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Biomedical subjects

R K Yu

Publications and source records attributed to R K Yu.

At least 91 records · Page 5Linked to original sources
PubMed1994

Activities of five different sialyltransferases in fish and rat brains.

To investigate the role of sialyltransferases in the metabolism of brain gangliosides, we examined activities of five different sialyltransferases (GM3-, GD3-, GT3-, GD1a-, and GT1a-synthase) using total membrane preparations from cichlid fish and Sprague-Dawley rat brains, and analyzed the relationship between the enzyme activities and the ganglioside compositions. The patterns of sialyltransferase activities in fish and rat brains differed from each other. In fish brain, the GM3-synthase activity was lower than GD3-synthase activity, whereas the opposite relationship was observed in rat brain. The GT3-synthase reaction with fish brain membranes produced radiolabeled GM3, GD3, and a ganglioside that was identified as GT3 based on mobility on TLC using two different solvent systems. No GT3-synthase activity was detected in rat brain. The GD1a- and GT1a-synthase activities in fish brain were higher than those in rat brain. Although GT1a was a single radiolabeled ganglioside in fish GT1a-synthase reaction, this ganglioside could not be detected in rat brain. The ratios of GM3-, GD3-, GT3-, GD1a-, and GT1a-synthase activities in fish and rat brain were 23:31:4:28:14 and 61:21:0:18:0, respectively. Ganglioside analysis showed that fish brain was enriched with c-series gangliosides including GT3 and polysialo-species, whereas a- and b-series gangliosides were major components in rat brain. These results suggest that the species-specific expression of gangliosides in brain tissues may be regulated, at least in part, at the level of sialyltransferase activities.

Animals↗
PubMed1994

Glycosphingolipid antigens in cultured bovine brain microvascular endothelial cells: sulfoglucuronosyl paragloboside as a target of monoclonal IgM in demyelinative neuropathy [corrected].

Since a number of anti-glycosphingolipid (GSL) antibody activities have been demonstrated in patients with various neurological disorders, the presence of common antigens between brain microvascular endothelial cells (BMECs) and the nervous tissues presents a potential mechanism for the penetration of macromolecules from the circulation to the nervous system parenchyma. We first investigated GSL composition of cultured bovine BMECs. Bovine BMECs express GM3(NeuAc) and GM3(NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b, as well as sialyl paragloboside and sialyl lactosaminylparagloboside as the minor species. Sulfoglucuronosyl paragloboside was also found to be a component of the BMEC acidic GSL fraction, but its concentration was lower in older cultures. On the other hand, the amounts of neutral GSLs were extremely low, consisting primarily of glucosylceramide. In addition, we analyzed the effect of anti-SGPG IgM antibody obtained from a patient of demyelinative polyneuropathy with macroglobulinemia against cultured BMECs. Permeability studies utilizing cocultured BMEC monolayers and rat astrocytes revealed that the antibody facilitated the leakage of [carboxy-14C]-inulin and 125I-labeled human IgM through BMEC monolayers. A direct cytotoxicity of this antibody against BMECs was also shown by a leakage study using [51Cr]-incorporated BMECs. This cytotoxicity depended on the concentration of the IgM antibody, and was almost completely blocked by preincubation with the pure antigen, sulfoglucuronosyl paragloboside. Our present study strongly supports the concept that immunological insults against BMECs induce the destruction or malfunction of the blood-nerve barrier, resulting in the penetration of the immunoglobulin molecule to attach peripheral nerve parenchyma.

Animals↗
PubMed1994

Endogenous phosphorylation of a 61,000 dalton hippocampal protein increases following traumatic brain injury.

Acute biochemical consequences of moderate traumatic brain injury (TBI) include activation of kinases, including protein kinase C (PKC). To determine the possible consequences of PKC activation at the substrate level, we have examined protein phosphorylation patterns 1 h following injury. Although the phosphorylation of most proteins remained unchanged following injury, we observed a significant increase in the phosphorylation of a 61,000 dalton protein (TBI61) in injured rat hippocampus (121% higher than sham control) in vitro. TBI61 phosphorylation could be enhanced by phosphatidyl serine and diacylglycerol or by addition of exogenous PKC. In addition, TBI61 phosphorylation was inhibited by the PKC inhibitor, staurosporine, suggesting further that this protein may be a PKC substrate. These data suggest that TBI increases the phosphorylation of a 61 kD hippocampal protein in vitro. Increases in the protein level and activity of PKC could contribute to this increased phosphorylation.

Alkaloids↗
PubMed1994

Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus.

Influenza C virus spike glycoprotein HEF specifically recognizes glycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid. The same protein also contains an esterase activity. Taking advantage of these two properties, influenza C virus was used as a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminic acid in human leucocytes. The binding of influenza C virus to leucocyte glycoproteins and gangliosides separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and thin-layer chromatography, respectively, was assayed using a chromogenic esterase substrate. In this way, glycoproteins of B-lymphocytes and T-lymphocytes were found to contain 9-O-acetylated sialic acids. Of the various 9-O-acetylated gangliosides detected, one had the characteristics of 9-O-acetylated GD3. The identification of 9-O-acetylated sialic acids on distinct glycoproteins and glycolipids should be helpful in assigning a physiological role to this sugar.

Acetylation↗
PubMed1994

Biosynthesis of glycosphingolipids is reduced in the absence of a vimentin intermediate filament network.

Our previous observations on the immunocytochemical colocalization of intermediate filaments and glycosphingolipids led us to analyze the role of filaments in the biosynthesis and intracellular transport of glycosphingolipids. Cells with (vim+) and without (vim-) vimentin intermediate filaments were cloned from the adrenal carcinoma cell line SW13. There was no difference between vim+ and vim- cells in the proportion of newly synthesized C6-NBD-glucosylceramide transported to the plasma membrane. The vim+ cells synthesized glycosphingolipids, especially lactosylceramide and globotriosylceramide, and to a lesser extent GM3 ganglioside, more rapidly than vim- cells. The altered rate of biosynthesis did not result from differences in the levels of the glycosyltransferases that synthesize those compounds. To determine whether the presence of a vimentin network was responsible for the differences in biosynthesis, mouse vimentin cDNA was transfected into vim- cells. Transfected cells that expressed a mouse vimentin network demonstrated a twofold or greater increase in the rate of biosynthesis of neutral glycosphingolipids and gangliosides. There was no difference between vim+ and vim- cells in the synthesis of ceramide or sphingomyelin, or in their content of phospholipids or cholesterol. The nature of the biochemical defect(s) underlying the diminished incorporation of radiolabeled sugars into glycosphingolipids is unclear. Possibilities include alterations in the ultrastructure of the Golgi and/or abnormalities in a portion of the endocytic pathway.

Adrenal Cortex Neoplasms↗
PubMed1994

Inhibition of LPS-mediated cell activation in vitro and in vivo by gangliosides.

Addition of purified GM1 gangliosides inhibited lipopolysaccharide (LPS)-stimulated proliferation of purified B cells by greater than 90%. Addition of gangliosides to B cells as late as 120 min after the addition of LPS still inhibited B-cell proliferation, suggesting that inhibition did not simply reflect direct binding of LPS to gangliosides. Gangliosides also inhibited proliferation of B cells stimulated by anti-Ig antibodies, albeit to a lesser degree than inhibition of the LPS-stimulated response. The finding that B-cell proliferation stimulated by the combination of PMA+ionomycin was also inhibited by gangliosides suggests that its inhibitory activity did not reflect interference with binding of the B-cell stimuli to membrane receptors. The inhibitory effect of gangliosides was not restricted to B cells, since LPS-induced TNF production by macrophages was also inhibited in vitro. The inhibitory activity of gangliosides was also seen in vivo, and mice injected with soluble gangliosides or implanted with slow-release pellets impregnated with gangliosides showed reduced TNF production in vivo in response to LPS. Mice that were implanted with these slow-release pellets were also protected from LPS-induced lethality. Thus, while only 10% of control mice survived injection with LPS+galactosamine, the experimental group showed a 64% survival. It is likely that this protective effect reflects the ability of gangliosides to suppress LPS-mediated TNF production. This model provides a basis for studying a regulatory role for gangliosides in B-cell activation in vitro and macrophage activation in vitro and in vivo. Furthermore, it suggests new approaches to suppress the toxic effects induced by LPS in vivo.

Animals↗
PubMed1993

Purification and characterization of CMP-N-acetylneuraminic acid:lactosylceramide (alpha 2-3) sialyltransferase (GM3-synthase) from rat brain.

CMP-N-acetylneuraminic acid:lactosylceramide (alpha 2-3) sialyltransferase (GM3-synthase) was purified to homogeneity from a Triton CF-54 extract of young rat brain. The enzyme was separated by affinity chromatography on CDP-Sepharose column and resolved by linear NaCl gradient elution from the same adsorbent. Final purification of GM3-synthase was achieved by chromatography on a "lactosylceramide acid"-Sepharose column and specific elution with lactosylceramide. The enzyme activity was highest at pH 6.5 and required the presence of Triton CF-54 (0.15%) and Mn2+ (10 mM) for its full activity. The product of the reaction catalyzed by the enzyme was identified as GM3 based on its mobility on thin layer chromatographic plates using two different solvent systems. Comparison with several glycolipid substrates showed high specificity of GM3-synthase for lactosylceramide. The apparent Km value for lactosylceramide and CMP-N-acetylneuraminic acid were 80 and 210 microM, respectively. The apparent molecular mass of the enzyme determined on SDS-polyacrylamide gel electrophoresis was 76 kDa.

Animals↗
PubMed1993

Possible role of myelin-associated neuraminidase in membrane adhesion.

Previous studies from our laboratory have demonstrated the presence of a specific interaction between myelin-associated neuraminidase and GM1 (Saito and Yu, J Neurochem 47:632-641, 1986). In the present study, we further characterized this neuraminidase-GM1 interaction and examined its role in the adhesion of rat oligodendroglial cells to GM1. Hydrolysis of N-acetylneuramin-lactitol by the enzyme was inhibited by GM1 in a competitive manner; GM1 itself was not hydrolyzed, suggesting that GM1 may serve as a competitive inhibitor of the enzyme. Asialo-GM1 had no inhibitory effect. When a soluble enzyme preparation was applied to a GM1-linked affinity column, the enzyme activity was retained on the column and was recovered from the column only by elution with a buffer containing 5 mM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (Neu2en5Ac), a competitive inhibitor of neuraminidase. A binding study with 51Cr-labeled rat oligodendroglial cells showed that oligodendroglial cells bound preferentially to GM1 developed on a thin-layer plate, but not to other gangliosides such as GM3, GD1a, GD1b, and GT1b. The binding reaction to GM1 was inhibited by Neu2en5Ac (5 mM). These results suggest that myelin-associated neuraminidase specifically interacts with GM1 and may be involved in adhesion of oligodendroglial cells to GM1. This neuraminidase-GM1 interaction may play an important role in the formation and stabilization of the multilamellar structure of the myelin sheath.

Animals↗
PubMed1993

Activation of UDP-galactose:globotriaosylceramide alpha 1-3-galactosyltransferase during PC12D cell differentiation induced by galactosylceramide.

We measured the activities of UDP-galactose:globotriaosylceramide alpha 1-3-galactosyltransferase (alpha-GalTase) and protein kinase C (PKC) in PC12D pheochromocytoma (PC12D) cells which were induced to differentiation by nerve growth factor (NGF), forskolin (FRK), staurosporine (STP), retinoic acid (RA), 2-chloroadenosine (ClAd), and/or galactosylceramide (GalCer). NGF, STP, FRK, and RA were found to be stimulators for the PKC activity, whereas ClAd appeared to be an inhibitor of the enzyme. At the concentration of 25 microM, GalCer having normal fatty acids was found to be a stimulator, whereas GalCer having hydroxy fatty acids was ineffective in modulating the PKC activity. Interestingly, all stimulators of PKC activities, including GalCer having normal fatty acids, appeared to be activators for the alpha-GalTase activity. On the other hand, GalCer having alpha-hydroxy fatty acids had no effect and ClAd was found to be a potent inhibitor for the alpha-GalTase activity. These data suggest that alpha-GalTase activity during PC12D cell differentiation may be regulated by a PKC-dependent process.

Alkaloids↗
PubMed1993

Alterations of protein kinase C in rat hippocampus following traumatic brain injury.

Calcium-dependent excitotoxic processes contribute significantly to pathologic responses to traumatic brain injury (TBI). TBI causes neuronal depolarization and excessive excitatory neurotransmitter release, which may lead to increases in intracellular calcium levels. However, responses of calcium-dependent enzymes such as protein kinase C (PKC) following TBI are poorly understood. Since PKC plays an important role in signal transduction and maintenance of normal neuronal function, we investigated changes in PKC activity and protein levels following fluid percussion brain injury in rats. We observed a 23.1% increase in PKC activity 1 h postinjury and 80.7% increase in PKC activity 3 h postinjury. There was no statistically significant change in PKC activity 5 min and 24 h after injury. PKC immunolabelling studies detected a significant increase in PKC levels in membrane fractions 3 h but not 1 h after injury. Thus PKC activation is transiently increased following TBI and may play an important role in pathophysiologic responses to TBI.

Animals↗
PubMed1993

Fucosyl-GM1 in human sensory nervous tissue is a target antigen in patients with autoimmune neuropathies.

Several gangliosides of human nervous tissues have been reported to be potential target antigens in autoimmune neuropathies. To explain the diversity of clinical symptoms in patients with antiganglioside antibodies, we have searched for ganglioside antigens that are specific to individual nervous tissues such as motoneurons, peripheral motor nerves, and sensory nerves. Although the major ganglioside compositions were not different among human peripheral motor and sensory nerves, fucosyl-GM1 was found to be expressed in sensory nervous tissue but not in spinal cord, motor nerve, and sympathetic ganglia. Sera from several patients with sensory nerve involvement also reacted with fucosyl-GM1 as well as GM1. Thus, fucosyl-GM1 may be a responsible target antigen for developing sensory symptoms in some patients with autoimmune neuropathies.

Animals↗
PubMed1993

Presence of cyclic AMP response element-binding protein in oligodendrocytes.

Several lines of evidence indicate that cyclic AMP (cAMP) induces oligodendrocytes differentiation. However, the mechanism(s) of this stimulation remains unknown. Because in several cell types the transcriptional activity of various cAMP-responsive genes is regulated through a cis-acting DNA sequence known as cAMP response element (CRE), we investigated the possible presence of a CRE binding (CREB) protein in myelinating oligodendrocytes. A double-stranded oligonucleotide containing a tandem repeat of the CRE sequence was labeled with T4 kinase in the presence of [32P]ATP and then incubated with a nuclear protein extract from 14-day-old rat brain oligodendrocytes. The reaction mixture was then electrophoresed on nondenaturing polyacrylamide gels. The results indicated the presence of a protein that specifically binds to the CRE sequence. The results were supported by southwestern blotting assays in which the CRE probe bound to a approximately 45-kDa protein species. In separate experiments, it was shown that the 45-kDa protein can be phosphorylated in vitro by the catalytic subunit of protein kinase A. Developmental analysis of CREB protein expression indicated a peak at 14 days of age, preceding the peak of myelinogenesis.

Animals↗
PubMed1993

GM3 regulates protein kinase systems in cultured brain microvascular endothelial cells.

The barrier function of endothelial cells is known to be positively regulated by protein kinase A (PKA) and negatively regulated by protein kinase C (PKC). We found that exogenously administered GM3(NeuAc) promoted PKA activity in cultured brain microvascular endothelial cells (BMECs). Other glycolipids, including GM1, sulfoglucuronyl paragloboside, and GM3(NeuGc), did not have any effect on the PKA activity of BMECs. PC12 cells did not respond to exogenously applied GM3(NeuAc). GM3(NeuAc) also suppressed the PKC activity of BMECs. Thus, GM3(NeuAc) may function as a modulator of blood-brain barrier function via the two different kinase systems.

Animals↗
PubMed1993

Sulfated glucuronyl glycolipids and gangliosides in the optic nerve of humans.

Pathologically delayed visual evoked potentials may be present in patients with neuropathy associated with IgM M-proteinemia, which is directed against myelin-associated glycoprotein and sulfated glucuronyl glycolipids (SGGLs), but there are no reports of these antigens in the optic nerve. We recently examined human optic nerve and occipital lobe tissues for the occurrence of SGGLs using the technique of immunostaining on thin-layer chromatographic plates and found them in the optic nerve, but not the occipital lobe. SGGLs in the optic nerve may represent target antigens for CNS involvement by the M-protein in patients with neuropathy. We also studied the ganglioside composition of the optic nerve and found it different from that of the brain. Human optic nerve is characterized by an abundance of the b-series gangliosides, including GD1b, GT1b, and GQ1b. GD1a, which is usually a major component of brain gangliosides, is only a minor species of the optic nerve ganglioside fraction.

Chromatography, Thin Layer↗
PubMed1993

Characterization of a hamster melanoma-associated ganglioside antigen as 7-O-acetylated disialoganglioside GD3.

We previously reported a hamster animal model of melanoma in which the tumor tissue expresses gangliosides GM3, GD3, and O-acetyl GD3. This ganglioside pattern is similar to that in human melanomas (Ren, S., A. Slominski, and R. K. Yu. 1989 Cancer Res. 49: 7051). In this study, we isolated and purified these gangliosides using chloroform-methanol extraction, Folch partition, chromatographies on DEAE-Sephadex A-25, and Iatrobeads columns. The yields of gangliosides GM3, GD3, and O-acetyl GD3 were 44.1 mg, 19.6 mg, and 9 mg per 100 g of Ma melanotic melanoma tissues, respectively. The structures of these gangliosides were characterized by periodate oxidation, gas chromatographic (GC) analysis, fast-atom bombardment-mass spectrometry (FAB-MS), and nuclear magnetic resonance (NMR) studies. The structure of hamster melanoma O-acetyl GD3 is different from the 9-O-acetyl GD3 previously reported in human melanoma. The major fatty acids of this ganglioside are C16:0, C18:0, C20:0, C22:0, and C24:0 and the long-chain base is C18-sphingosine.

Animals↗
PubMed1992

Modulation by glycosphingolipids of membrane-membrane interactions induced by myelin basic protein and melittin.

The effect of glycosphingolipids (GSLs) with oligosaccharide chains of different length and charge on membrane-membrane interactions induced by myelin basic protein (MBP) or melittin (Mel) was comparatively investigated with small unilamellar vesicles. MBP induces a fast vesicle aggregation and close membrane apposition. Merging of lipid bilayers and vesicle fusion induced by MBP are slower and less extensive processes compared to membrane apposition. The changes of membrane permeability concomitant to these phenomena are small. The Trp region of MBP remains in a rather polar environment when interacting with vesicles; its accessibility to NO3- or acrylamide quenching depends on the type of GSLs in the membrane. The Trp region of Mel is inserted more deeply into the lipid bilayer and its accessibility to the aqueous quenchers is less dependent on variations of the oligosaccharide chain of the GSLs. Mel induces a faster and more extensive membrane apposition and bilayer merging than does MBP. Extensive vesicle disruption occurs in the presence of Mel. Negatively charged GSLs facilitate membrane proximity and vesicle aggregation but an increase of the oligosaccharide chain length of either neutral or acidic GSLs decreases the interaction among vesicles that are induced by either protein. This effect is independent of the different mode of insertion of MBP and Mel into the membrane. Our results suggest that the modulation by the oligosaccharide chain on the protein-induced interactions between bilayers containing GSLs is probably exerted beyond the level of local molecular interactions between the basic proteins and the lipids.

Cell Membrane Permeability↗
PubMed1992

O-acetylated gangliosides in bovine buttermilk. Characterization of 7-O-acetyl, 9-O-acetyl, and 7,9-di-O-acetyl GD3.

Three O-acetylated gangliosides, G1, G2, and G3, were purified from bovine buttermilk by using chloroform/methanol extraction, Folch partitioning, chromatography on DEAE-Sephadex A-25, and Iatrobeads columns. The final yields of gangliosides G1, G2, and G3 were 2 mg, 37 mg, and 40 mg per 1.7 kg of the buttermilk powder, respectively. On the basis of immunostaining on high performance thin layer chromatography with specific monoclonal antibodies, mild alkaline treatment, gas-liquid chromatographic analysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance studies, G1 and G2 are characterized as O-acetylated GD3 and G3 as O-acetylated GT3, and the structures of these gangliosides are as follows: [formula: see text] The major fatty acids of these gangliosides were C18:0, C22:0, C23:0, and C24:0, and the long chain base was C18-sphingosine.

Acetylation↗