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R K Yu

Publications and source records attributed to R K Yu.

At least 19 recordsLinked to original sources
PubMed2006

Endogenous GD3 ganglioside induces apoptosis in U-1242 MG glioma cells.

GD3 ganglioside induces apoptosis in several cell types, but the molecular events through which this occurs are largely unknown. We investigated the apoptotic effects of GD3 expression using U-1242 MG glioblastoma cells, as these cells synthesize almost exclusively GM3 and GM2 but not GD3. To express GD3 under the control of the TetOn system with minimum leakage, we modified the system by constructing a single tri-cistronic retrovirus vector containing three genes separated by two internal ribosome entry sites: (a) transcriptional silencer, tTS; (b) mutant of reverse transcriptional activator, rtTA2(S)-M2 (provided by H. Bujard, Heidelberg, Germany); and (c) enhanced green fluorescent protein (EGFP), as an indicator of the tri-cistronic gene expression. Using flow cytometry, we selected glioma cells (U1242MG-GD3 clone) that express high levels of GD3 in response to doxycycline. Expression of GD3 was associated with apoptosis as verified by annexin-V binding, TdT-mediated dUTPnick end-labelling assay (TUNEL), and EGFP degradation. GD3-induced apoptosis occurred via caspase-8 activation, as GD3 caused cleavage of caspase-8 and inhibition of caspase-8 activation by zlETD-fmk minimized GD3-induced apoptosis.

Alkaline Phosphatase↗
PubMed2001

Regulation of apoptosis during neuronal differentiation by ceramide and b-series complex gangliosides.

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of b-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of ceramide-induced apoptosis. This was most likely due to an 80% reduced expression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone which only weakly stained for complex gangliosides. Whole cell patch clamping revealed a 2-fold increased calcium influx in ST2-transfected cells, the blocking of which with nifedipine restored apoptosis to the level of untransfected cells. In serum-free culture, supplementation of the medium with IGF-1 was required to maintain MAPK phosphorylation and the anti-apoptotic effect of BCG expression. BCG-enhanced calcium influx and the presence of insulin-like growth factor-1 may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression.

1-Deoxynojirimycin↗
PubMed2001

Recent studies on the roles of antiglycosphingolipids in the pathogenesis of neurological disorders.

Evidence is mounting to suggest a causal role of humoral immunity arising from antiglycosphingolipid (GSL) antibodies in a variety of neurological disorders. These disorders include the demyelinating and axonal forms of Guillain-Barre syndrome, multifocal motor neuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, and IgM paraproteinemia. Many claims have been made regarding other neurological disorders, which should be carefully scrutinized for their validity, based on several criteria proposed in this review. These criteria include 1) characterization of the causative antigens and immunoglobulins, 2) correlation of the pathological lesions and clinical manifestation of the antigens, 3) establishment of animal models using pure GSLs as the antigens, 4) immunopathogenic mechanisms of the neurodenerative process, 5) mechanisms for the malfunctioning of blood-nerve barrier and the ensuing leakage of circulating antibodies into peripheral nerve parenchyma, and 6) the roles of anti-GSL antibodies that may cause humorally mediated nerve dysfunction and injury as well as interference with ion channel function at the node of Ranvier, where carbohydrate epitopes are located. Finally, the origin of the anti-GSL antibodies is discussed in light of the recent circumstantial evidence pointing to a molecular mimicry mechanism with infectious agents. With a better understanding of the immunopathogenic mechanisms, it will then be possible to devise rational and effective diagnostic and therapeutic strategies for the treatment of these neurological disorders.

Antibodies↗
PubMed2001

Regulation of glycosyltransferases in ganglioside biosynthesis by phosphorylation and dephosphorylation.

The biosynthesis of gangliosides is known to be under strict metabolic control. One level of control is through post-translational modification of the glycosyltransferases responsible for their biosynthesis. Thus, the activities of several sialyltransferases have been demonstrated to be downregulated by the action of protein kinase C (PKC) in cell-free and intact cell systems. This modulatory effect can be reversed at least in part by the action of membrane-bound phosphatases. In contrast, the activity of N-acetylgalactosaminyltransferase can be upregulated by the action of protein kinase A (PKA) in cultured cells. In addition, studies from several laboratories have demonstrated that phosphorylation of certain glycosyltransferases can affect their intracellular processing and translocation. Thus, modulation of glycosyltransferases by phosphorylation and dephosphorylation should represent an important regulatory mechanism for ganglioside biosynthesis.

Animals↗
PubMed2001

Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter.

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at -747/-688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at -1325/-1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and gamma-IRE. A negative cis-acting domain localized in a far distal region at -1594/-1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene.

Base Sequence↗
PubMed2001

Metabolic studies of glycosphingolipid accumulation in mucopolysaccharidosis IIID.

Severe neurological deficits and mental retardation are frequently associated with disrupted ganglioside metabolism in a variety of gangliosidoses and lysosomal storage disorders. Accumulation of glycosphingolipids (GSLs) in the central nervous system (CNS) of humans and animals affected with several types of mucopolysaccharidoses (MPS) also correlates with the severity of neurological dysfunction. Mucopolysaccharidosis type IIID (MPS IIID) is characterized by deficiency in lysosomal N-acetylglucosamine 6-sulfatase activity and the accumulation and excretion of heparan sulfates and N-acetylglucosamine 6-sulfate. We investigated the metabolism of GSLs in the prenatal, neonatal, and adult MPS IIID caprine brains and an MPS experimental cell culture model. The amounts of total glycolipids in prenatal, neonatal, and adult MPS IIID caprine brains were about 2-fold higher than those in control samples. GM3, GD3, and lactosyl ceramide were the principal GSLs which abnormally accumulated in caprine MPS IIID brains. These changes may be, in part, due to the reduction of sialidase and UDP-N-acetylgalactosamine:GM3 N-acetylgalactosaminyltransferase (GalNAc-T) activities in MPS IIID caprine brain. To further examine the possible mechanism of GSL accumulation in MPS IIID brains, we employed a cell culture model using suramin-treated neuronal cultures of differentiated P19 cells. HPTLC analysis showed elevated GSLs in suramin-treated cells. Metabolic pulse-chase labeling study revealed that the GSL accumulation in suramin-treated cells may be attributed to both disturbed biosynthesis and significantly slower degradation of GSLs. In addition, the consistency of observations in the cell culture and caprine models supports the cell culture system as a means of evaluating GSL metabolic perturbations.

Age Factors↗
PubMed2001

Characterization of a phospholipid antigen reacting with serum antibody in patients with peripheral neuropathies and paraproteinemia.

A phospholipid antigen that reacted with the serum antibody from a patient with peripheral neuropathy and paraproteinemia with both impaired sensory and motor functions, but not with sera from patients with only impaired sensory functions and healthy controls, was purified from bovine cauda equina as a minor component with a concentration of about 0.6 microg per gram wet-weight tissue. The structure of the phospholipid was characterized as lysophosphatidylinositol by means of thin-layer chromatography, gas-liquid chromatography, and negative-ion fast-atom-bombardment mass spectrometry. The major fatty acid component of this phospholipid was stearic acid (> 81%). Our data suggest the possible involvement of a lysophospholipid antigen in the immunopathogenesis of peripheral neuropathies with severe motor and sensory dysfunctions. There is an intriguing possibility that the difference in immunoreactivity of serum antibodies may underlie the differential clinical manifestations in patients with peripheral neuropathy and paraproteinemia.

Animals↗
PubMed2001

Disialoganglioside GD3 is selectively expressed by developing and mature human mast cells.

BACKGROUND: Disialoganglioside GD3 is expressed on the surface of selected cell types. Anti-GD3 mAb administered to human subjects with malignant melanoma produces signs and symptoms of immediate hypersensitivity reactions. OBJECTIVE: The expression of GD3 by human mast cells was assessed during mast cell development in vitro and in samples of lung and skin. METHODS: GD3 on tissue- and in vitro-derived mast cells was analyzed after double labeling of cells for tryptase (G3 mAb) or Kit (YB5.B8 mAb) and GD3 (R24 mAb). Glycolipids in extracts of fetal liver-derived mast cells were examined by using high-performance thin-layer chromatography. RESULTS: Flow cytometry showed that the percentage of GD3+ cells increased in parallel to Kit+ cells during the recombinant human stem cell factor-dependent development of fetal liver-derived mast cells. Double-labeling experiments showed that GD3+ cells were also surface Kit+ and granule tryptase positive, identifying them as mast cells in preparations of lung-, skin-, fetal liver-, and cord blood-derived cells. The major acidic glycolipid detected was NeuAcalpha2-8NeuAcalpha2-3Galbeta1-4Glcbeta1-1'Cer (GD3). Among peripheral blood leukocytes, only basophils and about 10% of the T cells were labeled with anti-GD3 mAb. Anti-GD3 mAb-conjugated magnetic beads were used to purify mast cells to greater than 90% purity from dispersed skin cells enriched to approximately 12% purity by means of density-dependent sedimentation but were less proficient for dispersed human lung mast cells, most likely because of other cell types that express GD3. CONCLUSION: GD3 is expressed on the surface of developing human mast cells in parallel to tryptase in secretory granules and, like Kit, can serve as a target for their enrichment by immunoaffinity techniques.

Cells, Cultured↗
PubMed2000

Protein-ribosome-mRNA display: affinity isolation of enzyme-ribosome-mRNA complexes and cDNA cloning in a single-tube reaction.

An enzyme-ribosome-mRNA complex was specifically purified by binding to the immobilized enzyme substrate and the cDNA was cloned in a single-tube reaction by one-step reverse transcription-PCR. The ganglioside GM3, used by sialyltransferase II (ST-II) as a substrate, was coated on a 96-well microtiter plate and ST-II was in vitro transcribed and translated from a cDNA library. The isolation of an enzyme-specific protein-ribosome (PRIME) complex was achieved with as little as 0.1 ng ST-II-specific cDNA in 5 microg of a total plasmid preparation or with the cDNA prepared from sublibraries previously inoculated at a density of 2000 clones/culture well. The affinity purification of the PRIME complex was highly specific for GM3 and did not result in cDNA amplification when a different ganglioside (GM1) was used for coating of the microtiter plate. The amplified cDNA was used for cloning or a second round of ribosome display, providing a fast analysis of enzyme affinity to multiple substrates. PRIME display can be used for host-free cDNA cloning from mRNA or cDNA libraries and for binding site mapping of the in vitro translated protein. The use of a single-tube reaction in ligand-coated microtiter plates indicates the versatility of PRIME display for cDNA cloning by automated procedures.

Chromatography, Affinity↗
PubMed2000

Suppression of ganglioside GD3 expression in a rat F-11 tumor cell line reduces tumor growth, angiogenesis, and vascular endothelial growth factor production.

Ganglioside GD3 is overexpressed in many types of tumors and may be associated with tumor progression and the development of metastatic potential. In our previous study (G. Zeng et al., Biochemistry, 38: 8762-8769, 1999), we established a subclone of the rat dorsal root ganglion-derived F-11 cells in which the expression of ganglioside GD3 was inhibited by stable transfection of the antisense vector against CMP-NeuAc: GM3 alpha2-8 sialyltransferase (GD3-synthase) gene. This cell line exhibits markedly reduced rate of tumor growth in vivo. Here, we further characterized the antisense-transfected cell line, and the results showed that these cells formed small, minimally vascularized tumors exhibiting extensive necrosis. In vivo Matrigel assay revealed reduced vascularization and low hemoglobin content in the antisense xenografts. Significantly fewer new vessels were found on the antisense xenografts and the skin around them than those on/around the xenografts formed by the sense-transfected and untransfected F-11 cells. The hemoglobin content of the antisense xenografts was much lower than that of the xenografts formed by the control cells. The reduced angiogenesis in the antisense xenografts was correlated with a decrease in vascular endothelial growth factor (VEGF) production. The expression of VEGF was suppressed in the antisense xenografts and the conditioned culture media of the antisense-transfected F-11 cells as determined by Western blotting analysis. This was further confirmed by immunohistochemistry of the tumors using antibodies against VEGF and platelet/endothelial cell adhesion molecule (PECAM-1). Therefore, our results demonstrate that reduced tumor growth in nude mice by suppression of GD3-synthase expression in F-11 cells results from minimal angiogenesis of the tumors through down-regulation of the VEGF expression, which indicates an important role for GD3 in tumor angiogenesis.

Animals↗
PubMed2000

Expression of gangliosides in neuronal development of P19 embryonal carcinoma stem cells.

Gangliosides are constituents of the cell membrane and are known to have important functions in neuronal differentiation. We employed an embryonal carcinoma stem cell line P19 as an in vitro model to investigate the expression of gangliosides during neuronal development. After treatment with retinoic acid, these cells differentiate synchronously into neuron-like cells by a series of well-defined events of development. We examined several aspects of ganglioside metabolism, including the changes of ganglioside pattern, the activities and gene expression of several enzymes at different stages of differentiation, and the distribution of gangliosides in differentiating neurons. Undifferentiated P19 cells express mainly GM3 and GD3. After P19 cells were committed to differentiation, the synthesis of complex gangliosides was elevated more than 20-fold, coinciding with the stage of neurite outgrowth. During the maturation of differentiated cells, the expression of c-series gangliosides was downregulated concomitantly with upregulation of the expression of a- and b-series gangliosides. We also examined the distribution of gangliosides in differentiating neurons by confocal and transmission electron microscopy after cholera toxin B subunit and sialidase treatment. Confocal microscopic studies showed that gangliosides were distributed on the growth cones and exhibited a punctate localization on neurites and soma. Electron microscopic studies indicated that they also are enriched on the plasma membranes of neurites and the filopodia as well as on the lamellipodia of growth cones during the early stage of neurite outgrowth. Our data demonstrate that the expression of gangliosides in P19 cells during RA-induced neuronal differentiation resembles that of the in vivo development of the vertebrate brain, and hence validates it as an in vitro model for investigating the function of gangliosides in neuronal development.

Animals↗
PubMed2000

Reduced cell migration, tumor growth and experimental metastasis of rat F-11 cells whose expression of GD3-synthase is suppressed.

We previously established a rat F-11 cell line whose expression of ganglioside GD3 was inhibited by stable transfection of the anti-sense vector against the GD3-synthase gene, showing that specific inhibition of GD3-synthase expression in tumor cells greatly reduced their growth rate in nude mice. Here, we report that down-regulation of GD3-synthase expression in anti-sense-transfected F-11 cells correlates with reduced cell migration and invasion in vitro and tumor growth and metastasis in vivo. When cultures were denuded of cells in a 1-mm-wide strip, the anti-sense-transfected F-11 cells migrated very slowly into the denuded area. Differences in migration between anti-sense-transfected cells and control parental cells were easily apparent. In vitro invasion assay of F-11 cells revealed a 3-fold decrease in invasion ability from the GD3-synthase-suppressed cells; colony formation in soft agar was not affected. Injection (i.v.) of control sense-transfected and untransfected F-11 cells resulted in multiple, large metastatic nodules in each of the 12 mice, whereas i.v. injection of anti-sense-transfected F-11 cells formed a single, small metastatic nodule in only 2 of the 8 nude mice. In addition, even if metastasis occurred, the anti-sense-induced metastatic nodules were much smaller than the metastatic nodules formed by control F-11 cells. These results demonstrate that suppression of GD3-synthase expression, which results primarily in a marked decrease in the concentration of ganglioside GD3, greatly reduces cell spreading, invasion and both the incidence and growth rate of experimental metastasis of F-11 cells.

Animals↗
PubMed2000

N-acylated serinol is a novel ceramide mimic inducing apoptosis in neuroblastoma cells.

A novel structural analog of ceramide was synthesized by N-acylation of serinol (2-amino-1,3-propanediol) and studied for its effects on glycolipid biosynthesis and cell differentiation of neuroblastoma cells. Incubation with N-palmitoylated serinol (C16-serinol) increased the concentration of endogenous ceramide by 50-80% and caused apoptosis in rapidly dividing low density cells but not in confluent cells. Cell death was not suppressed by simultaneous incubation with phorbol ester, known to antagonize ceramide-induced apoptosis by activation of protein kinase C (PKC). Purification of potential target proteins of C16-serinol was achieved by affinity chromatography of a protein preparation from rat brain on immobilized C16-serinol. A gel activity assay revealed that the eluate from C16-serinol-Sepharose contained three serine/threonine-specific protein kinases with molecular masses of 50, 70, and 95 kDa. The 70-kDa protein was immunostained on a Western blot using a PKCzeta-specific antibody. The purified PKCzeta could be activated directly by C16-serinol in an in vitro phosphorylation assay. Induction of apoptosis in neuroblastoma cells was suppressed by inhibition of PKCzeta with Gö 6983. Our overall results indicate that apoptosis in neuroblastoma cells induced by C16-serinol was at least partially mediated by activation of PKCzeta on condition of ongoing cell division. N-Acylated serinols may thus be useful for induction of apoptosis in mitotic cells and may be of therapeutic potential for treatment of cancer in the nervous system.

Animals↗
PubMed2000

Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells.

Gangliosides are sialylated glycosphingolipids whose biosynthesis is catalyzed by a series of endoplasmic reticulum (ER)- and Golgi-resident glycosyltransferases. Protein expression, processing, and subcellular localization of the key regulatory enzymes for ganglioside biosynthesis, sialyltransferase II (ST-II) and N-acetylgalactosaminyltransferase I (GalNAcT), were analyzed upon transient expression of the two enzymes in the neuroblastoma cell lines NG108-15 and F-11. The enzymes were endowed with a C-terminal epitope tag peptide (FLAG) for immunostaining and immunoaffinity purification using a FLAG-specific antibody. Mature ST-II-FLAG and GalNAcT-FLAG were expressed as N-glycoproteins with noncomplex oligosaccharides. ST-II-FLAG was distributed to the Golgi apparatus, whereas GalNAcT-FLAG was found in the ER and Golgi. Inhibition of early N-glycoprotein processing with castanospermine resulted in a distribution of ST-II-FLAG to the ER, whereas that of GalNAcT-FLAG remained unaltered. In contrast to GalNAcT, the activity of ST-II and the amount of immunostained enzyme were reduced concomitantly by 75% upon incubation with castanospermine. This was due to a fourfold increased turnover of ST-II-FLAG, which was not found with GalNAcT-FLAG. The ER retention and increased turnover of ST-II-FLAG were most likely due to its inability to bind to calnexin upon inhibition of early N-glycoprotein processing. Calnexin binding was not observed for GalNAcT-FLAG, indicating a differential effect of N-glycosylation on the turnover and subcellular localization of the two glycosyltransferases.

Animals↗
PubMed2000

Glycosphingolipid composition of a new immortalized human cerebromicrovascular endothelial cell line.

Previous studies have demonstrated the involvement of glycosphingolipid (GSL) antigens in the pathogenesis of immune-mediated neurological disorders such as peripheral neuropathies and multiple sclerosis. To study the role of the blood-brain barrier (BBB) in these disorders, we used a new human cerebromicrovascular endothelial cell (HCEC) line that has been immortalized through transfection with the plasmid pSV3-neo encoding for the SV40 large T-antigen and the neomycin gene. The immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates but maintained phenotypic properties of early-passage control cells. Therefore, this human cell line may serve as a useful in vitro model for studying the properties of the human BBB. We first investigated the GSL composition of cultured SV-HCECs. The major gangliosides were GM3 (62% of total gangliosides), GM2 (18%), GM1 (3%), and GD1a (15%). The major neutral GSLs were glucosylceramide (15% of the total neutral glycolipids), lactosylceramide (36%), globotriaosylceramide (3%), and globoside (43%). Trace amounts of paragloboside, lactosaminyl paragloboside, and sulfoglucuronyl paragloboside could also be detected by TLC-immunostaining. These results provide the basis for further investigations of the expression of these cell surface antigens in cultured SV-HCECs on activation with inflammatory cytokines such as interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma, which have been implicated as playing an important role in the pathogenesis of many nervous system disorders.

Blood-Brain Barrier↗