Biomedical subjects

R K Yu

Publications and source records attributed to R K Yu.

At least 55 records · Page 3Linked to original sources

PubMed1998

Sulfoglucuronosyl glycolipids as putative antigens for autoimmune inner ear disease.

Autoimmune inner ear disease is diagnosed based on clinical history of fluctuating but progressive sensorineural hearing loss (SNHL) with or without vestibular symptoms occurring over weeks to months. An initial response to steroids or immunosuppressive drugs usually reverses the hearing loss. In search of specific diagnostic and therapeutic markers for autoimmune inner ear diseases, we investigated serum anti-glycolipid antibody activities in these patients by two different methods, HPTLC-immunoblotting and ELISA. We found that 37 out of 74 patients of clinically diagnosed autoimmune inner ear disease (30 of sensorineural hearing loss (SNHL) (group I), 14 of vestibular symptoms only (group II), 30 of Menieres symptoms (with both hearing loss and vestibular symptoms) (group III)) showed positive anti-sulfoglucuronosyl lactosaminyl paragloboside (SGLPG) antibody titers (p < 0.001). On the other hand, anti-sulfoglucuronosyl paragloboside (SGPG) titers were not elevated in these conditions. In contrast, only 3 out of 56 pathological control and 2 out of 28 healthy volunteers had measurable anti-SGLPG antibody titers. We further analyzed the localization of SGLPG in the auditory pathway and found that the antigens existed exclusively in inner ear and the eighth nerve, but not in pons, cerebellum, nor cerebrum. We conclude that the anti-SGLPG antibody represents a novel diagnostic marker for autoimmune inner ear disease and may participate in the pathogenesis of this disease.

Autoantibodies↗

PubMed1998

The role of globo-series glycolipids in neuronal cell differentiation--a review.

Alterations in glycolipid composition as well as glycosyltransferase activities during cellular differentiation and growth have been well documented. However, the underlying mechanisms for the regulation of glycolipid expression remain obscure. One of the major obstacles has been the lack of a well defined model system for studying these phenomena. We have chosen PC12 pheochromocytoma cells as a model because (a) the properties of these cells have been well characterized, and (b) they respond to nerve growth factor (NGF) by differentiating into sympathetic-like neurons and are amenable to well-controlled experimentation. Thus, PC12 cells represent a suitable model for studying changes in glycolipid metabolism in relation to cellular differentiation. We have previously shown that subcloned PC12 cells accumulate a unique series of globo-series neutral glycolipids which are not expressed in parental PC12 cells. This unusual change in glycolipid distribution is accompanied by changes in the activities of specific glycosyltransferases involved in their synthesis and is correlated with neuritogenesis and/or cellular differentiation in this cell line. We have further demonstrated that changes in the glycosyltransferase activities may be modulated by the phosphorylation states of the cells via protein kinase systems. We conclude that these unique globo-series glycolipids may play a functional role in the initiation and/or maintenance of neurite outgrowth in PC12 cells.

Animals↗

PubMed1998

Regulation of ganglioside metabolism by phosphorylation and dephosphorylation.

Cell differentiation is frequently accompanied by alterations in the composition of gangliosides in the plasma membrane resulting from a regulation of the enzyme activities involved. The regulation of CMP-NeuAc:GM1 alpha2-3-sialyltransferase (ST-IV) and UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase (Gal-NAc-T) by the degree of enzyme phosphorylation was analyzed by determination of the enzyme activity on incubation of NG108-15 cells with various protein phosphatase inhibitors (okadaic acid and orthovanadate) or protein kinase activators (phorbol ester and forskolin). Incubation with okadaic acid, but not with orthovanadate, inhibited the ST-IV activity to 45% of that of control cells with t(1/2) = 60 min for the inactivation reaction. This indicates a rapid hyperphosphorylation of ST-IV due to the inhibition of a serine/threonine-specific phosphatase. A similar rate of inactivation was found on stimulation of protein kinase C with phorbol ester. In contrast to ST-IV, the activity of GalNAc-T was increased on stimulation of intracellular phosphorylation systems. The fastest activation of GalNAc-T was achieved with forskolin, yielding up to 160% of the initial activity within 30 min of effector incubation. Up-regulation of GalNAc-T in conjunction with down-regulation of ST-IV by stimulation of phosphorylation is suggested to serve as a physiological mechanism to increase the concentration of GM1, which was found to be elevated in correlation with the cell density. This assumption was corroborated by metabolic labeling studies with radioactive ganglioside precursors indicating an enhancement of the relative amount of a-series gangliosides subsequent to GM3 on phosphorylation stimulation. In particular, the biosynthesis of GM1 was specifically elevated within 2 h of incubation with forskolin. We conclude from the overall data that the ganglioside composition during the cell differentiation of NG108-15 cells can be specifically regulated by both protein kinase A- and protein kinase C-related phosphorylation systems.

Animals↗

PubMed1998

Role of sphingolipid-mediated cell death in neurodegenerative diseases.

The metazoan nervous system gives rise intradevelopmentally to many more neurons than ultimately survive in the adult. Such excess cells are eliminated through programmed cell death or apoptosis. As is true for cells of other lineages, neuronal survival is sustained by an array of growth factors, such that withdrawal of neurotrophic support results in apoptotic cell death. Apoptosis is therefore believed to represent a beneficial process essential to normal development of central and peripheral nervous system (CNS and PNS) structures. Although the initiation of neuronal apoptosis in response to numerous extracellular agents has been widely reported, the regulatory mechanisms underlying this mode of cell death remain incompletely understood. In recent years, the contribution of lipid-dependent signaling systems, such as the sphingomyelin pathway, to regulation of cell survival has received considerable attention, leading to the identification of lethal functions for the lipid effectors ceramide and sphingosine in both normal and pathophysiological conditions. Moreover, the apoptotic capacities of several cytotoxic receptor systems (e.g., CD120a, CD95) and many environmental stresses (e.g., ionizing radiation, heat-shock, oxidative stress) are now known to derive from the activation of multiple signaling cascades by ceramide or, under some circumstances, by sphingosine. Inappropriate initiation of apoptosis has been proposed to underlie the progressive neuronal attrition associated with various neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and other neurological disorders that are characterized by the gradual loss of specific populations of neurons. In such pathophysiological states, neuronal cell death can result in specific disorders of movement and diverse impairments of CNS and PNS function. In some autoimmune neurological diseases such as Guillain-Barré syndrome, demyelinating polyneuropathy, and motoneuron disease, persistent immunological attack of microvascular endothelial cells by glycolipid-directed autoantibodies may lead to extensive cellular damages, resulting in increased permeability across brain-nerve barrier (BNB) and/or blood-brain barrier (BBB).

Animals↗

PubMed1997

Ganglioside GD3 and GD3-lactone mediated regulation of the intermolecular organization in mixed monolayers with dipalmitoylphosphatidylcholine.

The interactions of dpPC with ganglioside GD3 and two lactones. GD3LacI or GD3LacII, in lipid monolayers occur with reduced, unaltered, or increased molecular area and surface potential/molecule, respectively. dpPC is fully miscible with GD3 and GD3LacI but films with GD3LacII show immiscibility above 75 mol% lactone. At low proportions of GD3 in mixtures with dpPC, GD3 undergoes condensation and depolarization; dpPC is depolarized and its molecular area is reduced above 50 mol% GD3. GD3LacI forms ideally mixed films with dpPC. Mixtures of dpPC with GD3LacII at mole fractions below 0.3 show increased mean molecular area and surface potential/molecule mostly due to lactone alterations. Between mole fractions of 0.3 and 0.75 the surface parameters of dpPC are altered, and above these proportions both lipids are immiscible. Defined variations of molecular properties induced by ganglioside lactonization are selectively transduced to changes of the intermolecular organization and surface electrostatics in mixed interfaces with dpPC. Thus, changes in the relative proportions of a ganglioside and its lactone forms may act as sensitive biotransducers for membrane-mediated cellular functions, without the need for metabolically altering the concentration of gangliosides.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1997

Cloning, characterization, and expression of human ceramide galactosyltransferase cDNA.

Galactosylceramide (galactocerebroside, GalC) and its sulfated derivative, sulfatide, are major lipid components of the central and peripheral nervous system myelin sheath. The enzyme UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) catalyzes the final step of galactosylceramide synthesis. In this report we describe isolation of the complete copy of human CGT cDNA. Total RNA from N-370 FG cells, a human fetal glioma cell line, was reverse-transcribed and dG-tailed. Degenerate primers synthesized based on rat CGT cDNA sequence were used in 5'- and 3'- rapid amplification of cDNA ends reaction (RACE). The obtained sequence was used to synthesize the primers for the complete coding region to be amplified and cloned into a pCR 3.1 expression vector. Following transfection of the CHOP cells with the resulting vector, the cell homogenate was assayed for the galactosyltransferase activity. Northern blot hybridization was used to determine the length of CGT mRNA and Southern blot hybridization was used to determine the number of homologous genes. Our results indicate that human CGT retains all conservative features of rat and mouse CGT. It is a single copy gene with mRNA transcript of about 4 kb.

Amino Acid Sequence↗

PubMed1997

Cloning of the cDNA coding for rat brain CMP-NeuAc:GD3 alpha2-8 sialyltransferase.

The cDNA coding for GT3-synthase has been cloned from a rat fetal brain cDNA library. The complete nucleotide sequence and the deduced amino acid sequence of the rat GT3-synthase cDNA were highly homologous to those of the mouse GT3-synthase. Quantitative RT-PCR showed that, as a key enzyme for the synthesis of 'c'-series gangliosides, the expression of GT3-synthase was developmentally regulated in embryonic rat brains.

Amino Acid Sequence↗

PubMed1997

Glycosyltransferase activities in cultured endothelial cells of bovine brain microvascular origin.

Bovine brain microvascular endothelial cells (BMECs) express GM3 (NeuAc) and GM3 (NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b as well as sialosylparagloboside and sialosyllactosaminylparagloboside as the minor species. To investigate the metabolic basis of this ganglioside pattern, the activities of eight glycosyltransferases (GM3-, GD1a-, GD3-, LM1-, GM2 (NeuAc)-, GM2 (NeuGc)-, LacCer-, and GM1-synthases) in cultured BMECs were studied. It was found that BMECs possessed high activities of GM3- and GD1a-synthases, and low activities of GM2-, GM1-, and GD3-synthases. Thus, the present study provides evidence that endothelial cells are capable of synthesizing gangliosides in situ and that the high content of GM3 in BMEC is closely associated with high activities of GM3-synthase and low activities of GM2-, GM1-, and GD3-synthases.

Animals↗

PubMed1997

Unusual gangliosidosis in emu (Dromaius novaehollandiae).

A previous study has demonstrated an unusual gangliosidosis in emu that is characterized by the accumulation of gangliosides in the brain tissues with GM3 and GM1 predominating. To provide insight into this unique disorder of emu gangliosidosis, the current study focused on analysis of neutral glycosphingolipids and gangliosides from brain and liver tissues of affected birds and healthy controls. We found not only that the total lipid-bound sialic acid content was increased three- and fourfold in the affected brain and liver, respectively, but also that the ganglioside pattern was rather complex as compared with the control. The absolute ganglioside sialic acid content was significantly increased in the diseased tissues, with the highest elevation levels of GM3 (14-fold) and GM1 (ninefold) in the affected brain. Relative increases in content of these monosialogangliosides were also significant. GM2 was only detected in the affected brain, but not in normal controls. The neutral glycosphingolipid fraction showed accumulation of many oligosylceramides, with six- and 5.5-fold increases in lactosylceramide levels for brain and liver, respectively. The level of myelin-associated galactosylceramide (GalCer) in the brain was decreased to only 41% of that in the healthy control, whereas no difference was found in liver tissues from both groups. Besides GalCer, the brain content of sulfatide (cerebroside-sulfate esters), another myelin-associated glycolipid, decreased to only 16% of the control. The loss of myelin-associated GalCer and sulfatide strongly suggests demyelination in the affected emu brain. Our overall data are consistent with the presence of a unique form of sphingolipidosis in the affected emus, perhaps with secondary demyelination, and suggest a metabolic disorder related to total sphingolipid activator deficiency.

Animals↗

PubMed1997

Antibodies to glycosphingolipid antigens in patients with immune-mediated cochleovestibular disorders.

HYPOTHESIS: To determine the presence of antibodies against the glycosphingolipid antigen sulfated glucuronic lactosominyl paragloboside (SGLPG) in the sera of patients suspected of immune-mediated cochleovestibular disorders (IMCVD). BACKGROUND: Glycospingolipids are molecules present on the surface of normal nerve cells and are considered antigenic. Previous studies have isolated these antigens in vestibular neuroepithelia, cochleovestibular nerves and endolymphatic sacs. METHODS: The sera of 22 patients suspected of IMCVD were tested for antibodies against the antigen SGLPG. Thin-layer chromatography-immunostaining method was used. RESULTS: Antibody titers were elevated in 63.6% of patients tested. Statistical significance (p < 0.0001) was achieved since reactivity was seen in only 7% of 43 age-matched healthy controls. CONCLUSIONS: Antibodies to SGLPG antigens are present in some patients with IMCVD. Because SGLPD antigens have been previously isolated in the inner ear and the cochleovestibular nerve, these structures can potentially become targets for anti-SGLPG antibodies.

Adult↗

PubMed1996

Molecular cloning of cDNA for rat brain GD3-synthase.

The complete nucleotide sequence of the cDNA coding for rat GD3-synthase was determined for the first time using PCR and the rapid amplification of cDNA ends (RACE) procedures. The deduced amino acid sequence consists of 359 residues. The protein contains a potential transmembrane domain with a short N-terminus and such highly conserved regions as the sialylmotifs. An antisense oligonucleotide derived from the rat GD3-synthase cDNA was applied to the cultures of rat cerebellar neurons and results showed a decrease in the synthesis of ganglioside GD3, indicating that the expression of GD3-synthase was modulated by the antisense sequence.

Amino Acid Sequence↗

PubMed1996

Association of a 14-3-3 protein with CMP-NeuAc:GM1 alpha 2,3-sialyltransferase.

CMP-NeuAc:GM1 alpha 2,3-sialyltransferase (ST-IV) was purified to homogeneity from rat brain. Microsequencing of the tryptic peptides derived from the purified enzyme revealed two amino acid sequences homologous to the 14-3-3 proteins. A polyclonal antibody was raised against purified ST-IV. A 33 kDa protein was co-immunoprecipitated from rat brain extracts with the anti-(ST-IV) antibody as detected by Western blot analysis. This protein was identified as a subtype of 14-3-3 family by an anti-(14-3-3) antibody. Screening of a rat brain lambda gt11 library using the anti-(ST-IV) antibody resulted in the identification of a cDNA clone coding for the subtype of 14-3-3 protein. These results indicate an association of the 14-3-3 protein with the sialyltransferase. Since the 14-3-3 protein has PKC inhibitor activities and the activity of sialyltransferases is, at least in part, regulated by PKC, the association of the 14-3-3 protein with ST-IV may indicate a role for this protein in the post-translational regulation of the sialyltransferase activity through the processes of phosphorylation and dephosphorylation.

14-3-3 Proteins↗

PubMed1996

Generation and characterization of anti-sulfoglucuronosyl paragloboside monoclonal antibody NGR50 and its immunoreactivity with peripheral nerve.

Sulfoglucuronosyl paragloboside (SGPG) is a member of the sulfated glucuronic acid-containing glycolipid (SGGL) family found primarily in peripheral nerves. These glycolipids contain the HNK-1 carbohydrate epitope and are recognized by monoclonal IgM from patients with chronic demyelinating neuropathy and paraproteinemia. Recent studies indicate that SGGLs may serve as ligands for selectins, amphoterin, and laminin, suggesting that these glycolipids may play an important role in cellular adhesion. To elucidate the biological function of these glycolipids, we produced a murine monoclonal antibody (mAb) and studied its antigenic specificity. Using an enzyme-linked immunosorbent assay (ELISA), we found that the mAb designated as NGR50 belonged to the IgG2a subclass, and that the minimal titer (2 SD above the mean optical density value of control) of this mAb was 1:640, with 20 ng of purified SGPG as the antigen. Thin-layer chromatography (TLC) immunoblotting revealed that this mAb reacted specifically with SGPG and sulfoglucuronosyl lactosaminyl paragloboside (SGLPG), which is a structural analogue of the former, but not with other glycolipids. Desulfated derivates of SGPG and SGLPG did not react with mAb NGR50. Western blot analysis showed crossreactivity with human myelin-associated glycoprotein (MAG), but not with rat MAG or rat glycoprotein P0. Unlike anti-HNK-1 monoclonal antibody, however, NGR50 reacted only weakly with several proteins in the 20-30-kD regions, including human P0, suggesting that mAb50 has a different fine specificity as an anti-HNK-1 antibody. Immunocytochemical study of rat sciatic nerve using mAb NGR50 revealed positive staining at the outer surface of the myelin sheath and Schwann cells, as well as in the intervening connective tissues. Faint staining was also visible at the axolemmal-myelin interface; however, compact myelin was not stained.

Animals↗

PubMed1996

Sensitization of Lewis rats with sulfoglucuronosyl paragloboside: electrophysiological and immunological studies of an animal model of peripheral neuropathy.

Antibodies against sulfoglucuronosyl glycosphingolipids (SGGLs) are known to be present in sera of patients with chronic polyneuropathy associated with IgM paraproteinemia. We recently studied rats sensitized with sulfoglucuronosyl paragloboside (SGPG), a major SGGL species, emulsified with keyhold limpet hemocyanin and Freund's adjuvant. The titer of the IgM class antibodies against SGPG increased up to 1:1,600, while that of the IgG class increased up to 1:800 2 weeks after sensitization. The antibodies showed a high degree of antigenic specificity; no cross-reactivity with other brain glycolipids could be detected. They, however, reacted with human myelin-associated glycoprotein (MAG) by Western blot analysis, but not with rat MAG. These animal models showed minor but clear clinical signs of neuropathy, consisting of mild tail muscle tone loss and walking disabilities. Electrophysiological examination of the sciatic nerves revealed nerve conduction abnormalities which consisted of conduction block and mild decrease in conduction velocity. Thus, our results support the concept that anti-SGPG antibodies may play an important pathogenetic role in this type of chronic neuropathy.

Action Potentials↗

PubMed1996

Expression and localization of Lewis(x) glycolipids and GD1a ganglioside in human glioma cells.

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 micrograms of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis(x) (fucosylneolactonorpentaosyl ceramide; Le(x)), difucosylneolactonorhexaosyl ceramide (dimeric Le(x)), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 micrograms of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le(x) and the complex type of sialyl-Le(x) derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le(x) glycolipids and sialyl-Le(x) were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le(x) glycolipid was located on the tip of fine cellular processes. The unique localization of the Le(x) glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.

Animals↗

PubMed1996

Expression of a unique globo-series glycolipid in cultured rat dorsal root ganglion neurons: relationship with neuronal development.

Previous studied from the laboratory demonstrated the presence of a UDP-galactose:Gb3Cer alpha 1-3galactosyltansferase activity responsible for the synthesis of a unique glycosphingolipid (GSL), Gal alpha 1-3Gb3Cer, in cultured PC12 pheochromocytoma cells (21). In this investigation, we examined the presence of this enzyme activity in isolated rat embryonic dorsal root ganglion neurons (DRGN), which, like pheochromocytoma cells, originate from the neural crest cells. DRGN exhibited the alpha-galactosyltransferase activity and the activity was comparable to that of the PC12 cells while several other rat tissues, with the exception of kidney, showed minimal activity. In order to define the spatial and temporal expression of Gal alpha 1-3Gb3Cer in DRGN, we examined the expression of Gal alpha 1-3Gb3Cer in cultured DRGN derived from embryonic day 16 rat embryos. Using a polyclonal antibody raised against Gal alpha 1-3Gb3Cer, we examined the localization of this glycolipid in DRGN cells after 5, 8, 12, and 15 days in culture. Immunostaining was restricted to the neurons while Schwann cells were negative. At day 5, the immunostaining was weak and confined to the cell body of the DRGN, though neurites were present at this stage. The period between days 5 and 15 represented a period of rapid neuritic growth and continued enlargement of the cell bodies. Immunoreactivity in the cell bodies increased dramatically by day 8. By day 12, immunoreactivity was present in neurites, and by day 15, was strong in both cells bodies and neurites. The expression of Gal alpha 1-3Gb3Cer in vivo was confirmed by immunostaining of frozen sections of dorsal root ganglia. Our present studies which demonstrate neuron-specific expression of Gal alpha 1-3Gb3Cer during neurotigenesis combined with previous observations for its expression in PC12 cells, strongly implicates this GSL in neuronal development.

Animals↗

PubMed1996

Sialidase activity in nuclear membranes of rat brain.

A highly purified nuclear membrane preparation was obtained from adult rat brain and examined for sialidase activity using GM3, GD1a, GD1b, or N-acetylneuramin lactitol as the substrate. The nuclear membranes contained an appreciable level of sialidase activity; the specific activities toward GM3 and N-acetylneuramin lactitol were 20.5 and 23.8% of the activities in the total brain homogenate, respectively. The sialidase activity in nuclear membranes showed substrate specificity distinct from other membrane-bound sialidases localized in lysosomal membranes, synaptosomal plasma membranes, or myelin membranes. These results strongly suggest the existence of a sialidase activity associated with the nuclear membranes from rat brain.

Animals↗

PubMed1996

Transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the RelA transcription factor as a mechanism producing airway mucosal inflammation.

The most common cause of epidemic pediatric respiratory disease, respiratory syncytial virus (RSV), stimulates interleukin-8 (IL-8) synthesis upon infecting airway epithelium, an event necessary for the development of mucosal inflammation. We investigated the mechanism for enhanced IL-8 production in human A549 type II pulmonary epithelial cells. Infection with sucrose-purified RSV (pRSV) produced a time-dependent increase in the transcriptional initiation rate of the IL-8 gene. Transient transfection of the human IL-8 promoter mutated in the binding site for nuclear factor-kappaB (NF-kappaB) demonstrated that this sequence was essential for pRSV-activated transcription. Gel mobility shift assays demonstrated pRSV induction of sequence-specific binding complexes; these complexes were supershifted only by antibodies directed to the potent NF-kappaB transactivating subunit RelA. Both Western immunoblot and indirect immunofluorescence assays showed that cytoplasmic RelA in uninfected cells became localized to the nucleus after pRSV infection. RelA activation requires replicating RSV, because neither conditioned medium nor UV-inactivated pRSV was able to stimulate its translocation. We conclude that RelA undergoes changes in subcellular distribution in airway epithelial cells upon pRSV infection. The ability of replicating RSV to activate RelA translocation may play an important role in activating IL-8 and other inflammatory gene products necessary for airway mucosal inflammation seen in RSV disease.

Cell Nucleus↗