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Biomedical subjects

S Gasa

Publications and source records attributed to S Gasa.

At least 37 records · Page 2Linked to original sources
PubMed1995

Novel modification of ceramide: rat glioma ganglioside GM3 having 3-O-acetylated sphingenine.

A novel O-acetylated GM3 containing 3-O-acetyl 4-sphingenine was isolated with one having a non-acetylated base from transplanted rat glioma tissue. The presence and position of the acetyl group were estimated by one- and two-dimensional proton nuclear magnetic resonance, and fast atom bombardment-mass spectrometries. In addition, the O-acetyl GM3 showed higher immunological activity toward anti-melanoma antibody in the presence of non-acetylated GM3 in complement-dependent liposome lysis than did non-acetylated or acetylated GM3 alone in the liposome, suggesting enhancement of immunological reactivity of the intact tumor cells by a small amount of O-acetyl GM3.

Animals↗
PubMed1995

Interleukin 4 enhances ganglioside GD3 expression on the human fibroblast cell line WI-38.

Human fibroblast cell line WI-38 cultured in vitro was treated with a human recombinant IL-4 at concentrations of 1 to 100 U/ml to examine the alteration of glycosphingolipid (GSL) expression of the cells. Neutral GSL of non-treated WI-38 cells consisted of CMH (GlcCer), CDH, CTH, and Gb4Cer; CMH and CTH were the major components. The acidic GSL were composed of GM3 as the predominant component and other minor gangliosides including GD3. The neutral GSLs did not change in profile during the treatment with IL-4, while the acidic GSLs showed a prominent change, an increase of GD3 content. The increase of GD3 was detectable with IL-4 concentrations over 1 U/ml, and reached a plateau at 10 U/ml, where the amount of GD3 was almost equal to that of GM3. The GD3 increase occurred at 24 h after the IL-4 treatment, and lasted for at least 96 h, as long as IL-4 remained present in the culture media. The GD3 synthase (sialyltransferase) level was found to be increased in an IL-4 dose-dependent manner. IL-4 did not influence the growth or morphological appearance of WI-38 cells. The results demonstrate a novel biological effect of IL-4, modulating GSL in non-hematopoietic cells.

Cell Line↗
PubMed1995

Enzymatic sulfation of gangliotriaosylceramide in human renal cancer cells.

Biosynthesis of sulfoglycolipid is markedly increased in a human renal cancer cell line, SMKT-R3. We investigated the sulfotransferase catalyzing the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gangliotriaosylceramide (Gg3Cer) in SMKT-R3 cells. On thin-layer chromatography, the reaction product comigrated with Gg3Cer III3-sulfate (SM2b), not with Gg3Cer II3-sulfate (SM2a). To examine which monosaccharide of Gg3Cer was sulfated, the product was treated with beta-hexosaminidase A. Unlike authentic SM2a, of which the non-reducing terminal N-acetylgalactosamine was cleaved off, the product was not hydrolyzed. These results suggest that sulfate was transferred to the non-reducing terminal N-acetylgalactosamine. Gg3Cer sulfotransferase activity was independent of divalent cations but stimulated by Fe2+, and the optimal pH was approximately 6.5. The apparent Km values were 102 microM for PAPS and 348 microM for Gg3Cer. Gg3Cer II3,III3-bis-sulfate (SB2) and a sulfotransferase activity synthesizing SB2 from SM2a were also detected in SMKT-R3 cells.

Acetylgalactosamine↗
PubMed1994

Hepatocyte growth factor elevates the activity levels of glycolipid sulfotransferases in renal cell carcinoma cells.

Accumulation of sulfoglycolipids associated with markedly elevated activity levels of glycolipid sulfotransferases has previously been demonstrated in the human renal cell carcinoma cell line, SMKT-R3. To elucidate the regulatory mechanisms of sulfoglycolipid synthesis in SMKT-R3 cells, the effects of various growth factors on the metabolic enzymes of sulfoglycolipids were investigated. Hepatocyte growth factor (HGF) significantly increased the activity levels of the sulfotransferases in a dose-dependent manner, but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. Scatchard analysis of 125I-HGF binding to SMKT-R3 cells indicated that the cells expressed high-affinity receptors for HGF with a Kd of 36 pM and 750 sites/cell. Furthermore, metabolic labeling with [35S]sulfate revealed that the addition of HGF to the culture medium of the cells resulted in an increment of sulfoglycolipid synthesis. Therefore, these observations suggest that HGF can function as a regulatory factor in sulfoglycolipid synthesis through the modulation of the sulfotransferase activity levels in renal cell carcinoma cells. In addition, HGF stimulated the proliferation and motility of SMKT-R3 cells, suggesting that HGF has multiple biological activities in renal cell carcinoma cells.

Animals↗
PubMed1994

Serum levels of steroid sulfatase protein in gynecologic carcinomas.

Steroid sulfatase (STS) desulfates a number of 3 beta-hydroxysteroid sulfates, converting inactive steroid hormone to the active form. We have established an enzyme-linked immunosorbent assay (ELISA) of STS by using polyclonal antibody against STS purified from human placenta to measure the amount of the enzyme protein in sera. ELISA was performed by a 'Sandwich' method using a peroxidase conjugated anti-STS IgG Fab' fragment. A range of STS of 10-1,500 ng/ml in serum was assayed by this method. When the serum STS from the patients with gynecologic carcinomas was assayed by the ELISA, the level was significantly elevated in endometrial carcinoma (P < 0.05) and ovarian carcinoma (P < 0.01), respectively, as compared with that of normal healthy women.

Adult↗
PubMed1994

Photoaffinity labeling of lipoproteins in human cerebrospinal fluid with a heterobifunctional derivative of galactosylsphingosine.

The binding of a galactosylceramide (GalCer) analog to proteins in normal human cerebrospinal fluid (CSF) was examined by photo-affinity labeling using a radioiodolabeled 2-(p-azido-salicylamido) ethyl-1,3-dithiopropionate (ASD) derivative of galactosylsphingosine (GalSph) as a probe. The affinity-bound peptides appeared at 66, 36 and 28 kDa as radiolabeled bands. The latter two peptides, 36 and 28 kDa, were independently identified by immunostaining of the isolated peptides using biotinylated GalSph, and immobilized Avidin, and by immunoprecipitation of the photolabeled peptide, to apolipoproteins (ALPs) E and A-I, respectively. The direct binding of the GalCer analog to these apo-peptides suggested that the existence in the body fluids or the transfer between the fluids and cells of the glycolipid was related to high density lipoprotein (HDL) constructions.

Affinity Labels↗
PubMed1993

Regulation of activity levels of glycolipid sulfotransferases by transforming growth factor alpha in renal cell carcinoma cells.

Accumulation of sulfolipids associated with markedly elevated levels of glycolipid sulfotransferase activities was previously demonstrated in human renal cell carcinoma cells. To explore the regulation mechanisms of sulfoglycolipid synthesis in renal cancer, effects of various growth factors on the metabolic enzymes of sulfoglycolipids were investigated by using a human renal cell carcinoma cell line, SMKT-R3. Among the growth factors tested, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to increase the sulfotransferase activity markedly (about 300%), but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. The augmented effects of TGF-alpha was abolished by cycloheximide. Since TGF-alpha is known to bind to the same receptor as EGF, SMKT-R3 cells were investigated for the EGF receptor by affinity cross-linking with 125I-EGF. A radiolabeled protein with a molecular mass of 175 kDa corresponding to the ligand-receptor complex was immunoprecipitated with a monoclonal anti-EGF receptor antibody. When production of the growth factors was examined immunochemically, the cells were found to secrete TGF-alpha at a low level and retain it in a membrane-bound form, whereas EGF was not detected. These observations suggest that the sulfotransferase activities are regulated through the autocrine, paracrine, and/or juxtacrine modes of intercellular stimulation by TGF-alpha in human renal cancer cells.

Carcinoma, Renal Cell↗
PubMed1993

Expression, purification and binding to the receptor of human insulin-like growth factor II.

Human insulin-like growth factor II (IGF-II) was expressed as a fused protein with 14 additive amino acids in Escherichia coli with a high yield by an expression system using T7 RNA polymerase. Purification of the expressed protein was simply performed using only differential ultrafiltrations, giving a homogeneous preparation upon polyacrylamide gel electrophoresis and high-performance liquid chromatography. The expressed peptide was reacted with a monoclonal antibody raised against native IGF-II on a blotted membrane. Furthermore, the peptide was bound to IGF-II receptor in solubilized rat fetus membrane, though the affinity was slightly inferior to that of native IGF-II. In addition, fusion IGF-II immobilized on a gel matrix was useful for one-step purification of the IGF-II receptor with a high yield from solubilized rat fetus membranes.

Amino Acid Sequence↗
PubMed1993

Epidermal growth factor elevates the activity levels of glycolipid sulfotransferases in renal-cell-carcinoma cells.

Accumulation of sulfoglycolipids associated with markedly elevated levels of glycolipid-sulfotransferase activity was previously demonstrated in human renal-cell-carcinoma cells. To elucidate the regulatory mechanisms of sulfoglycolipid metabolism in renal-cell carcinoma, effects of various growth factors on the sulfotransferase-activity levels were investigated using a human renal-cell-carcinoma cell line, SMKT-R3. Exogenous epidermal growth factor (EGF) significantly increased the activity levels of the sulfotransferases in a dose-dependent manner, but did not change that of arylsulfatase A, which hydrolyzes sulfoglycolipids. Furthermore, metabolic labeling with 35S-sulfate revealed that the addition of EGF to the culture medium of the cells resulted in an increment of sulfoglycolipid synthesis. The expression of the EGF receptor on SMKT-R3 cells was demonstrated by affinity cross-linking with 125I-EGF. These observations suggest that EGF can regulate sulfotransferase-activity levels in renal-cell-carcinoma cells, and function as one of the regulatory factors of sulfoglycolipid synthesis in these carcinoma cells.

Carcinoma, Renal Cell↗
PubMed1993

Human blood group B-active ganglio-glycosphingolipid in rat glioma.

A human blood group B-active glycosphingolipid, belonging to the ganglio-series, was isolated from rat glioma cell line RG2 subcutaneous isografts. The oligosaccharide structure of the glycosphingolipid was completely characterized as Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1- 1'ceramide by NMR spectrometry, negative fast atom bombardment-mass spectrometry, sequential degradation by glycosidases and methylation analysis. Human blood group B antigenicity and the activity of this glycosphingolipid were confirmed by immunostaining on thin-layer chromatography and the inhibition of hemagglutination, respectively. Although the lipid has been detected in rat granuloma, bone marrow cells, spleen, thymus, ascites hepatoma cells and gastric mucosa, this is the first report of the occurrence of the B-active lipid in glioma.

ABO Blood-Group System↗
PubMed1993

gamma-Glutamylcysteine synthetase and active transport of glutathione S-conjugate are responsive to heat shock in K562 erythroid cells.

Effect of heat shock on a glutathione-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and ATP-dependent outward transport of glutathione S-conjugate was characterized using K562 erythroid cells. When K562 cells grown at 37 degrees C were shifted to 42 degrees C for 2 h, an approximate 1.7-fold increase in the activity of gamma-GCS was observed. Treatment of K562 cells with erythropoietin (EP) for 12 h resulted in a decrease in the activity of gamma-GCS to 64% of the control. However, responsiveness of this enzyme activity in the cells treated with EP to heat shock was similar to that in untreated cells. Changes in the immunological activity of gamma-GCS were also observed in parallel with those in the enzymatic activity. On Northern blot analysis of total RNAs isolated from the cells with human cDNA for gamma-GCS, a substantial induction of mRNA level was found by heat shock and a reduction of EP. These changes were modest but correlated to the mRNA expression of a heat shock protein, HSP 70. Heat shock also had an effect of 1.8-fold stimulation on glutathione S-conjugate transport in K562 cells previously incubated with 1-chloro-2,4-dinitrobenzene. Treatment of the cells with EP resulted in a decrease in this transport by 62%. Similarly, the levels of glutathione S-conjugate-stimulated Mg(2+)-ATPase, which enzyme is thought to be involved in the transport of glutathione S-conjugate, were responsive to heat shock and EP. These results suggest that glutathione synthesis and transport process of glutathione metabolites are responsive to heat shock and play a role in the defense system against stresses. It is also suggested that the regulatory site of the expression of these enzymes by heat shock is independent of that by EP.

Biological Transport, Active↗
PubMed1993

Involvement of protein kinase C in the regulation of glycolipid sulfotransferase activity levels in renal cell carcinoma cells.

Accumulation of sulfolipids associated with elevated levels of glycolipid sulfotransferase activities has previously been demonstrated in renal cell carcinoma cells. To investigate the role of protein kinase C in the synthesis of sulfolipids, the effects of 12-O-tetradecanoylphorbol-13-acetate and protein kinase C inhibitors on glycolipid sulfotransferase activity levels were examined in a human renal cell carcinoma cell line, SMKT-R3. Continuous treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate caused a dose- and time-dependent reduction of the sulfotransferase activity levels. Similarly, protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and staurosporine, reduced the enzyme activities in a dose-dependent manner. These observations suggest that the glycolipid sulfotransferase activity levels are regulated by protein kinase C in SMKT-R3 cells. Furthermore, long-term 12-O-tetradecanoylphorbol-13-acetate treatment resulted in a reduction of sulfolipid synthesis and a decrease of the expression of sulfolipids on the cell surface. Taken together, it is suggested that protein kinase C is involved in the synthesis of sulfolipids through the regulation of the glycolipid sulfotransferase activity levels in renal cell carcinoma cells.

Carcinoma, Renal Cell↗
PubMed1993

An adult-type metachromatic leukodystrophy caused by substitution of serine for glycine-122 in arylsulfatase A.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase A (ASA). We have identified a new mutation in the ASA gene of a patient with adult-type MLD. In this mutation, the glycine at position 122, a highly conserved residue in the AS gene family, was replaced by serine. In a transient expression study, COS cells transfected with the mutant cDNA carrying 122Gly-->Ser did not show an increase of ASA activity and produced little material immunoreactive to an anti-ASA antibody, despite normal mRNA levels.

Adult↗
PubMed1993

Effect of endurance training on angiogenic activity in skeletal muscles.

The effect of endurance training on angiogenic activity in skeletal muscle was investigated using cell culture. The exercised rats were trained on a rodent treadmill, 5 days a week for 9 weeks. After the training program, a significant increase (7.8%) in mass was found in soleus muscle (MS), although we noted no apparent increase in gastrocnemius muscle and extensor digitorum longus muscle. The extracts from all the muscles (especially from the MS) of trained rats considerably increased the capillary growth in an in vitro angiogenesis model, in which microvascular fragments and myofibroblastic (Mf) cells isolated from lipid tissues were grown in co-culture. In addition, the extract from the MS of trained rats increased the DNA synthesis and growth of bovine capillary endothelial (BCE) cells, although the extract did not stimulate the growth of Mf cells. Moreover, the extract from the MS of trained rats notably enhanced the activity of plasminogen activator in the conditioned medium of BCE cells compared with that of sedentary rats. On the other hand, extracts from the MS of both sedentary and trained rats increased the synthesis of collagen by Mf cells and metalloproteinase activity in the conditioned medium of BCE cells to the same degree. These results suggest that endurance training increases the angiogenic activity in skeletal muscles.

Animals↗
PubMed1993

Iron responsive element-binding protein (IRE-BP) in leukemic cells: analysis using enzyme-linked immunosorbent assay and semiquantitative polymerase chain reaction.

The level of iron responsive element-binding protein (IRE-BP) in leukemic cells, which is essential for iron homeostasis and plays an important role in cell metabolism and cell growth, was measured using an enzyme-linked immunosorbent assay (ELISA). Comparing the levels in different clinical stages, the levels in CML cells in the chronic phase (mean +/- S.E., 0.270 +/- 0.110 U/mg protein, n = 9) and those in AML cells (0.150 +/- 0.104 U/mg) protein, n = 21) were significantly lower than that in normal granulocytes (0.628 +/- 0.216 U/mg protein, n = 9, p < 0.001 vs CML and AML). Analysis of IRE-BP mRNA expression in leukemic cells using semiquantitative polymerase chain reaction showed suppressed expression of mRNA as compared to normal bone marrow cells. These observations suggest that there may be dysregulation of IRE-BP expression and production in leukemic cells.

Adult↗
PubMed1993

Sulfolipids and glycolipid sulfotransferase activities in human renal cell carcinoma cells.

A cell line (SMKT-R3) established from human renal cell carcinoma was characterized for the presence of sulfolipids and glycolipid sulfotransferases. Sulfolipids were found to constitute a large part of the acidic glycolipid fraction in SMKT-R3 cells. These findings were confirmed by metabolic labelling with 35S-sulfate. These sulfolipids were expressed at the surface of SMKT-R3 cells as ascertained by cytofluorometry using a monoclonal antibody directed to sulfolipids. Furthermore, markedly high activity levels of glycolipid sulfotransferases were observed in SMKT-R3 cells compared with other cell lines. These results suggest that the increased synthesis of sulfolipids in renal cell carcinoma tissue (Sakakibara et al., 1989. Cancer Res., 49, 335-339) is due to the elevation of the sulfotransferase activities of renal carcinoma cells themselves.

Carcinoma, Renal Cell↗
PubMed1993

Preparation of a photoaffinity probe for the Man6P/IGF-II receptor.

A photoaffinity probe, 2-(4-phosphopentamanniminophenyl)ethyl-(4-azido) salicylamide, was prepared for photolabelling of the mannose 6-phosphate/insulin-like growth factor II receptor. The probe was synthesized from phosphopentamannan and 2-(4-aminophenyl)ethylamine followed by coupling with photosensitive 4-azidosalicylic acid. The 125I-labelled probe was reacted with receptor purified from rat liver under irradiation with ultraviolet light. Fluorography of the reaction product on a polyacrylamide gel effectively detected the receptor coupled to the photoprobe as a molecular mass of 250 kDa. Specific binding of the probe or of radiolabelled insulin-like growth factor II was not inhibited by the growth factor or by mannose 6-phosphate, confirming different binding sites to the receptor between the ligands.

Affinity Labels↗