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Biomedical subjects

N Sugiyama

Publications and source records attributed to N Sugiyama.

At least 73 records · Page 4Linked to original sources
PubMed1997

Immortalized gastric epithelial cell line GSM06 synthesizes hyaluronan under the influence of simian virus 40 large T-antigen expression.

GSM06 is a cell line established from the stomach of transgenic mouse harboring a temperature-sensitive simian virus 40 (SV40) large T-antigen gene. 3H-labeled macromolecules produced by the cells incubated with [3H] glucosamine were characterized to examine whether or not GSM06 cells synthesize mucin (mucus glycoprotein). The GSM06 cells grew until a confluent monolayer formed at 33 degrees C (the permissive temperature for SV40 large T-antigen expression), and the 3H-labeled macromolecules appeared in both cell extract and medium during culture for at least 1 week. Unexpectedly, almost all 3H-labeled macromolecules, which were excluded from a column of Sepharose CL-4B, were identified as hyaluronan by analyses using Sepharose CL-2B chromatography, cesium trifluoroacetate equilibrium centrifugation, treatment with dithiothreitol, and trypsin, hyaluronidase, and chondroitinase ABC digestion. At a nonpermissive temperature (39 degrees C), GSM06 cells grew only slightly, but produced much more hyaluronan than at 33 degrees C. The results indicate that GSM06 cells produce not mucin, but hyaluronan, and that the expression of large T-antigen may influence hyaluronan synthesis in GSM06 cells.

Animals↗
PubMed1997

Neuroleptic malignant syndrome and hydroxylase gene mutations: no association with CYP2D6A or CYP2D6B.

To examine a possible association between debrisoquine 4-hydroxylase gene mutations and neuroleptic malignant syndrome, we assessed frequencies of wild type and A and B mutant alleles of the CYP2D6 gene in 24 patients with a history of neuroleptic malignant syndrome, 50 patients with neuroleptic-treated schizophrenia but no history of neuroleptic malignant syndrome, and 50 healthy controls. Allele frequencies did not differ significantly between these groups. Homozygotes for CYP2D6A and for CYP2D6B, which indicate a poor-metabolizer phenotype for the CYP2D6 substrate, were not detected among the neuroleptic malignant syndrome cases. This result indicates no excess of poor CYP2D6 metabolizers in neuroleptic malignant syndrome. The aetiology of neuroleptic malignant syndrome is not explainable in terms of CYP2D6 gene mutations.

Alleles↗
PubMed1997

Insulin stimulates production of glycoconjugate layers on the cell surface of gastric surface mucous cell line GSM06.

The mechanism of regulation of mucus production in the gastric mucosa remains unclear. Recently, we established a gastric surface mucous cell line GSM06, which produces periodic acid-Shiff (PAS)-positive glycoconjugate (mucus) layers on the cell surface, from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen gene. In this study, GSM06 cells were examined for its production of PAS-positive glycoconjugate layers to acid secretagogues and growth factors. The cells were cultured at nonpermissive temperature (39 degrees C) for 3-18 days and stained with PAS. Insulin (1-30 microg/ml; 0.29-8.6 microM) time- and dose-dependently increased production of glycoconjugates on the cell surface. When glycoconjugate layers produced by stimulation of insulin (3-30 microg/ml; 0.86-8.6 microM) were removed from the cell surface of GSM06 cells by a mild trypsin treatment, PAS-positive materials were remarkably decreased (day 18). In addition, morphological findings indicate that a high concentration of insulin (30 microg/ml; 8.6 microM) produced thick PAS-positive glycoconjugate layers just like normal gastric surface mucosa on the cell surface on day 18. In contrast, histamine (0.1-100 microM), carbachol (0.1-100 microM), gastrin-17 (0.1-100 nM), epidermal growth factor (0.01-10 ng/ ml; 1.7-1,700 pM), transforming growth factor-alpha (0.01-10 ng/ml; 1.8-1,800 pM), and fetal bovine serum (1-10%) did not increase glycoconjugate production. These findings suggest that insulin is a stimulator of glycoconjugate production, and stimulates production of glycoconjugate layers on the cell surface in the gastric surface mucous cell line GSM06.

Animals↗
PubMed1997

Mutations in the proteolipid protein gene in Japanese families with Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a rare X-linked dysmyelinating disorder of the CNS resulting from abnormalities in the proteolipid protein (PLP) gene. Exonic mutations in the PLP gene are present in 10 to 25% of all cases. In investigating genotype-phenotype correlations, we screened five Japanese families with PMD for PLP gene mutations and compared their clinical manifestations. We identified two novel nucleotide substitutions in exon 5, at V208N and at P210L, in two families. In the remaining three families, there were no mutations detected. Although all patients satisfied the criteria for the classical form of PMD, two families not carrying the mutations showed milder clinical manifestations than those with the mutations. Since linkage analysis has shown homogeneity at the PLP locus in patients with PMD, our findings suggest that there may be genetic abnormalities other than exonic mutations that cause milder forms of PMD.

Apoproteins↗
PubMed1997

[Highly reproducible technique for the estimation of trabecular bone density: three slice method on peripheral quantitative CT].

The reproducibility of bone mineral density (BMD) measurements with peripheral quantitative CT has been limited by the repositioning error. In this study, 1 mm-step 3-slice scan data were used to compensate for this error in trabecular BMD of the distal radius. The assessment was based on the liner relations between the cross-sectional area and trabecular bone mineral content and trabecular area ratio, on the condition that the trabecular bone is defined by a BMD value. The estimated reproducible errors of less than 2% under clinical conditions indicate that the method is reliable for follow-up examination.

Adult↗
PubMed1996

Anionic residue in the alpha-subunit of the nicotinic acetylcholine receptor contributing to subunit assembly and ligand binding.

To ascertain the anionic sites on the nicotinic receptor to which acetylcholine and other quaternary ammonium ligands bind, we have examined the role of an aspartyl residue (Asp-152) in the alpha-subunit. Prior photolytic labeling with agonist analogues of the neighboring residues Trp-149 and Tyr-151 suggests that their side chains reside on the binding face (also termed the (+)- or counterclockwise face) of the alpha-subunit. Asp-152 presents an anionic charge in the vicinity of these aromatic residues. Modification of the aspartate to asparagine (D152N) creates a glycosylation signal (Asn-152-Gly-Ser), and we find, on the basis of altered electrophoretic migration, that glycosylation occurs at this position upon cotransfection of the mutant alpha-subunit with beta-, gamma-, and delta-subunits. Glycosylation results in a reduction in the capacity of the receptor to assemble; this reduction is manifest in the initial step of dimer formation between the alphagamma- and alphadelta-subunits. The alpha-subunit mutant receptor reaching the assembled pentamer exhibits an altered selectivity for certain ligands. Little reduction in alpha-bungarotoxin binding is observed, whereas affinities for agonists and competitive alkaloid antagonists are reduced substantially. Separation of the contributions of charge removal and glycosylation addition shows that both factors affect agonist affinity, with the charge influence being far more predominant. These findings raise the possibility that a component of the coulombic attraction stabilizing the binding of agonists comes from the aspartyl residue at position 152 in the alpha-subunit.

Amino Acid Sequence↗
PubMed1996

Biochemical bases in differentiation of a mouse cell line GSM06 to gastric surface cells.

A mouse gastric surface cell line GSM06 established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene was subjected to the lipid and glycoprotein analysis. When GSM06 cells were cultured for a long time after formation of a confluent monolayer, they differentiated to resemble foveolar epithelial cells morphologically. Biochemical changes during culture were studied in cells harvested just when a monolayer had formed (day 0), on day 7, and on day 21. Content of total phospholipids, cholesterol, cholesterol sulfate, total sugar and sialic acid increased about 1.5-fold from day 0 to 7 and remained elevated till day 21. The fatty acid composition of phospholipids revealed increased relative levels of oleic acid in phosphatidylcholine and phosphatidylethanolamine, and an increased level of plasmenylethanolamine from day 0 to 7. The level of dolichylphosphate continued to increase in a time-dependent manner. Glycosylation of various proteins, detected with lectins, was enhanced from day 7. In addition, greater resistance to taurodeoxycholate and acetylsalicylic acid was observed on days 7 and 21 than on day 0. Thus, enhanced glycosylation of proteins and an overall increase in the area of cellular membranes were the major changes in GSM06 cells during culture, and they were accompanied by an enhancement of cytoprotective potential.

Animals↗
PubMed1996

Biological characterization of gastric surface mucous cell line GSM06 from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.

We investigated the biological features of gastric surface mucous cell line GSM06, established from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. GSM06 cells grew until confluent monolayers were formed at the permissive temperature (33 degrees C), showing that the cessation of cell growth may be due to contact inhibition. Moreover, the cells did not grow in a soft agar gel. However, chromosome numbers of the cells were not normal. When GSM06 cells were cultured in Daigo's T medium supplemented with growth factors and 10% fetal bovine serum at 33 degrees C for 1, 3 or 9 days, the cells gradually grew to confluent monolayers (day 9). Morphological observations revealed that the cells time-dependently formed microvilli-like structures and yielded periodic acid-Schiff-positive glycoproteins on the cell surface with the growth of the structures. When GSM06 cells were cultured on a membrane filter for 1-9 days, elevated transepithelial resistance was noted in a culture period-dependent fashion. On day 9, junctional complexes such as tight junctions and desmosomes were observed between the cells. In addition, prostaglandin E2 production was evoked from the cells from day 1. In order to determine cell viability, GSM06 cells were labeled with the fluorescence dye 2',7'-bis(carboxyethyl)-carboxyfluorescein. Exposure of GSM06 cells to ethanol (7.5-17.5%) elicited cell injuries in a concentration-related manner on day 1, whereas these cytotoxic effects were attenuated on days 3 and 9, suggesting that this protection may be, at least in part, related to the increased glycoproteins and transepithelial resistance. GSM06 cells possessing these unique characteristics should be highly useful as an in vitro model of gastric epithelium.

Animals↗
PubMed1996

Secondary carnitine palmitoyltransferase deficiency in chronic renal failure and secondary hyperparathyroidism.

A 14-year-old girl, having mental and growth retardation with end stage renal disease, was affected by a stroke-like attack. The attack was associated with transient low density areas at both sides of the parietal portion on head CT. Lactic acidosis, hypertrophic cardiomyopathy, angina pectoris-like attacks, hypertension and hyperparathyroidism were also observed and they were supposedly due to mitochondrial cytopathy. No morphological or biochemical abnormalities were found on the mitochondrial respiratory chain. However, muscle carnitine palmitoyltransferase (CPT) activity was significantly low, which was restored to a normal level after hyperparathyroidism was controlled by alphacalcidol administration. Furthermore, we also found two more chronic renal failure patients with secondary hyperparathyroidism, as well as the primary hyperparathyroidism patient showing markedly low muscle CPT activity. These findings suggest the possible contribution of parathyroid hormone to lipid metabolism in skeletal muscle and to the myopathic manifestations often seen in hyperparathyroidism.

Adolescent↗
PubMed1996

Effect of carnitine administration on glycine metabolism in patients with isovaleric acidemia: significance of acetylcarnitine determination to estimate the proper carnitine dose.

In isovaleric acidemia (IVA), accumulated isovaleryl-CoA in the mitochondrion induces variable metabolic disturbances. To remove intramitochondrial isovaleryl groups, glycine therapy has been advocated primarily. On the other hand, secondary carnitine deficiency has been documented in this disorder and carnitine supplementation alone has been reported to be effective. In the present study, we administered carnitine and glycine to patients with IVA, and investigated serum carnitine and urinary excretion of total and free carnitine, acylcarnitine profile (i.e., isovalerylcarnitine and acetylcarnitine), and isovalerylglycine. By adding carnitine to glycine supplementation, more isovalerylglycine, not only isovalerylcarnitine, was excreted in the urine. Acetylcarnitine was detected in the urine only when sufficient carnitine was supplemented. We concluded that combined therapy of glycine and carnitine is more effective and safer to eliminate isovaleryl-CoA in IVA than conventional therapy using either glycine or carnitine. Urinary acetylcarnitine concentration might be a good marker indicating the optimal dose of L-carnitine supplementation.

Acetylcarnitine↗
PubMed1996

A duplicated PLP gene causing Pelizaeus-Merzbacher disease detected by comparative multiplex PCR.

Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused by abnormalities in the proteolipid protein (PLP) gene, which is essential for oligodendrocyte differentiation and CNS myelin formation. Although linkage analysis has shown the homogeneity at the PLP locus in patients with PMD, exonic mutations in the PLP gene have been identified in only 10%-25% of all cases, which suggests the presence of other genetic aberrations, including gene duplication. In this study, we examined five families with PMD not carrying exonic mutations in PLP gene, using comparative multiplex PCR (CM-PCR) as a semiquantitative assay of gene dosage. PLP gene duplications were identified in four families by CM-PCR and confirmed in three families by densitometric RFLP analysis. Because a homologous myelin protein gene, PMP22, is duplicated in the majority of patients with Charcot-Marie-Tooth 1A, PLP gene overdosage may be a important genetic abnormality in PMD and affect myelin formation.

Base Sequence↗
PubMed1996

Direct evidence of the production of IgA by tonsillar lymphocytes and the binding of IgA to the glomerular mesangium of IgA nephropathy patients.

Tonsils were obtained by tonsillectomy from a 33-year-old woman with IgA nephropathy. Hetero-hybridoma cells of human tonsillar B-lymphocytes with mouse myeloma cells (NS-1) were made, and cultured in HAT medium supplemented with 10% fetus bovine serum and hybridoma cloning factor. The culture medium was analyzed by Western blot analysis using anti-human IgA antibody, and both IgA1 and IgA2 were demonstrated to be produced. The specimens of the biopsied kidney tissue of IgA nephropathy were washed with 0.02 M citrate buffer (pH 3.2) to remove deposited IgA from glomerulus. The specimens were then incubated with the culture media of hybridoma cells, and immuno-fluorescence analysis using FITC-conjugated anti-human IgA antibody was performed. IgA deposit was efficiently removed by washing with citrate buffer and was recovered after incubation with the culture medium of hybridoma cells. Direct evidence of binding of IgA produced by tonsillar B-lymphocytes to the glomerular mesangium of IgA nephropathy was demonstrated.

Adult↗
PubMed1995

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric analysis of glycine conjugates and urinary isovalerylglycine in isovaleric acidemia.

n-Acetylglycine, n-propionylglycine, n-butyrylglycine, isobutyrylglycine, n-valerylglycine, isovalerylglycine, heptanoylglycine, phenylacetylglycine and isovalerylglucuronide were identified based on their liquid chromatographic-atmospheric pressure chemical ionization mass spectra (LC-APCI-MS). We were able to detect the presence of urinary isovalerylglycine in two cases of isovaleric acidemia using LC-APCI-MS. Membrane-filtered urine samples were injected into the LC-APCI-MS system in the negative-ion mode without any further pretreatment, and large amounts of isovalerylglycine were detected as the [M-H]- ion. The urinary excretion of isovalerylglycine appeared to increase after L-carnitine therapy. This analytical method is quick and easy and it may be a useful tool in understanding dysfunctional conditions in isovaleric acidemia.

Acidosis↗
PubMed1995

Ebselen, a seleno-organic compound, protects against ethanol-induced murine gastric mucosal injury in both in vivo and in vitro systems.

The inhibitory effect of the seleno-organic compound ebselen on ethanol-induced murine gastric mucosal injury was examined. In an in vivo study, absolute ethanol (50 microliters/mouse, oral) produced marked gastric mucosal necrosis along with hemorrhage or edema and elevations in both lipid peroxide and peptidoleukotriene levels in the fundic mucosa. Pretreatment with ebselen (30 and 100 mg/kg, oral) significantly prevented this gastric mucosal injury and, further, remarkably decreased the elevated lipid peroxide and peptidoleukotriene levels. In an in vitro study using a murine gastric surface mucous cell line GSM06, exposure to ethanol concentration dependently elicited cell damage (7.5-17.5% ethanol) and an increase in lipid peroxides without alterations in peptidoleukotrienes (15% ethanol). Addition of ebselen (10 and 100 microM) to this system (15% ethanol) significantly inhibited the cell damage and completely prevented the increase in lipid peroxide level. These results indicate that ebselen protects against murine gastric mucosal injury both in vivo and in vitro, and that this protection may be related at least in part to its inhibitory action on lipid peroxides.

Animals↗