Biomedical subjects

M Ueda

Publications and source records attributed to M Ueda.

At least 109 records · Page 6Linked to original sources

PubMed2001

Microcirculatory changes in right lobe grafts in living-donor liver transplantation: a near-infrared spectrometry study.

BACKGROUND: A continuing shortage of cadaveric liver even for adult patients has motivated not a few centers to proceed to living-donor liver transplantation using right lobe grafts. One of controversies is potential congestion in the graft anterior segment by the deprivation of the middle hepatic vein. METHODS: Hepatic tissue oxygenation and hemoglobin concentration were investigated with a near-infrared spectroscopy in the course of harvesting and implantation in living-donor liver transplantation. Twenty adult recipients of right lobe graft were involved in the study. The aim of the analysis was to detect tissue congestion or ischemia. RESULTS: No significant change in mean hepatic tissue oxygenation and hemoglobin was noted in the right lobe during donor operation even after hepatic parenchymal transection, although some trend for relative congestion, i.e., increased tissue hemoglobin, compared with the left lobe was observed. After graft reperfusion in the recipient, both mean hepatic tissue oxygen saturation and hemoglobin decreased significantly in the anterior segment, which was accompanied by increased heterogeneity of tissue hemoglobin and oxygenation. Increased heterogeneity of oxygenation and decreased tissue hemoglobin were observed also in the posterior segment. CONCLUSIONS: The anterior segment in right lobe living-donor liver transplantation is sensitive to ischemia, rather than congestion, at least in the immediate phase after graft reperfusion. The anterior segment seems to be also more prone to circulatory disturbance than the other part of the graft.

Adult↗

PubMed2001

Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction.

Myelodysplastic syndrome (MDS) is a slowly progressing hematologic malignancy associated with a poor outcome. Despite the relatively high incidence of MDS in the elderly, differentiation of MDS from de novo acute myeloid leukemia (AML) still remains problematic. Identification of genes expressed in an MDS-specific manner would allow the molecular diagnosis of MDS. Toward this goal, AC133 surface marker-positive hematopoietic stem cell (HSC)-like fractions have been collected from a variety of leukemias in a large-scale and long-term genomics project, referred to as "Blast Bank," and transcriptome of these purified blasts from the patients with MDS were then compared with those from AML through the use of oligonucleotide microarrays. A number of genes were shown to be expressed in a disease-specific manner either to MDS or AML. Among the former found was the gene encoding the protein Delta-like (Dlk) that is distantly related to the Delta-Notch family of signaling proteins. Because overexpression of Dlk may play a role in the pathogenesis of MDS, the disease specificity of Dlk expression was tested by a quantitative "real-time" polymerase chain reaction analysis. Examination of the Blast Bank samples from 22 patients with MDS, 31 with AML, and 8 with chronic myeloid leukemia confirmed the highly selective expression of the Dlk gene in the individuals with MDS. Dlk could be the first candidate molecule to differentiate MDS from AML. The proposal is made that microarray analysis with the Blast Bank samples is an efficient approach to extract transcriptome data of clinical relevance for a wide range of hematologic disorders.

DNA↗

PubMed2001

Mutations of the beta- and gamma-catenin genes are uncommon in human lung, breast, kidney, cervical and ovarian carcinomas.

Beta-catenin forms complexes with Tcf and Lef-1 and functions as a transcriptional activator in the Wnt signalling pathway. Although recent investigations have been focused on the role of the adenomatous polyposis coli (APC)/ beta-catenin/Tcf pathway in human tumorigenesis, there have been very few reports on mutations of the beta-catenin gene in a variety of tumour types. Using PCR and single-strand conformational polymorphism analysis, we examined 93 lung, 9 breast, 6 kidney, 19 cervical and 7 ovarian carcinoma cell lines for mutations in exon 3 of the beta-catenin gene. In addition, we tested these same samples for mutations in the NH2-terminal regulatory region of the gamma-catenin gene. Mutational analysis for the entire coding region of beta-catenin cDNA was also undertaken in 20 lung, 9 breast, 5 kidney and 6 cervical carcinoma cell lines. Deletion of most beta-catenin coding exons was confirmed in line NCI-H28 (lung mesothelioma) and a silent mutation at codon 214 in exon 5 was found in HeLa (cervical adenocarcinoma). A missense mutation at codon 19 and a silent mutation at codon 28 in the NH2-terminal regulatory region of the gamma-catenin gene were found in H1726 (squamous cell lung carcinoma) and H1048 (small cell lung carcinoma), respectively. Neither deletions nor mutations of these genes were detected in the other cell lines examined. These results suggest that beta- and gamma-catenins are infrequent mutational targets during development of human lung, breast, kidney, cervical and ovarian carcinomas.

Cytoskeletal Proteins↗

PubMed2001

Antitumor effect of beta2-microglobulin in leukemic cell-bearing mice via apoptosis-inducing activity: activation of caspase-3 and nuclear factor-kappaB.

We have reported previously that beta2-microglobulin (beta2m) induces apoptosis in leukemic cells in vitro, and that an interaction between beta2m and HLA class I antigen induces apoptosis. Here we examined whether beta2m can induce apoptosis in leukemic cells in vivo and whether it has an antitumor effect in tumor-bearing mice. Daily administration of 50 or 250 microg of beta2m induced apoptosis and an antitumor effect on K562 leukemia cell-bearing mice in the same manner as tumor necrosis factor-alpha. In tumor tissues in beta2m-treated mice, both caspase-3 and nuclear factor-kappaB (NF-kappaB) were stained more strongly than in control mice by anti-caspase-3 and anti-NF-kappaB p65/Rel A polyclonal antibodies. We also observed the in vivo immunological effects of beta2m on lymphoid and hematopoietic organs, such as thymus, bone marrow, Peyer's patches, liver, and spleen in normal mice. Using antibodies against caspase-3 and NF-kappaB, immunohistochemical staining showed that no specific tissues were damaged or stained in normal mice. We conclude that beta2m stimulates caspase-3 and NF-kappaB pathways to induce apoptosis, making it a useful approach to a new therapy for leukemia.

Animals↗

PubMed2001

Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes.

BACKGROUND: There is accumulating data that acute coronary syndromes relate to recent onset activation of inflammation affecting atherosclerotic plaques. Increased blood levels of oxidized low density lipoprotein (ox-LDL) could play a role in these circumstances. METHODS AND RESULTS: Ox-LDL levels were measured in 135 patients with acute myocardial infarction (AMI; n=45), unstable angina pectoris (UAP; n=45), and stable angina pectoris (SAP; n=45) and in 46 control subjects using a sandwich ELISA method. In addition, 33 atherectomy specimens obtained from a different cohort of patients with SAP (n=10) and UAP (n=23) were studied immunohistochemically for ox-LDL. In AMI patients, ox-LDL levels were significantly higher than in patients with UAP (P<0.0005) or SAP (P<0.0001) or in controls (P<0.0001) (AMI, 1.95+/-1.42 ng/5 microgram LDL protein; UAP, 1.19+/-0.74 ng/5 microgram LDL protein; SAP, 0.89+/-0.48 ng/5 microgram LDL protein; control, 0.58+/-0.23 ng/5 microgram LDL protein). Serum levels of total, HDL, and LDL cholesterol did not differ among these patient groups. In the atherectomy specimens, the surface area containing ox-LDL-positive macrophages was significantly higher in patients with UAP than in those with SAP (P<0.0001). CONCLUSIONS: This study demonstrates that ox-LDL levels show a significant positive correlation with the severity of acute coronary syndromes and that the more severe lesions also contain a significantly higher percentage of ox-LDL-positive macrophages. These observations suggest that increased levels of ox-LDL relate to plaque instability in human coronary atherosclerotic lesions.

Angina Pectoris↗

PubMed2001

Accumulation of LOX-1 ligand in plasma and atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits: identification by a novel enzyme immunoassay.

LOX-1 (lectin-like oxidized LDL receptor) is a newly identified cell surface receptor for oxidized LDL mainly expressed in endothelial cells. Recombinant soluble LOX-1(LOX-Fc) was generated by fusing the extracellular domain of LOX-1 with the Fc portion of IgG. A novel sandwich enzyme immunoassay specific for LOX-1 ligand is designed, using LOX-Fc and anti-apoB antibody. This immunoassay was used to determine LOX-1 ligand activity in normal and Watanabe heritable hyperlipidemic (WHHL) rabbit plasma. LOX-Fc was further applied for staining of atherosclerotic lesions of WHHL rabbits. LOX-1 ligand levels were significantly elevated in the plasma of hyperlipidemic rabbits compared with controls. Furthermore, LOX-1 ligand activity was detected in the atherosclerotic lesions in situ. These results support the potential roles of LOX-1 interacting with its ligand in the pathogenesis of atherosclerosis, which is enhanced in hyperlipidemia.

Animals↗

PubMed2001

Activation-dependent surface expression of LOX-1 in human platelets.

Lectin-like oxidized LDL receptor-1 (LOX-1) was initially identified as an oxidized LDL receptor in aortic endothelial cells. Here we identified LOX-1 mRNA and protein in human platelets in addition to recent findings on the expression in macrophages and smooth muscle cells. The presence of LOX-1 was further confirmed in the megakaryocytic cell lines. Flow cytometric analyses revealed that LOX-1 was exposed on the surface of platelets in an activation-dependent manner. Consistently, the activation-dependent binding of OxLDL to platelets was mostly inhibited by anti-LOX-1 antibody. Immunohistochemistry of the atherosclerotic plaque from a patient with unstable angina pectoris (UAP) revealed accumulation of LOX-1 protein at the site of thrombus. As LOX-1 recognizes and binds activated platelets, exposure of LOX-1 on activated platelets surface might assist thrombosis formation.

Animals↗

PubMed2001

Correlation between thymidine phosphorylase expression and invasion phenotype in cervical carcinoma cells.

The correlation between thymidine phosphorylase (dThdPase) expression and invasion phenotype in human uterine cervical carcinoma cells was investigated using 10 cervical carcinoma cell lines. Semi-quantitative reverse transcription-polymerase chain reaction analysis was performed to investigate the mRNA levels of dThdPase and matrix metalloproteinase (MMP)-2 with beta-actin coamplified as an internal standard. dThdPase protein expression levels were detected by highly sensitive enzyme-linked immunosorbent assay. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were evaluated by haptotactic migration and invasion assay. Although dThdPase mRNA and protein expression levels differed remarkably among the cell lines, there was a statistical correlation between them (r = 0.743, p = 0.0139). dThdPase gene and protein expression levels were well correlated with the number of cells that migrated and invaded (p < 0.05). Moreover, there was a close correlation between MMP-2 gene and dThdPase gene and protein expression levels (p < 0.05). Tumor cells that produce dThdPase may have a higher invasive and metastatic potential because of their capacity to pass through tissue barriers.

Carcinoma, Squamous Cell↗

PubMed2001

Activation of BMK1 via tyrosine 1062 in RET by GDNF and MEN2A mutation.

Big mitogen-activated protein kinase 1 (BMK1) is a new member of mitogen-activated protein kinase (MAPK) family. In the present study, we investigated whether glial cell line-derived neurotrophic factor (GDNF) can induce activation of BMK1 through RET tyrosine kinase. Its activation reached a maximal level at 30 min and continued at least for 120 min after GDNF stimulation. In addition, we detected BMK1 activation in NIH3T3 cells expressing RET with a multiple endocrine neoplasia (MEN) 2A mutation. The level of BMK1 activation markedly decreased by replacement of tyrosine 1062 with phenylalanine (designated Y1062F) in RET, indicating the importance of downstream signaling via tyrosine 1062. However, although both RAS/MAPK and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways are activated via tyrosine 1062, BMK1 activation by GDNF was not significantly impaired by treatment with an MEK1 inhibitor, PD98059, or two distinct PI3-K inhibitors, LY294002 and wortmannin, suggesting that the RAS and PI3-K signaling pathways are not crucial for BMK1 activation by GDNF. Moreover, luciferase reporter assays revealed that RET-MEN2A mutant proteins can activate the MEF2C transcription factor that is known to be a cellular target for BMK1, and that its activation is impaired by the Y1062F mutation or by expression of a dominant negative form of MEK5.

Animals↗

PubMed2001

Intermittent implosion and pattern formation of trapped Bose-Einstein condensates with an attractive interaction.

The collapsing dynamics of a trapped Bose-Einstein condensate with attractive interaction are revealed to exhibit two previously unknown phenomena. During the collapse, the condensate undergoes a series of rapid implosions that occur intermittently within a very small region. When the sign of the interaction is suddenly switched from repulsive to attractive, e.g., by the Feshbach resonance, density fluctuations grow to form various patterns such as a shell structure.

Journal Article↗

PubMed2001

Field-inversion electrophoresis on a microchip device.

We have proposed a field-inversion electrophoresis on a microchip for a shorter effective length, and investigated the external frequency for the DNA analysis based on the field-inversion electrophoresis device. By using the optimized frequency, we demonstrated that the field-inversion electrophoresis has great potentials for the separation of DNA fragments with shorter effective length.

DNA, Single-Stranded↗

PubMed2001

Direct measurement of conformational changes on DNA molecule intercalating with a fluorescence dye in an electrophoretic buffer solution by means of atomic force microscopy.

By means of atomic force microscopy (AFM), we performed the direct imaging of DNA molecules (200, 500, 1000 bp) in a Tris-borate buffer solution, and measured the contour length and the end-to-end distance of DNA. Processing the data according to the worm-like chain model, we calculated the persistence length of the double-stranded DNA. Based on the analysis of the contour length and the persistence length, we discussed the interactions between DNA and an intercalating fluorescence dye (YO-PRO-1). YO-PRO-1 stacks between the base pairs and extends the contour length of DNA, changing the electric charge and the persistence length of DNA. From AFM measurement, we investigated directly the relationship between the persistence length and the number of the YO-PRO-1 intercalating to DNA. We will discuss on the relationship between the effect of an intercalating dye on the electrophoretic behavior and the conformational changes of DNA with an intercalating dye.

Benzoxazoles↗

PubMed2001

Vascular smooth muscle cell growth-promoting factor/F-spondin inhibits angiogenesis via the blockade of integrin alphavbeta3 on vascular endothelial cells.

Vascular smooth muscle cell growth-promoting factor (VSGP) was originally isolated from bovine ovarian follicular fluid as a stimulator of vascular smooth muscle cell proliferation. Homology searches indicate that bovine and human VSGPs are orthologs of rat F-spondin. Here, we examined whether recombinant human VSGP/F-spondin affected the biological activities of endothelial cells. VSGP/F-spondin did not affect the proliferation of human umbilical vein endothelial cells (HUVECs); however, it did inhibit VEGF- or bFGF-stimulated HUVEC migration. To clarify the mechanism of this inhibitory effect, we examined the adhesion of HUVECs to extracellular matrix proteins. VSGP/F-spondin specifically inhibited the spreading of HUVECs on vitronectin via the functional blockade of integrin alphavbeta3. As a result, VSGP/F-spondin inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) when HUVECs were plated on vitronectin. Moreover, VSGP/F-spondin inhibited the activation of Akt when HUVECs on vitronectin were stimulated with VEGF. VSGP/F-spondin inhibited tube formation by HUVECs in vitro and neovascularization in the rat cornea in vivo. These results indicate that VSGP/F-spondin inhibits angiogenesis at least in part by the blockade of endothelial integrin alphavbeta3.

Animals↗

PubMed2001

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection.

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.

Animals↗

PubMed2001

Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat.

The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37 degrees C for a period of 48 hr. Cells were subsequently cultured at 33 degrees C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 approximately 9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33 degrees C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39 degrees C due to the reduced expression of large T-antigen, supporting temperature-dependent cell growth. TR-iBRBs expressed GLUT1 and exhibited 3- O -methyl- D -glucose (3-OMG) uptake activity. This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 +/- 0.51 m M and a maximum uptake rate of 45.3 +/- 2.6 nmol min(-1) mg protein(-1). P-Glycoprotein, with a molecular weight of approximately 180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Serotonin modulates expression of VIP and GRP mRNA via the 5-HT(1B) receptor in the suprachiasmatic nucleus of the rat.

The expression of vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) in the suprachiasmatic nucleus (SCN) changes depending on light. VIP mRNA increases and GRP mRNA decreases in the light phase, while they do not show change without light. In the present study we investigated the involvement of serotonin (5-HT) in the expression of VIP and GRP messenger RNA in the SCN of the rat. The decrease in VIP mRNA and the increase in GRP mRNA in the light phase were amplified by 5-HT depletion using 5,6-dihydroxytryptamine injected into the lateral ventricle. These enhancements due to 5-HT depletion were reversed to control levels by applying 5-HT(1B) agonists TFMPP and CGS12066A, but not a 5-HT(1A)/5-HT(7) agonist, 8-OH-DPAT. The 5-HT(1B) receptor is known to exist on the terminals of the retinohypothalamic tract (RHT). Therefore, next we investigated the morphological relationship of RHT and 5-HT terminals by double-labeling immunocytochemistry and demonstrated that 5-HT-immunoreactive fibers and cholera toxin B subunit-labeled RHT terminals were intermingled in the ventrolateral SCN, and 5-HT axon processes had close contact with RHT terminals. Collectively, these pharmacological and morphological results suggest that 5-HT afferents from raphe nuclei modulate VIP and GRP expression in neurons of the ventrolateral SCN by activating the 5-HT(1B) receptor in the RHT.

5,6-Dihydroxytryptamine↗

PubMed2001

Vascular endothelial growth factor C gene expression is closely related to invasion phenotype in gynecological tumor cells.

OBJECTIVE: The correlation between the gene expression of various angiogenic factors and in vitro invasive activity in 16 human gynecological cancer cell lines was investigated. METHODS: Semiquantitative reverse transcription polymerase chain reaction analysis was performed to investigate the mRNA expression levels of vascular endothelial growth factors (VEGF-A, -B, -C, and -D), basic fibroblast growth factor (bFGF), and matrix metalloproteinase (MMP)-2 with beta-actin coamplified as an internal standard. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were evaluated by haptotactic migration and invasion assay. RESULTS: Expression of VEGF-A mRNA was detected in all 16 cell lines, whereas the relative expression levels of other VEGF family members and bFGF, differed markedly among the cell lines. There was a statistical correlation between VEGF-C gene expression and the number of cells that migrated and invaded (P < 0.01). However, expression of mRNAs of other angiogenic factors did not correlate with motility and invasive activity of the cells. Moreover, there was a close correlation between VEGF-C and MMP-2 gene expression levels (P < 0.05). CONCLUSION: Tumor cells that produce VEGF-C may have a higher invasive and metastatic potential because of their capacity to pass through tissue barriers.

DNA, Neoplasm↗

PubMed2001

Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene.

We established a novel and convenient method to construct a ura3 strain (ura3/ura3) of the asporogenous and diploid yeast, Candida tropicalis, that produces dicarboxylic acid. One copy of the URA3 gene was disrupted using a mutated hygromycin B resistance gene (HYG#). The obtained hygromycin-resistant strain was further transformed with a URA3 disruption cassette and selected on a plate containing 5-fluoroorotic acid. The obtained strains were analyzed and the disruption of the gene was confirmed by PCR and Southern blot analysis. The results showed that the strains were obtained in which allelic URA3 genes were simultaneously disrupted. Furthermore, we established a cotransformation method for this gene disruption, using HYG# in C. tropicalis. In order to disrupt the allelic POX4 genes (encoding acyl-CoA oxidase) of dicarboxylic acid-producing strains, the ARS plasmid (which contained HYG#) and a POX4 disruption cassette (which carried the LAC4 gene encoding beta-galactosidase of Kluyveromyces lactis) were simultaneously introduced by transformation. As a result, the allelic POX4 gene was successfully disrupted.

Anti-Bacterial Agents↗