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Biomedical subjects

E Takai

Publications and source records attributed to E Takai.

At least 19 recordsLinked to original sources
PubMed2006

Substrate modulation of osteoblast adhesion strength, focal adhesion kinase activation, and responsiveness to mechanical stimuli.

Osteoblast interactions with extracellular matrix (ECM) proteins are known to influence many cell functions, which may ultimately affect osseointegration of implants with the host bone tissue. Some adhesion-mediated events include activation of focal adhesion kinase, and subsequent changes in the cytoskeleton and cell morphology, which may lead to changes in adhesion strength and cell responsiveness to mechanical stimuli. In this study we examined focal adhesion kinase activation (FAK), F-actin cytoskeleton reorganization, adhesion strength, and osteoblast responsiveness to fluid shear when adhered to type I collagen (ColI), glass, poly-L-lysine (PLL), fibronectin (FN), vitronectin (VN), and serum (FBS). In general, surfaces that bind cells through integrins (FN, VN, FBS) elicited the highest adhesion strength, FAK activation, and F-actin stress fiber formation after both 15 and 60 minutes of adhesion. In contrast, cells attached through non-integrin mediated means (PLL, glass) showed the lowest FAK activation, adhesion strength, and little F-actin stress fiber formation. When subjected to steady fluid shear using a parallel plate flow chamber, osteoblasts plated on FN released significantly more PGE2 compared to those on glass. In contrast, PGE2 release of osteoblasts attached to FN or glass was not different in the absence of fluid shear, suggesting that differences in binding alone are insufficient to alter PGE2 secretion. The increased adhesion strength as well as PGE2 secretion of osteoblasts adhered via integrins may be due to increased F-actin fiber formation, which leads to increased cell stiffness.

Actins↗
PubMed2001

A high-speed atomic force microscope for studying biological macromolecules.

The atomic force microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and placed in aqueous solution. At present, however, it is limited by the speed at which it can successively record highly resolved images. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed a high-speed scanner, free of resonant vibrations up to 60 kHz, small cantilevers with high resonance frequencies (450-650 kHz) and small spring constants (150-280 pN/nm), an objective-lens type of deflection detection device, and several electronic devices of wide bandwidth. Integration of these various devices has produced an AFM that can capture a 100 x 100 pixel(2) image within 80 ms and therefore can generate a movie consisting of many successive images (80-ms intervals) of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica (see http://www.s.kanazawa-u.ac.jp/phys/biophys/bmv_movie.htm).

Animals↗
PubMed2001

Differential splenic migration of dendritic cells after immunologic unresponsiveness in rat hepatic allografts induced by pretransplant donor-specific transfusion.

BACKGROUND: Donor dendritic cells migrate into the recipient spleen after hepatic transplantation. We previously reported that immunologic unresponsiveness to rat hepatic allografts can be induced by prior donor-specific blood transfusion (DST). We investigated the phenotype and splenic distribution of donor dendritic cells after allografting and DST. METHODS: Donor dendritic cells were identified with anti-rat dendritic cell (OX-62) and anti-donor class II MHC (RT1B(a)) (OX-76) antibodies. The phenotype of dendritic cells was determined with antibodies to CD45RC, CD62L, and the maturation markers CD80 (B7-1) and CD86 (B7-2). The cytokine profile of sorted CD45RC(+) OX-62(+) and CD45RC(-) OX-62(+) dendritic cells was analyzed by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Pretransplant DST significantly prolonged rat hepatic allograft survival. Immunostaining revealed OX76(+)/OX-62(+) cells in the splenic red pulp of animals receiving pretransplant DST and in the white pulp of untreated animals after transplantation. The ratio of splenic CD45RC(-) OX-62(+) cells to CD45RC(+) OX-62(+) cells was significantly higher in DST recipients than in untreated animals. CD62L, CD80, and CD86 were lower on CD45RC(-) OX-62(+) than CD45RC(+) OX-62(+) cells. RT-PCR revealed that sorted CD45RC(-) OX-62(+) cells expressed interleukin (IL)-4 and IL-10. In contrast, sorted CD45RC(+) OX-62(+) cells expressed only IL-2 and interferon gamma (IFN-gamma). CONCLUSION: Differential splenic migration of CD45RC(-) dendritic cells is associated with immunologic unresponsiveness to rat hepatic allografts.

Animals↗
PubMed2001

Immunohistochemical analysis of distribution of RON receptor tyrosine kinase in human digestive organs.

The immunohistochemical distribution of RON receptor tyrosine kinase in digestive organs of both human fetus and adult, including the esophagus, stomach, duodenum, small intestine, colon, rectum, liver, gallbladder, pancreas, and spleen, was investigated semiquantitively using an affinity-purified rabbit polyclonal antibody. RON was observed to be widely distributed throughout various digestive organs and cell types in humans. The immunoreactivity for RON was observed in the epithelium of the esophagus, small intestine, colon, hepatocytes, Kupffer cells, and splenic macrophages both in the adult and the fetus, suggesting that the MSP/RON signaling pathway possesses the proper biological properties to possibly be involved in morphogenesis or differentiation of cells in these organs and cell types. Several organs differed in immunoreactivity between adult and fetus. No immunoreactive cells were found in the pancreas of adults; however, immunoreactivity was observed in acinar cells and in some of the duct or ductular cells and endocrine cells of the islet of the fetus. Similarly, immunoreactivity was not observed in gastric mucosa except in the intestinal metaplastic cells in adults; however, immunoreactivity was found in the foveolar epithelium of the stomach of the fetus. Although the biological significance of RON in malignancy is unclear, the presence of RON immunoreactivity in the fetus and it lack in the adult may indicate that RON is a oncofetal substance in human pancreas and stomach.

Adult↗
PubMed2001

Frequency and clinical characteristics of dilated cardiomyopathy caused by desmin gene mutation in a Japanese population.

AIMS: Dilated cardiomyopathy is partly caused by a mutation of some cytoskeletal or nuclear envelope proteins. It has been confirmed recently that a missense mutation of the gene encoding desmin, a cytoskeletal protein, can cause dilated cardiomyopathy. This study was aimed at elucidating the frequency and clinical characteristics of dilated cardiomyopathy caused by desmin mutation. METHODS AND RESULTS: We examined 265 Japanese patients with dilated cardiomyopathy (217 sporadic cases and 48 probands of familial dilated cardiomyopathy). The exon 8 of the desmin gene, the critical region for the pathogenesis of dilated cardiomyopathy, was analysed by polymerase chain reaction, single-strand conformation polymorphism and sequencing. The same missense mutation (Ile451Met) as reported previously was detected in three patients (1.1%). All these patients were male and sporadic, and more likely to be accompanied by characteristics such as younger age at diagnosis, lower fractional shortening and ejection fraction than each mean value of sporadic cases. The chronological changes in cardiac function were inconsistent in the three patients. CONCLUSION: The missense mutation (Ile451Met) of the desmin gene can be the genetic cause of dilated cardiomyopathy, although with very low frequency. The ages at diagnosis were younger and the cardiac function had deteriorated further than general cases of sporadic dilated cardiomyopathy.

Adult↗
PubMed2001

CD45RC gammadelta T-cell infiltration is associated with immunologic unresponsiveness induced by prior donor-specific blood transfusion in rat hepatic allografts.

Little is known regarding the role of gammadelta(+) T cells in organ transplantation. We previously reported that immunologic unresponsiveness is induced by prior donor-specific blood transfusion (DST) in rat hepatic allografts. We investigated the phenotype and distribution of gammadelta(+) T cells in the hepatic allograft, spleen, and peripheral blood of recipient rats with immunologic unresponsiveness induced by DST. gammadelta(+) T cells were enumerated in allograft livers and spleens by immunostaining and in blood by flow cytometric analysis. The phenotype of gammadelta(+) T cells was determined using CD45RC isoforms derived from alternative mRNA splicing. The cytokine profile of CD45RC(+) and CD45RC(-) gammadelta(+) T cells was analyzed by reverse transcription polymerase chain reaction. The number of gammadelta(+) T cells in hepatic infiltrates in recipient rats pretreated with DST was significantly greater than in untreated animals. This correlated with significantly higher levels of gammadelta T cell receptor (TCR) mRNA in hepatic allografts of DST-treated rats as compared with untreated animals. The gammadelta(+) T cell/alphabeta(+) T-cell ratio increased in hepatic infiltrates in DST-treated recipient rats but not in untreated animals. CD45RC(-)gammadelta(+) T cells were predominantly increased in DST-treated hepatic allografts compared with untreated allografts. Most of the intestinal intraepithelial T cells were CD45RC(-)gammadelta(+). Interleukin (IL)-10 and IL-4 mRNA were detected more in CD45RC(-)gammadelta(+) T cells than CD45RC(+)gammadelta(+) T cells. CD45RC(-)gammadelta(+) T cells infiltrating liver allografts produce Th2-type cytokines and are associated with immunologic unresponsiveness induced by DST.

Acute Disease↗
PubMed2001

Asymptomatic pyuria in diabetic women.

The aim of the present study was to determine the prevalence of and the host factors for asymptomatic pyuria (ASP) in women with type 2 diabetes. The study included 179 type 2 diabetic women and consecutive 455 non-diabetic women attending as out-patients in 1996. Patients with symptoms of a urinary tract infection were excluded. ASP was defined as the presence of more than 10 leukocytes/high-power field in a random urine sample. Diabetic women more often had ASP than non-diabetic women (27.9 vs. 15.8%, P<0.001). The prevalence of ASP was significantly increased in patients with a duration of diabetes exceeding 15 years (0 approximately 4 years; 20.3%, 5 approximately 9 years; 24.3%, 10 approximately 14 years; 23.8%, and > or =15 years; 46.3%). No differences were evident in HbA(1C) between diabetic patients without ASP and those with ASP. Diabetic women with ASP more often had diabetic retinopathy, neuropathy, nephropathy, cerebrovascular disease, ischemic heart disease, and hyperlipidemia than those without ASP. However, no statistically significant differences were evident in the prevalence of hypertension, constipation, or dementia. As the degree of neuropathy increases, it is accompanied by an increasing prevalence of ASP (none, 21.4%; blunt tendon reflexes, 24.5%; symptomatic, 50.0%; and gangrene, 66.6%). The prevalence of ASP was significantly increased in the patients with proliferative diabetic retinopathy (none, 23.2%; background, 29.4%; pre-proliferative, 18.2%; and proliferative, 50.0%). As the degree of nephropathy increases, it is accompanied by an increasing prevalence of ASP (none, 20.0%; microalbuminuria, 31.9%; macroalbuminuria, 37.0%; and renal failure, 60.0%). Thus, the prevalence of ASP is increased in women with diabetes and increased with longer duration of diabetes but was not affected by glucose control. The incidence of ASP increases significantly as diabetic microangiopathy becomes severer.

Aged↗
PubMed2001

CA19-9 epitope a possible marker for MUC-1/Y protein.

It has been reported that MUC-1 molecules devoid of the tandem repeat region (MUC-1/Y) are detected preferentially in carcinoma cells and are associated with their progression. However, its clinical significance is still unknown. We constructed a mouse colon adenocarcinoma cell line (MC-38) transduced with either MUC-1/Y cDNA defecting the tandem repeat region (Y-MC-38) or MUC-1/R cDNA containing ten tandem repeats (R-MC-38). RT-PCR of mRNAs derived from Y-MC-38 cells using the specific primers to MUC-1/Y mRNAs, proved the existence of 600 bp RT-PCR products generated only from MUC-1/Y mRNAs. DF3 and CA19-9 epitopes out of the MUC-1-related tumor-associated antigens, have been reported to be involved in the prognosis of cancer patients. We examined the expression of DF3 and CA19-9 epitopes on Y-MC-38 and R-MC-38 cells. Fluorescence-activated cell sorting (FACS) analysis of R-MC-38 and Y-MC-38 cells using two monoclonal antibodies against DF3 (mAb DF3) and CA19-9 (mAb CA19-9) epitopes revealed that R-MC-38 cells expressed DF3 but not CA19-9 [DF3(+)CA19-9(-)], while Y-MC-38 cells expressed CA19-9 but not DF3 [DF3(-)CA19-9(+)]. On Western blot, a 40 kDa protein product was recognized by mAb CA19-9 but not by mAb DF3 on cell lysates of Y-MC-38 cells, whereas a 70 kDa protein product was recognized by mAb DF3 but not by mAb CA19-9 on the cell lysates of R-MC-38. Further, we analyzed the expression of MUC-1/Y mRNAs by RT-PCR on various human cancer cell lines: the gastric cancer cell line AZ521, the pancreatic cancer cell lines PANC-1 and Capan-1, the gall bladder cell line GBK-1, the breast cancer cell line MCF-7, and the colon cancer cell lines HT-29 and Colo205. HT-29 and Capan-1 cells producing the 600 bp RT-PCR product, were positive for mAb CA19-9. These results demonstrate that CA19-9 epitope is produced only on MUC-1/Y core protein, suggesting that CA19-9 epitope may be a specific marker for MUC-1/Y protein.

Adenocarcinoma↗
PubMed2001

CD3+CD56+CD8+ cells demonstrating a suppressor T cell-like function in the peripheral blood of colon cancer patients.

We previously reported that HLA-unrestricted CTLs against MUC-1 were induced from colon cancer patients by stimulating peripheral blood lymphocytes (PBLs) with recombinant MUC-1 vaccinia virus (rVMUC-1). We have performed adoptive immunotherapy (AI) for two gastric and two colon cancer patients, using the rVMUC-1-stimulated T lymphocytes. A significant level of HLA-unrestricted cytotoxicity against MUC-1 was induced in the two colon cancer patients (pA and pB) during the first adoptive immunotherapy, but extremely reduced during the second AI. During the second stimulation phase, the rate of CD3+CD56+CD8+ cells were significantly increased and that of CD3+CD56-CD4+ cells were significantly decreased in the two colon cancer patients as compared to the first AI. CD3+CD56+CD8+ and CD3+CD56-CD4+ cells were isolated from the second AI of the colon cancer patient (pB) and designated as D856 and D4, respectively. The D4 cells demonstrated a high level of HLA-unrestricted CTL activity against MUC-1, but D856 cells did not. When D856 cells were mixed with D4 cells at a D856/D4 ratio of 1:3, 1:2, and 1:1 and used as effector cells, the HLA-unrestricted and MUC-1-specific CTL activity of D4 cells was suppressed in a D856/D4 ratio-dependent manner. Further, D856 cells were highly lytic for the D4 cells demonstrating HLA-unrestricted cytotoxicity against MUC-1. It is concluded that the reduction in HLA-unrestricted cytotoxicity against MUC-1 during the second AI is attributed to the D856 cells killing MUC-1-specific CTLs (D4). Thus, the CD3+CD56+ CD8+ cells seem likely to behave as a suppressor T cell.

Adenocarcinoma↗
PubMed1999

Systemic endothelial function is preserved in men with both active and inactive variant angina pectoris.

To test the hypothesis that coronary spasm could be a coronary manifestation of systemic endothelial dysfunction and that the activity of coronary spasm could influence systemic endothelial function, we examined brachial flow-mediated, endothelium-dependent vasodilation and nitroglycerin-induced endothelium-independent vasodilation with high-resolution ultrasound in 11 men with variant angina pectoris (6 active and 5 inactive) without established coronary atherosclerosis. Endothelium-dependent vasodilation in peripheral circulation was preserved in men with active and inactive variant angina pectoris, suggesting that systemic endothelial dysfunction is not involved in either the pathogenesis or the activity of coronary spasm.

Aged↗
PubMed1999

Modification of acetylcholine release by nociceptin in conscious rat striatum.

Nociceptin (NOC), an endogenous ligand for the orphan opioid receptor ORL1 (ORL1), has recently been recognized as a neuropeptide. We used brain microdialysis and on-line high performance liquid chromatography (HPLC) to examine the effect of NOC on the basal outflow of acetylcholine (ACh) in the freely moving rat striatum in vivo. ACh release was reduced by nociceptin at a concentration of 10(-5) M to 79% of control release. This effect of NOC was attenuated by [Phe1Psi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 (PhePsi), suggesting that NOC activates the ORL1 receptor and (PhePsi) acts as an antagonist on ORL1 in rat striatum in vivo. These findings indicate that NOC may act as a neuropeptide which inhibits ACh release in the striatum via ORL1.

Acetylcholine↗
PubMed1999

Mutational analysis of the cardiac actin gene in familial and sporadic dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) results in part from genetic disorders. Recently, missense mutations of the cardiac actin gene have been reported to cause DCM. We studied 136 Japanese DCM cases to elucidate how frequently the gene mutations are involved in its pathogenesis. Genomic DNA samples were obtained from 136 DCM cases (107 males, 29 females), containing 30 familial DCM (5 confirmed and 25 suspected). All six exons of the cardiac actin gene were analyzed by polymerase chain reaction, single-strand conformation polymorphism, and sequencing. We detected no mutations of the disease causation previously reported (G867A or A1014G) but two silent mutations (G979C and C1018T) in exon 6 and one point mutation (T1080A) in the 3'-untranslated region. As a result of screening 128 healthy subjects, these novel silent mutations were found to be mere genetic polymorphisms, not responsible for the disease. Although some genetic polymorphisms exist in the cardiac actin gene, mutations of the gene are rarely responsible for DCM, at least in the Japanese patients.

3' Untranslated Regions↗
PubMed1999

Stromelysin promoter 5A/6A polymorphism is associated with acute myocardial infarction.

BACKGROUND: Rupture of the fibrous cap of an atherosclerotic plaque is a key event that predisposes to acute myocardial infarction (AMI). Matrix metalloproteinases (MMPs) may contribute to weakening of the cap, which favors rupture. Stromelysin, a member of MMP family, is identified extensively in human coronary atherosclerotic lesions. It can degrade most of the constituents of extracellular matrix as well as activating other MMPs, which suggests that it may play an important role in plaque rupture. Recently, a common variant (5A/6A) in the promoter of the stromelysin gene has been identified. The 5A/6A polymorphism could regulate the transcription of the stromelysin gene in an allele-specific manner. METHODS AND RESULTS: To investigate the relation between the 5A/6A polymorphism in the promoter of the stromelysin gene and AMI, we conducted a case-control study of 330 AMI patients and 330 control subjects. The prevalence of the 5A/6A+5A/5A genotype was significantly more frequent in the patients with AMI than in control subjects (48.8% vs 32.7%, P<0.0001). In logistic regression models, the odds ratio of the 5A/6A+5A/5A was 2.25 (95% CI, 1.51 to 3.35). The association of 5A/6A polymorphism with AMI was statistically significant and independent of other risk factors. CONCLUSIONS: The 5A/6A polymorphism in the promoter of the stromelysin gene is a novel pathogenetic risk factor for AMI.

Alleles↗
PubMed1999

Regulation of squid visual phospholipase C by activated G-protein alpha.

Phospholipase C (PLC) is the key enzyme in the phototransduction cascade of invertebrate rhabdomeric photoreceptors. In addition to 130 kDa PLC, a 95 kDa protein recognized by antibody against the catalytic site of PLC was found in the squid retina. The PLC-like 95 kDa protein (95 kDa PLC) was produced from 130 kDa PLC by an intrinsic protease in the presence of calcium. The 130 kDa PLC was stimulated by the active form of Gq-class G-protein alpha (Gq alpha), but the 95 kDa PLC was not, although their PLC activity was similar. A 35 kDa fragment, the counterpart of 95 kDa PLC, was not recognized by antibodies against catalytic site or N-terminal site of the 130 kDa PLC, indicating that the cleavage site is on the C-terminal side beyond the catalytic site. In the presence of a large excess of the 35 kDa fragment, 95 kDa PLC was stimulated by Gq alpha to a similar extent as intact 130 kDa PLC. These results indicate that the C-terminal polypeptide of PLC is necessary for regulation of its enzyme activity by Gq alpha. The uncoupling of PLC from Gq alpha, caused by limited proteolysis, is therefore a candidate regulatory mechanism of the phototransduction cascade in rhabdomeric photoreceptors.

Animals↗
PubMed1999

Prognostic values of MUC-1 molecule expressing cytokine receptor-like epitope and DF3 in patients with gastric carcinoma.

As recently reported, DF3/MUC-1 molecules having cytokine receptor-like sequences (CRL) at the extracellular region, are likely to function in signal transduction pathways. To elucidate the functional significance of CRL expressed on the DF3/MUC1 molecule, immunohistochemical localization of CRL and/or DF3 was investigated in cases of 115 patients with gastric carcinomas, treated by surgical resection. CRL was detected in 65 of 115 patients (56.5%), DF3 in 85 (73.9%), and both DF3 and CRL in 52 (45.2%). The combined immunohistochemical analysis of CRL and/or DF3, revealed that simultaneous expression of DF3 and CRL (DF3+/CRL+) significantly correlated to lymph node metastasis and to blood vessel invasion, and that patients with DF3+/CRL+-tumors survived for a significantly shorter period after surgery than did the other three groups (DF3+/CRL-, DF3-/CRL+, and DF3-/CRL-). Multivariate analysis showed independent prognostic significance for DF3+/CRL+ expression (hazard ratio [HR]=2.733, P=0.0085), and surgical cure (HR=4.334, P=0.003). To investigate the biological role of the simultaneous expression of DF3 and CRL, we constructed DF3-/CRL+ (NR-MC-38) and DF3+/CRL+ (R-MC-38) cells by transducing a mouse colon adenocarcinoma cell line MC-38 expressing neither DF3 nor CRL with MUC-1 cDNA containing ten tandem repeats (R-MC-38) or MUC-1 cDNA devoid of tandem repeats (NR-MC-38). R-MC-38 (DF3+/CRL+) cells were more invasive than NR-MC-38 (DF3-/CRL+) and MC-38 (DF3-/CRL-) cells. When these transfectants were incubated with pAb CRL, the invasiveness of R-MC-38 (DF3+/CRL+) was strikingly elevated over the case with native MC-38 (DF3-/CRL-) and NR-MC-38 (DF3-/CRL+) cells. The pAb CRL-induced invasiveness of R-MC-38 cells was inhibited by adding mAb DF3 or CRL peptides together with pAb CRL. These results suggest that an expression of DF3/MUC1 is highly associated with cell-invasiveness, and the DF3/MUC1-associated invasiveness is amplified by CRL. Thus DF3+/CRL+-MUC-1 molecule seems to be closely involved in a poor prognosis for gastric cancer patients.

Adult↗
PubMed1999

Presence of RON receptor tyrosine kinase and its splicing variant in malignant and non-malignant human colonic mucosa.

The presence of RON and its variant isoform in malignant and non-malignant human colonic tissues was examined by immunohistochemistry using paraffin-embedded sections and RT-PCR analysis followed by direct sequencing of PCR product using RNAs isolated from frozen tissues. In normal colonic mucosa, RON was uniformly expressed in crypt cells, especially in the bottom of crypta. On the other hand, the expression was distributed heterogeneously in adenomas and in colon cancer. The expression of RON was significantly related to the degree of differentiation of colon cancer and the deletion of the expression was observed in colon cancer specimens with high incidence. The RT-PCR analysis of RNA isolated from non-malignant and malignant colonic tissue revealed the presence of two RON mRNA isoforms with 432-bp and 286-bp. Direct sequencing of major product of 432-bp was revealed to be identical to that of human wild-type RON. On the other hand, major product of 286-bp was revealed to be almost identical to that of a splicing variant of RON transcript which has been found in human gastric cancer cell line, KATO-III. The results obtained in this study may indicate that both wild-type RON and its variant isoform play an important role in regulating the normal function of colonic mucosa such as differentiation and motile activity and the expression of both wild-type RON and its variant isoform could be considered to be reduced during malignancy of human colonic mucosa.

Adenocarcinoma↗