Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The EBV/lipoplex is a nonviral gene delivery system composed of a cationic lipid and Epstein-Barr virus (EBV)-based plasmid vector that carries the EBV oriP and EBV nuclear antigen 1 (EBNA1) gene. Because the EBNA1 supports retention, nuclear localization, and transcriptional upregulation of the oriP-bearing plasmid, cells transfected with the EBV/lipoplex express the transgene at a very high level. We hypothesized that tumor cells genetically manipulated with the EBV/lipoplex may be used as a tumor vaccine without drug selection, strongly contributing to immunotherapy of patients with malignancies. The cytokines interleukin (IL)-12 and IL-18 exert a variety of immune-regulatory functions including interferon (IFN)-gamma production and cytotoxic T lymphocyte (CTL) and natural killer (NK) activation. Here, we investigated the possible therapeutic effects of an autologous tumor cell vaccine in the B16 melanoma model. The vaccine was engineered to secrete IL-12 and IL-18 by means of the EBV/lipoplex. B16 cells were subcutaneously implanted into syngenic mice followed by repetitive immunization with irradiated B16 cells that had been transfected 3 days earlier by TFL2-3, a novel cationic lipid, with EBV-plasmid vectors encoding IL-12 and/or IL-18 genes (B16/mIL-12, B16/mIL-18, and B16/mIL-12+mIL-18). The mice vaccinated with B16/mIL-12 underwent strong tumor suppression accompanied by a high IFN-gamma production. Both CTL and NK activities were significantly elevated in these mice. When the tumor cell vaccine was prepared by means of conventional (non-EBV) plasmid vectors combined with the same cationic lipid, the therapeutic outcome was not as good, suggesting the superiority of the EBV-based plasmid in engineering these types of tumor vaccines. Vaccination with B16/mIL-18 was not effective in suppressing tumors, whereas B16/mIL-12+mIL-18 showed comparable antitumor therapeutic validity as B16/mIL-12 did. When IFN-gamma mutant (IFN-gamma(-/-) mice were treated, B16/mIL-12 vaccine did not show any therapeutic activity, suggesting the necessity of IFN-gamma in the anti-melanoma immune responses. In contrast, the antitumor effect was not affected by NK depletion in mice that received repetitive injections with anti-asialo GM1 antibody. Furthermore, vaccination with B16/mIL-12 significantly suppressed pulmonary metastases in mice that had been intravenously injected with parental B16. Our results suggest that the EBV/lipoplex is quite useful in generating an autologous tumor cell vaccine and that IL-12 is an important component of the vaccine.
Mol Ther 2002 May
PMID:Significant antitumor effects obtained by autologous tumor cell vaccine engineered to secrete interleukin (IL)-12 and IL-18 by means of the EBV/lipoplex. 1199 52

The studies on the inhibitory effect exerted by Cholera Toxin (CT) on cell growth and proliferation indicate a remarkable heterogeneity of cell response suggesting that the inhibition represents the final event of many different ways or mechanisms. After CT binding, cAMP accumulation may not occur (as in L1210 leukemia cells) or, when occurring (as in SR-4987 stromal cells), may not be coupled with the antiproliferative effect of CT. In WEHI-3B cells CT binds a Gal-GalNac-GM1b receptor and the anticlonogenic effect of CT seems correlated with cAMP accumulation. To demonstrate the central role of cAMP in WEHI-3B cells, starting from the sensitive cell strain we selected and established a clone of WEHI-3B resistant to CT. This revertant clone (WEHI-3B/CT/REV) is currently cultured in the absence of CT and in the proliferation assay shows a dramatic resistance (>46,000 than the parental cells). Stimulation ofWEHI-3B/CT/REV cells by cholera toxin failed to enhance cAMP and the ganglioside-CT binding studied on Thin Layer Chromatography (TLC) blots showed that the resistant cells lost the spot correspondent to the migration of Gal-GalNac-GM1b ganglioside. Both the lines respond at the same level to the adenylate cyclase stimulation by forskolin and the incorporation of GM1a did not decrease the resistance of WEHl-3B/CT/REV. These data confirm that Gal-GalNac-GM1b is the most important functional receptor for CT in WEHI-3B cells able to transduce the signal by enhancing cAMP which in turn inhibits cell proliferation (probably by cAMP dependent protein kinase activation). Our study describes the first cell line resistant to CT originated from a susceptible parental strain and provides a new interesting cell model for studying the cAMP dependent mechanisms involved in cell growth regulation.
Mol Cell Biochem 2002 Apr
PMID:Selection of a WEHI-3B leukemia cell subclone resistant to inhibition by cholera toxin. 1208 75

The regulation of receptor tyrosine kinases (RTKs) is important in several cellular events, including proliferation, differentiation, and apoptosis. Gangliosides are sialic acid-containing glycosphingolipids that can regulate RTK activity. The addition of ganglioside GM1 to the medium of Swiss 3T3 fibroblasts inhibits both platelet-derived growth factor (PDGF)-mediated tyrosine phosphorylation of PDGF receptor beta (PDGFRbeta) and receptor-mediated endocytosis. However, GM1 did not affect PDGF-mediated receptor phosphorylation, neuritogenesis, or endocytosis in PC12 cells stably transfected with the gene for PDGFRbeta. The ability of GM1 to modulate PDGFRbeta in 3T3 cells but not in transfected PC12 cells indicates a cell context-dependent response. We hypothesized that this inhibition of PDGFRbeta by GM1 must map to one or more domains of the receptor. Thus, a chimeric receptor was created that possessed the extracellular and transmembrane domains of the nerve growth factor (NGF) receptor TrkA and the cytoplasmic domain of PDGFRbeta (TTbeta). In 3T3 cells transfected with the TTbeta construct, GM1 did not inhibit NGF-induced tyrosine phosphorylation of the chimeric receptor or of Erk1/2 in this cell line. GM1 still inhibited PDGF-mediated tyrosine phosphorylation of endogenous PDGFRbeta and of Erk1/2 in Swiss TTbeta cells. Thus, the cytoplasmic domain of PDGFRbeta is not required for GM1-dependent inhibition of PDGFRbeta in 3T3 cells. This suggests that the inhibition of PDGFRbeta by GM1 in Swiss 3T3 fibroblasts maps to either the extracellular and/or transmembrane domain of PDGFRbeta.
J Mol Neurosci 2003 Apr
PMID:Domain-dependent modulation of PDGFRbeta by ganglioside GM1. 1279 4

We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80-90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM1, was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.
Mol Biol Cell 2003 Aug
PMID:Selective caveolin-1-dependent endocytosis of glycosphingolipids. 1292 61

Water dynamics in samples of ceramide tetrasaccharide (Gg4Cer) vesicles and GM1 ganglioside micelles at 300:1 water/lipid mole ratio were studied by using deuterium nuclear magnetic resonance (2H-NMR). GM1 imposes a different restriction on water dynamics that is insensitive to temperatures either above or below its phase transition temperature or below the freezing point of water. The calculated correlation times are in the range of 10(-10) s, typical of water molecules near to the polar groups. Pure GM1 micelles have two distinct water microenvironments dynamically characterized. Their dynamic parameters remain constant with temperature ranging from -18 to 32 degrees C, but the amount of strongly associated water is modified. By contrast, a mixture of single soluble carbohydrates corresponding to GM1 polar head group does not preserve the dynamic parameters of water hydration when the temperature is varied. Incorporation of cholesterol or lysophosphatidylcholine into GM1 micelles substantially increases the mobility of water molecules compared with that found in pure GM1 micelles. The overall results indicate that both the supramolecular organization and the local surface quality (lipid-lipid interaction) strongly influence the interfacial water mobility and the extent of hydration layers in glycosphingolipid aggregates.
Mol Membr Biol
PMID:Mixed lipid aggregates containing gangliosides impose different 2H-NMR dynamical parameters on water environment depending on their lipid composition. 1457 47

Hedgehog interacting protein (Hip) is considered as a membrane protein implicated in sequestering the hedgehog (hh) morphogens during embryonic development. Here, we demonstrate that Hip transcription also occurs in cells scattered in discrete brain areas of adult rodents and we identify the presence of membrane-associated and soluble forms of Hip in the mature brain. Moreover, we show that soluble forms of Hip, present in the conditioned medium of HEK293 cells overexpressing Hip, inhibit Sonic hedgehog (Shh)-induced differentiation of C3H10T1/2 cells, a well-characterised response associated with Shh signalling. After transfection in HEK293 cells, Hip partitions with the raft component ganglioside GM1 during density gradient centrifugation. Analysis of tagged Hip constructs reveals that the putative transmembrane domain of Hip is not cleaved suggesting that other mechanisms are implicated in the release of its soluble forms. Taken together, these data are consistent with the involvement of both membrane-associated and soluble Hip in the regulation of Shh signalling in adult neural tissues.
Mol Cell Neurosci 2004 Feb
PMID:Hedgehog interacting protein in the mature brain: membrane-associated and soluble forms. 1501 48

Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, the ganglioside GM1, palmitoylated LAT, and cross-linked IgE receptors, FcepsilonRI. This study addresses the relationship of fractionation data to the organization of raft markers in native membranes. Immunogold labeling and electron microscopy shows there is little or no colocalization of the raft markers Thy-1, GM1, and LAT with each other or with FcepsilonRI on native membrane sheets prepared from unstimulated cells. External cross-linking of Thy-1 promotes coclustering of Thy-1 with LAT, but not with GM1. Thy-1 and LAT clusters occur on membrane regions without distinctive features. In contrast, external cross-linking of FcepsilonRI and GM1 causes their redistribution to electron-dense membrane patches independently of each other and of Thy-1. The distinctive patches that accumulate cross-linked FcepsilonRI and GM1 also accumulate osmium, a stain for unsaturated lipids, and are sites for coated vesicle budding. Electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes.
Mol Biol Cell 2004 Jun
PMID:Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes. 1503 44

Plaques containing amyloid beta-peptides (Abeta) are a major feature in Alzheimer's disease (AD), and GM1 ganglioside is an important component of cellular plasma membranes and especially enriched in lipid raft. GM1-bound Abeta (GM1/Abeta), found in brains exhibiting early pathological changes of AD including diffuse plaques, has been suggested to be involved in the initiation of amyloid fibril formation in vivo by acting as a seed. However, the role of GM1 in amyloid beta-protein precursor (APP) processing is not yet defined. In this study, we report that exogenous GM1 ganglioside promotes Abeta biogenesis and decreases sAPPalpha secretion in SH-SY5Y and COS7 cells stably transfected with human APP695 cDNA without affecting full-length APP and the sAPPbeta levels. We also observe that GM1 increases extracellular levels of Abeta in primary cultures of mixed rat cortical neurons transiently transfected with human APP695 cDNA. These findings suggest a regulatory role for GM1 in APP processing pathways.
Mol Psychiatry 2004 Oct
PMID:GM1 ganglioside regulates the proteolysis of amyloid precursor protein. 1505 75

The plasma membrane of T cells is made of a combination of glycosphingolipids and protein receptors organized in glycolipoprotein microdomains termed lipid rafts. The structural assembly of lipid rafts was investigated by various physical and biochemical assays. Depending on the differentiation status of T cells, the lipid rafts seclude various protein receptors involved in T cell signaling, cytoskeleton reorganization, membrane trafficking, and the entry of infectious organisms into the cells. This review article summarizes the most common methods, and their limits and advantages for analyzing the composition and assembly of lipid rafts with protein receptors into lipid rafts microdomains in plasma membrane of T cells. It also includes new methods such as ELISA/Polysorp and flow cytometry, and a combined sucrose gradient centrifugation-FPLC-Western blot strategy developed in our laboratory to study non-covalent interactions between the GM1 glycosphingolipid and protein receptors in plasma membrane of T cells.
Mol Immunol 2004 Jun
PMID:Analysis of lipid rafts in T cells. 1516 37

Endogenous expression of human membrane type ganglioside sialidase (Neu3) was examined in various cell lines including NB-1, U87MG, SK-MEL-2, SK-N-MC, HepG2, Hep3B, Jurkat, HL-60, K562, ECV304, Hela and MCF-7. Expression was detected in the neuroblastoma cell lines NB-1 and SK-N-MC, and also in erythroleukemia K562 cells, but not in any other cells. We isolated a Neu3 cDNA from K562 cells and expressed a His-tagged derivative in a bacterial expression system. The purified recombinant product of approximately 48 kDa had sialidase activity toward 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH of the purified Neu3 protein for GD3 ganglioside was 4.5. The enzyme also efficiently hydrolyzed GD3, GD1a, GD1b and GM3 whereas sialyllactose, 4MU-NeuAc, GM1 and GM2 were poor substrates, and it had no activity against sialylated glycoproteins such as fetuin, transferrin and orosomucoid. We conclude that the sialidase activity of Neu3 is specific for gangliosides.
Mol Cells 2004 Apr 30
PMID:Molecular characterization of membrane type and ganglioside-specific sialidase (Neu3) expressed in E. coli. 1517 41


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>