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Jin-ichi Inokuchi

Publications and source records attributed to Jin-ichi Inokuchi.

13 recordsLinked to original sources
PubMed2007

Regulation of the transport and protein levels of the inositol phosphorylceramide mannosyltransferases Csg1 and Csh1 by the Ca2+-binding protein Csg2.

Complex sphingolipids in yeast are known to function in cellular adaptation to environmental changes. One of the yeast complex sphingolipids, mannosylinositol phosphorylceramide (MIPC), is produced by the redundant inositol phosphorylceramide (IPC) mannosyltransferases Csg1 and Csh1. The Ca2+-binding protein Csg2 can form a complex with either Csg1 or Csh1 and is considered to act as a regulatory subunit. However, the role of Csg2 in MIPC synthesis has remained unclear. In this study, we found that Csg1 and Csh1 are N-glycosylated with core-type and mannan-type structures, respectively. Further identification of the glycosylated residues suggests that both Csg1 and Csh1 exhibit membrane topology with their C termini in the cytosol and their mannosyltransferase domains in the lumen. After complexing with Csg2, both Csg1 and Csh1 function in the Golgi, and then are delivered to the vacuole for degradation. However, uncomplexed Csh1 cannot exit from the endoplasmic reticulum. We also demonstrated that Ca2+ stimulates IPC-to-MIPC conversion, because of a Csg2-dependent increase in Csg1 levels. Thus, Csg2 has several regulatory functions for Csg1 and Csh1, including stability, transport, and gene expression.

Calcium-Binding Proteins↗
PubMed2006

The synthetic ceramide analog L-PDMP partially protects striatal dopamine levels but does not promote dopamine neuron survival in murine models of parkinsonism.

A number of previous studies have demonstrated a positive effect of exogenously administered monosialoganglioside GM1 on striatal dopamine (DA) levels and DA neuron survival in animal models of parkinsonism. However, due to low bioavailability of peripherally administered GM1, the present study investigated the neuroprotective/neurorestorative potential of enhancing endogenous GM1 biosynthesis by administration of the synthetic ceramide analog L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (L-PDMP) in two mouse models of Parkinsonism produced by acute or subacute 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. L-PDMP treatment caused an increase in brain GM1 levels in both Parkinson models and resulted in a partial sparing of striatal DA levels in the subacute MPTP model but not in the acute MPTP model. L-PDMP treatment had no effect on DA neuron survival in either model. These data suggest that the administration of L-PDMP as a means to enhance endogenous brain GM1 levels may hold limited promise as a potential neuroprotective or neurorestorative therapeutic strategy for Parkinson's disease.

Animals↗
PubMed2006

Endogenously produced ganglioside GM3 endows etoposide and doxorubicin resistance by up-regulating Bcl-2 expression in 3LL Lewis lung carcinoma cells.

The ganglioside patterns have been shown to dramatically change during cell proliferation and differentiation and in certain cell-cycle phases, brain development, and cancer malignancy. To investigate the significance of the ganglioside GM3 in cancer malignancy, we established GM3-reconstituted cells by transfecting the cDNA of GM3 synthase into a GM3-deficient subclone of the 3LL Lewis lung carcinoma cell line (Uemura, S. (2003) Glycobiology, 13, 207-216). The GM3-reconstituted cells were resistant to apoptosis induced by etoposide and doxorubicin. There were no changes in the expression levels of topoisomerase IIalpha or P-glycoprotein, or in the uptake of doxorubicin between the GM3-reconstituted cells and the mock-transfected cells. To understand the mechanism of the etoposide-resistant phenotype acquired in the GM3-reconstituted cells, we investigated their apoptotic signaling. Although no difference was observed in the phosphorylation of p53 at serine-15-residue site by etoposide between the GM3-reconstituted cells and mock-transfected cells, the activation of both caspase-3 and caspase-9 was specifically inhibited in the former. We found that the anti-apoptotic protein B-cell leukemia/lymphoma 2 (Bcl-2) was increased in the GM3-reconstituted cells. Moreover, wild-type 3LL Lewis lung carcinoma cells, which have an abundance of GM3, exhibited no DNA fragmentation following etoposide treatment and expressed higher levels of the Bcl-2 protein compared with the J5 subclone. Thus, these results support the conclusion that endogenously produced GM3 is involved in malignant phenotypes, including anticancer drug resistance through up-regulating the Bcl-2 protein in this lung cancer cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Insulin resistance as a membrane microdomain disorder.

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling, but their role in the pathogenesis of insulin resistance has not been investigated. Detergent-resistant membrane microdomains (DRMs), isolated in the low density fractions, are highly enriched in cholesterol, glycosphingolipids and various signaling molecules. TNFalpha induces insulin resistance in type 2 diabetes, but its mechanism of action is not fully understood. We have found a selective increase in the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytes treated with TNFalpha, suggesting a specific function for GM3. We were able to extend these in vitro observations to living animals using obese Zucker fa/fa rats and ob/ob mice, in which the GM3 synthase mRNA levels in the white adipose tissues are significantly higher than in their lean controls. In the DRMs from TNFalpha-treated 3T3-L1 adipocytes, GM3 levels were doubled, compared to results in normal adipocytes. Additionally, insulin receptor (IR) accumulations in the DRMs were diminished, while caveolin and flotillin levels were unchanged. GM3 depletion was able to counteract the TNFalpha-induced inhibition of IR accumulation into DRMs. Together, these findings provide compelling evidence that in insulin resistance the insulin metabolic signaling defect can be attributed to a loss of IRs in the microdomains due to an accumulation of GM3.

3T3-L1 Cells↗
PubMed2005

Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme.

The sialyltranferase ST3Gal-V transfers a sialic acid to lactosylceramide. We investigated the role of each of the N-glycans modifying mouse ST3Gal-V (mST3Gal-V) by measuring the in vitro enzyme activity of Chinese hamster ovary (CHO) cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagine was replaced with glutamine (N180Q, N224Q, N334Q), we determined that all three sites are N-glycosylated and that each N-glycan is required for enzyme activity. Despite their importance, N-glycosylation sites in ST3Gal-V are not conserved among species. Therefore, we considered whether the function in the activity that is performed in mST3Gal-V by the N-glycan could be substituted for by specific amino acid residues selected from the ST3Gal-V of other species or from related sialyltransferases (ST3Gal-I, -II, -III, and -IV), placed at or near the glycosylation sites. To this end, we constructed a series of interspecies mutants for mST3Gal-V, specifically, mST3Gal-V-H177D-N180S (medaka or tetraodon type), mST3Gal-V-N224K (human type), and mST3Gal-V-T336Q (zebrafish type). The ST3Gal-V activity of these mutants was quite similar to that of the wild-type enzyme. Thus, we have demonstrated here that the N-glycans on mST3Gal-V are required for activity but can be substituted for specific amino acid residues placed at or near the glycosylation sites. We named this method SUNGA (substitution of N-glycan functions in glycosyltransferases by specific amino acids). Furthermore, we verified that the ST3Gal-V mutant created using the SUNGA method maintains its high activity when expressed in Escherichia coli thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo.

Amino Acid Sequence↗
PubMed2005

Structure and function of lipid rafts in human activated T cells.

Lipid rafts, specialized membrane microdomains enriched in sphingolipids and cholesterol, have been shown to function as signaling platforms in T cells. Surface raft expression is known to be increased in human T cells upon activation, and this increased raft expression may account for efficient signaling capability and decreased dependency for co-stimulation in effector and/or activated T cells. However, raft-mediated signaling ability in activated T cells remains to be clarified. In this study, we analyzed the structure and function of lipid rafts in human activated T cells. We demonstrated that raft protein constituents are dramatically changed after activation along with an increase in lipid contents. T cells stimulated with anti-CD3 plus anti-CD28 antibodies showed an increase not only in surface monosialoganglioside GM1 expression but also in total amounts of raft-associated lipids such as sphingomyelin, cholesterol and glycosphingolipids. Raft proteins increased after activation include Csk, Csk-binding protein and Fyn, the molecules known to be involved in negative regulation of T cell activation. Consistent with the increase in expression of these proteins, TCR-mediated Ca(2+) response, a response dependent on raft integrity, was clearly inhibited in activated T cells. Thus, the structure and function of lipid rafts in human activated T cells seem to be quite distinct from those in naive T cells. Further, human activated T cells are relatively resistant to signaling, at least transiently, by TCR re-stimulation even though their raft expression is increased.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Glycosphingolipids govern gene expression.

To elucidate the biological significance of the lactosylceramide (LacCer) branching in glycosphingolipid (GSL) biosynthesis, we established ganglioside GM3- and lactosylsulfatide SM3-reconstituted cells by introducing the GM3 synthase gene and the sulfotransferase gene, respectively. In SM3-expressing cells, the reduction of beta1 integrin mRNA expression, the reduced adhesivity to fibronectin and laminin, and the suppression of anchorage-independent growth (tumorigenic potential) were observed. On the other hand, in GM3-expressing cells, anchorage-independent growth was promoted and the expression of PDGF alpha receptor mRNA was specifically reduced. Interestingly enough, no change in anchorage-dependent growth was observed in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions. Thus, the spatio-temporal, gene expression control mechanism by individual GSL molecules accumulating in the cell membrane microdomain (raft) has been proven.

Animals↗
PubMed2004

Apoptosis of human carcinoma cells in the presence of inhibitors of glycosphingolipid biosynthesis: I. Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP.

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells by anti-cancer drugs and biosynthetic inhibitors of cells surface glycolipids in the human colon carcinoma cells (Colo-205) are of interest in recent years. In our present studies, we have employed different stereoisomers of PPMP and PDMP (inhibit GlcT-glycosyltransferase (GlcT-GLT)) to initiate apoptosis in Colo-205 cells grown in culture in the presence of (3)H-TdR and (3)H/or (14)C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 microM) initiated apoptosis with induction of Caspase-3 activities and phenotypic morphological changes in a dose-dependent manner. We have observed an increase of radioactive ceramide formation in the presence of a low concentration (1-4 microM) of these reagents in these cell lines. However, high concentrations (4-20 microM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0-20 microM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer beta 1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 beta 1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Le(x) formation had been detected in these colon carcinoma (or Colo-205) cells (Basu M et al. Glycobiology 1, 527-35 (1991)). However, during progression of apoptosis in Colo-205 cells with increasing concentrations of L-PPMP, the GalT-4 activity was decreased significantly. These changes in the specific activity of GalT-4 in the total Golgi-membranes could be the resultant of decreased gene expression of the enzyme.

Antineoplastic Agents↗
PubMed2004

Triglyceride accumulation and altered composition of triglyceride-associated fatty acids in the skin of tenascin-X-deficient mice.

Tenascin-X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. Here, we observed abnormalities in the skin of TNX-deficient mice in comparison with that of wild-type mice. Histological analysis with Oil Red O staining demonstrated that there was considerable accumulation of lipid in the skin of TNX-deficient (TNX-/-) mice. By thin-layer chromatography of total lipids, it was found that the level of triglyceride was significantly increased in TNX-/- mice. The mRNA levels of most of the lipogenic enzyme genes examined were remarkably increased in TNX-/- mice. By gas chromatography-mass spectrometry analysis of triglyceride-associated fatty acids in the skin, saturated fatty acid palmitoic acid was decreased, whereas unsaturated fatty acids palmitoleic acid and oleic acid were increased in TNX-/- mice compared with those in wild-type mice. Conversely, fibroblast cell lines transfected with TNX showed a significant decrease in the amount of triglyceride. An increase in the saturated fatty acid stearic acid and decreases in the unsaturated fatty acids palmitoleic acid, oleic acid and linoleic acid, compared to those in mock-transfected cells were also caused by over-expression of TNX. These results indicate that TNX is involved in the regulation of triglyceride synthesis and the regulation of composition of triglyceride-associated fatty acids.

Adipose Tissue↗
PubMed2003

Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor.

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.

Animals↗
PubMed2003

A synthetic ceramide analog ameliorates spatial cognition deficit and stimulates biosynthesis of brain gangliosides in rats with cerebral ischemia.

A synthetic ceramide analog, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (L-PDMP) upregulates ganglioside biosynthesis in several cell lines. In cultured cortical neurons, neurotrophic effects of L-PDMP on neurite outgrowth and synaptic activity were demonstrated. In addition, it was found that L-PDMP could ameliorate the spatial cognition deficit in rats with ischemia. To elucidate this effect, we evaluated the effect of L-PDMP on brain ganglioside biosynthesis and its therapeutic efficacy against spatial cognition deficit in rats made ischemic. Rats were trained for 2 weeks, using an 8-arm radial maze task, and then forebrain ischemia was induced. L-PDMP was injected i.p. at 40 mg/kg twice a day starting from day 1 or 3 after ischemia induction for 6 or 4 days, respectively. The first study showed significantly reduced spatial cognition deficit at 12 h after the final drug administration, and L-PDMP tended to attenuate apoptosis in hippocampal CA1. To examine the effect of L-PDMP on brain ganglioside biosynthesis, N-[3H]acetyl-D-mannosamine was infused into the lateral ventricle via an injection cannula at 12 h after the final drug administration. After 4 h, the brain gangliosides were purified and analyzed. Upregulation of ganglioside biosynthesis by L-PDMP was observed on days 3 and 5 after ischemia. These results are an indication that L-PDMP may ameliorate spatial cognition deficit by upregulating ganglioside biosynthesis in ischemic brain.

Animals↗
PubMed2002

Effects of the mono- and tetrasialogangliosides GM1 and GQ1b on ATP-induced long-term potentiation in hippocampal CA1 neurons.

The effects of the mono- and tetrasialogangliosides, GM1 and GQ1b, on ATP-induced long-term potentiation (LTP) were studied in CA1 neurons of guinea pig hippocampal slices. Application of 5 or 10 microM ATP for 10 min resulted in a transient depression followed by a slow augmentation of synaptic transmission, leading to LTP. LTP induced by treatment with 5 microM ATP was facilitated in hippocampal slices prepared from animals treated for 6 days with a ceramide analog, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propranol, which stimulates ganglioside biosynthesis. In addition, LTP induced by 5 microM ATP was significantly enhanced when naive slices were incubated with GQ1b but not with GM1. These results suggest that a cooperative effect between extracellular ATP and GQ1b enhances ATP-induced LTP in hippocampal CA1 neurons. In addition, the LTP induced by 10 microM ATP was blocked by coapplication of the NMDA antagonist AP5 (5 microM or 50 microM), and this effect was partially inhibited by GQ1b pretreatment of the slices, suggesting that in hippocampal CA1 neurons, the enhancing effect of GQ1b on ATP-induced LTP is mediated by modulation of NMDA receptors/Ca(2+) channels.

Adenosine Triphosphate↗