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Biomedical subjects

Robert K Yu

Publications and source records attributed to Robert K Yu.

At least 19 recordsLinked to original sources
PubMed2007

Chemical validation of molecular mimicry: interaction of cholera toxin with Campylobacter lipooligosaccharides.

It is generally believed that molecular mimicry between bacterial lipooligosaccharide (LOS) and nerve glycolipids may play an important pathogenic role in immune-mediated peripheral neuropathy. One of the putative infectious agents is Campylobacter jejuni (C. jejuni). To elucidate the structural basis for the molecular mimicry, we investigated the structure of the lipooligosaccharide (LOS) fraction of C. jejuni, strain HS19, and found that it includes at least two components, characterized as fast-and slow-moving bands (LF and LS) by thin-layer chromatography as revealed by cholera toxin B subunit (Ctxb) overlay. Structural analysis of the oligosaccharide portion of LS established that it had the following structure: Gal-GalNAc-(NeuAc)Gal-Hep-(Glc;PO(3)H)Hep-Kdo. The GM1-like epitope was validated by a terminal tetrasaccharide unit within this structure. On the other hand, analysis of LF revealed an entirely different structure: 1, 4'-bisphosphoryl glucosamine disaccharide N, N'-acylated by 3-(2-hydroxytetracosanoyloxy)octadecanoic acid at 2- and 2'-positions, which is consistent with that of lipid A. No GM1-like epitope was observed in LF. Both LS and LF interacted with Ctxb as demonstrated by TLC-overlay and sucrose density gradient centrifugation. Surprisingly, LF does not have the basic GM1 structure for interacting with Ctxb. Instead, the affinity of LF to Ctxb required that one or both of the phosphate groups be present in the glucosamine disaccharide residue because after alkaline phosphatase treatment the dephosphorylated LF was unable to bind to Ctxb. We conclude that LS is likely the component contributing to GM1-mimicry in autoimmune peripheral neuropathy and that the role of LF is not clear but may be associated with the initial activation of autoreactive T cells.

Campylobacter jejuni↗
PubMed2006

Fucosyl-GM1 expression and amyloid-beta protein accumulation in PC12 cells.

Gangliosides, sialic acid-containing glycosphingolipids, are ubiquitously expressed in all eukaryotic cells and are localized primarily in the plasma membrane. For a rat pheochromocytoma cell line, PC12, which has been used frequently as a model for investigating events leading to neuronal differentiation, it is generally thought that GM1 is a major ganglioside, based on reactivity with the probe cholera toxin B subunit (Ctxb). From a series of biochemical studies, however, it has been reported that no GM1 is expressed in PC12 cells. In this study, we have reevaluated GM1 expression and Ctxb reactivity in PC12 cells and a subcloned line, PC12D cells. Flow cytometric analysis with Ctxb revealed that about 30-50% of PC12 cells were reactive with Ctxb. However, a detailed biochemical analysis showed that PC12 cells express abundantly a different ganglioside, fucosyl-GM1, instead of GM1, and the reactivity of Ctxb in the PC12 cells actually arose from its interaction with fucosyl-GM1, which also interacts with this ligand. Because it has been claimed that amyloid-beta protein (Abeta) interacts with GM1 in PC12 cells to provide "seeding" for amyloid to accumulate, we further evaluated this possibility and found that Abeta is mostly likely interacting with fucosyl-GM1 in this cell line. Our data thus suggest that a specific interaction may occur between Abeta and fucosyl-GM1 for the accumulation of amyloid in PC12 cells.

Amyloid beta-Peptides↗
PubMed2006

Down-regulation of WNK1 protein kinase in neural progenitor cells suppresses cell proliferation and migration.

WNK1, a Ser/Thr protein kinase, is widely expressed in many tissues. Its biological functions are largely unknown. Disruption of the WNK1 gene in mice leads to embryonic lethality at day 13, implicating a critical role of WNK1 in embryonic development. To investigate this potential function, we used antisense strategy to knock down the expression of WNK1 in a mouse neural progenitor cell line, C17.2. Down-regulation of WNK1 in C17.2 cells greatly reduced cell growth. Addition of epidermal growth factor (EGF), a mitogen for C17.2 cells, had no effect on growth. The WNK1-knockdown cells showed a flat and rounded morphology, characteristic of the immature and non-differentiated phenotype of the progenitor cells; this was further demonstrated by immunostaining for the progenitor and neuronal markers. Migration of the WNK1-knockdown C17.2 cells was reduced as tested in culture dishes or Matrigel-covered chambers. Moreover, activation of extracellular signal-regulated kinase (ERK1)/2 and ERK5 by EGF in the WNK1-knockdown cells was suppressed. These results demonstrate a novel function of WNK1 in proliferation, migration, and differentiation of neural progenitor cells, likely by mechanisms involving activation of the mitogen-activated protein (MAP) kinase ERK1/2 and/or ERK5 pathways.

Animals↗
PubMed2006

Human and chicken antibodies to gangliosides following infection by Campylobacter jejuni.

Campylobacteriosis is frequently associated with Guillain-Barré syndrome. Poultry are frequently highly colonized with Campylobacter jejuni and are a major foodborne vehicle for campylobacteriosis. In this study, high titer anti-GM1 antibodies were found in the serum of a laboratory worker who developed campylobacteriosis. The microbiologically confirmed strain VLA2/18 (non-serotyped) was isolated from the worker and subsequently inoculated into chickens, resulting in high titers of serum antibodies to GM1. However, none of the immunized chickens in our study showed any noticeable neurological symptoms, such as paralysis or cramping. High titer anti-GM1 antibodies in chicken and human sera strongly inhibited spontaneous muscle action potential in an in vitro system of spinal cord and muscle cell co-culture. In addition, infection of chickens with C. jejuni strains 81116 (HS6) and 99/419 (HS21) or immunization with purified GM1, GM2, and GM3 resulted in elevation of serum anti-ganglioside antibodies with an inhibitory effect on spontaneous muscle action potential. Immunoabsorption studies demonstrated that this inhibitory activity is due to anti-ganglioside antibodies. On the other hand, anti-GM1 is the only specific human serum antibody to induce an inhibitory effect on neuromuscular junctions. Chicken anti-GM1 antibodies showed a strong inhibitory effect, but anti-GM2 and -GM3 had weaker activities. Taken together, our data suggest that campylobacteriosis in chickens may provide a strong link between infection and the development of anti-ganglioside antibody-mediated peripheral nerve dysfunctions.

Action Potentials↗
PubMed2006

Molecular mimicry: sensitization of Lewis rats with Campylobacter jejuni lipopolysaccharides induces formation of antibody toward GD3 ganglioside.

Recently we have reported cases of demyelinating inflammatory neuropathy showing elevated titers of anti-GD3 antibodies, which occurs rarely in Guillain-Barré syndrome. To examine the correlation between the anti-GD3 antibody titer and Campylobacter jejuni infection, we sensitized female Lewis rats with lipopolysaccharides (LPSs) from serotype HS19 of C. jejuni and examined changes in nerve conduction velocity and nerve conduction block (P/D ratio). After 16 weeks of sensitization, animals revealed decreases of nerve conduction velocity and conduction block (P/D ratio) and high titer of anti-GD3 antibodies. These anti-GD3 antibodies also blocked transmission in neuromuscular junctions of spinal cord-muscle cells cocultures. The GD3 epitope was verified to be located on the Schwann cell surface and nodes of Ranvier in rat sciatic nerve. To determine the target epitope for GD3 antibodies in causing nerve dysfunction, the LPS fraction containing the GD3 epitope was purified from the total LPS by using an anti-GD3 monoclonal antibody-immobilized affinity column. Subsequently, chemical analysis of the oligosaccharide portion was performed and confirmed the presence of a GD3-like epitope as having the following tetrasaccharide structure: NeuAcalpha2-8NeuAc2-3Galbeta1-4Hep. Our data thus support the possibility of a contribution of GD3 mimicry as a potential pathogenic mechanism of peripheral nerve dysfunction.

Animals↗
PubMed2006

Further characterization of embryonic stem cell-derived radial glial cells.

Previously, we showed that radial glia-like (RG) cells differentiated from embryonic stem (ES) cells after retinoic acid induction (Liour and Yu, 2003: Glia 42:109-117). In the present study, we demonstrate that the production of RG cells from ES cells is independent of the neural differentiation protocol used. These ES cell-derived RG (ES-RG) cells are similar in morphology to RG cells in vivo and express several characteristic RG cell markers. The processes of these ES-RG cells are organized into radial arrays similar to the RG scaffold in developing CNS. Expression of Pax6, along with other circumstantial data, suggests that at least some of these ES-RG cells are neural progenitors. The progression of neurogenesis into gliogenesis during the in vitro neural differentiation of ES cells recapitulates the in vivo developmental process. The identification of two cell surface markers, SSEA-1 and GM1, on both the native embryonic RG cells and ES-RG cells, may facilitate purification of radial glial cells for future studies and cell therapy. Overall, our study suggests that differentiation of radial glial cells is a common pathway during the neural differentiation of ES cells.

Animals↗
PubMed2006

Cholera toxin B subunit binding does not correlate with GM1 expression: a study using mouse embryonic neural precursor cells.

Gangliosides, sialic acid-containing glycosphingolipids, are ubiquitously expressed in all eukaryotic cells and are primarily localized in the plasma membrane. Cholera toxin B subunit (Ctxb), a component of a heat-labile enterotoxin produced by Vibrio cholerae, has been frequently used as a probe to detect GM1 ganglioside because of its high affinity for this glycolipid. In this study, we evaluated the reactivity of Ctxb and the expression of GM1 in mouse embryonic neuroepithelial cells (NECs). Analysis of Ctxb reactivity of NECs based on flow cytometry revealed that about 80% of the cells are Ctxb positive. A detailed biochemical analysis, however, indicated that GM1 was expressed in NECs in barely detectable quantities. Thus, it was thought that reactivity of Ctxb in the NECs could arise from high-affinity interaction with GM1. Because Ctxb is commonly used as a reagent for flow cytometry and GM1 cell staining, we recommend that using this reagent alone would be inconclusive and that biochemical analysis of GM1 should also be performed to avoid overestimation of GM1 expression and/or mischaracterization of the ganglioside species.

Animals↗
PubMed2006

Glycobiology of neural stem cells.

The mammalian central nervous system is organized by a variety of cells, such as neurons and glial cells, that are generated from a common progenitor, the neural stem cell (NSC). NSCs are defined as undifferentiated neural cells that are characterized by their high proliferative potential while retaining the capacity for self-renewal and multipotency. NSCs and their progeny may be distinguished by the expression of glycoconjugates (e.g., glycoproteins, glycolipids, and proteoglycans). The carbohydrate antigens carried by glycoconjugates are mainly localized on the plasma membrane surface of the cells and they serve as excellent biomarkers for various stages of cellular differentiation. Thus, they have been utilized as ligands for sorting NSCs or their progeny by cell cytometry. Methods have been established for utilizing polysialic acid-neural cell adhesion molecule (PSA-NCAM), stage-specific embryonic antigen-1 (SSEA-1), and gangliosides for cell sorting. Furthermore, glycoconjugates have also been suggested to have a wide range of receptor and signaling functions in NSCs. For example, basic fibroblast growth factor, an important mitogen of NSCs, requires heparan sulfate proteoglycans and glycosylated cystatin C for activity. Notch signaling, which regulates a wide variety of developmental processes in various cells including NSCs, is modulated by the O-fucose glycan modification. In peripheral nervous system (PNS), the human natural killer-1 (HNK-1) antigen regulates the migration of neural crest cells, cell populations containing the stem cells. Thus, glycoconjugates serve not only as marker molecules, but also as functional molecules as well. In the present review, we discuss the expression pattern and possible functions of glycoconjugates in NSCs.

Animals↗
PubMed2005

Spatiotemporal expression of GM1 in murine medial pallial neural progenitor cells.

The expression of gangliosides is developmentally regulated in the central nervous system. The expression of GM1 in the neural progenitor cells of the telencephalonic ventricular zone (VZ) has been reported in several studies. However, information on the spatial and temporal regulation of GM1 expression in the VZ is still lacking. In this study, we characterized the expression of GM1 in the developing mouse telencephalon. At E13, GM1 is expressed in neuronal cells as well as in the VZ. The initial expression of GM1 in the VZ is restricted to regions close to the medial pallium. Fluorescence-activated cell sorting (FACS) analysis and characterization of E14 GM1-positive cells showed that they contain progenitor cells that proliferate in response to epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF) stimulation. The results obtained from quantitative gene expression analysis of region-specific genes (Emx1, Lhx2, Ngn1, Ngn2, Pax6, Dlx2, Gsh2, Mash1, and Nkx2.1), using real-time polymerase chain reaction indicate that FACS of GM1-expressing cells in the fetal forebrain enriches for the medial pallial neural progenitor cells.

Animals↗
PubMed2005

Characterization of glycoconjugate antigens in mouse embryonic neural precursor cells.

Neuronal and glial cells organizing the central nervous system (CNS) are generated from common neural precursor cells (NPCs) during neural development. However, the expression of cell-surface glycoconjugates that are crucial for determining the properties and biological function of these cells at different stages of development has not been clearly defined. In this study, we investigated the expression of several stage-specific glycoconjugate antigens, including several b-series gangliosides GD3, 9-O-acetyl GD3, GT1b and GQ1b, stage-specific embryonic antigen-1 (SSEA-1) and HNK-1, in mouse embryonic NPCs employing immunocytochemistry and flow cytometry. In addition, several of these antigens were positively identified by chemical means for the first time. We further showed that the SSEA-1 immunoreactivity was contributed by both glycoprotein and glycolipid antigens, and that of HNK-1 was contributed only by glycoproteins. Functionally, SSEA-1 may participate in migration of the cells from neurospheres in an NPC cell culture system, and the effect could be repressed by anti-SSEA-1 antibody. Based on this observation, we identified beta1 integrin as one of the SSEA-1 carrier glycoproteins. Our data thus provide insights into the functional role of certain glycoconjugate antigens in NPCs during neural development.

Animals↗
PubMed2005

Glial-guided neuronal migration in P19 embryonal carcinoma stem cell aggregates.

During development of the nervous system, neuronal precursors that originated in proliferative regions migrate along radial glial fibers to reach their final destination. P19 embryonal carcinoma (EC) stem cells exposed to retinoic acid (RA) differentiate into neurons, glia, and fibroblast-like cells. In this work, we induced P19 aggregates for 4 days with RA and plated them onto tissue culture dishes coated with poly-L-lysine. Several cells migrated out of and/or extended processes from the aggregates after 24 hr. Some cell processes were morphologically similar to radial glial fibers and stained for glial fibrillar acidic protein (GFAP) and nestin. Large numbers of migrating cells showed characteristics similar to those of bipolar migrating neurons and expressed the neuronal marker microtubule-associated protein 2. Furthermore, scanning electron microscopy analysis revealed an intimate association between the radial fibers and the migrating cells. Therefore, the migration of neuron-like cells on radial glia fibers in differentiated P19 aggregates resembled some of the migration models used thus far to study gliophilic neuronal migration. In addition, HPTLC analysis in this system showed the expression of 9-O-acetyl GD3, a ganglioside that has been associated with neuronal migration. Antibody perturbation assays showed that immunoblockage of 9-O-acetyl GD3 arrested neuronal migration in a reversible manner. In summary, we have characterized a new cell culture model for investigation of glial-guided neuronal migration and have shown that 9-O-acetyl GD3 ganglioside has an important role in this phenomenon.

Animals↗
PubMed2005

Sp1 and AP2 enhance promoter activity of the mouse GM3-synthase gene.

Promoters of the glycosyltransferase genes for ganglioside synthesis are TATA-less and often have multiple binding sites for transcription factors Sp1 and AP2 in their proximal regions. However, the function of Sp1 and AP2 in the promoters has not yet been defined. Here, we cloned 5'-flanking fragments of the mouse GM3-synthase gene and assessed the promoter activity of these fragments in mouse Neuro-2a cells. This promoter is TATA-less and contains a number of potential transcription factor-binding sites. Multiple putative transcriptional initiation sites for this gene were identified, including several downstream initiation sites. We then set out to dissect the regulatory elements important for GM3-synthase promoter function. We found that a 5'-flanking 254-bp DNA fragment of the gene contained regulatory elements including two Sp1-binding and six AP2-binding sites that were essential for the basal activity of the promoter in mouse Neuro-2a cells. The effects of the individual Sp1- and AP2-binding sites on basal activity of the GM3-synthase gene were investigated. Mutations in the juxtaposed Sp1/AP2-binding site and in an AP2-binding site decreased the activity of the proximal promoter to approximately 50%. In vitro and in vivo interactions between transcription factors Sp1 and AP2 and these regulatory elements were confirmed by EMSA and the chromatin immunoprecipitation approach, respectively. Therefore, our results demonstrate that Sp1 and AP2 enhance the basal activity of the TATA-less mouse GM3-synthase promoter.

5' Flanking Region↗
PubMed2005

AIDP and CIDP having specific antibodies to the carbohydrate epitope (-NeuAcalpha2-8NeuAcalpha2-3Galbeta1-4Glc-) of gangliosides.

Anti-ganglioside antibodies were investigated in plasma exchange solutions (PEs) from two patients with acute and chronic inflammatory demyelinating neuropathies (AIDP and CIDP). Both cases show markedly elevated antibody titers against the lacto-series gangliosides, GM3, GD3, and GT3. In the CIDP patient, the IgG antibody titer to GD3 was remarkably elevated (titer, 1:10,000), indicating maximal avidity to the tetrasaccharide epitope (-NeuAcalpha2-8NeuAcalpha2-3Galbeta1-4Glc-). There were also activities toward GM4 and GM2 with the affinity higher to GM4 than to GM2, indicating that the antibody activity was not highly specific. In contrast, the antibody activities in the AIDP patient showed similar avidity to GM3, GD3, and GT3. These two patients are very rare cases that have not previously encountered in GBS. The effects on co-cultured cells of rat spinal cord and muscle differed according to which PE was used. PE from the AIDP patient produced an inhibitory effect (reduction to 26.8%) on the spontaneous muscle action potential of the neuromuscular junction (NMJ), but the PE from the CIDP patient did not. Thus, in AIDP, the common epitope of GM3, GD3, or GT3 may be shared with certain antigens localized in the peripheral nervous system (PNS) and may participate in a component of conduction-related molecules in the NMJ. High titers of anti-GD3 antibody and the distortion of antibody recognition found in CIDP seem to have no immediate effect on electrophysiologic function in the PNS.

Action Potentials↗
PubMed2005

Antiglycolipid antibodies in Guillain-Barré syndrome and related diseases: review of clinical features and antibody specificities.

Guillain-Barré syndrome (GBS) is an acute inflammatory polyradiculoneuropathy that usually develops following a respiratory or intestinal infection. Although the pathogenic mechanisms of GBS have not been fully established, both humoral and cell-mediated immune factors have been shown to contribute to the disease process. Several antiglycosphingolipid (anti-GSL) antibodies have been found in the sera of patients with GBS or related diseases. Measurements of these antibody titers are very important in the diagnosis of GBS and in evaluating the effectiveness of treatments in clinical trials. The most common treatment strategies for these disorders involve plasmapheresis and the use of steroids for reducing anti-GSL antibody titers to ameliorate patients' clinical symptoms. Administration of intravenous immunoglobulin may also be beneficial in the treatment of neuropathies by suppressing the immune-mediated processes that are directed against antigenic targets in myelin and axons. In certain demyelinating neuropathies, the destruction or malfunctioning of the blood-nerve barrier, which results in the leakage of circulating antibodies into the peripheral nerve parenchyma, has been considered to be an initial step in development of the disease process. In addition, anti-GSL antibodies, such as anti-GM1, may cause nerve dysfunction and injury by interfering with the ion channel function at the nodes of Ranvier, where carbohydrate epitopes of glycoconjugates are located. These malfunctions thus contribute to the pathogenic mechanisms of certain demyelinating neuropathies.

Animals↗
PubMed2005

Screening and sequencing of complex sialylated and sulfated glycosphingolipid mixtures by negative ion electrospray Fourier transform ion cyclotron resonance mass spectrometry.

A protocol for negative ion nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (-)nanoESI-FTICR MS, investigation of complex biological mixtures consisting of sialylated or sulfated glycosphingolipids (GSL) expressing high heterogeneity in the ceramide portion is described. Different instrumental and solvent conditions were explored and optimized to promote efficient ionization, reduce the in-source fragmentation and consequently enhance the detection of intact molecular species from complex mixtures. Using the novel optimized (-)nanoESI-FTICR MS protocol, a reliable and detailed compositional fingerprint of the polysialylated ganglioside mixture isolated from human brain was obtained. Sustained off-resonance irradiation collision-induced dissociation mass spectrometry (SORI-CID MS2) was introduced for the first time for structural elucidation of polysialylated gangliosides. Under well-defined conditions, an informative fragmentation pattern of the trisialylated ganglioside GT1 was obtained. The compositional mapping of a complex mixture of sulfated glucuronic acid containing neolacto-series GSLs extracted from bovine Cauda equina provided hard evidence upon previously described components and new structures not identified before by any other analytical method. Negative ion nanoESI-FTICR MS at 9.4 T is shown here to represent a valuable method in glycolipidomics, allowing a high resolution and mass accuracy detection of major and minor GSL glycoforms and identification of known and novel biologically relevant structures.

Animals↗
PubMed2004

Expression of the mouse WNK1 gene in correlation with ganglioside GD3 and functional analysis of the mouse WNK1 promoter.

WNK1 is one of WNK (With No K=Lysine) protein kinases which comprise a newly described subfamily. Our studies showed that expression of the mouse WNK1 gene was dramatically suppressed in a tumor cell line when its phenotype was altered by suppression of the GD3-synthase gene expression. The mouse WNK1 gene was expressed at a high level at early stage of embryonic brain and its expression decreased as brain developed, similar to the expression pattern of the GD3-synthase gene. To study transcriptional regulation, we cloned a 5'-flanking 1239-bp fragment of the mouse WNK1 gene. This fragment contains a number of potential consensus binding sites for transcription factors, including Sp1, AP2, CCAAT, Est-1, Oct-1, CNBP, and NFkB, but lacks a TATA box. Primer extension identified multiple putative transcriptional initiation sites, including several sites downstream of the ATG codon. Activities of the promoter fragments were assessed in mouse breast Sa/R-MT cells by transient transfection and the results showed that the promoter elements between -700 and -977 is required for maintaining a high level of promoter activity of the TATA-less mouse WNK1 gene.

5' Flanking Region↗
PubMed2004

Lack of apparent neurological abnormalities in rabbits sensitized by gangliosides.

Two very high titer polyclonal antibodies against two ganglioside antigens, GM1 and GD1a, have been raised in New Zealand white rabbits using a homogeneous suspension of the highly purified antigens in Keyhole Limpet Hemocyanin and Freund's adjuvant. The antisera were prepared over a period of 6 months with repeated injections of the ganglioside suspension, followed by an intravenous injection of the purified ganglioside solution, and collecting the serum (approximately 50 ml) at defined time intervals. The GM1-antibody, thus prepared, showed a cross reactivity toward GDlb and asialo-GM1 (GA1), while the GDla-antibody reacted with GD1a, GM1 and GA1 and GD1b as determined by immuno-overlay and ELISA methods. The titer for GM1 antiserum, determined by'ELISA, was greater than 1/10,000 dilution while the titer for GD1a antibody was greater than 1/5000 dilution. No neurological or behavioral abnormality was observed during the period of antiserum production. To evaluate any likely pathological damage caused by such a high titer ganglioside-antibody, autopsy of CNS as well PNS tissues from the rabbits were carried out after the final bleeding. No obvious pathological changes, including demyelination, were noted in any of the four rabbits. These observations cast doubt as to the direct effect of anti-ganglioside antibody induced neurological and pathological disorders.

Animals↗