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Biomedical subjects

Masatsugu Ueda

Publications and source records attributed to Masatsugu Ueda.

At least 37 records · Page 2Linked to original sources
PubMed2004

Catalase in manipulation buffer enhances the developmental competence of DNA-injected embryos.

To improve the efficiency of transgenesis, we investigated the effects of a radical scavenger during microinjection on the development to blastocysts or pups of mouse pronuclear embryos, microinjected with the enhanced green fluorescent protein (EGFP) transgene. When embryos were microinjected in medium containing 0-1,000 units/ml catalase, the developmental rate to blastocysts was significantly higher (P<0.01) in 100-units/ml catalase (81%) than those in 0 and 1,000 units/ml (56 and 65%). To investigate the ontogenetic ability of DNA-injected embryos, EGFP-injected embryos manipulated under 0 or 100 units/ml catalase were transferred separately to recipient mice. The proportion of fetuses derived from EGFP-injected embryos manipulated under 100 units/ml catalase (29%) was significantly higher (P<0.05) than that manipulated under 0 units/ml catalase (19%). Furthermore, the numbers of transgenic pups were 17 in 100 units/ml catalase and 14 in 0 units/ml catalase. The results of the present study indicate that scavenging reactive oxygen species during in vitro micromanipulation is beneficial for the development of DNA-injected embryos.

Animals↗
PubMed2004

Refined porcine follicle stimulating hormone promotes the responsiveness of rabbits to multiple-ovulation treatment.

We investigated whether refined follicle stimulating hormone (FSH) with only a little contaminating LH can promote the responsiveness of rabbits to multiple-ovulation treatment. One group of female rabbits was stimulated with refined porcine FSH (pFSH), an FSH source with low LH activity, and another group was treated with pFSH. The mean number of eggs recovered from donors stimulated with refined pFSH (27 +/- 3) was significantly greater (P<0.05) than that with pFSH (20 +/- 2). Furthermore, the mean number of remaining follicles of donors stimulated with refined pFSH (19 +/- 4) was significantly greater (P<0.05) than that with pFSH (12 +/- 1). To decrease the number of remaining follicles in donors treated with refined pFSH, the dose of human chorionic gonadotropin (hCG) was increased from 75 to 150. However, there were no differences in the numbers of eggs and remaining follicles. The results of the present study suggest that refined pFSH with little contaminating LH promotes the responsiveness of rabbits to multiple-ovulation treatment compared with pFSH.

Animals↗
PubMed2004

Transporter mRNA expression in a conditionally immortalized rat small intestine epithelial cell line (TR-SIE).

Small intestine epithelial cell lines (TR-SIE), which are established from the small intestine of transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), were used to characterize the mRNA expression of small intestine transporters. TR-SIE cells had a polygonal morphology and expressed cytokeratin protein and villin mRNA. Although the large T-antigen was strongly expressed at 33 degrees C, this was reduced at 37 and 39 degrees C. Concomitantly, the cell growth was arrested at 37 and 39 degrees C compared with that at 33 degrees C, suggesting that TR-SIE cells are conditionally immortalized cell lines. RT-PCR analysis revealed that TR-SIE cells expressed ABCB1 (mdr1a and mdr1b), ABCB4 (mdr2), ABCC2 (mrp2), ABCC6 (mrp6), ABCG1, ABCG2 (bcrp/mxr), Slc21a7 (Oatp3), Slc15a1 (PepT1), and Slc16a1 (Mct1). Conditionally immortalized rat small intestine epithelial cell lines were established from tsA58 Tg rats and expressed the mRNA of intestinal transporters.

Animals↗
PubMed2003

Inducible liver injury in the transgenic rat by expressing liver-specific suicide gene.

Suicide gene expression in specific tissue of transgenic animals has been used for cell-specific ablation. To examine the influence of hepatocyte removal, we produced the herpes simplex virus thymidine kinase (HSVtk) transgenic rat, whose gene was regulated by an albumin enhancer promoter. The liver presence of HSVtk was demonstrated in one line of the transgenic rats. We injected ganciclovir (GCV, 50mg/kg) into the rat on alternate days. After 28 days of GCV administration, liver tissues, and blood of the rats were collected. The histological investigation revealed infiltration of T cells, macrophages, granulocytes/neutrophils, and hepatocyte cell death. The biochemistry analysis demonstrated elevated levels of AST, ALT, and total bilirubin in transgenic rat. In conclusion, the transgenic rat with expressed albumin-specific HSVtk developed experimental hepatitis with administration of GCV, and will be a useful model to facilitate the evaluation of drug effects for clinical control of liver disease.

Animals↗
PubMed2003

Establishment of lacZ-transgenic rats: a tool for regenerative research in myocardium.

Animals transgenic (Tg) for reporter genes would be useful to following a given cell lineage during differentiation and regeneration processes. Here, we established a beta-galactosidase (lacZ) Tg rat to use as a tool for regenerative research. Strong lacZ expression was observed in the skeletal muscles, myocardium, pancreas, and skin obtained from these lacZ-Tg rats, and moderate lacZ expression was observed in the liver, spleen, kidney, and cartilage. In contrast, brain, vessels, lung, adrenal gland, small intestine, blood leukocytes, bone marrow (BM) cells, and peripheral blood cells showed no lacZ expression. To test whether this lacZ-Tg rat could be used for regenerative research in myocardium, we induced myocardial injury after a lacZ-Tg BM transplant (BMT) into wild-type rats. The results show that lacZ-positive cardiomyocytes were found in the peri-infarct and uninjured myocardium in the BMT recipient rats. These findings suggest that lacZ-Tg rats are useful tool for regenerative research in the myocardium.

Animals↗
PubMed2003

Analysis of the flanking regions of the human alpha-lactalbumin gene responsible for position-effect independent expression.

Transgenic rats with the 130 kb bacterial artificial chromosome construct bLA, including the alpha-lactalbumin gene, had position-independent and copy number-dependent expression, which confirmed previous experiments using the 210 kb yeast artificial construct, yLALBA. To identify elements that confer a position effect, we compared the yLALBA and bLA sequences. yLALBA was chimeric. A common 32 kb region was identified and the total nucleotide sequence was determined. We previously analyzed transgenic rats using polymerase chain reaction to compare the integrity and expression of the transgenes. The -6 to +9 kb region is considered to be necessary for position-independent expression. Transgenic rats lacking the -3.4 to -0.85 kb region had a severe position effect. This 2.5 kb region contains two DNaseI hypersensitive sites at -1.0 and -2.8 kb. The 2.5 kb region is proposed to be a locus control region of the human alpha-lactalbumin gene.

5' Flanking Region↗
PubMed2003

Expression and regulation of L-cystine transporter, system xc-, in the newly developed rat retinal Müller cell line (TR-MUL).

The purpose of the present study was to elucidate the expression and regulation of the L-cystine transporter, system x(c) (-), in Müller cells. In this study, newly developed conditionally immortalized rat Müller cell lines (TR-MUL) from transgenic rats harboring the temperature-sensitive SV 40 large T-antigen gene were used as an in vitro model. TR-MUL cells express large T-antigen and grow well at 33 degrees C with a doubling time of 30 h, but do not grow at 39 degrees C. TR-MUL cells express typical Müller cell markers such as S-100, glutamine synthetase, and EAAT1/GLAST, whereas EAAT2/GLT-1 and EAAT5 are not detected. TR-MUL cells also exhibit little or no expression of glial fibrillary acidic protein. We found that TR-MUL5 cells exhibited [(14)C]L-cystine uptake activity and expressed xCT and 4F2hc, which involve system x(c) (-). The uptake of [(14)C]L-cystine was significantly inhibited by L-glutamic acid and L-aspartic acid, whereas L-leucine had no effect. Following diethyl maleate (DEM) treatment, the glutathione concentration in TR-MUL5 cells was reduced in the first 24 h, then gradually recovered for more than 24 h. The L-cystine uptake rate and the xCT expression level in TR-MUL5 cells were enhanced by DEM treatment. In contrast, the 4F2hc expression level was unchanged. In conclusion, TR-MUL cells have the properties of Müller cells and exhibit system x(c) (-)-mediated L-cystine uptake activity. The oxidative stress conditions following DEM treatment activate L-cystine transport in TR-MUL cells due to the enhanced transcription of the xCT gene.

Animals↗
PubMed2003

Homeobox gene expression and mutation in cervical carcinoma cells.

An association between deregulation of homeobox (HOX) gene expression and oncogenic transformation has been recently reported in human tumors. In this study, we investigated HOX gene expression and mutation in cervical carcinoma cells. Using reverse transcription-PCR, 11 human cervical carcinoma cell lines and 14 normal cervical tissue samples were examined for mRNA expression of the 39 class I HOX genes. DNA samples from 11 cell lines were tested for mutations in exons 1 and 2 of the HOXA10 and A13 genes using overlapping primer pairs which also cover intron 1 of these genes. HOXA1, B2, B4, C5, C10 and D13 genes were expressed in 8, 7, 9, 9, 9 and 11 of 11 cervical carcinoma cell lines, respectively, but not in any of the normal cervical tissues. HOXA9, A11, A13, B5, C4, D3 and D9 genes were expressed in all cell lines and normal tissues. In contrast, 13 of 39 HOX genes were silent in all materials examined. Single-strand conformational polymorphism and sequence analysis revealed a C insertion after base 1042 and/or a G to C substitution at base 1113 in intron 1 of the HOXA13 gene in 4 of 11 cell lines, however, neither deletions nor mutations were detected in exons 1 and 2 of the HOX A10 and A13 genes. Our data suggest that the expression of HOXA1, B2, B4, C5, C10 and D13 genes might be involved in the process leading to the transformation of normal cervical cells.

Carcinoma, Squamous Cell↗
PubMed2003

Effect of 5-fluorouracil and epidermal growth factor on cell growth and dihydropyrimidine dehydrogenase regulation in human uterine cervical carcinoma SKG-IIIb cells.

We previously demonstrated that epidermal growth factor (EGF) induces a decrease in dihydropyrimidine dehydrogenase (DPD), which is the first and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU), in EGF receptor (EGFR)-positive human SKG-IIIb uterine cervical carcinoma cells, and thereby increased the sensitivity of the cells to 5-FU. In the present study, we examined the individual and combined effects of 5-FU and EGF on growth and DPD activity in SKG-IIIb cells, and also investigated the mode of regulation of DPD activity. The cells showed sensitivity to 5-FU, and growth was stimulated by EGF. When the agents were used in combination, the sensitivity of SKG-IIIb cells to 5-FU was increased roughly sixfold at maximum, as judged in terms of the 50% growth-inhibitory concentration. We then examined the effects of 5-FU and EGF on DPD. Either agent inhibited DPD activity and protein expression in a concentration-dependent manner. Expression of DPD mRNA was concentration-dependently inhibited by treatment with 5-FU and by EGF at a concentration that strongly stimulated cell growth. Further, combination treatment inhibited DPD activity, as well as DPD protein and mRNA expression, more strongly than did treatment with 5-FU or EGF alone. These results suggest that inhibition of DPD activity by EGF or 5-FU is regulated at least at the level of protein expression and that regulation via mRNA is also involved. The above findings indicate that 5-FU and EGF act synergistically in suppressing DPD activity and that the use of 5-FU against tumors in which EGF plays an important role would maximize the potential of 5-FU as an anticancer drug.

Antimetabolites, Antineoplastic↗
PubMed2003

Glutathione S-transferase GSTM1, GSTT1 and p53 codon 72 polymorphisms in human tumor cells.

The genes of the glutathione S-transferase (GST) family encode enzymes that appear to be critical in cellular protection against the cytotoxic effects, whereas p53 is a tumor suppressor gene. Despite a large number of studies on germline polymorphisms of GSTM1, GSTT1 and p53 genes, there have been very few reports on genotyping of these genes in human malignant tumor cells. In this study, we investigated GSTM1, GSTT1 and p53 codon 72 polymorphisms in a variety of human tumor cell lines originating from different organs to clarify tissue-specific polymorphic frequency of these genes in human solid tumors. The GSTM1 and GSTT1 genetic polymorphisms were evaluated using multiplex PCR techniques and PCR-RFLP analysis was conducted to identify p53 codon 72 genotypes. Gene expression of GSTM1 or GSTT1 was detected by RT-PCR in the cells with respective present genotype for each. Polymorphisms of p53 codon 72 detected by PCR-RFLP were also confirmed using SSCP and sequence analyses. GSTM1 and GSTT1 genotypes were various in 104 cell lines examined. Null GSTM1 genotype was dominant in small cell lung, kidney and ovarian carcinoma cells, whereas null GSTT1 genotype was dominant in cervical and endometrial carcinoma cells. GSTM1 and GSTT1 genotypes in ovarian carcinoma cells were quite similar to those in small cell lung carcinoma cells. Polymorphic frequency of p53 codon 72 was also various among the cells, however, the Pro allele was found in only 1 of 6 kidney, 14 cervical and 4 endometrial carcinoma cell lines. There was a significant difference in GSTM1 and p53 genotypes between 34 small cell and 24 non small cell lung carcinoma cells (P < 0.01). Combined study on the distribution of GSTM1, GSTT1 and p53 genotypes revealed that null GSTM1 genotype was associated with the Arg allele of p53 codon 72 in 58 lung carcinoma cells and null GSTT1 genotype was associated with the Pro/Pro homozygote in 104 tumor cell lines examined. This is the first study examining GSTM1, GSTT1 and p53 codon 72 polymorphisms in a variety of human solid tumor cells and suggesting that polymorphic frequency of these genes may be tissue- and organ-specific. The molecular interaction between GST gene defects and p53 codon 72 genotype in the development of human malignant tumors should be further investigated.

Codon↗
PubMed2003

Establishment of conditionally immortalized rat retinal pericyte cell lines (TR-rPCT) and their application in a co-culture system using retinal capillary endothelial cell line (TR-iBRB2).

The purpose of this study was to establish and characterize a retinal pericyte cell line from retinal capillaries of transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), and to apply this to the co-culture with a retinal capillary endothelial cell line. The conditionally immortalized rat retinal pericyte cell lines (TR-rPCTs), which express a temperature-sensitive large T-antigen, were obtained from two tsA58 Tg rats. These cell lines had a multicellular nodule morphology and reacted positively with von Kossa staining, a marker of calcification. TR-rPCTs cells expressed mRNA of pericyte markers such as rat intercellular adhesion molecule-1, platelet-derived growth factor-receptor beta, angiopoietin-1, and osteopontin. Western blot analysis indicated that alpha-smooth muscle actin (alpha-SMA) was expressed in TR-rPCT3 and 4 cells. In contrast, alpha-SMA was induced by transforming growth factor-beta1 and its enhancement was reduced by basic fibroblast growth factor in TR-rPCT1 and 2 cells. When TR-rPCT1 cells were cultured with a rat retinal endothelial cell line (TR-iBRB2) in a contact co-culture system, the number of TR-iBRB2 cells were significantly reduced in comparison with that of a single culture of TR-iBRB2 cells, suggesting that physical contact between pericytes and retinal endothelial cells is important for the growth of retinal endothelial cells. In conclusion, conditionally immortalized retinal pericyte cell lines were established from tsA58 Tg rats. These cell lines exhibited the properties of retinal pericytes and can be applied in co-culture systems with a retinal capillary endothelial cell line.

Actins↗
PubMed2003

Development of a conditionally immortalized testicular Sertoli cell line RTS3-3 from adult transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene.

Transgenic mice and rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene are useful for establishing cell lines from tissues. We succeeded in establishing a conditionally immortalized testicular Sertoli cell line, RT3-3, from adult transgenic rats harboring the oncogene. The cells grew at permissive (33 degrees C) and intermediate (37 degrees C) temperatures but not at nonpermissive temperature (39 degrees C). Large T-antigen was expressed at 33 and 37 degrees C, whereas the expression level was gradually decreased at 39 degrees C, suggesting that the temperature-sensitive growth characteristics arise as a result of the function of tsSV40 large T-antigen. The cells showed biochemical features associate with normal Sertoli cells including expressions of mRNAs of sulfated glycoprotein-2 (SGP-2), transferrin (TF) and steel factor. Quantitative polymerase chain reaction revealed that nonpermissive temperature induced increase in the level of SGP-2. Moreover, levels of SGP-2 and/or TF were significantly elevated in the cells treatment with sodium butyrate and retinoic acid, inducers of cellular differentiation. To our knowledge, this is the first report of the establishment of a testicular Sertoli cell line from the transgenic rats. Thus, the conditionally immortalized cell line RTS3-3 with unique characteristics may serve as good experimental in vitro models for basic and applied biology of testicular Sertoli cells.

Animals↗
PubMed2003

Cryopreservation of spermatozoa from closed colonies, and inbred, spontaneous mutant, and transgenic strains of rats.

We attempted to cryopreserve spermatozoa from closed colonies (Jcl:SD and Jcl:Wistar), and inbred (BN/Crj, F3441 DuCrj, LEW/Crj, Long-Evans and WKY/NCrj), mutant (Zitter [WTC.ZI-zi] and Tremor [TRM]), transgenic (human A-transferase [A], and green fluorescent protein [GFP]) strains of rats. Rat epididymal spermatozoa suspended in cryopreservation solution (23% egg yolk, 8% lactose monohydrate, and 0.7% Equex Stm, pH 7.4, adjusted with 10% Tris [hydroxymethy] aminomethane) were frozen and stored at -196 degrees C. After thawing at 37 degrees C, the spermatozoa were instilled into the tip of each uterine horn of the recipients. A total of five recipient females for each strain were inseminated with cryopreserved spermatozoa, and normal live offspring of all strains (Jcl:SD: 11, Jcl:Wistar: 13, BN/Crj: 9, F344/DuCrj: 28, LEW/Crj: 4, Long-Evans: 6, WKY/NCrj: 8, TRM: 24, WTC.ZI-zi: 27, A: 30 and GFP: 20) were obtained.

Animals↗
PubMed2002

Identification of the coding sequences responsible for Tsc2-mediated tumor suppression using a transgenic rat system.

Hereditary renal carcinomas in the Eker rat are caused by germline retrotransposon insertion in the tuberous sclerosis-2 (Tsc2) gene. We established previously a transgenic Eker rat model into which was introduced a wild-type Tsc2 gene. The embryonic lethality of mutant homozygotes and renal carcinogenesis of heterozygotes were completely suppressed by this transgene (Tg). The function of the Tsc2 product (tuberin) is not fully understood, although several findings have been obtained mainly in vitro. Therefore, to elucidate the functional domains of Tsc2 in vivo, we generated transgenic Eker rats carrying deletion mutants of the Tsc2 gene. A Tg coding for the C-terminal region (amino acids 1425-1755) suppressed renal carcinogenesis in the Eker rat and interestingly the degree of this suppression correlated with the level of expression of the Tg. Notably, the product of this Tg lacks the ability to bind to the Tsc1 product (hamartin). Surprisingly, while a Tg lacking the C-terminus of tuberin (amino acids 1-1755) completely suppressed renal carcinogenesis, it partially rescued homozygous mutants from embryonic lethality. In conclusion, we have determined the minimal region of tuberin necessary for tumor suppression but the suppressive effect was quantitative. Tuberin could function as a tumor suppressor without binding to hamartin. The requirement of the functional domain(s) of tuberin might differ for prevention of embryonic lethality and for suppression of renal carcinogenesis.

Analysis of Variance↗
PubMed2002

Correlation between vascular endothelial growth factor-C expression and invasion phenotype in cervical carcinomas.

The correlation between vascular endothelial growth factor (VEGF)-C gene expression and in vitro invasive activity and matrix metalloproteinase (MMP)-2 or 9 gene expression and proteolytic activity in 11 cervical carcinoma cell lines, was investigated. Immunohistochemical expression of VEGF-C in 52 cervical carcinoma tissues was also correlated with tumor aggressiveness with respect to clinicopathologic features, tumor vascularity, MMP-2 expression and patient outcome. Expression of VEGF-C mRNA differed remarkably among the cell lines and there was a statistical correlation between VEGF-C gene expression and the number of invaded tumor cells (p = 0.0009) and MMP-2 gene expression and activity (p < 0.05). Anti-VEGF-C antibody inhibited the invasive and proteolytic activity of tumor cells in a concentration-dependent manner. VEGF-C or MMP-2 expression in clinical tissue samples was well correlated with depth of myometrial invasion, endometrial invasion, pelvic lymphnode metastasis and tumor vascularity (p < 0.05) and there was a close relation between VEGF-C and MMP-2 expression (p < 0.0001) in cervical carcinomas. Overall survival rates for 14 patients with strong VEGF-C staining tumors were lower than those for 38 patients with weak VEGF-C staining tumors (p = 0.0132) and VEGF-C tissue status emerged as an independent prognostic parameter (p = 0.0232). These results suggest that VEGF-C expression is closely related to invasion phenotype and affects the patient's survival in cervical carcinomas.

Adenocarcinoma↗
PubMed2002

Polarized glucose transporters and mRNA expression properties in newly developed rat syncytiotrophoblast cell lines, TR-TBTs.

In this study, we have established new syncytiotrophoblast cell lines (TR-TBTs) from the recently developed transgenic rat harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg-rat). Four conditionally immortalized syncytiotrophoblast cell lines (TR-TBT 18d-1 approximately 4) were obtained from pregnant Tg-rats at gestational day 18. These cell lines had a syncytium-like morphology, could be prepared as monolayers, expressed cytokeratins and rat syncytiotrophoblast markers, and exhibited apical or basal GLUT1 localizations and apical GLUT3 localizations. TR-TBTs express large T-antigen and grow well at 33 degrees C with a doubling time of about 30 h. TR-TBTs have processes for the uptake of dehydroepiandrosteron-3-sulfate (DHEAS) and these are predominantly located on the basal side, and this is the first report of an in vitro model of blood placental barrier (BPB) able to incorporate DHEAS. Therefore, TR-TBTs are an appropriate in vitro model for investigating carrier-mediated transport functions at the BPB. Moreover, TR-TBTs express betaine/GABA transporter (GAT-2/BGT-1), concentrative nucleoside transporter 2 (CNT2), equilibrative nucleoside transporter 1 (ENT1), and ENT2 and the expression of these transporters has been reported in blood-brain barrier (BBB). Thus, the expression patterns of nucleoside and neurotransmitter transporters examined are quite similar in both the BPB and BBB.

Animals↗
PubMed2002

Rescue of infertile transgenic rat lines by intracytoplasmic injection of cryopreserved round spermatids.

Transgenic male rats carrying human alpha-lactalbumin with thymidine kinase gene (line name; LAC3) were found to be infertile due to expression of the transgene in the testes. Furthermore, it was not possible to maintain the line even by the use of intracytoplasmic sperm injection (ICSI). Therefore, round spermatids prepared from the LAC3 rats were microinjected into strontium-activated oocytes using a Piezo-driven micromanipulator. Of 263 oocytes microinjected with LAC3 spermatids, 244 (92.8%) survived the injection and 96 (39.3%) developed to the 2-cell stage. Three viable offspring were born after transfer (1.4%, 3/219), and two offspring carried the LAC3 transgene. In the control experiment using spermatids of Wistar rats, similar proportions of post-injection survival (91.3%, 241/264), cleavage (40.2%, 97/241), and development into offspring (0.5%, 1/206) were obtained. Thus, this paper reports not only the first rat offspring derived from round spermatid injection but also the practical application of the microinsemination technique to the rescue of transgenes of infertile transgenic male rats.

Animals↗