254,316 research outputs found

    Identification of a carbohydrate-based endothelial ligand for a lymphocyte homing receptor.

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    Lymphocyte attachment to high endothelial venules within lymph nodes is mediated by the peripheral lymph node homing receptor (pnHR), originally defined on mouse lymphocytes by the MEL-14 mAb. The pnHR is a calcium-dependent lectin-like receptor, a member of the LEC-CAM family of adhesion proteins. Here, using a soluble recombinant form of the homing receptor, we have identified an endothelial ligand for the pnHR as an approximately 50-kD sulfated, fucosylated, and sialylated glycoprotein, which we designate Sgp50 (sulfated glycoprotein of 50 kD). Recombinant receptor binding to this lymph node-specific glycoprotein requires calcium and is inhibitable by specific carbohydrates and by MEL-14 mAb. Sialylation of the component is required for binding. Additionally, the glycoprotein is precipitated by MECA-79, an adhesion-blocking mAb reactive with lymph node HEV. A related glycoprotein of approximately 90 kD (designated as Sgp90) is also identified

    Effects of HIV and Drugs of Abuse on the Blood-Brain Barrier

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    Despite effective systemic therapy, HIV-1 infection within the brain results in neuronal degradation and neurocognitive dysfunction. This neurocognitive dysfunction is worsened in the setting of opiate abuse. The central nervous system (CNS) is protected by the blood-brain barrier (BBB), a selective barrier regulating the passage of substances from peripheral circulation into the CNS. The BBB is composed of microvascular endothelial cells encased by basal lamina, pericytes, and perivascular astrocyte endfeet. Intracellular junctional complexes comprising of adherens and tight junctions are located between the endothelial cells and form tight barrier, preventing traffic of compounds between cells (paracellular flux). Clinical and in vitro data suggest that BBB integrity is compromised in HIV infection, which leads to a leaky barrier. Brain microvascular endothelial cells also express efflux transporters that are responsible for the extrusion of substances from the brain back into the blood. P-glycoprotein is a drug efflux transporter involved in the efflux of many antiretroviral drugs and overexpression of P-glycoprotein can limit therapeutic concentrations of substrate drugs within the brain. Additionally, P-glycoprotein expression and/or function may be altered in the setting of HIV infection and in the setting of drug abuse. In order to study the impact of morphine, a commonly used opiate drug of abuse, on drug-efflux proteins at the BBB, we measured the effects of morphine and the HIV-1 protein Tat on P-glycoprotein expression and function. hCMEC/D3 cells, which are human derived brain microvascular endothelial cells, were pre-treated for 24 hours with Tat (100nM), morphine (500nM), or Tat (100nM) + morphine (500nM). P-glycoprotein function was evaluated by measuring intracellular accumulation of the prototypical P-glycoprotein substrate, rhodamine-123. Compared to control, statistically significant increases in cellular accumulation of rhodamine-123 were observed in both the morphine (mean±SEM; 118±6.5%, p\u3c0.05) and Tat+morphine (118 ±13.1%, p\u3c0.05) groups, suggesting decreased efflux activity of P-glycoprotein. Protein expression of P-glycoprotein was measured using western blot analysis. Significant decreases in P-glycoprotein expression was observed in all treatment groups as compared to control; Tat (63±4.2%, p\u3c 0.05), morphine (64±13.5%, p\u3c0.05) and Tat+morphine (69±15.6%, p\u3c0.05). Understanding the factors that influence efflux transporter function and expression in the BBB are crucial in optimizing antiretroviral penetration into the brain, even in the setting of drug abuse.https://scholarscompass.vcu.edu/uresposters/1263/thumbnail.jp

    The use of chitin binding proteins for glycoprotein analysis

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    The focus of the pharmaceutical industry has dramatically shifted in the past number of years. Traditional drugs were synthesised using chemical reactions have been replaced by recombinant glycoprotein molecules. These potential recombinant glycoprotein therapeutics display oligosaccharide structures on their surfaces that are recognised by their target host. The specific glycan moieties on the surface of the molecules vary dramatically and have a large impact on the efficacy of the drug. The development of bioanalytical tools to identify and separate the species of glyco-forms present in a preparation of the glycoprotein therapeutic will significantly help to advance the quality and effectiveness of recombinant glycoprotein molecules. Traditionally lectins, isolated from plants, had been used to profile sugar species displayed on glycoproteins. I have explored the use of prokaryotic chitin binding proteins (CBPs) to investigate structures on glycoproteins

    Amino acid sequence elucidation of human acrosin-trypsin inhibitor (HUSI-II) reveals that Kazal-type proteinase inhibitors are structurally related to β-subunits of glycoprotein hormones

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    The amino acid sequence of the acrosin-trypsin inhibitor HUSI-II from human seminal plasma is presented which unequivocally identifies HUSI-II as being of Kazal-type. In addition, the HUSI-II sequence shows a striking similarity to the middle part of glycoprotein hormone β-subunits thus revealing a hitherto unknown structural and evolutionary relationship between Kazal-type inhibitors and glycoprotein hormone

    Vesicular stomatitis virus glycoprotein is necessary for H-2-restricted lysis of infected cells by cytotoxic T lymphocytes

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    Vesicular stomatitis virus (VSV) elicited cytotoxic thymus-derived lymphocytes (CTLs) in mice of the BALB/c and three congenic strains (BALB.b, BALB.k, BALB.HTG). CTL lysis of VSV-infected fibroblasts from the four strains was restricted by the target cells' major histocompatibility complex (H-2). Target cells were also infected with two temperature-sensitive mutants of VSV, tsM and tsG in which, respectively, the viral matrix protein and glycoprotein are not expressed at 39 degrees (restrictive temperature) on the infected cell's surface membrane. At the restrictive temperature, cells infected with wild-type VSV or tsM were lysed by CTLs, but cells infected with tsG were not. The requirement for the glycoprotein on the target cell was also evident from the ability of antisera to the glycoprotein to block completely CTL lysis of VSV-infected cells

    Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

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    Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein

    Lectin based glycoprotein analysis

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    Many of the biopharmaceutical therapeutics entering the market and currently in clinical trails are recombinant glycoprotein molecules, the glycan moieties of which have a significant impact on efficacy and immunogenicity. The cell culture techniques required to produce these glycoproteins often result in products that are heterogeneous with respect to glycan content. This inconsistency ultimately leads to increased production costs and restricts patient accessibility to these therapeutics. To overcome these difficulties novel analytical platforms facilitating rapid in-process monitoring and product quality control are essential. Work undertaken within the Centre for Bioanalytical Sciences (CBAS) seeks to exploit the microbial world as a source of novel biorecognition elements to produce such platforms

    Functional Divergence of Glycoprotein Hormone Receptors

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    Two lamprey glycroprotein hormone receptors (lGpH-R I and II) highly similar with gnathostome GpH-Rs were cloned from sea lamprey testes and thyroid, respectively. Vertebrate glycoprotein protein receptors have a large extracellular domain (ED) containing a leu rich domain (LRD) linked to a rhodopsin-like transmembrane domain (TMD) through a highly divergent linker region (signal specificity domain, SSD or \u27hinge\u27 region) and a third major segment, the intracellular domain. To determine the potential roles of the different domains in the activation of the receptor following ligand-receptor binding, functional assays were performed on lGpH-R I/rat luteinizing hormone (LH)-R domain swapped chimeric receptors. These results show that the functional roles of the lamprey glycoprotein-receptor I (lGpH-R I) domains are conserved compared with its Gnathostome homologs. The ability of different glycoprotein hormones to activate chimeric lamprey/rat receptors suggests that the selectivity of the GpH-Rs in respect to their ligands is not controlled exclusively by a single domain but is the result of specific interactions between domains. We hypothesize that these interactions were refined during millions of years of co-evolution of the receptors with their cognate ligands under particular intramolecular, intermolecular and physiological constraints
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