Biomedical subjects

Masatsugu Ueda

Publications and source records attributed to Masatsugu Ueda.

43 records · Page 3Linked to original sources

PubMed2002

Influence of severe endometriosis on gene expression of vascular endothelial growth factor and interleukin-6 in granulosa cells from patients undergoing controlled ovarian hyperstimulation for in vitro fertilization-embryo transfer.

OBJECTIVE: To evaluate how endometriosis affects expression of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in granulosa cells. DESIGN: Prospective study. SETTING: IVF-ET program at Osaka Medical College. PATIENT(S): Seventeen patients with revised American Fertility Society stage IV endometriosis and 17 patients with tubal infertility and no endometriosis. INTERVENTION(S): Granulosa cells obtained at oocyte retrieval were examined for VEGF and IL-6 gene expression. MAIN OUTCOME MEASURE(S): Serum E(2) and P levels at hCG administration, number of oocytes, fertilization rate, high-quality embryo rate, and pregnancy rate, and expression of VEGF and IL-6 genes. RESULT(S): Total hMG and FSH levels were statistically significantly higher in patients with endometriosis; however, the number of retrieved oocytes and the fertilization rate were lower compared with patients with tubal infertility. Serum E(2) levels and expression of VEGF in patients with tubal infertility were statistically significantly higher than those in patients with endometriosis. Interleukin-6 gene expression did not differ between the groups. CONCLUSION(S): In severe endometriosis, lower VEGF gene expression in granulosa cells may adversely affect oocyte development and maturation.

Chorionic Gonadotropin↗

PubMed2002

Offspring derived from intracytoplasmic injection of transgenic rat sperm.

The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at -20 degrees C until use. When the sperm heads were aspirated into injection pipettes 7-10 microm in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2-4 microm in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague-Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed.

Animals↗

PubMed2002

Newly developed rat brain pericyte cell line, TR-PCT1, responds to transforming growth factor-beta1 and beta-glycerophosphate.

Brain pericytes form an incomplete envelope around endothelial cells and within the microvascular basement membrane of capillaries and postcapillary venules. Recently, it has been reported that brain pericytes exhibit pluripotency, regulation of endothelial cell activity, and macrophage activity. However, many molecular and cellular aspects of brain pericytes remain unclear. In this study, we have tried to establish a conditionally immortalized brain pericyte cell line (TR-PCT) derived from the brain capillary of a transgenic rat harboring a temperature-sensitive simian virus 40 T antigen gene. One of the clones was named TR-PCT1, and we established 6 clones of pericyte-like cells from a 16 week-old tsA58 transgenic rat. For comparison, primary pericytes from a Wistar rat were also studied. The expression of platelet-derived growth factor receptor beta, angiopoietin-1, osteopontin, and intercellular adhesion molecule-1 in TR-PCT1 was determined by reverse transcription-polymerase chain reaction. Transforming growth factor-beta1 enhanced a-smooth muscle actin expression in TR-PCT1, but this expression was reduced by subsequent treatment with basic fibroblast growth factor. When TR-PCT1 was seeded on type I collagen plates and treated with beta-glycerophosphate, nodules developed in the cells and these nodules reacted positively to von Kossa stain used as a marker of calcification. We believe that TR-PCT1 will help us gain a better understanding of the physiological and/or pathophysiological role of pericytes.

Actins↗

PubMed2002

Human hypoxic signal transduction through a signature motif in hepatocyte nuclear factor 4.

We identified a human hypoxic signal transduction pathway acting through a signature motif in the carboxyl terminal of hepatocyte nuclear factor 4 (HNF-4), by functional comparison of the transcriptional and protein-protein interaction activities of the wild type and mutants. It was previously shown that HNF-4 functions as a tissue-specific and hypoxia-activated transcription factor for the erythropoietin (Epo) gene. Human HNF-4 (465 amino acid residues) has DNA-binding, ligand-binding, and transactivation domains. The deletion mutant without the carboxyl terminal transactivation domain (amino acids 369-465) has been shown to be a dominant-negative mutant that repressed Epo transcriptional activity in hypoxia. Further characterization of the hypoxia-responsive domain by site-directed mutagenesis indicated that a TKQE motif of the carboxyl terminal (amino acids 460-463) in HNF-4 was essential for hypoxia-inducible Epo gene expression. We also found, by means of immunoprecipitation and a mammalian two-hybrid system, direct interactions between HNF-4 and hypoxia-inducible factor 1 (HIF-1), a heterodimer composed of alpha and beta subunits. HNF-4 was observed to interact with HIF-1alpha and HIF-1beta (arylhydrocarbon receptor nuclear translocator, ARNT) during hypoxia. In addition, the TKQE motif of HNF-4 was essential for protein-protein interactions with HIF-1alpha and ARNT. These results indicate that the human hypoxic signal of HIF-1 is transduced through interactions with the signature TKQE motif of the carboxyl terminal of HNF-4, resulting in Epo gene expression as a response to hypoxia.

Amino Acid Motifs↗

PubMed2002

Survivin gene expression in endometriosis.

Survivin is a novel inhibitor of apoptosis and is expressed during fetal development and in cancer tissues, but its expression has not been reported in normal adult tissues or benign diseases. We investigated survivin gene and protein expression in a tumor-like benign disease, endometriosis, and correlated them with apoptosis and invasive phenotype of endometriotic tissues. Gene expression levels of survivin, matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1 (MT1)-MMP in 63 pigmented or nonpigmented endometriotic tissues surgically obtained from 35 women with endometriosis were compared with those in normal eutopic endometrium obtained from 12 women without endometriosis. Survivin, MMP-2, MMP-9, and MT1-MMP mRNA expression levels in clinically aggressive pigmented lesions were significantly higher than those in normal eutopic endometrium, and survivin gene expression in pigmented lesions was also higher than that in nonpigmented lesions (P < 0.05). There was a close correlation between survivin and MMP-2, MMP-9, or MT1-MMP gene expression levels in 63 endometriotic tissues examined (P < 0.01). Apoptotic cells detected by the dUTP nick-end labeling were rare in 11 ovarian endometriotic tissues, which showed positive immunohistochemical expression for survivin and MMPs. Our findings suggest that up-regulation of survivin and MMPs may cooperatively contribute to survival and invasion of endometriosis.

Adult↗

PubMed2002

Development and characterization of conditionally immortalized gastric epithelial cell lines from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene.

Conditionally immortalized gastric epithelial cell lines were established from transgenic rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene. Gastric mucosal cells and epithelial tissues isolated from the stomach of the transgenic rats were cultured at permissive temperature (33 degrees C), and proliferative cells were cloned by colony formation. Six cell lines (designated as RGE1-01, RGE1-02, RGE1-03, RGE1-21, RGE1-22 and RGE2-01) showing epithelial-like morphology have been established. All cells grew at 33 degrees C, but did not at nonpermissive temperature (39 degrees C). High expression level of large T-antigen in the nuclei was observed at 33 degrees C, whereas the expression level was gradually decreased in a time-dependent manner at 39 degrees C. These results suggest that the temperature-sensitive growth characteristics arise as a result of a function of the tsSV40 large T-antigen. None of the cell lines were transformed as judged by anchorage-independent growth assay. Immunocytochemical findings indicated that all cells expressed epithelial cell markers including cytoskeletal (cytokeratin and actin), basement membrane (laminin and collagen type IV) and junctional complex (ZO-1 and desmoplakin I+II) proteins at 33 degrees C. All cells expressed mRNA of cathepsin E, a pit cell marker. Moreover, transepithelial resistance was observed between apical and basolateral sides in the cells. RGE1-22 cells produced prostaglandin E(2). Levels of mRNA for cathepsin E, transepithelial resistance and prostaglandin E(2) were influenced by the nonpermissive temperature. Thus, these conditionally immortalized gastric cell lines which preserve some epithelial cell characteristics will provide a useful in vitro model of gastric epithelium.

Animals↗

PubMedDate not supplied

MS-818 accelerates mobilization of endothelial progenitor cells and differentiation to endothelial cells.

MS-818 that is a synthetic pyrimidine compound and shown to have neurotrophic actions, enhanced basic fibroblast growth factor (bFGF)-induced angiogenesis in vivo. However, the mechanism and whether MS-818 affects endothelial cells (ECs) directly is not known. Here, the authors investigated whether MS-818 alone could induce angiogenesis and tried to clarify the mechanism of neovascularization by MS-818 in terms of angiogenesis and vasculogenesis. The authors show that MS-818 affects ECs directly and induces migration of and tube formation by ECs in vitro (angiogenesis). Furthermore, the authors demonstrate that MS-818 mobilizes endothelial progenitor cells (EPCs) from the bone marrow and potentiates their differentiation to ECs (vasculogenesis). The effect of MS-818 on the endothelial differentiation was further confirmed with an in vitro differentiation system using mouse embryonic stem cells. MS-818 activates the mitogen-activated protein kinase (MAPK) pathway but not the phosphoinositol 3-kinase (PI3K)-Akt pathway in ECs. These results indicate that MS-818, a synthetic compound, promotes both angiogenesis and vasculogenesis.

Animals↗