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Biomedical subjects

M Fujino

Publications and source records attributed to M Fujino.

378 records · Page 21Linked to original sources
PubMedDate not supplied

Difference in O6-methylguanine methyltransferase activity among transformed NIH3T3 cell clones.

We examined the sensitivity to the lethal effects of methylating agents and the O6-methylguanine methyltransferase (MTR) activities of in vitro transformed NIH3T3 cell clones. The sensitivities to the lethal effects of MNNG were not different among all 49 transformed cell clones examined and do not correlate with the MTR activities. All 8 spontaneously transformed cell clones showed the same sensitivities to ACNU as the parental cell line. 2 of 20 transformants induced by UV or MNNG showed higher sensitivities to the ACNU although the MTR activity was normal. One cell clone transformed by UV was sensitive to ACNU and showed about half MTR activity. 5 of 19 cell clones transformed by oncogenes (Ha-ras or SV40 ori-) were sensitive to the lethal effects of ACNU and showed the low MTR activities, but were not as much sensitive as a Ha-MuSV transformed cell clone, Ha821.

Animals↗
PubMedDate not supplied

Characteristics of thyrotropin releasing hormone (TRH) receptors in rat brain.

Characteristics of TRH-receptors were studied in the rat central nervous system (CNS). Ion species, pH and temperature importantly influenced TRH-receptor binding. Subcellular distribution of TRH-receptor binding revealed that synaptic membranes had the greatest percentage of total sites. Scatchard analysis suggested that the rat CNS had two distinct TRH binding sites with apparent dissociation constants (Kd) of 5 X 10(09) M and 13 X 10(-8) M. Receptor activity is sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for the binding of [3H]TRH. Thiol reagents reduced the binding activity of the receptor, suggesting that an intrachain disulfide bond may form an important constituent of the binding site for TRH. The TRH-receptor in the rat brain was successfully solubilized with non-ionic detergent Triton X-100. On gel chromatography with Sepharose 6B column, the solubilized TRH-receptor molecule eluted at the fraction corresponding to an apparent molecular weight of 300,000 daltons, with Stokes' radius of 5.8 nm. The regional distribution of TRH-receptor binding was examined to clarify the site of TRH action. The highest level of binding was in the hypothalamus, cerebral cortex and hippocampus, indicating that TRH affects the CNS function mainly through the limbic system, cerebral cortex and hypothalamus. Moreover, tricyclic anti-depressants and Li+ decreased the binding of [3H]TRH. These findings suggest that endogenous TRH and TRH receptor may play the role of a neurotransmission modulator in the brain to control emotional and mental functions.

Animals↗
PubMedDate not supplied

Primary structure and characterization of the precursor to human pituitary adenylate cyclase activating polypeptide.

Human cDNAs encoding the precursor to pituitary adenylate cyclase activating polypeptide (PACAP) were cloned from human testis and cerebral cortex cDNA libraries. Nucleotide sequencing revealed that cDNA from the testis library encoded the entire precursor for PACAP, while cDNA from the brain library represented only the carboxy-terminal half of the precursor. The predicted human PACAP precursor consisted of 176 amino acid residues and was very similar to the ovine one (82%). Both human and ovine precursors contained both PACAP and another peptide, PACAP-related peptide (PRP), having 29 amino acids. PACAP and PRP were preceded and followed by paired basic amino acids, recognized as important for post-translational processing. The PACAP precursor resembles the vasoactive intestinal peptide (VIP) precursor, which contains VIP and peptide histidine methionine/isoleucine amide (PHM/PHI). Structurally, PRP had some similarity to PHM/PHI, growth hormone-releasing hormone (GRH) and PACAP. Northern blot analysis indicated that a 3.0-kb transcript was expressed in the ovine hypothalamus. Tissue distribution of PACAP mRNA was also clarified in the rat. Southern blot analysis of human genomic DNA gives single bands with six restriction enzymes, indicating that a single copy of the PACAP gene is contained in a haploid genome. The cDNA for human PACAP precursor was expressed using COS-7 and Chinese hamster ovary (CHO) cells. Immunoreactive PACAP was secreted into the culture media of both transfected cell lines.

Amino Acid Sequence↗
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

An attempt to add biological functions by genetic engineering in order to produce high-performance bioreactor cells for hybrid artificial liver: transfection of glutamine synthetase into Chinese hamster ovary (CHO) cell.

In the course of immortalization, hepatocyte cell lines lose their original differentiated functions, such as ammonia removal and urea formation, drug metabolism, serum protein synthesis, etc. (Enosawa et al., Cell Transplant. 5:S39-S40; 1996). With the aim of adding lost or deficient functions and producing cell lines for the bioreactor of a hybrid artificial liver, rat glutamine synthetase (GS) gene was transfected into Chinese hamster ovary (CHO) cells, because it is able to lower the ammonia level. The GS gene-inserted pSV2 plasmid was transfected into the CHO-K1 line by electroporation. Transfected CHO (GS-CHO) cells were cultured in a glutamine-free medium containing ammonia, glutamic acid, and the GS inhibitor methionine sulfoximine (MSX). The MSX concentration was increased stepwise from 25 mumol/L to 1600 mumol/L to amplify the GS gene. In several GS-CHO sublines resistant to 300-1600 mumol/L of MSX, the specific activities of GS were increased from 0.2 x 10(4) to 1.7-2.9 x 10(4) unit/10(6) cells. When the amplified GS-CHO cells were cultured in the ammonia-containing medium, a slow but steady decrease of the ammonia level was observed when the level was high. Finally, the prospect of genetically modulated cells for bioreactors in the hybrid artificial liver is discussed.

Ammonia↗
PubMedDate not supplied

Higher efficiency of retrovirus transduction in the late stage of primary culture of hepatocytes from nontreated than from partially hepatectomized rat.

The aim of this investigation was to optimize the conditions for gene transduction by a retroviral vector into primary cultured hepatocytes. Because a retrovirus infection is dependent upon the proliferative activity of the target cells, we examined the thymidine incorporation of the primary culture, using cells from nontreated and partially hepatectomized donor rats. Partial hepatectomy 1 day before cell isolation greatly enhanced the thymidine incorporation, and similarly, the transduction of LacZ, the gene for E. coli beta-galactosidase, although the percentage of transduced cells remained low. With cells from the normal rat, the thymidine incorporation increased gradually after the beginning of the culture and reached 16 times the initial activity on day 2, while hepatocytes from the partially hepatectomized donor showed no increase at that stage of the culture. Correspondingly, the number of gene-transduced hepatocytes was increased when the vector was added to the cells 1 or 2 days after isolation. Therefore, hepatocytes cultured for 1 or 2 days after isolation are suitable for retroviral transduction.

Animals↗
PubMedDate not supplied

Evaluation of an optical system in the fibercolonoscope: trial manufacture of an instrument with 30 degrees deviation optical system.

To evaluate the best optical system for colonoscopy a colonofibrescope was manufactured in which the optical system had a 30 degrees prograde deviation. In this test model, frontal view was easily obtained and close-up observations were possible. At areas with prominent semilunar folds, the zone behind them also came into full view. The bowel lumen could be visualized by angulation, the same as with conventional instruments. The passage trough the sigmoid-descending junction was easy, and visualization or the colon up to the distal transverse colon was accomplished within a short time in the majority of cases without fluoroscopic control.

Colonic Diseases↗