recombinant CrkII protein was phosphorylated in vitro by EGF receptor. These results strongly suggest that EGF receptor directly phosphorylates CrkII. Mutational analysis revealed that the src homology 2 domain was essential for the phosphorylation of CrkII by EGF receptor but not by c-Abl, arguing that these kinases phosphorylate CrkII by different phosphorylation mechanisms. Finally, we found that the CrkII protein phosphorylated upon EGF stimulation did not bind to the phosphotyrosine-containing peptide and that CrkII initiated dissociation from EGF receptor within 3 min even with the sustained tyrosine phosphorylation of EGF receptor. This result implicated phosphorylation of Tyr221 in the negative regulation of the src homology 2-mediated binding of CrkII to EGF receptor.

Animals↗