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Biomedical subjects

M Minemura

Publications and source records attributed to M Minemura.

At least 19 recordsLinked to original sources
PubMed2002

Attenuated acute liver injury in mice by naked hepatocyte growth factor gene transfer into skeletal muscle with electroporation.

BACKGROUND: Hepatocyte growth factor (HGF) plays an essential role in hepatic development and regeneration, and shows proliferative and antiapoptotic activity in hepatocytes. AIMS: To establish an effective new method for HGF gene transfer in vivo and to investigate its effects in acute experimental liver injury. ANIMALS: Eight week old female mice were used. METHODS: Rat HGF gene in a modified pKSCX plasmid was transferred to the tibialis anterior muscle by electroporation using a pulse generator. Four days later, plasma HGF concentrations were determined by enzyme linked immunosorbent assay every two days for three weeks. To confirm the efficacy of electroporation, a plasmid bearing green fluorescence protein (GFP) was transferred similarly. Four days after electroporation, carbon tetrachloride (CCl(4)) was administered to mice to induce acute liver injury. Plasma alanine aminotransferase (ALT) activity was measured. Hepatic apoptosis was assessed by Hoechst 33258 staining and the TUNEL method. RESULTS: Fluorescence microscopy showed strong green fluorescence where the GFP gene had been transferred into muscle. In mice given the HGF gene, HGF in plasma was increased up to fourfold from pretreatment amounts, peaking 6-9 days after electroporation and quickly decreasing within three weeks. Compared with the group without HGF transfer, the percentage of apoptotic hepatocytes after CCl(4) intoxication was significantly lower, as was ALT activity. In addition, ALT activity normalised more rapidly in the HGF gene transfer group. CONCLUSIONS: Naked DNA injection and transfer by electroporation efficiently brings about HGF expression in vivo, which can attenuate acute liver injury.

Alanine Transaminase↗
PubMed2001

Complementing yeast rho1 mutation groups with distinct functional defects.

Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-beta-glucan synthase, respectively. Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-beta-glucan synthase activity.

Base Sequence↗
PubMed2001

Yeast 1,3-beta-glucan synthase activity is inhibited by phytosphingosine localized to the endoplasmic reticulum.

1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.

Base Sequence↗
PubMed2001

A mutation in SPC42, which encodes a component of the spindle pole body, results in production of two-spored asci in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, SPC42 is an essential gene, which encodes one of the major components of the spindle pole body (SPB). We report on a mutation in the SPC42 gene (spc42-102) that results in a sporulation-specific defect. Mitotic growth of haploid and diploid spc42-102 strains is normal and both exhibit the same growth rates as the isogenic wild-type strains. Many diploid spc42-102/spc42-102 cells undergo normal meiotic nuclear divisions, producing four haploid nuclei. However, a significant fraction of meiotic spc42-102/spc42-102 cells contain two immature SPBs and aberrant nuclei that are not surrounded by a prospore membrane. Some 40% of the resultant asci contain only two spores, while wild-type diploid cells almost always produce four-spored asci. Segregation of auxotrophic markers that are tightly linked to the centromere reveals that two-spore asci formed from spc42-102/spc42-102 diploid cells exclusively contain nonsister haploid spores. Western analysis and measurements of the fluorescent signal from an Spc42p-GFP (green fluorescent protein) fusion reveal that the mutant strain fails to accumulate Spc42p at meiosis. Thus, our results suggest that insufficiency of Spc42p during meiosis results in a pair of immature nonsister SPBs that are not enclosed by prospore membrane.

Blotting, Western↗
PubMed2001

Helicobacter pylori alters n-6 fatty acid metabolism and prostaglandin E2 synthesis in rat gastric mucosal cells.

BACKGROUND AND AIMS: Little is known about whether Helicobacter pylori infection alters fatty acid metabolism in gastric mucosal cells. By using cultured rat gastric mucosal cells (RGM-1), we investigated the effect of H. pylori broth culture filtrates on this point. Furthermore, our study aimed to find out whether n-6 long chain polyunsaturated fatty acids from linoleic acid are formed in RGM-1 cells. METHODS: Rat gastric mucosal cells were incubated with 10, 20 and 40 microg/mL of linoleic acid or medium alone. Phosphatidylcholine content extracted from whole RGM-1 cells was quantitated by using a densitometer, and its fatty acid composition was analyzed by using gas chromatography. Prostaglandin E2 concentration in the culture medium was measured by using radioimmunoassay. The expression of cyclooxygenase (COX)-1 and COX-2 was examined by using reverse transcription-polymerase chain reaction. In addition, after incubation with [1-14C] linoleic acid, radioactivities of both linoleic acid and arachidonic acid components of the PC fraction were counted. The effects of H. pylori broth culture filtrates on PC content, its fatty acid composition and prostaglandin (PG)E2 synthesis were also assessed. RESULTS: Linoleic acid addition caused an increase in the composition of arachidonic acid, as well as linoleic acid, and also in PGE2 concentration. Cyclo-oxygenase-2 expression was induced in RGM-1 cells by the addition of linoleic acid. In addition, [1-14C] linoleic acid added to the culture medium was converted to [1-14C] arachidonic acid in RGM-1 cells. Helicobacter pylori broth culture filtrates decreased linoleic acid composition and increased arachidonic acid composition. Moreover, after incubation with H. pylori broth culture filtrates, PGE2 concentrations were higher than that of the controls. CONCLUSIONS: These findings suggest the presence of fatty acid elongase and Delta5- and Delta6-desaturases synthesize arachidonic acid from linoleic acid in RGM-1 cells. Thus, H. pylori infection may enhance PGE2 synthesis and accelerate n-6 fatty acid metabolism in gastric mucosal cells, which could make the gastric mucosal barrier more fragile.

Animals↗
PubMed2001

Antisense oligonucleotides specific to mutated K-ras genes inhibit invasiveness of human pancreatic cancer cell lines.

BACKGROUND/AIMS: Point mutations of the K-ras gene are detected in > 90% of human pancreatic cancers and may play an important role in tumorigenesis. However, correlations between mutant K-ras and the invasive activity of the tumor have remained unclarified. METHODS: 17-merphosphorothioate antisense oligonucleotides targeting K-ras point mutations were transfected into three kinds of human pancreatic cancer cell lines (MIAPaCa-2, PANC-1, and BxPC-3), and the invasive activity was investigated using an in vitro chemoinvasion assay. RESULTS: Antisense oligonucleotides strongly inhibited the invasive activity of the cell lines with mutant K-ras genes (MIAPaCa-2, PANC-1), but not in that with a wild-type K-ras (BxPC-3). CONCLUSION: Antisense oligonucleotides specific to mutated K-ras genes inhibited the invasiveness of human pancreatic cancer cell lines. Specific antisense therapy to the point mutation of K-ras might be a new anticancer strategy for pancreatic cancer.

Blotting, Western↗
PubMed2001

Appearance of poor-fermenting variants in brewing yeast culture.

A class of yeast variants appears after cultivation of a bottom-fermenting brewing yeast strain, IFO2003. Although IFO2003 fails to grow well above 33 degrees C, the variants can grow up to 34 degrees C. Temperature-resistance and an acquired phenotype of maltose poor-fermentation ability are strictly correlated in the bottom-fermenting brewing yeast, enabling us to develop easy estimation of the fermentation ability of the variants.

Colony Count, Microbial↗
PubMed1999

Functional B-cell response in intrahepatic lymphoid follicles in chronic hepatitis C.

Intrahepatic lymphoid follicle (ILF) formation is one of the most characteristic and commonly observed histological features in patients with chronic hepatitis C. However, little is known regarding whether follicles in the liver belong to functional lymphoid tissues, where B cells are activated, differentiated, and proliferated, or if the lymphocytes are merely infiltrated after recruitment from the secondary lymphoid organs. To ascertain this possibility, we examined the expression of markers for B-cell activation, differentiation, and proliferation in ILFs in patients with chronic hepatitis C using surgically resected specimens, and compared them with specimens of perihepatic lymph nodes by an immunohistochemical technique. Germinal center (GC) formation in the ILFs was frequently found in HCV-positive cases. The distribution of immunoglobulin M (IgM)-, IgD-, and IgG-positive cells and the expression patterns of Ki-67, CD23, or bcl-2 and bcl-6 gene products in the follicles with GC formation in the liver of patients with chronic hepatitis C were similar to those of lymph nodes, indicating that B cells are activated, proliferated, and differentiated in the ILFs with GC formation in patients with chronic hepatitis C. Oligoclonal expansion of B cells in the livers with ILFs was confirmed by an analysis of immunoglobulin heavy chain (IgH) gene rearrangement determined by polymerase chain reaction (PCR). These data strongly suggest that ILFs with GC formation, which are frequently found in patients with chronic hepatitis C, may functionally be the same as those found in lymph nodes with respect to B-cell expansion and maturation.

Adult↗
PubMed1999

Overexpression of multidrug resistance genes MDR1 and cMOAT in human hepatocellular carcinoma and hepatoblastoma cell lines.

Overexpression of either of the multidrug resistance genes MDR1 and MRP is associated with resistance of tumors to multiple chemotherapeutic agents. Overexpression of MDR1 has been reported in some cell lines derived from human hepatocellular carcinomas (HCC) and hepatoblastomas (HB). The human gene for cMOAT ( ), a homologue of MRP, is thought to mediate hepatobiliary excretion of organic anions and to be associated with cisplatin resistance. In this study, expression levels of MDR1 and cMOAT were examined in 9 human HCC and HB cell lines and 10 other human cancer cell lines. Overexpression of the cMOAT gene was observed in all 9 HCC and HB cell lines and 3 of 10 other cancer cell lines. Co-overexpression of the cMOAT and MDR1 genes was observed in 7 of 9 HCC and HB cell lines, but in none of the 10 other cancer cell lines. Seven of the HCC and HB cell lines that had overexpression of the cMOAT gene were shown to be highly resistant to cisplatin compared to 2 HCC cell lines with low levels of cMOAT expression. These findings suggest that overexpression of cMOAT could contribute to cisplatin resistance in HCC and HB.

Biological Transport, Active↗
PubMed1998

Phosphatidylinositol-4-phosphate 5-kinase localized on the plasma membrane is essential for yeast cell morphogenesis.

Phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2), an important element in eukaryotic signal transduction, is synthesized either by phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P 5K) from phosphatidylinositol 4-phosphate (PtdIns(4)P) or by phosphatidylinositol-5-phosphate 4-kinase (PtdIns(5)P 4K) from phosphatidylinositol 5-phosphate (PtdIns(5)P). Two Saccharomyces cerevisiae genes, MSS4 and FAB1, are homologous to mammalian PtdIns(4)P 5Ks and PtdIns(5)P 4Ks. We show here that MSS4 is a functional homolog of mammalian PtdIns(4)P 5K but not of PtdIns(5)P 4K in vivo. We constructed a hemagglutinin epitope-tagged form of Mss4p and found that Mss4p has PtdIns(4)P 5K activity. Immunofluorescent and fractionation studies of the epitope-tagged Mss4p suggest that Mss4p is localized on the plasma membrane, whereas Fab1p is reportedly localized on the vacuolar membrane. A temperature-sensitive mss4-1 mutant was isolated, and its phenotypes at restrictive temperatures were found to include increased cell size, round shape, random distribution of actin patches, and delocalized staining of cell wall chitin. Thus, biochemical and genetic analyses on Mss4p indicated that yeast PtdIns(4)P 5K localized on the plasma membrane is required for actin organization.

Actins↗
PubMed1998

p53 overexpression in small hepatocellular carcinomas containing two different histologic grades.

There is evidence to suggest that a focus of less-differentiated hepatocellular carcinoma (HCC) may arise within a pre-existing well-differentiated HCC, eventually replacing it. In the present study, the p53 tumor suppressor gene was analyzed by immunohistochemistry in 31 hepato-cellular carcinomas (HCCs) containing two or more regions in the same nodule with different histologic grades. p53 was overexpressed in the nucleus in 13 of 31 HCCs (42%), in seven of which p53 overexpression was seen only in the less-differentiated area of the tumor. This suggests that overexpression of presumed mutant p53 may have contributed to dedifferentiation during the development of HCC.

Adult↗
PubMed1997

Analysis of T-cell receptor Vbeta repertoire in liver-infiltrating lymphocytes in chronic hepatitis C.

BACKGROUND/AIMS: To examine the T-cell repertoire which is involved in the immunopathogenesis of chronic hepatitis, we analyzed the T-cell receptor Vbeta gene usage in liver-infiltrating lymphocytes by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical technique. METHODS: Complementary DNA was synthesized from RNA which was extracted from 26 liver biopsy specimens and from peripheral blood lymphocytes from eight subjects, and amplified by RT-PCR. Radioactivity of each amplified product using 32P-labeled primers was measured and the percentage of each Vbeta expression was calculated. RESULTS: The mean frequency of Vbeta5.1 (11.1%) in liver-infiltrating lymphocytes of chronic hepatitis C was highest among those of all Vbeta regions, and was significantly higher than that in both peripheral blood lymphocytes of chronic hepatitis C and liver-infiltrating lymphocytes of chronic hepatitis B. In the immunohistochemical analysis, Vbeta5.1-positive cells were mostly observed in portal areas where inflammatory reactions occurred. The sequences of the complementarity determining region (CDR)3 on T-cell receptor expressing Vbeta5.1 were examined in six patients with chronic hepatitis C. The sequences were similar to each other and all had one common amino acid (valine) irrespective of different HLA haplotype. CONCLUSIONS: These data suggest that Vbeta5.1-positive cells are preferentially accumulated in the liver of chronic hepatitis C and are involved in the immunopathogenesis of the disease. Sequence analysis showed that Vbeta5.1-positive cells recognize a common conventional antigen and valine recognized at the same position of the CDR3 may be a key residue in determining an antigen/major histocompatibility complex contact point.

Adult↗
PubMed1997

[Expression of ATP binding cassette superfamily (multidrug resistance-1, multidrug resistance-associated protein, human canalicular multispecific organ anion transporter) mRNA in etoposide and m-AMSA resistant cell lines].

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. To further understand resistance to topoisomerase (topo) II inhibitors, 50 sublines were isolated as single clones from parental cells by exposure to etoposide or m-AMSA. Subsequently, a population of cells from each sublines was exposed to three-fold higher drug concentrations allowing 16 stable sublines to be established at higher extracellular drug concentration. Quantitative aspects of MRP and C-MOAT were studied by Northern blotting in 66 resistant cell lines. Increased MRP mRNA was observed in 48.5% of resistant cell lines (64.7% of etoposide resistant cells and 31.3% of m-AMSA resistant cell lines). Increased C-MOAT mRNA was also observed in 39.4% of resistant cell lines (41.2% in etoposide resistant cell lines and 37.5% in m-AMSA resistant cell lines). To characterize the function of C-MOAT, cellular accumulation assay for 3H-etoposide was performed in three resistant cell lines which overexpress C-MOAT but do not express MRP. Accumulation of etoposide was reduced in the cell lines. Our findings suggest that increased MRP and O-MOAT mRNA seems to be an important mechanism of resistance to topo II inhibitors.

ATP-Binding Cassette Transporters↗
PubMed1996

Expression and localization of basic fibroblast growth factor (bFGF) in the repair process of rat liver injury.

BACKGROUND/AIMS: To clarify the expression and localization of basic fibroblast growth factor in the repair process of liver injury, acute liver injury was induced by administration of carbon tetrachloride, D-glactosamine hydrochloride, or dimethylnitrosamine to rats. METHODS: We measured basic fibroblast growth factor protein in the liver tissue by radioimmunoassay, evaluated the expression of basic fibroblast growth factor mRNA by the reverse transcriptase polymerase chain reaction, and identified basic fibroblast growth factor-positive cells by immunostaining. RESULTS: In the carbon tetrachloride injured liver, the basic fibroblast growth factor protein contents began to increase 2 days after administration when liver injury was most marked, and reached a peak after 4 days, decreasing thereafter. In the carbon tetrachloride-injured liver, basic fibroblast growth factor mRNA expression was observed from 12 h after administration, prior to an increase in the protein content. In the D-galactosamine hydrochloride-injured liver, basic fibroblast growth factor protein also increased. On the other hand, in the dimethylnitrosamine-injured liver, the basic fibroblast growth factor protein content decreased 2 days after administration when liver injury was marked, but increased after 7 days. In the regenerating liver after partial hepatectomy, the basic fibroblast growth factor protein content did not increase. Among cell fractions, the Ito cell fraction obtained from the carbon tetrachloride-injured liver after 4 days showed expression of basic fibroblast growth factor mRNA. In cells cultured for 24 h, this fraction was immunopositive for basic fibroblast growth factor. Ito cells in the liver tissue markedly increased in the carbon tetrachloride-injured liver and increased after 7 days in the dimethylnitrosamine-injured liver. CONCLUSIONS: This study confirmed basic fibroblast growth factor production in the liver tissue in the repair process of liver injury. Our results suggest that basic fibroblast growth factor is primarily produced in Ito cells, acts on sinusoidal wall cells including Ito cells by the autocrine and paracrine mechanisms, and promotes extracellular matrix production and vascularization, involving the repair process of liver injury.

Animals↗
PubMed1996

Exogenous phytohormone-independent growth and regeneration of tobacco plants transgenic for the 6b gene of Agrobacterium tumefaciens AKE10.

The 6b gene of Agrobacterium tumefaciens AKE10 (AK-6b) induces crown gall tumors on certain plants but so far there have been no reports of the gene being able to induce tumors on culture medium. We cloned T-DNA segments containing the 6b gene but lacking the auxin and cytokinin biosynthesis genes from A. tumefaciens AKE10. Tobacco (Nicotiana tabacum) leaf discs infected with A. tumefaciens LBA4404 carrying the clones produced shooty calli on hormone-free Murashige-Skoog medium. The relevant T-DNA segment was integrated into plant DNA as determined by Southern hybridization. Some of these immature shoots spontaneously developed into mature shoots, of which several leaves displayed morphological abnormalities. When leaf discs of these mature plants were placed onto the same medium numerous shoots developed from the wounding sites, indicating that the transgenic plants possessed a high regenerative potential. Northern blot and reverse transcriptase-polymerase chain reaction analyses showed a large accumulation of the AK-6b transcripts in the shooty calli, but only a limited degree in mature plants, demonstrating that AK-6b expression is regulated in plants and essential for the early stages of regeneration. Cytokinin levels in the shooty calli were comparable to those in normal shoots, suggesting that shoot regeneration is not mediated by the modulation of cytokinin content.

Agrobacterium tumefaciens↗
PubMed1995

Serum concentration of intercellular adhesion molecule-1 in patients with hepatocellular carcinoma is a marker of the disease progression and prognosis.

Serum levels of soluble forms of intracellular adhesion molecule-1 (sICAM-1) and lymphocyte function-associated antigen-3 (sLFA-3) in 122 patients with chronic liver disease including hepatocellular carcinoma (HCC) were measured by enzyme-linked immunosorbent assays. Serum levels of sICAM-1 in patients with HCC were significantly higher than those of chronic hepatitis (CH) and cirrhosis. On the other hand, serum levels of sLFA-3 in patients with HCC were almost the same as those of cirrhosis. Western blot analyses showed that molecular sizes of sICAM-1 and sLFA-3 detected in the sera were 90 kd and 50 kd, respectively, indicating that both molecules include whole extracellular domains. In patients with HCC, circulating sICAM-1 levels were significantly (P < .001) correlated with tumor volume (r = .50), total bilirubin (r = .38), serum aspartate aminotransferase levels (r = .51), and gamma-globulin (r = .63). Furthermore, serum sICAM-1 levels were significantly elevated in patients with multiple HCC (tumor number > 3) or HCC with tumor embolus in the first branch or trunk of portal vein. Survival periods were analyzed in relation to serum sICAM-1 levels in patients with HCC who had been treated by transcatheter arterial chemoembolization. The HCC patients with < 1,000 ng/mL of serum ICAM-1 showed significantly (P = .0005) longer survival than those with higher levels of the molecule. The same results were obtained when only patients with moderately differentiated HCC were analyzed (P = .02). Analyses by Cox's proportional hazard model showed that sICAM-1 is a significant (P = .032) prognostic factor for patients with HCC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗