Biomedical subjects

J Fujimoto

Publications and source records attributed to J Fujimoto.

At least 127 records · Page 7Linked to original sources

PubMed1998

HVJ-liposome mediated gene transfer into hepatocytes in vivo.

BACKGROUND/AIMS: The efficient transduction of appropriate target cells will be critical for gene therapy. We evaluated the suitability of hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer for gene therapy of liver diseases. METHODS: The Escherichia coli beta-galactosidase (beta-gal) gene was introduced into rat liver by HVJ-liposome to examine gene transfer efficacy and persistence of expression with or without partial hepatectomy prior to transfection. RESULTS: About 30% of hepatocytes were transduced after portal vein injection. Gene expression was transient, with only 2% of hepatocytes expressing beta-gal after 4 weeks. However, partial hepatectomy performed 24 h prior to injection resulted in persistently high levels of beta-gal for 4 weeks after injection. A 247-bp beta-gal polymerase chain reaction fragment transcript was detected in livers of transfected rats, but not in livers of control rats. The rat livers following gene transfer were histologically normal, and serum glutamic-pyruvic transaminase was not found to be elevated in rats. CONCLUSIONS: Our results demonstrate that HVJ-liposome-mediated gene transfer produced high gene transduction and persistent gene expression in the liver.

Alanine Transaminase↗

PubMed1998

Progression of hepatocellular carcinoma as reflected by nuclear DNA ploidy and cellular differentiation.

BACKGROUND/AIMS: Intratumor heterogeneity of DNA ploidy within a single hepatocellular carcinoma is not well understood. The present study was designed to examine the histologic distribution of intratumor DNA ploidy in hepatocellular carcinomas of different growth types in relation to cell differentiation. METHODS: Twenty patients (16 men and four women; mean age, 60.2 years) with hepatocellular carcinoma (mean diameter, 4.3 cm) were studied. One hundred and twenty-seven samples from different sites of each tumor were analyzed by determination of the nuclear DNA content and histological examination. RESULTS: The DNA ploidy was heterogeneous in nine (45%) of the 20 tumors. Five tumors had a mixture of diploid and aneuploid regions, and the remaining four consisted of aneuploid regions with different DNA indices. There was no significant difference in patient characteristics between the heterogeneous and homogeneous groups. A significant correlation was found between tumor growth type and the incidence of heterogeneity. Only 16% of single nodular carcinomas without intratumor septal formation exhibited heterogeneity, while single nodular tumors with septal formation or confluent multinodular tumors were associated with high incidences of different DNA ploidy patterns or DNA indices. There was no aneuploidy in well-differentiated foci, while aneuploidy was frequently found in moderately or poorly differentiated foci (incidences of 67% and 74%, respectively). CONCLUSIONS: Heterogeneity of DNA ploidy may develop along with changes in growth pattern and cell dedifferentiation or by confluence of nodules originating from different tumor cell clones.

Adult↗

PubMed1998

Sex steroidal regulation of vessel permeability associated with vessel endothelial cadherin (V-cadherin).

In an attempt to understand the roles of cadherins in the placenta, mRNA expression and biological function of cadherins in 3A(tPA-30-1) cells (derived from human term placenta and transformed by SV40), and in HUV-EC-C cells (derived from the endothelial cells in human umbilical cord) were studied under the influence of sex steroids. Estradiol transiently decreased the endothelial cell barrier properties (ECBP) of HUV-EC-C cells, and progesterone reversed the changes induced by estradiol. However, neither estradiol nor progesterone demonstrated any effect on cell aggregation of either 3A(tPA-30-1) or HUV-EC-C cells. Estradiol transiently decreased the level of V-cadherin and its mRNA in HUV-EC-C cells, and progesterone reversed the level decreased by estradiol. However, neither estradiol nor progesterone demonstrated any effect on the level of E-cadherin mRNA in 3A(tPA-30-1) cells. Therefore, a sex steroidal role for placental development and function related to cadherins seems to focus on the endothelial cells, plausibly via vessel permeability for the utilization of placental products.

Antigens, CD↗

PubMed1998

Synthesis and characterization of isotopically enriched pyrimidine deoxynucleoside oxidation damage products.

Oxidative damage to DNA is an established source of genomic instability. In this paper, we describe the synthesis and characterization of several pyrimidine deoxynucleoside oxidation damage products, enriched with stable isotopes. These products include the 2'-deoxynucleoside derivatives of 5-(hydroxymethyl)uracil, 5-formyluracil, 5-hydroxyuracil, 5-(hydroxymethyl)cytosine, 5-formylcytosine, and 5-hydroxycytosine. The common precursor is 2'-deoxy-2"-deutero[1,3-15N]uridine. Additional stable isotopes are added during functional group conversions. Characterization of these derivatives includes mass spectrometry and 1H and 15N NMR spectroscopy. Proton and nitrogen NMR studies reported here allow an examination of the influence of the modification on sugar conformation and tautomeric equilibrium, properties likely to be important in understanding the biological consequences of these DNA damage products.

DNA Adducts↗

PubMed1998

Persistent gene expression in rat liver in vivo by repetitive transfections using HVJ-liposome.

Most viral vectors are highly immunogenic and are of limited use for somatic gene therapy that requires repetitive administrations. We have developed a highly efficient gene transduction procedure useful for repetitive transfections using liposome containing hemagglutinating virus of Japan (HVJ-liposome). The Escherichia coli beta-galactosidase (beta-gal) gene was embodied in HVJ-liposome, and introduced directly into the caudal lobe of rat liver that was transiently isolated from a systemic circulation. A 116 kDa beta-gal protein was detected in transfected rat liver tissues by Western blot analysis and it was expressed in more than two-thirds of the liver by histological staining. It was found that the transfection efficiency was not affected by repetitive transfections. In support of these findings, antibody response to HVJ-liposome detected in the rat sera was weak and transient. Furthermore, cytotoxic T lymphocytes were not elicited against autologous rat hepatocytes that were transfected in vivo using HVJ-liposome. Thus, our results demonstrate that the isolation of a target liver from systemic circulation and the direct administration of foreign genes using HVJ-liposomes are useful for high gene transduction and persistent gene expression in the liver.

Animals↗

PubMed1998

Efficient transfer of oligonucleotides and plasmid DNA into the whole heart through the coronary artery.

Several of the current techniques for transfer of both oligonucleotide and plasmid DNA into the myocardium are impaired by low efficiency and toxicity. To improve gene transfer techniques, especially into the whole heart, a gene transfer method involving liposome in conjunction with a viral envelope (HVJ-liposome) was essayed as an alternative. FITC-labeled oligonucleotide (F-ODN) and the cDNA of beta-galactosidase (beta-gal) were introduced into the myocardium by coronary infusion of HVJ-liposome during cardioplegic arrest of adult Sprague-Dawley rat hearts. Then, transfected heart was ectopically transplanted into another rat abdomen of the same strain to maintain the transfected heart long enough to allow for protein synthesis. After 3 days of transfection, transfected heart was excised and the efficiency of gene transfection was evaluated. FITC was detected in the nuclei of more than 70% of the myocytes and endothelial cells both in the epicardium and endocardium. beta-Gal was expressed in the cytosol of more than 50% of the myocytes. beta-Gal expression was demonstrated by Western blotting analysis at day 3 after transfection and continued for at least 14 days. No significant histological damage of the myocardium or leakage of CPK were detected in the rats transfected by the HVJ-liposome method. These results clearly demonstrate that the hearts were efficiently transfected both by oligonucleotide and plasmid DNA as a result of coronary infusion of HVJ-liposome during cardioplegic arrest. This thus appears to be an efficient method for gene transfer into the whole heart, providing a new tool for research and therapy for heart diseases.

Animals↗

PubMed1998

Regulation of cytokine expression by an autoreactive B cell clone derived from MRL/MP-lpr/lpr mice.

The B cell line, MRL159.5, was established by somatic hybridization between splenic MRL/MP-lpr/lpr (lpr) mice B cells and 2.52M, a hypoxanthine-aminopterine-thymidine (HAT) medium-sensitive B cell line mutant. It possessed a receptor molecule for mouse erythrocytes treated with bromelain (Br-MRBC) on its surface, likely to be an autoreactive B cell clone specific for Br-MRBC as detected by rosette-forming assay with Br-MRBC. MRL159.5 spontaneously produced IL-6 and secreted IgM, and was induced to augment IgM secretion when treated with Br-MRBC or IL-6. Triggering of CD40 led to an augmentation of IgM secretion as well as IL-6 expression. Blocking the binding of IL-6 to its cellular receptor through the use of inhibitory antibodies inhibited CD40-induced IgM secretion, suggesting a possible autocrine role of IL-6 for CD40-induced differentiation of this B cell hybridoma. Addition of IL-4 or Br-MRBC augmented IL-6 expression as well as IgM secretion by CD40-activated MRL159.5 cells. CD40 also augmented tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression but resulted in decreased IL-10 expression. Furthermore, under conditions where IL-6 expression was augmented, IL-6R alpha (gp80) expression was down-regulated, suggesting a negative feedback mechanism of an IL-6 autocrine loop in this hybridoma. These results demonstrate a role by which T cell-dependent activation through CD40 regulates an IL-6 autocrine loop, controlling differentiation of autoreactive B cells in autoimmune disease.

Animals↗

PubMed1998

Regulation of interleukin-6 and interleukin-6R alpha (gp80) expression by murine immunoglobulin-secreting B-cell hybridomas.

We have examined the contribution of endogenous interleukin-6 (IL-6) to the differentiation of murine B-cell hybridomas. AT73 was established by somatic hybridization between BALB/c mice B cells and 2.52M, a hypoxanthine-aminopterine-thymidine (HAT) medium-sensitive B-cell line mutant. It spontaneously secreted IgM, and addition of exogenous IL-6 augmented IgM secretion. Triggering of CD40 led to an augmentation of IL-6 expression and IgM secretion. Blocking the binding of IL-6 to its cellular receptor through the use of inhibitory monoclonal antibodies inhibited CD40-induced IgM secretion, suggesting a possible autocrine role of IL-6 for the differentiation of a CD40-activated B-cell hybridoma. Co-triggering with CD40 and B-cell receptor or activation through CD40 and IL-4 led to a synergistic augmentation of IL-6 expression as well as additive IgM secretion; this was followed by a marked decrease in the expression of B-cell surface markers on the cell membrane. Furthermore, under conditions where IL-6 expression was augmented, gp80 expression was down-regulated, suggesting a negative feedback mechanism in this B-cell hybridoma. These findings provide a role by which T-cell-dependent activation through CD40 regulates an IL-6 autocrine loop, controlling B-cell differentiation.

Animals↗

PubMed1998

Verotoxins induce apoptosis in human renal tubular epithelium derived cells.

Apoptosis mediated by verotoxins (VTs) has been identified in a renal carcinoma cell line, ACHN cells, which are an in vitro model of renal tubular epithelial cells. ACHN cells express the renal tubular marker CD24 as well as globotriaosyl ceramide/CD77, the receptor for VTs. VT binding to the ACHN cell surface was confirmed by positive staining with antibodies to the VTs. Treatment of ACHN cells with VTs induced prompt growth inhibition and cell death, and fragmentation of the genomic DNA in cells, typical of apoptosis, was observed. The expression of apoptotic antigen 7A6 detected by APO2.7 antibody in ACHN cells further supports the occurrence of apoptosis as a result of VT treatment. Cycloheximide enhanced VT-mediated apoptosis of ACHN cells, suggesting a strong correlation between the inhibition of protein synthesis and VT-mediated apoptosis. Moreover, tumor necrosis factor-alpha had a synergistic effect on VT-mediated apoptosis in ACHN cells. Considering the above evidence together with the clinical evidence showing the presence of apoptosis in the renal epithelium of a HUS patient, our results suggest a VT-induced apoptotic mechanism in normal renal tubular epithelium that may contribute to the pathogenesis of hemolytic uremic syndrome.

Antigens, Surface↗

PubMed1998

Induction of apoptosis in normal human renal tubular epithelial cells by Escherichia coli Shiga toxins 1 and 2.

The cytotoxicity of Shiga toxin (Stx) 1 and Stx2 produced by Escherichia coli to human renal cortical epithelial cells (HRCEC) in primary culture was investigated. HRCEC express CD24, the marker of renal distal tubules, as well as globotriaosyl ceramide/CD77, the receptor for Stxs. Binding of Stxs to HRCEC was confirmed by positive staining with specific antibodies to Stxs. Treatment of HRCEC with Stxs induced rapid cell death, which was reversed in the presence of neutralizing antibody specific for Stx. DNA fragmentation was found to be accompanied by Stx-mediated cell death in HRCEC, indicating that apoptosis was part of the process. These data and previous reports indicate that a variety of renal cell types, including tubular epithelial cells as well as glomerular capillary endothelial cells, may be targets for Stx-mediated apoptosis, which could contribute to the pathogenesis of hemolytic-uremic syndrome caused by Stx-producing E. coli infection.

Adult↗

PubMed1998

Expression of progesterone receptor isoforms in corpora lutea of human subjects: correlation with serum oestrogen and progesterone concentrations.

To understand the biological significance of progesterone receptor forms A (PR-A) and B (PR-B) in human corpus luteum, the expression of mRNA and serum steroid hormone concentrations were determined simultaneously in the luteal stages. The expression of PR-A mRNA predominated over PR-B mRNA in all samples analysed. Total PR (PR-AB) and PR-B mRNA concentrations at the late secretory phase were significantly (P < 0.01) lower than those at the early and mid secretory phases of the menstrual cycle. The ratio of PR-B to PR-AB mRNA concentration showed no significant change during the secretory phase. In the early and mid secretory phases, there was a negative correlation between PR-B mRNA concentration and serum progesterone concentration, and between the ratio of PR-B to PR-AB mRNA concentrations and serum progesterone concentration (P < 0.01). These findings suggest that human corpus luteum might intracellularly synthesize PR-A and PR-B, and thus be involved in the steroid functional regulation of the corpus luteum, especially at the early and mid secretory phases, and that progesterone might regulate the synthesis of PR-A and PR-B.

Adult↗

PubMed1998

Expression of platelet-derived endothelial cell growth factor and its mRNA in uterine endometrium during the menstrual cycle.

Steroid hormones, e.g. progesterone and oestradiol, may be responsible for the production and expression of a variety of angiogenic growth factors present in endometrial tissue. The expression of platelet-derived endothelial cell growth factor (PD-ECGF) in neovascularization after regression of the microvessels in the endometrium was examined. PD-ECGF protein expression in the endometrium during the menstrual cycle was determined by a sandwich enzyme immunoassay. Transcription levels of PD-ECGF were measured by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) Southern blot technique. The data show that levels of PD-ECGF protein and mRNA in uterine endometrium did not alter during the proliferative phase prior to ovulation. During the midcycle phase a sharp transient fall in mRNA levels accompanied by a gradual drop in protein levels was observed. After ovulation transcription of PD-ECGF recovered with a sharp increase in mRNA levels which persisted during the ovulatory phase. PD-ECGF protein levels were temporarily low after ovulation, but increased remarkably through the late secretory phase. PD-ECGF expression in the endometrium seems to be inversely correlated with oestradiol concentrations during the menstrual cycle.

Adult↗

PubMed1998

Relatively high expression ratio of sex hormone-binding globulin exon VII splicing variant to wild-type mRNA in human uterine cervical cancers.

We have demonstrated the intracellular expression of sex hormone-binding globulin (SHBG) exon VII splicing variant mRNA in human uterine cervical cancer using reverse transcription-polymerase chain reaction-Southern blot and DNA sequencing analyses. Analysis of the missing base pairs proved they corresponded to the entire exon VII, which is considered to encode a portion of the steroid-binding site, suggesting that the steroid-binding affinity of the variant protein might be different from that of the wild-type SHBG. In uterine cervical cancers, the wild-type mRNA levels were lower (P<0.01) and the ratio of the SHBG variant to wild-type mRNA levels was higher (P<0.01) than in the normal cervix. In cervical adenocarcinomas, the wild-type mRNA levels were higher (P<0.05) and the ratio of the SHBG variant to wild-type mRNA levels was lower (P<0.05) than in cervical keratinizing squamous cell carcinomas. There was no difference in expression among the clinical stages of cervical cancers. These results suggest that a relative increase of intracellular variant SHBG protein in human uterine cervical cancers might be involved in the disruption of the normal estrogen dependence.

Adenocarcinoma↗

PubMed1998

Expression of telomerase catalytic component, telomerase reverse transcriptase, in human gastric carcinomas.

Telomerase activity is believed to be crucial for cellular immortality, which is considered to participate in the development of a majority of human cancers. Human telomerase reverse transcriptase (TERT) has recently been identified as a catalytic subunit of telomerase. We examined the expression of TERT and other telomerase components such as human telomerase RNA component (hTR, encoded by TERC) and human telomerase-associated protein (TEP1) by reverse transcription-polymerase chain reaction in human gastric carcinomas and non-neoplastic mucosa, in addition to measuring the telomerase activity. Of 20 gastric carcinomas examined, 18 (90%) and 18 (90%) showed increased expression of TERT and higher telomerase activity in comparison with corresponding non-neoplastic mucosa, respectively. Increased expression of hTR/TERC was also observed in 15 (75%) of the gastric carcinomas. Immunohistochemically, strong expression of TERT protein was detected in the nuclei of the tumor cells of all carcinoma tissues, while the expression of TERT in non-neoplastic mucosal cells as well as stromal elements (except lymphocytes) was weak or negative. These findings suggest that increased TERT expression associated with telomerase activity may serve as a novel marker for the diagnosis of stomach cancer.

Aged↗

PubMed1998

Effects of tamoxifen on endometrial carcinogenesis in mice.

Two experiments were conducted to determine the effect of tamoxifen (TAM) in mouse endometrium in comparison with that of 17beta-estradiol (E2). In a medium-term assay, TAM as well as E2 treatment semi-dose-dependently increased the levels of fos/jun mRNA and their oncoproteins (Fos/Jun). The long-term effect of TAM on mouse endometrial carcinogenesis was also examined in the following model. A total of 150 female ICR mice, 12-13 weeks of age, were used. Of these, 125 mice received an injection of N-methyl-N-nitosourea (MNU) solution (1 mg/100 g body weight) into their left uterine tube and saline into the right. One week later, they were divided into four groups: groups 1 (35 mice) and 2 (30 mice) were given 25 ppm and ppm E2-containing diet, respectively, while group 3 (30 mice) was fed 5 ppm TAM-containing diet. Group 5 (30 mice) was fed basal diet alone. The remaining 25 mice (group 4) received 5 ppm TAM-containing diet alone. At the termination of the experiment (30 weeks), endometrial carcinomas were confirmed to be present in the groups exposed to MNU. TAM increased the incidence of preneoplastic lesions of the endometrium, while E2 enhanced the occurrence of the carcinoma. No carcinomas were found in the group given TAM alone. In the ovaries, corpora lutea were lacking in most of the mice exposed to TAM, suggesting that the animals were not cycling. Such findings indicated that TAM has an enhancing effect on endometrial carcinogenesis in mice, probably via a mechanism involving overexpression of Fos/Jun proteins.

Adenocarcinoma↗

PubMed1998

Steroid receptor mRNA levels in human corpus luteum.

To understand the biology of sex steroids in the human corpus luteum, the expression of estrogen receptor alpha, progesterone receptor, and androgen receptor mRNA levels was determined by semiquantitative reverse-transcription polymerase chain reaction-Southern blot analysis. Expression of all receptor mRNAs was detected in all samples analyzed. Each steroid receptor mRNA level was significantly lower (p < 0.05) during the late secretory phase than that during the early or the mid-secretory phase of the endometrium. These findings support the concept of a local role for sex steroids in modulating the function and life span of the human corpus luteum.

Adult↗

PubMed1998

Expression of progesterone receptor form A and B mRNAs in uterine leiomyoma.

This study was designed to determine the expression pattern of progesterone receptor form A (PR-A) and B (PR-B) mRNAs in uterine leiomyoma. Approximately equal expression of PR-A and PR-B mRNAs was designated as type AB and dominant expression of PR-B mRNA as type B. In all cases of normal uterine myometrium, PR mRNA expression was type AB. Alteration to type B in the superficial part of uterine leiomyoma was observed in approximately 40% of cases. Therefore, the relative overexpression of PR-B mRNA in the surface of uterine leiomyoma might reveal an activated phenotype of progestational proliferation, plausibly related to the growth of uterine leiomyoma.

Adult↗

PubMed1998

Significance of sex steroids in roles of cadherin subfamily and its related proteins in the uterine endometrium and placenta.

To know the biological functions of the adherens junction in uterine endometrium, mRNA expressions of E-cadherin and alpha- and beta-catenin. which mainly comprise the adherens junction, were determined. Furthermore, to understand various functions related to V-cadherin in the placenta. vessel permeability was determined by assessing V-cadherin mRNA expression in HUV-EC-C cells, derived from the endothelial cells in human umbilical cord. The levels of E-cadherin and alpha- and beta-catenin mRNAs in the endometria of the proliferative phase were significantly less than those of the secretory phase. Treatment with estradiol dipropionate significantly reduced their levels in the endometria of the secretory phase. These suggest that the functions of the adherens junction in endometrial epithelial cell are controlled by sex steroids. On the other hand, estradiol decreased the endothelial cell barrier properties in HUV-EC-C cells, whereas progesterone partly reversed the changes induced by estradiol. While estradiol decreased the level of V-cadherin mRNA in HUV-EC-C cells, progesterone partly reversed the level decreased by estradiol. Therefore, sex steroids play a role in placental development and function related to cadherins on the endothelial cells, probably via placental vessel permeability.

Adult↗