Biomedical subjects

Masatsugu Ueda

Publications and source records attributed to Masatsugu Ueda.

At least 19 recordsLinked to original sources

PubMed2007

Transposon-tagged mutagenesis in the rat.

Although the laboratory rat (Rattus norvegicus) is an indispensable experimental animal for biomedical research and drug development, the lack of embryonic stem cell lines hampers gene-knockout studies. Here we report the successful generation of insertional mutant rats using the Sleeping Beauty (SB) transposon system. This would benefit a variety of biomedical research fields for which the rat model is better suited than the mouse model.

Adenosine Triphosphatases↗

PubMed2006

Establishment and characterization of CAG/EGFP transgenic rabbit line.

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP fluorescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.

Animals↗

PubMed2006

Disruption of genetic interaction between two autosomal regions and the X chromosome causes reproductive isolation between mouse strains derived from different subspecies.

Reproductive isolation that initiates speciation is likely caused by incompatibility among multiple loci in organisms belonging to genetically diverging populations. Laboratory C57BL/6J mice, which predominantly originated from Mus musculus domesticus, and a MSM/Ms strain derived from Japanese wild mice (M. m. molossinus, genetically close to M. m. musculus) are reproductively isolated. Their F1 hybrids are fertile, but successive intercrosses result in sterility. A consomic strain, C57BL/6J-ChrX(MSM), which carries the X chromosome of MSM/Ms in the C57BL/6J background, shows male sterility, suggesting a genetic incompatibility of the MSM/Ms X chromosome and other C57BL/6J chromosome(s). In this study, we conducted genomewide linkage analysis and subsequent QTL analysis using the sperm shape anomaly that is the major cause of the sterility of the C57BL/6J-ChrX(MSM) males. These analyses successfully detected significant QTL on chromosomes 1 and 11 that interact with the X chromosome. The introduction of MSM/Ms chromosomes 1 and 11 into the C57BL/6J-ChrX(MSM) background failed to restore the sperm-head shape, but did partially restore fertility. This result suggests that this genetic interaction may play a crucial role in the reproductive isolation between the two strains. A detailed analysis of the male sterility by intracytoplasmic sperm injection and zona-free in vitro fertilization demonstrated that the C57BL/6J-ChrX(MSM) spermatozoa have a defect in penetration through the zona pellucida of eggs.

Animals↗

PubMed2006

Inverse relationship between serum erythropoietin and blood lead concentrations in Kathmandu tricycle taxi drivers.

OBJECTIVE: Kathmandu tricycle taxi drivers, whose environmental lead (Pb) exposure is ascribable mainly to vehicular exhaust, were studied to examine a dose-response relationship between blood Pb (Pb-B) and serum erythropoietin (sEPO) concentrations. METHODS: Subjects were 27 drivers and 9 non-drivers. They were non-anemic healthy men with normal renal function. Pb-B was measured by an atomic absorption spectrometer with a graphite furnace, and sEPO was determined with a sandwich-type enzyme-linked immunosorbent assay. RESULTS: sEPO levels in drivers were lower than those of non-drivers, while Pb-B levels in drivers were higher than those of non-drivers. There was an inverse relationship between Pb-B and sEPO. CONCLUSIONS: The data suggest that Pb inhibits renal EPO production in a dose-dependent manner in persons with subclinical Pb toxicity. sEPO may serve as an early biochemical marker of subclinical Pb toxicity.

Adolescent↗

PubMed2006

Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats.

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33 degrees C. A temperature shift to 37 degrees C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.

Animals↗

PubMed2006

Transcriptional expression of survivin and its splice variants in endometriosis.

Survivin is a novel inhibitor of apoptosis (IAP), and the two splice variants of survivin (survivin-2B and survivin-EX3) have been identified. Gene expression levels of survivin, survivin-2B and survivin-EX3 in 56 ectopic (16 peritoneal red and 16 peritoneal black lesions and 24 ovarian endometriomata) and 13 eutopic endometrial tissues surgically obtained from 42 women with endometriosis (group A) were compared with those in 16 control eutopic endometrium from 16 women without endometriosis (group B) by quantitative RT-PCR analysis. Survivin mRNA expression levels in ectopic endometriotic tissues were significantly higher than those in eutopic endometrium of groups A and B over the whole cycle. Red peritoneal lesions had higher gene expression levels of survivin than black lesions. In contrast, all tissue samples examined showed relatively lower gene expression levels of survivin-2B and survivin-EX3. No cyclic variation was found in survivin and the two splice variants, both in ectopic and in eutopic endometrium. Although there was no significant difference in the ratio of survivin-2B/survivin between ectopic and eutopic endometrium, the ratio of survivin-EX3/survivin in peritoneal endometriotic lesions was significantly higher than that of eutopic endometrium of groups A and B. These results suggest that survivin and survivin-EX3 may be closely linked to escape from apoptosis and the development of endometriosis.

Adult↗

PubMed2006

Constitutive expression of the Period1 gene impairs behavioral and molecular circadian rhythms.

Three mammalian Period (Per) genes, termed Per1, Per2, and Per3, have been identified as structural homologues of the Drosophila circadian clock gene, period (per). The three Per genes are rhythmically expressed in the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. The phases of peak mRNA levels for the three Per genes in the SCN are slightly different. Light sequentially induces the transcripts of Per1 and Per2 but not of Per3 in mice. These data and others suggest that each Per gene has a different but partially redundant function in mammals. To elucidate the function of Per1 in the circadian system in vivo, we generated two transgenic rat lines in which the mouse Per1 (mPer1) transcript was constitutively expressed under the control of either the human elongation factor-1alpha (EF-1alpha) or the rat neuron-specific enolase (NSE) promoter. The transgenic rats exhibited an approximately 0.6-1.0-h longer circadian period than their wild-type siblings in both activity and body temperature rhythms. Entrainment in response to light cycles was dramatically impaired in the transgenic rats. Molecular analysis revealed that the amplitudes of oscillation in the rat Per1 (rPer1) and rat Per2 (rPer2) mRNAs were significantly attenuated in the SCN and eyes of the transgenic rats. These results indicate that either the level of Per1, which is raised by overexpression, or its rhythmic expression, which is damped or abolished in over expressing animals, is critical for normal entrainment of behavior and molecular oscillation of other clock genes.

Animals↗

PubMed2006

Infection of human papillomavirus and overexpression of dihydrodiol dehydrogenase in uterine cervical cancer.

OBJECTIVE: To determine the association of human papillomavirus (HPV) infection with the expression of dihydrodiol dehydrogenase (DDH) in uterine cervical cancer (UCC). METHODS: In situ hybridization (ISH) and immunohistochemistry were applied to examine pathological specimens of 145 patients with UCC. RESULTS: By ISH, HPV16/18 DNA was detected in 108 (74.5%) UCC cases. DDH expression determined by immunohistochemistry was detected in 81 (75%) lesions among 108 HPV-positive cases. In contrast, of 37 HPV-negative cases, DDH was only detected in 16 (43.2%) of the lesions. A significant correlation was found between DDH expression and the presence of HPV (P < 0.001), FIGO stage (P = 0.004), lymph node involvement (P < 0.001), as well as patients' survival (P = 0.002). In vitro, DDH expression was also found closely associated with HPV infection, and DDH content was proportional to cell sensitivity for cisplatin and doxorubicin. CONCLUSIONS: HPV infection provokes local inflammation, which can then induce DDH expression and drug resistance in UCC. The detailed biological relationship among HPV infection, expression of DDH and drug resistance, however, remains to be clarified.

Cell Line, Tumor↗

PubMed2005

Diagnostic and therapeutic laser conization for cervical intraepithelial neoplasia.

OBJECTIVE: The clinical efficacy of conservative treatment using Nd-YAG laser technique for cervical intraepithelial neoplasia (CIN) was evaluated in a large series of CIN patients based on the long-term follow-up results. METHODS: We have treated 2107 women preoperatively diagnosed as having CIN by Nd-YAG contact laser conization with vaporization of the base. Their postoperative histologic findings and clinical outcomes were evaluated. RESULTS: The cone specimen was reported as showing that 1956 (92.8%) of 2107 cases had no CIN or CIN 1-3 and 151 (7.2%) cases had invasive diseases. 1956 cases without invasive diseases were followed up for 16 to 252 months. Incomplete excision occurred in 230 (12.3%) of 1874 patients with CIN lesion in the cone specimen, but failure rate (persistence or recurrence) was only 1.2%. 192 (83.5%) of 230 postoperative CIN patients with positive surgical margins showed no abnormal cytology or colposcopy during follow-up period. Preoperative underdiagnosis of biopsy results compared with cytologic or colposcopic findings elevated the risk for incomplete excision and failure rate. CONCLUSION: The combination of laser excision and vaporization of the base was useful to detect unexpected invasive disease and revealed excellent therapeutic effects for CIN. Preoperative cytologic or colposcopic findings should be taken into account for the conservative treatment of CIN.

Conization↗

PubMed2005

Germline polymorphism of p53 codon 72 in gynecological cancer.

OBJECTIVE: To investigate the biological significance of single nucleotide polymorphism at codon 72 of the p53 gene in the development of gynecological cancer. METHODS: p53 codon 72 polymorphism was examined in a total of 354 blood samples from 95 normal, 83 cervical, 108 endometrial and 68 ovarian cancer cases using polymerase chain reaction and restriction fragment length polymorphism techniques. RESULTS: When p53 codon 72 genotype was classified into two subgroups of Arg/Arg and Arg/Pro + Pro/Pro, the Arg/Arg genotype was associated with an increased risk for the development of endometrial cancer (OR = 1.86, 95% CI = 1.06 to 3.26) compared with the Arg/Pro + Pro/Pro genotype (P = 0.0301). The Arg allele also increased the risk of endometrial cancer (OR = 1.42, 95% CI = 0.93 to 1.52) compared with the Pro allele, but no statistical difference was found (P = 0.1031). There was no significant difference in the genotype or allele prevalence between control subjects and cervical or ovarian cancer patients. CONCLUSION: Homozygous Arg at codon 72 of the p53 gene may be a risk factor for developing endometrial cancer in a Japanese population.

Adult↗

PubMed2005

Vascular endothelial growth factor-C expression and invasive phenotype in ovarian carcinomas.

PURPOSE: To investigate the biological correlation between vascular endothelial growth factor (VEGF)-C expression and invasive phenotype in ovarian carcinomas. EXPERIMENTAL DESIGN: Gene and protein expression levels of VEGF-C in 10 ovarian carcinoma cell lines were correlated with invasive activity of the cells. The correlation between immunohistochemical expression of VEGF-C and tumor aggressiveness in 73 ovarian carcinomas was also examined with respect to clinicopathologic features and patient outcome. RESULTS: VEGF-C gene and protein expression differed remarkably among the cell lines, and there was a statistical correlation among VEGF-C expression, in vitro invasive activity, and matrix metalloproteinase-2 (MMP-2) gene expression and its activity. Anti-VEGF-C and anti-MMP-2 antibodies inhibited the invasive activity of tumor cells. VEGF-C expression in clinical tissue samples was well correlated with clinical stages, retroperitoneal lymph node metastasis, MMP-2 expression, angiogenesis, lymphangiogenesis, and low apoptotic index (AI). The patients whose tumors had strong VEGF-C expression and low AI underwent a poorer prognosis than did those with weak VEGF-C expression and high AI. CONCLUSION: VEGF-C expression is closely related to invasive phenotype and affects the patient's survival in ovarian carcinomas.

Antibodies↗

PubMed2005

Glutathione-S-transferase and p53 polymorphisms in cervical carcinogenesis.

OBJECTIVE: To investigate the clinical significance of glutathione-S-transferase GSTM1, GSTT1 and p53 codon 72 polymorphisms in cervical carcinogenesis. METHODS: GSTM1, GSTT1 and p53 codon 72 polymorphisms together with human papillomavirus (HPV) types were examined in a total of 198 cervical smear samples using multiplex polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (RFLP) techniques. RESULTS: Forty-two patients with high-grade squamous intraepithelial lesion (HSIL) had higher frequency of high-risk HPV and null GSTT1 genotype than 102 with low-grade SIL (LSIL) and 54 controls. Thirty-one patients with HSIL had also statistically higher frequency of null GSTT1 genotype than 28 with LSIL among 69 patients with high-risk HPV. There was no statistical difference in p53 Arg, Arg/Pro and Pro genotypes between SILs and controls with or without high-risk HPV. CONCLUSION: GSTT1 null genotype in cervical cell samples may be associated with more severe precancerous lesions of the cervix in a Japanese population. The p53 codon 72 polymorphism is unlikely to be related to HPV status and the onset of cervical cancer.

Cell Transformation, Neoplastic↗

PubMed2005

Fas gene promoter -670 polymorphism (A/G) is associated with cervical carcinogenesis.

OBJECTIVE: To investigate the biological significance of single nucleotide polymorphism (SNP) at Fas gene promoter in cervical carcinogenesis. METHODS: SNP at -670 of Fas gene promoter (A/G) together with human papillomavirus (HPV) types were examined in a total of 279 cervical smear samples and 8 human cervical squamous carcinoma cell lines using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques. RESULTS: 49 patients with high-grade squamous intraepithelial lesion (HSIL) had higher frequency of high-risk HPV and GA + GG genotype than 167 with low-grade SIL (LSIL) and 63 controls. G allele frequency was also higher in HSIL than in LSIL and controls. There was an increased OR (6.00; CI, 1.32-27.37; P = 0.021) for GA + GG genotype in HSIL cases compared to controls among 96 patients with high-risk HPV. 7 of 8 cervical carcinoma cell lines also showed GA or GG genotype. CONCLUSION: Fas gene promoter -670 polymorphism (A/G) may be closely associated with cervical carcinogenesis in a Japanese population.

Carcinoma, Squamous Cell↗

PubMed2005

Transcriptional expression of the genes implicated in angiogenesis and tumor invasion in cervical carcinomas.

OBJECTIVE: Co-expression patterns of the genes implicated in angiogenesis and tumor invasion in cervical carcinoma cells were investigated together with invasive activity of tumor cells. Transcript levels of those genes were also compared between tumor cells and normal cervical tissues. METHODS: Real-time quantitative RT-PCR analysis was conducted on selected 11 genes (total VEGF-A, VEGF(121), VEGF(165), VEGF(189), VEGF-B, C and D, bFGF, dThdPase, MMP-2 and uPA) using 11 cervical carcinoma cell lines and 14 normal cervical tissues. Protein expression of VEGF-C and MMP-2 and invasive activity of tumor cells were evaluated for each cell line by sandwich ELISA and haptoinvasion assay, respectively. RESULTS: Gene co-expression analysis revealed the significant correlation between angiogenic factors and proteinases in malignant but not in normal cervical samples. Gene or protein expression levels of VEGF-C and MMP-2 were well correlated with the number of invaded tumor cells. VEGF-A splicing variants were increased in malignant compared to normal cervical samples but not associated with the invasive activity of the cells. CONCLUSION: VEGF-C and MMP-2 were closely related to invasive phenotype of tumor cells, whereas VEGF-A isoforms were considered to be involved in cervical carcinogenesis.

Angiogenic Proteins↗

PubMed2005

Tumor angiogenesis and molecular target therapy in ovarian carcinomas.

Growth of solid tumors depends on angiogenesis, the process by which new blood vessels develop from the endothelium of a pre-existing vasculature. Tumors promote angiogenesis by secreting various angiogeneic factors, and newly formed blood vessels induce tumor cell proliferation and invasiveness. Ovarian carcinomas have a poor prognosis, often associated with multifocal intraperitoneal dissemination accompanied by intense neovascularization. The degree of angiogenesis of ovarian carcinomas may directly influence the clinical course of the disease. Although a growing body of evidence indicates that angiogenic intensity may play a prognostic role in gynecological malignancies including ovarian carcinomas, the related biological mechanisms remain to be further elucidated. In this review, we describe current knowledge pertaining to mechanisms and regulation of angiogenesis in ovarian carcinomas with special reference to our recent research results.

Angiogenesis Inducing Agents↗

PubMed2004

Restoration of copper metabolism and rescue of hepatic abnormalities in LEC rats, an animal model of Wilson disease, by expression of human ATP7B gene.

Hepatic abnormalities in Long-Evans Cinnamon (LEC) rats, an animal model of Wilson disease (WD), were restored by the expression of the human ATP7B cDNA under the control of CAG promoter. Expression of ATP7B transcript and protein in the liver of the transgenic rats resulted in the restoration of biosynthesis of holoceruloplasmin and biliary copper excretion. Meanwhile, transgenic rats showed striking improvements in their hepatic abnormalities, i.e., rescue from fulminant hepatitis, late onset of hepatic cholangiofibrosis, suppression of hepatocellular carcinoma and much improved survival rates. Moreover, dramatic decreases were noted both in the levels of hepatic copper and iron in transgenic rats before the occurrence of hepatitis. These results indicated that the human ATP7B product compensated for the deficiency of the endogenous rattus protein and did function in intrahepatic copper transport by secreting copper into the plasma via incorporation into ceruloplasmin and by the excretion of copper into the bile, and that ATP7B is critical to hepatic dysfunctions in WD. This first successful transgenic rescue has important implications for the gene therapy of WD.

Adenosine Triphosphatases↗

PubMed2004

Vascular endothelial growth factor A and C gene expression in endometriosis.

Angiogenesis is essential for the pathogenesis of endometriosis. Gene expression levels of vascular endothelial growth factor (VEGF) A and C in 10 eutopic endometrial, 23 normal peritoneal, and 62 endometriotic tissues surgically obtained from 47 women with endometriosis (group 2) were compared with those in 12 control eutopic endometrial and 9 normal peritoneal tissues from 15 women without endometriosis (group 1). VEGF-A mRNA expression levels in eutopic endometrium of group 2 were higher than those of group 1 throughout the menstrual cycle (P <0.01) and increased in the secretory phase. VEGF-A gene expression in peritoneal endometriotic lesion was statistically higher than that in normal peritoneum (P <0.01) and similar to that in eutopic endometrium of group 2. In contrast, gene expression levels of VEGF-C were relatively lower than those of VEGF-A in each lesion, and no cyclic variation was found. VEGF-A and C mRNA expression levels were significantly higher in ovarian endometriomas >6 cm in size than in those <6 cm in size. Immunohistochemical expression of VEGF-A and C was detected in the cytoplasm of glandular epithelial and stromal cells of ovarian endometrioma. These results suggest that endometriosis may arise from eutopic endometrium with higher levels of angiogenic activity possibly induced by VEGF-A in women with endometriosis. Moreover, VEGF-C as well as VEGF-A may be involved in the pathogenesis of ovarian endometrioma.

DNA Primers↗

PubMed2004

Conservative excisional laser conization for early invasive cervical cancer.

OBJECTIVE: To investigate the possibility of conservative excisional laser conization for early invasive cervical cancer. METHODS: Four hundred one women with early invasive squamous cell cancer were treated by laser conization and semiradical or radical hysterectomy with pelvic lymphadenectomy. Their histologic findings and clinical outcomes were evaluated retrospectively. RESULTS: Two hundred Ia1 cases without confluent invasion or vessel permeation receiving only laser therapy had no recurrent disease. There was no lymph node metastasis in 123 Ia1 and 24 Ia2 cases with stromal invasion of under 4 mm in depth regardless of confluent invasion and vessel permeation. However, lymph node metastasis was detected in 1 of 13 Ia2 cases with stromal invasion of over 4 mm in depth and in 5 of 41 Ib1 cases. All of these six cases had vessel permeation in the resected specimens. CONCLUSION: Conservative excisional laser conization may be possible for stage Ia cervical cancer with stromal invasion of under 4 mm in depth. However, the risk of lymph node metastasis should be still considered for those lesions with vessel permeation.

Carcinoma, Squamous Cell↗