Biomedical subjects

M Kosuga

Publications and source records attributed to M Kosuga.

24 records · Page 2Linked to original sources

PubMed1998

Fas-mediated apoptosis is involved in the elimination of gene-transduced hepatocytes with E1/E3-deleted adenoviral vectors.

Gene-transduced hepatocytes with E1/E3-deleted adenoviral vectors are eliminated immediately and the expression of transduced genes disappears rapidly following the vector administration. In this report, we analysed the involvement of apoptotic cell death in the elimination of hepatocytes infected with adenoviral vectors. An E1/E3-deleted adenoviral vector expressing Escherichia coli beta-galactosidase (LacZ) was injected via the portal vein into congenitally Fas-deficient mice (lpr), Fas ligand-deficient mice (gld) and their control mice, MRL and C3H. 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal) staining of the liver specimens showed that 80-100% of hepatocytes were LacZ positive at 7 days after virus administration, suggesting that most of the hepatocytes received the injected adenoviral vectors. In normal mice, the number of LacZ-positive cells decreased dramatically at 14 and 21 days after transduction and few positive cells were observed at day 28. Beta-galactosidase activity, quantified by the O-nitrophenyl-beta-D-galactopyranoside assay, gave comparable results to X-gal staining. At days 14 or 21, many apoptotic hepatocytes and apoptotic infiltrating cells were detected with the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) in situ apoptosis detection method. This observation suggested that the apoptotic process was associated with the elimination of adenovirus-infected hepatocytes. To test the involvement of the Fas-Fas ligand interaction in this apoptotic process, the period of transgene expression was measured in lpr and gld mice, which had received the same amount of AxCALacZ. X-Gal histochemical analysis detected many LacZ-positive cells in lpr or gld mice liver even at 21 or 28 days after AxCALacZ injection. There were significant differences in the reduction rates of beta-galactosidase activity of liver homogenates between lpr and MRL, or gld and C3H mice. Based on these observations, we conclude that the Fas-mediated apoptotic process is involved in the elimination of hepatocytes infected with E1/E3-deleted adenoviral vectors.

Adenoviridae↗

PubMed1991

Neuronal differentiation of Ewing's sarcoma induced by cholera toxin B and bromodeoxyuridine--establishment of Ewing's sarcoma cell line and histochemical study.

An Ewing's sarcoma (ES) cell line was established from a metastatic bone marrow specimen in a patient with advanced disease, and some histochemical characteristics were investigated by neuronal differentiation induced with cholera toxin B (CTB) and bromodeoxyuridine (BrdU). Neuronal differentiation was investigated by the expression of neurofilament and Leu-7, and glial differentiation was observed by expression of S-100 protein. Neurofilament (NF) and Leu-7 were positive in ES cells and these were expressed more intensively by induction with CTB than with BrdU. There was no expression of S-100 protein in untreated or differentiated ES cells. ES cells became differentiated to neuronal cells with CTB and BrdU, but it was not observed, that ES cells had the potential to differentiate to glial cells. It appears that ES is of more primitive neural origin than neuroblastoma, primitive neuroectodermal tumors and other related neural tumors.

Antigens, Differentiation↗

PubMed1983

Characterization of a novel ceramide octasaccharide isolated from whole tissue of a fresh-water bivalve, Corbicula sandai.

A novel glycosphingolipid containing two mannose residues, provisionally named GL-1 in a previous paper (Itasaka, O. and Hori, T. (1979) J. Biochem. 85, 1469-1481), was isolated from whole tissue of a fresh-water bivalve, Corbicula sandai, and the structure of the glycolipid was determined to be: Galp4Me(beta1 more than 3)GalpNAc(beta1 more than 3)Fucp(alpha1 more than 4)GlcpNAc(beta1 more than 2)Manp(alpha1 more than 3)(Xylp(alpha1 more than 2))Manp(beta1 more than 4)Glcp(beta1 more than 1)Cer. Glycosphingolipid-1 seems to be a biosynthetic precursor of glycosphingolipid-3, because they have an identical octasaccharide structure, except for the presence of an aminoethylphosphoryl residue in glycospingolipid-3. However, in contrast to glycosphinogolipid-3, glycosphingolipid-1 has a more complex ceramide pattern. This fact suggests that glycosphingolipid-1 as isolated is not a degradation product of glycosphingolipid-3, and it can be presumed that the restricted molecular species of glycosphingolipid-1 which has the same ceramide constitution as glycosphingolipid-3 may be utilized preferentially for glycosphingolipid-3 biosynthesis.

Animals↗

PubMedDate not supplied

Strong, long-term transgene expression in rat liver using chicken beta-actin promoter associated with cytomegalovirus immediate-early enhancer (CAG promoter).

For successful gene therapy in hepatic enzyme deficiencies, it is essential to use promoters that can maintain strong transcriptional activity for the long term in the liver. Using Gunn rats, a model animal for Crigler-Najjar syndrome type I, the long-term transcriptional function of the CAG promoter (a combination of chicken beta-actin promoter and cytomegalovirus immediate-early enhancer) was evaluated in the rat liver. We constructed a plasmid pCAGGHUGT, containing expression cassettes of human bilirubin UDP-glucuronosyltransferase (BUGT) and hygromycin phosphotransferase, under the control of the CAG promoter and murine phosphoglycerate kinase promoter, respectively. Conditionally immortalized Gunn rat hepatocytes (IGRH), which had been established using mutant SV40 large T antigen ((TS)T), were transfected with pCAG-GHUGT. A stably transfected clone IGRHUGT, expressing a high level of BUGT, was obtained after selection with hygromycin. At 33 degrees C, the cells doubled in number in approximately 72 h; however, at 37 degrees C, cell proliferation stopped, indicating that the characteristic of temperature-dependent proliferation was retained in this clone. Ten million cells were injected into the spleen of syngeneic Gunn rats five times at 10-day intervals. Serum bilirubin levels were reduced by 45-50% at 70 days after the first transplantation and remained so throughout the duration of the study (120 days). These results suggested that the CAG promoter was able to maintain strong transcriptional activity in rat liver for at least 120 days.

Actins↗

PubMedDate not supplied

Phenotype correction in murine mucopolysaccharidosis type VII by transplantation of human amniotic epithelial cells after adenovirus-mediated gene transfer.

Cell therapy with human amniotic epithelial (HAE) cells was developed as an alternative method for enzyme replacement therapy in congenital lysosomal storage disorders, but only limited therapeutic efficacy has been reported. A major drawback is insufficient production and secretion of lysosomal enzymes from HAE cells. In this study, we infected HAE cells with an E1-deleted adenoviral vector expressing human beta-glucuronidase (GUSB), and generated cells overexpressing GUSB by a hundred times as much as endogenous GUSB in untreated HAE cells. GUSB secreted from the gene-transferred HAE cells were efficiently transported to murine fibroblasts with endocytosis mediated by mannose-6-phosphate receptors. The cells were administered into the spleen of the mice with the lysosomal storage disease mucopolysaccharidosis type VII (B6/MPSVII). Approximately 10-15% of the normal GUSB activity was detected in both liver and spleen 7 days after the cell administration. Histopathological examination showed that lysosomal enlargement in tissue macrophages in the liver and the spleen had disappeared by day 14. These results suggest that transplantation of the HAE cells transduced with adenoviral vectors can be employed for the treatment of congenital lysosomal storage disorders.

Adenoviridae↗

PubMedDate not supplied

Myocardial metabolism of spontaneously hypertensive rat.

Oxidative phosphorylation of myocardial mitochondria isolated from spontaneously hypertensive rats (SHR) was determined polarographically. The concentrations of high-energy phosphate compounds, the concentrations of intermediate metabolites, and the activities of the enzymes within the metabolic systems of glycolysis, the pentose phosphate (PPh) cycle, and the tricarboxylic acid (TCA) cycle of the myocardium of SHR were determined. The gradual development of cardiac hypertrophy was observed in SHR in comparison with normotensive Wistar rats. There were no significant differences in the function of oxidative phosphorylation of the mitochondria, in concentrations of high-energy phosphate compounds and carbohydrate metabolites, or in activities of glycolytic enzymes between SHR and normotensive Wistar rats.

Adenine Nucleotides↗