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Sample records for cell-surface molecules identifies

  1. Advances in targeting cell surface signalling molecules for immune modulation

    Science.gov (United States)

    Yao, Sheng; Zhu, Yuwen; Chen, Lieping

    2013-01-01

    The past decade has witnessed a surge in the development of immunomodulatory approaches to combat a broad range of human diseases, including cancer, viral infections, autoimmunity and inflammation as well as in the prevention of transplant rejection. Immunomodulatory approaches mostly involve the use of monoclonal antibodies or recombinant fusion proteins that target cell surface signalling molecules on immune cells to drive immune responses towards the desired direction. Advances in our understanding of the human immune system, along with valuable lessons learned from the first generation of therapeutic biologics, are aiding the design of the next generation of immunomodulatory biologics with better therapeutic efficacy, minimized adverse effects and long-lasting clinical benefit. The recent encouraging results from antibodies targeting programmed cell death protein 1 (PD1) and B7 homolog 1 (B7H1; also known as PDL1) for the treatment of various advanced human cancers show that immunomodulatory therapy has come of age. PMID:23370250

  2. Methods To Identify Aptamers against Cell Surface Biomarkers

    Directory of Open Access Journals (Sweden)

    Frédéric Ducongé

    2011-09-01

    Full Text Available Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment. During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometry or microscopy. Some have also been used as drugs by inhibiting or activating functions of their targets in a manner similar to neutralizing or agonistic antibodies. Additionally, it is straightforward to conjugate aptamers to other agents without losing their affinity and they have successfully been used in vitro and in vivo to deliver drugs, siRNA, nanoparticles or contrast agents to target cells. Hence, aptamers identified against cell surface biomarkers represent a promising class of ligands. This review presents the different strategies of SELEX that have been developed to identify aptamers for cell surface-associated proteins as well as some of the methods that are used to study their binding on living cells.

  3. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  4. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  5. Methods To Identify Aptamers against Cell Surface Biomarkers

    OpenAIRE

    Frédéric Ducongé; Daniel Miotto Dupont; Agnes Cibiel

    2011-01-01

    Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment). During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometr...

  6. Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules.

    OpenAIRE

    Tal-Singer, R; Peng, C.; Ponce de Leon, M; Abrams, W R; Banfield, B W; Tufaro, F; Cohen, G H; Eisenberg, R J

    1995-01-01

    The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the bacu...

  7. Microassay for measurement of binding of radiolabelled ligands to cell surface molecules

    International Nuclear Information System (INIS)

    An improved technique for measuring the binding of radiolabelled ligands to cell surface molecules has been developed by modification of a procedure using centrifugation through a water-immiscible oil to separate free and cell-bound ligand. It maximises the percentage of ligand bound since cell-bound and free ligand can be separated easily and reproducibly even when very small reaction volumes are used. This permits low levels of ligand radiolabelling and relatively low numbers of cells to be used

  8. Microassay for measurement of binding of radiolabelled ligands to cell surface molecules.

    Science.gov (United States)

    Woof, J M; Burton, D R

    1988-07-22

    An improved technique for measuring the binding of radiolabelled ligands to cell surface molecules has been developed by modification of a procedure using centrifugation through a water-immiscible oil to separate free and cell-bound ligand. It maximises the percentage of ligand bound since cell-bound and free ligand can be separated easily and reproducibly even when very small reaction volumes are used. This permits low levels of ligand radiolabelling and relatively low numbers of cells to be used. PMID:2840465

  9. A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing

    Energy Technology Data Exchange (ETDEWEB)

    Giblin, P.; Kavathas, P. (Yale Univ. School of Medicine, New Haven, CT (USA)); Ledbetter, J.A. (Oncogen, Seattle, WA (USA))

    1989-02-01

    The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. The authors demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8{sup +} T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern observed differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation.

  10. A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing

    International Nuclear Information System (INIS)

    The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. The authors demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8+ T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern observed differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation

  11. An Innovative Method to Identify Autoantigens Expressed on the Endothelial Cell Surface: Serological Identification System for Autoantigens Using a Retroviral Vector and Flow Cytometry (SARF

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Shirai

    2013-01-01

    Full Text Available Autoantibodies against integral membrane proteins are usually pathogenic. Although anti-endothelial cell antibodies (AECAs are considered to be critical, especially for vascular lesions in collagen diseases, most molecules identified as autoantigens for AECAs are localized within the cell and not expressed on the cell surface. For identification of autoantigens, proteomics and expression library analyses have been performed for many years with some success. To specifically target cell-surface molecules in identification of autoantigens, we constructed a serological identification system for autoantigens using a retroviral vector and flow cytometry (SARF. Here, we present an overview of recent research in AECAs and their target molecules and discuss the principle and the application of SARF. Using SARF, we successfully identified three different membrane proteins: fibronectin leucine-rich transmembrane protein 2 (FLRT2 from patients with systemic lupus erythematosus (SLE, intercellular adhesion molecule 1 (ICAM-1 from a patient with rheumatoid arthritis, and Pk (Gb3/CD77 from an SLE patient with hemolytic anemia, as targets for AECAs. SARF is useful for specific identification of autoantigens expressed on the cell surface, and identification of such interactions of the cell-surface autoantigens and pathogenic autoantibodies may enable the development of more specific intervention strategies in autoimmune diseases.

  12. Assessing the role of cell-surface molecules in central synaptogenesis in the Drosophila visual system.

    Directory of Open Access Journals (Sweden)

    Sandra Berger-Müller

    Full Text Available A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps, is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements.

  13. Adhesion molecule expression stimulated by Bacteroides thetaiotaomicron cell-surface antigens.

    Science.gov (United States)

    Rokosz, A; Meisel-Mikołajczyk, F; Malchar, C; Nowaczyk, M; Górski, A

    1999-01-01

    Bacteroides thetaiotaomicron, a Gram-negative anaerobic rod belonging to the Bacteroides fragilis group (BFG), is involved in many systemic and local, most frequently suppurative infections in man. The cell envelope of these rods is composed of two carbohydrate-containing antigens: lipopolysaccharide (LPS) and capsular polysaccharide (CPS). Adhesion molecules ICAM-1, VCAM-1 and E-selectin (ELAM-1) are induced on the endothelial cells by mediators of inflammation. The aim of this study was to assay the ability of B. thetaiotaomicron surface antigens to induce adhesion molecule expression on the endothelial cells. The influence of LPS and CPS on the expression of adhesion molecules on HMEC-1 cell line was examined in an ELISA test. ELISA was performed with monoclonal mouse anti-human: ICAM-1, VCAM-1 and E-selectin antibodies of the IgG class. B. thetaiotaomicron lipopolysaccharides revealed the ability to induce ICAM-1, VCAM-1 and E-selectin expression on the endothelial cells. Their activities were similar, but lower than the activity of Eschericha coli LPS. ICAM-1 was the most stimulated adhesion molecule. The strongest activation by LPS was achieved at the concentrations of 10.0 and 1.0 micrograms/ml. The ability of capsular polysaccharide to induce the expression of adhesion molecules was considerably weaker.

  14. Cell surface adhesion molecules and cytokine profiles in primary progressive multiple sclerosis

    DEFF Research Database (Denmark)

    Ukkonen, Maritta; Wu, Xingchen; Reipert, Birgit;

    2007-01-01

    OBJECTIVE: We evaluated the utility of adhesion molecule (AM) and cytokine/chemokine expressions in blood and cerebrospinal fluid (CSF) as markers of disease activity in primary progressive multiple sclerosis (PPMS). METHODS: The expressions of AMs and the levels of 17 cytokines in patients......) and intercellular adhesion molecule 1 (ICAM-1) in blood and CSF were higher in PPMS than in controls. Comparison between PPMS and SPMS showed higher levels of ICAM-1 in blood and CSF in PPMS, while the level of the vascular adhesion molecule (VCAM-1) was higher only in blood. There was no difference in the levels...... of cytokines in serum or CSF between PPMS and SPMS or controls, but evidence suggesting intrathecal synthesis of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) was found in PPMS. The expressions of CSF VLA-4 in PPMS correlated with the total volume of cerebral lesions and the number...

  15. Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins

    OpenAIRE

    1982-01-01

    Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to r...

  16. Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Huang, Xiaohua [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Biochemistry, College of Laboratory Medicine, Dalian Medical University, Dalian 116044 (China); An, Yue [Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Ren, Feng [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Yang, Zara Zhuyun; Zhu, Hongmei; Zhou, Lei [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); He, Xiaowen; Schachner, Melitta [Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, New Brunswick, NJ (United States); Xiao, Zhicheng, E-mail: zhicheng.xiao@monash.edu [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); Ma, Keli, E-mail: makeli666@aliyun.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Li, Yali, E-mail: yalilipaper@gmail.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Anatomy, National University of Singapore, Singapore 119078 (Singapore)

    2013-10-25

    Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.

  17. Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

    International Nuclear Information System (INIS)

    Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression

  18. Role of soluble and cell surface molecules in the pathogenesis of autoimmune skin diseases.

    Science.gov (United States)

    Drosera, M; Facchetti, F; Landolfo, S; Mondini, M; Nyberg, F; Parodi, A; Santoro, A; Zampieri, S; Doria, A

    2006-01-01

    The skin is one of the most commonly involved tissue in rheumatic autoimmune diseases. Different mechanisms are thought to be implicated in the pathogenesis of skin lesions. In genetically predisposed individuals, ultraviolet (UV) light can contribute to the induction of skin lesions via an inflammatory process. UV light promotes the release of cytokines by keratinocytes and the induction of adhesion molecules on the surface of epidermal cells initiating a cascade of inflammatory events and recruiting immunoinflammatory cells into the skin. In this review data regarding the expression of TNF-alpha in lesional skin tissue from subacute cutaneous lupus erythematosus patients and the role of interferons in the pathogenesis of skin manifestations of rheumatic autoimmune diseases are reported. In addition, an overview on the expression of cellular adhesion molecules in these diseases is provided.UV light can also induce apoptosis in keratinocytes. During this cell death several enzymes became activated. Among them, desoxyribonuclease (DNase) is an enzyme involved in degrading DNA during apoptosis. Data regarding the activity of DNAse in patients with cutaneous lupus erythematosus as a possible risk factor for the development of systemic disease are here reported. PMID:16466628

  19. Indexing molecules with chemical graph identifiers.

    Science.gov (United States)

    Gregori-Puigjané, Elisabet; Garriga-Sust, Rut; Mestres, Jordi

    2011-09-01

    Fast and robust algorithms for indexing molecules have been historically considered strategic tools for the management and storage of large chemical libraries. This work introduces a modified and further extended version of the molecular equivalence number naming adaptation of the Morgan algorithm (J Chem Inf Comput Sci 2001, 41, 181-185) for the generation of a chemical graph identifier (CGI). This new version corrects for the collisions recognized in the original adaptation and includes the ability to deal with graph canonicalization, ensembles (salts), and isomerism (tautomerism, regioisomerism, optical isomerism, and geometrical isomerism) in a flexible manner. Validation of the current CGI implementation was performed on the open NCI database and the drug-like subset of the ZINC database containing 260,071 and 5,348,089 structures, respectively. The results were compared with those obtained with some of the most widely used indexing codes, such as the CACTVS hash code and the new InChIKey. The analyses emphasize the fact that compound management activities, like duplicate analysis of chemical libraries, are sensitive to the exact definition of compound uniqueness and thus still depend, to a minor extent, on the type and flexibility of the molecular index being used.

  20. A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis.

    Science.gov (United States)

    Sugii, Yuh; Kasai, Tomonari; Ikeda, Masashi; Vaidyanath, Arun; Kumon, Kazuki; Mizutani, Akifumi; Seno, Akimasa; Tokutaka, Heizo; Kudoh, Takayuki; Seno, Masaharu

    2016-01-01

    To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer.

  1. High-risk human papillomavirus E7 expression reduces cell-surface MHC class I molecules and increases susceptibility to natural killer cells

    DEFF Research Database (Denmark)

    Bottley, G; Watherston, O G; Hiew, Y-L;

    2007-01-01

    killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy...

  2. Immunological network activation by low-dose rate irradiation. Analysis of cell populations and cell surface molecules in whole body irradiated mice

    Energy Technology Data Exchange (ETDEWEB)

    Ina, Yasuhiro; Sakai, Kazuo [Central Research Inst. of Electric Power Industry, Low Dose Radiation Research Center, Komae, Tokyo (Japan)

    2003-07-01

    The effects of low-dose rate whole body irradiation on biodefense and immunological systems were investigated using female C57BL/6 (B6) mice. These B6 mice were exposed continuously to {gamma}-rays from a {sup 137}Cs source in the long-term low-dose rate irradiation facility at CRIEPI for 0 - 12 weeks at a dose rate of 0.95 mGy/hr. In the bone marrow, thymus, spleen, lymph nodes, and peripheral blood of the irradiated mice, changes in cell populations and cell surface molecules were examined. The cell surface functional molecules (CD3, CD4, CD8, CD19, CD45R/B220, ICAM-1, Fas, NK-1.1, CXCR4, and CCR5), and activation molecules (THAM, CD28, CD40, CD44H, CD70, B7-1, B7-2, OX-40 antigen, CTLA-4, CD30 ligand, and CD40 ligand) were analyzed by flow cytometry. The percentage of CD4{sup +} T cells and cell surface CD8 molecule expressions on the CD8{sup +} T cells increased significantly to 120-130% after 3 weeks of the irradiation, compared to non-irradiated control mice. On the other hand, the percentage of CD45R/B220{sup +} CD40{sup +} B cells, which is one of the immunological markers of inflammation, infection, tumor, and autoimmune disease, decreased significantly to 80-90% between the 3rd to 5th week of irradiation. There was no significant difference in other cell population rates and cell surface molecule expression. Furthermore, abnormal T cells bearing mutated T cell receptors induced by high-dose rate irradiation were not observed throughout this study. These results suggest that low-dose rate irradiation activates the immunological status of the whole body. (author)

  3. The influence of galvanic currents and voltage on the proliferation activity of lymphocytes and expression of cell surface molecules.

    Science.gov (United States)

    Podzimek, S; Hána, K; Miksovský, M; Pousek, L; Matucha, P; Meloun, M; Procházková, J

    2008-01-01

    Release of metal ions from dental metal fillings supported by galvanism can cause local or general pathological problems in sensitive and genetically susceptible individuals. We aimed to investigate in vitro lymphocyte responses and expression of surface molecules influenced by galvanic currents and voltage. Human peripheral blood lymphocytes were influenced by galvanic currents and voltages and lymphocyte proliferation was measured. Control samples were not exposed to the influence of galvanism. We also studied the expression of surface molecules by the FACS analysis. A 15-h and shorter influence of almost all tested currents and voltages caused a significant decrease in lymphocyte proliferation and the 15-h influence of 20 microA currents significantly increased expression of surface molecules CD 19, 11a/18, 19/69 and 19/95. An influence of 10 and 3 microA currents led to a significant decrease in the expression of surface molecules CD 3, 11a/18, 3/69 and 3/95 and to a significant increase in CD 19 expression. An 80 mV voltage influence led to a significant decrease in the expression of surface molecules CD 3, 11a/18, 3/69, 3/95, 19/69 and 19/95, and 200 and 300 mV voltages significantly decreased the expression of surface molecules CD 3, 19, 11a/18, 3/95 and 19/95 and significantly increased CD 19/69 expression. A long-lasting influence of galvanism can, in sensitive and genetically susceptible individuals, influence lymphocyte proliferation and surface molecule expression. The threshold for pathological values of 5 microA for galvanic currents and 100 mV for galvanic voltage was confirmed.

  4. Functional role of HLA class I cell-surface molecules in human T-lymphocyte activation and proliferation.

    OpenAIRE

    Taylor, D S; Nowell, P C; Kornbluth, J

    1986-01-01

    This investigation addressed the role of major histocompatibility complex-encoded class I molecules in the activation and proliferation of human lymphocytes. We studied the effect of antibodies specific for HLA-A and HLA-B locus gene products on mitogen-stimulated peripheral blood mononuclear cell (PBMC) subpopulations. Three individually derived, well-characterized anti-HLA class I monoclonal antibodies were demonstrated to inhibit the proliferation of human PBMC stimulated by either OKT3 or...

  5. A tyrosine-rich cell surface protein in the diatom Amphora coffeaeformis identified through transcriptome analysis and genetic transformation.

    Directory of Open Access Journals (Sweden)

    Matthias T Buhmann

    Full Text Available Diatoms are single-celled eukaryotic microalgae that are ubiquitously found in almost all aquatic ecosystems, and are characterized by their intricately structured SiO2 (silica-based cell walls. Diatoms with a benthic life style are capable of attaching to any natural or man-made submerged surface, thus contributing substantially to both microbial biofilm communities and economic losses through biofouling. Surface attachment of diatoms is mediated by a carbohydrate- and protein- based glue, yet no protein involved in diatom underwater adhesion has been identified so far. In the present work, we have generated a normalized transcriptome database from the model adhesion diatom Amphora coffeaeformis. Using an unconventional bioinformatics analysis we have identified five proteins that exhibit unique amino acid sequences resembling the amino acid composition of the tyrosine-rich adhesion proteins from mussel footpads. Establishing the first method for the molecular genetic transformation of A. coffeaeformis has enabled investigations into the function of one of these proteins, AC3362, through expression as YFP fusion protein. Biochemical analysis and imaging by fluorescence microscopy revealed that AC3362 is not involved in adhesion, but rather plays a role in biosynthesis and/or structural stability of the cell wall. The methods established in the present study have paved the way for further molecular studies on the mechanisms of underwater adhesion and biological silica formation in the diatom A. coffeaeformis.

  6. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    Science.gov (United States)

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties. PMID:26060069

  7. Detecting and identifying small molecules in a nanopore flux capacitor

    Science.gov (United States)

    Bearden, Samuel; McClure, Ethan; Zhang, Guigen

    2016-02-01

    A new method of molecular detection in a metallic-semiconductor nanopore was developed and evaluated with experimental and computational methods. Measurements were made of the charging potential of the electrical double layer (EDL) capacitance as charge-carrying small molecules translocated the nanopore. Signals in the charging potential were found to be correlated to the physical properties of analyte molecules. From the measured signals, we were able to distinguish molecules with different valence charge or similar valence charge but different size. The relative magnitude of the signals from different analytes was consistent over a wide range of experimental conditions, suggesting that the detected signals are likely due to single molecules. Computational modeling of the nanopore system indicated that the double layer potential signal may be described in terms of disruption of the EDL structure due to the size and charge of the analyte molecule, in agreement with Huckel and Debye’s analysis of the electrical atmosphere of electrolyte solutions.

  8. A Potent Activator of Melanogenesis Identified from Small Molecule Screening

    OpenAIRE

    McNaughton, Brian R.; Gareiss, Peter C.; Jacobs, Stacey E.; Fricke, Alex F.; Scott, Glynis A.; Miller, Benjamin L.

    2009-01-01

    Small molecules that increase the cellular level of melanin can be used to study melanogenesis, and have therapeutic potential for melanin-related diseases such as albinism. We describe the identification of a potent activator of melanogenesis from a targeted combinatorial library. Treating melanocytes with our most active molecule results in a 1.8-fold increase in melanin, and an increase in tyrosinase-catalyzed oxidation of L-tyrosine, a key step in melanin biosynthesis.

  9. Small molecule screening identifies targetable zebrafish pigmentation pathways

    DEFF Research Database (Denmark)

    Colanesi, Sarah; Taylor, Kerrie L; Temperley, Nicholas D;

    2012-01-01

    Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish......, including pigment cells, are conserved between zebrafish and other vertebrates, we present these chemicals as molecular tools to study developmental processes of pigment cells in living animals and emphasize the value of zebrafish as an in vivo system for testing the on- and off-target activities...

  10. High-Throughput Screening of Small Molecules Identifies Hepcidin Antagonists

    OpenAIRE

    Fung, Eileen; Sugianto, Priscilla; Hsu, Jason; Damoiseaux, Robert; Ganz, Tomas; Nemeth, Elizabeta

    2013-01-01

    Anemia of inflammation (AI) is common in patients with infection, autoimmune diseases, cancer, and chronic kidney disease. Unless the underlying condition can be reversed, treatment options are limited to erythropoiesis-stimulating agents with or without intravenous iron therapy, modalities that are not always effective and can cause serious adverse effects. Hepcidin, the iron regulatory hormone, has been identified as a pathogenic factor in the development of AI. To explore new therapeutic o...

  11. Stimulators of translation identified during a small molecule screening campaign.

    Science.gov (United States)

    Shin, Unkyung; Williams, David E; Kozakov, Dima; Hall, David R; Beglov, Dmitri; Vajda, Sandor; Andersen, Raymond J; Pelletier, Jerry

    2014-02-15

    In screening a library of natural and synthetic products for eukaryotic translation modulators, we identified two natural products, isohymenialdisine and hymenialdisine, that exhibit stimulatory effects on translation. The characterization of these compounds led to the insight that mRNA used to program the translation extracts during high-throughput assay setup was leading to phosphorylation of eIF2α, a potent negative regulatory event that is mediated by one of four kinases. We identified double-stranded RNA-dependent protein kinase (PKR) as the eIF2α kinase that was being activated by exogenously added mRNA template. Characterization of the mode of action of isohymenialdisine revealed that it directly acts on PKR by inhibiting autophosphorylation, perturbs the PKR-eIF2α phosphorylation axis, and can be modeled into the PKR ATP binding site. Our results identify a source of "false positives" for high-throughput screen campaigns using translation extracts, raising a cautionary note for this type of screen.

  12. Functions of proteoglycans at the cell surface

    DEFF Research Database (Denmark)

    Höök, M; Woods, A; Johansson, S;

    1986-01-01

    Proteoglycans (primarily heparan sulphate proteoglycans) are found at the surface of most adherent eukaryotic cells. Earlier studies suggest that these molecules can be associated with the cell surface principally by two different mechanisms. Proteoglycans may occur as membrane-intercalated glyco......Proteoglycans (primarily heparan sulphate proteoglycans) are found at the surface of most adherent eukaryotic cells. Earlier studies suggest that these molecules can be associated with the cell surface principally by two different mechanisms. Proteoglycans may occur as membrane......-intercalated glycoproteins, where the core protein of the proteoglycan is anchored in the lipid interior of the plasma membrane, or they may be bound via the polysaccharide components of the molecule to specific anchoring proteins present at the cell surface. A number of functions have been proposed for cell surface...

  13. Mapping the energy and diffusion landscapes of membrane proteins at the cell surface using high-density single-molecule imaging and Bayesian inference: application to the multi-scale dynamics of glycine receptors in the neuronal membrane

    CERN Document Server

    Masson, Jean-Baptiste; Salvatico, Charlotte; Renner, Marianne; Specht, Christian G; Triller, Antoine; Dahan, Maxime

    2015-01-01

    Protein mobility is conventionally analyzed in terms of an effective diffusion. Yet, this description often fails to properly distinguish and evaluate the physical parameters (such as the membrane friction) and the biochemical interactions governing the motion. Here, we present a method combining high-density single-molecule imaging and statistical inference to separately map the diffusion and energy landscapes of membrane proteins across the cell surface at ~100 nm resolution (with acquisition of a few minutes). When applying these analytical tools to glycine neurotransmitter receptors (GlyRs) at inhibitory synapses, we find that gephyrin scaffolds act as shallow energy traps (~3 kBT) for GlyRs, with a depth modulated by the biochemical properties of the receptor-gephyrin interaction loop. In turn, the inferred maps can be used to simulate the dynamics of proteins in the membrane, from the level of individual receptors to that of the population, and thereby, to model the stochastic fluctuations of physiologi...

  14. The cell surface proteome of human mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Christian Niehage

    Full Text Available BACKGROUND: Multipotent human mesenchymal stromal cells (hMSCs are considered as promising biological tools for regenerative medicine. Their antibody-based isolation relies on the identification of reliable cell surface markers. METHODOLOGY/PRINCIPAL FINDINGS: To obtain a comprehensive view of the cell surface proteome of bone marrow-derived hMSCs, we have developed an analytical pipeline relying on cell surface biotinylation of intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin to enrich the plasma membrane proteins and mass spectrometry for identification with extremely high confidence. Among the 888 proteins identified, we found ≈200 bona fide plasma membrane proteins including 33 cell adhesion molecules and 26 signaling receptors. In total 41 CD markers including 5 novel ones (CD97, CD112, CD239, CD276, and CD316 were identified. The CD markers are distributed homogenously within plastic-adherent hMSC populations and their expression is modulated during the process of adipogenesis or osteogenesis. Moreover, our in silico analysis revealed a significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells reported previously. CONCLUSIONS/SIGNIFICANCE: Collectively, our analytical methods not only provide a basis for further studies of mechanisms maintaining the multipotency of hMSCs within their niches and triggering their differentiation after signaling, but also a toolbox for a refined antibody-based identification of hMSC populations from different tissues and their isolation for therapeutic intervention.

  15. Utilizing Yeast Surface Human Proteome Display Libraries to Identify Small Molecule-Protein Interactions.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact with small bioactive molecules is a critical but often difficult and time-consuming step in understanding cellular signaling pathways or molecular mechanisms of drug action. Numerous methods for identifying small molecule-interacting proteins have been developed and utilized, including affinity-based purification followed by mass spectrometry analysis, protein microarrays, phage display, and three-hybrid approaches. Although all these methods have been used successfully, there remains a need for additional techniques for analyzing small molecule-protein interactions. A promising method for identifying small molecule-protein interactions is affinity-based selection of yeast surface-displayed human proteome libraries. Large and diverse libraries displaying human protein fragments on the surface of yeast cells have been constructed and subjected to FACS-based enrichment followed by comprehensive exon microarray-based output analysis to identify protein fragments with affinity for small molecule ligands. In a recent example, a proteome-wide search has been successfully carried out to identify cellular proteins binding to the signaling lipids PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Known phosphatidylinositide-binding proteins such as pleckstrin homology domains were identified, as well as many novel interactions. Intriguingly, many novel nuclear phosphatidylinositide-binding proteins were discovered. Although the existence of an independent pool of nuclear phosphatidylinositides has been known about for some time, their functions and mechanism of action remain obscure. Thus, the identification and subsequent study of nuclear phosphatidylinositide-binding proteins is expected to bring new insights to this important biological question. Based on the success with phosphatidylinositides, it is expected that the screening of yeast surface-displayed human proteome libraries will be of general use for the discovery of novel small

  16. Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Sean P Sherman

    Full Text Available Differentiated cells from human embryonic stem cells (hESCs provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

  17. Enhancing bull sexual behavior using estrus-specific molecules identified in cow urine.

    Science.gov (United States)

    Le Danvic, Chrystelle; Gérard, Olivier; Sellem, Eli; Ponsart, Claire; Chemineau, Philippe; Humblot, Patrice; Nagnan-Le Meillour, Patricia

    2015-06-01

    Deficiencies in bull mating behavior have implications for bovine artificial insemination activities. The aim of this study was to identify the compounds present in fluids emitted by cows during estrus, which could enhance bull libido. Chemical analysis of urine samples from cows led to the characterization of molecules varying specifically at the preestrous and estrous stages. The synthetic counterpart molecules (1,2-dichloroethylene, squalene, coumarin, 2-butanone, oleic acid) were used to investigate the biological effects on male sexual behavior and sperm production. When presented to males, 2-butanone and oleic acid synthetic molecules significantly lowered mounting reaction time and ejaculation time (-33% and 21% after 2-butanone inhalation, respectively, P increase of sperm quantity (P < 0.05). This study suggests that the identified estrous-specific molecules could be part of the chemical signals involved in male and female mating behavior and may be used for a wide range of applications. The identification of these molecules may have implications for the cattle breeding industry. PMID:25817331

  18. Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

    Science.gov (United States)

    Wu, Zining; Graybill, Todd L; Zeng, Xin; Platchek, Michael; Zhang, Jean; Bodmer, Vera Q; Wisnoski, David D; Deng, Jianghe; Coppo, Frank T; Yao, Gang; Tamburino, Alex; Scavello, Genaro; Franklin, G Joseph; Mataruse, Sibongile; Bedard, Katie L; Ding, Yun; Chai, Jing; Summerfield, Jennifer; Centrella, Paolo A; Messer, Jeffrey A; Pope, Andrew J; Israel, David I

    2015-12-14

    DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

  19. Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

    Science.gov (United States)

    Gal, Maayan; Bloch, Itai; Shechter, Nelia; Romanenko, Olga; Shir, Ofer M

    2016-01-01

    Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

  20. Cell surface recycling in yeast: mechanisms and machineries.

    Science.gov (United States)

    MacDonald, Chris; Piper, Robert C

    2016-04-15

    Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeastSaccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.

  1. Machine learning models identify molecules active against the Ebola virus in vitro.

    Science.gov (United States)

    Ekins, Sean; Freundlich, Joel S; Clark, Alex M; Anantpadma, Manu; Davey, Robert A; Madrid, Peter

    2015-01-01

    The search for small molecule inhibitors of Ebola virus (EBOV) has led to several high throughput screens over the past 3 years. These have identified a range of FDA-approved active pharmaceutical ingredients (APIs) with anti-EBOV activity in vitro and several of which are also active in a mouse infection model. There are millions of additional commercially-available molecules that could be screened for potential activities as anti-EBOV compounds. One way to prioritize compounds for testing is to generate computational models based on the high throughput screening data and then virtually screen compound libraries. In the current study, we have generated Bayesian machine learning models with viral pseudotype entry assay and the EBOV replication assay data. We have validated the models internally and externally. We have also used these models to computationally score the MicroSource library of drugs to select those likely to be potential inhibitors. Three of the highest scoring molecules that were not in the model training sets, quinacrine, pyronaridine and tilorone, were tested in vitro and had EC 50 values of 350, 420 and 230 nM, respectively. Pyronaridine is a component of a combination therapy for malaria that was recently approved by the European Medicines Agency, which may make it more readily accessible for clinical testing. Like other known antimalarial drugs active against EBOV, it shares the 4-aminoquinoline scaffold. Tilorone, is an investigational antiviral agent that has shown a broad array of biological activities including cell growth inhibition in cancer cells, antifibrotic properties, α7 nicotinic receptor agonist activity, radioprotective activity and activation of hypoxia inducible factor-1. Quinacrine is an antimalarial but also has use as an anthelmintic. Our results suggest data sets with less than 1,000 molecules can produce validated machine learning models that can in turn be utilized to identify novel EBOV inhibitors in vitro. PMID:26834994

  2. Functional dynamics of cell surface membrane proteins

    Science.gov (United States)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  3. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation.

    Science.gov (United States)

    Yang, Zijiang; Concannon, John; Ng, Kelvin S; Seyb, Kathleen; Mortensen, Luke J; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P; Glicksman, Marcie A; Karp, Jeffrey M

    2016-07-26

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy.

  4. Bayesian Source Separation Applied to Identifying Complex Organic Molecules in Space

    CERN Document Server

    Knuth, Kevin H; Choinsky, Joshua; Maunu, Haley A; Carbon, Duane F

    2014-01-01

    Emission from a class of benzene-based molecules known as Polycyclic Aromatic Hydrocarbons (PAHs) dominates the infrared spectrum of star-forming regions. The observed emission appears to arise from the combined emission of numerous PAH species, each with its unique spectrum. Linear superposition of the PAH spectra identifies this problem as a source separation problem. It is, however, of a formidable class of source separation problems given that different PAH sources potentially number in the hundreds, even thousands, and there is only one measured spectral signal for a given astrophysical site. Fortunately, the source spectra of the PAHs are known, but the signal is also contaminated by other spectral sources. We describe our ongoing work in developing Bayesian source separation techniques relying on nested sampling in conjunction with an ON/OFF mechanism enabling simultaneous estimation of the probability that a particular PAH species is present and its contribution to the spectrum.

  5. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    Directory of Open Access Journals (Sweden)

    Stephen R Spindler

    Full Text Available Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR, platelet-derived growth factor (PDGF/vascular endothelial growth factor (VEGF receptors, G-protein coupled receptor (GPCR, Janus kinase (JAK/signal transducer and activator of transcription (STAT, the insulin and insulin-like growth factor (IGFI receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38, c-Jun N-terminal kinase (JNK and protein kinase C (PKC. If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  6. Hypothetical granulin-like molecule from Fasciola hepatica identified by bioinformatics analysis.

    Science.gov (United States)

    Machicado, Claudia; Marcos, Luis A; Zimic, Mirko

    2016-01-01

    Fasciola hepatica is considered an emergent human pathogen, causing liver fibrosis or cirrhosis, conditions that are known to be direct causes of cancer. Some parasites have been categorized by WHO as carcinogenic agents such as Opisthorchis viverrini, a relative of F. hepatica. Although these two parasites are from the same class (Trematoda), the role of F. hepatica in carcinogenesis is unclear. We hypothesized that F. hepatica might share some features with O. viverrini and to be responsible to induce proliferation of host cells. We analyzed the recently released genome of F. hepatica looking for a gene coding a granulin-like growth factor, a protein secreted by O. viverrini (Ov-GRN-1), which is a potent stimulator of proliferation of host cells. Using computational biology tools, we identified a granulin-like molecule in F. hepatica, here termed FhGLM, which has high sequence identity level to Ov-GRN-1 and human progranulin. We found evidence of an upstream promoter compatible with the expression of FhGLM. The FhGLM architecture showed to have five granulin domains, one of them, the domain 3, was homologue to Ov-GRN-1 and human GRNC. The structure of the FhGLM granulin domain 3 resulted to have the overall folding of its homologue the human GRNC. Our findings show the presence of a homologue of a potent modulator of cell growth in F. hepatica that might have, as other granulins, a proliferative action on host cells during fascioliasis. Future experimental assays to demonstrate the presence of FhGLM in F. hepatica are needed to confirm our hypothesis. PMID:27386259

  7. Hypothetical granulin-like molecule from Fasciola hepatica identified by bioinformatics analysis.

    Science.gov (United States)

    Machicado, Claudia; Marcos, Luis A; Zimic, Mirko

    2016-01-01

    Fasciola hepatica is considered an emergent human pathogen, causing liver fibrosis or cirrhosis, conditions that are known to be direct causes of cancer. Some parasites have been categorized by WHO as carcinogenic agents such as Opisthorchis viverrini, a relative of F. hepatica. Although these two parasites are from the same class (Trematoda), the role of F. hepatica in carcinogenesis is unclear. We hypothesized that F. hepatica might share some features with O. viverrini and to be responsible to induce proliferation of host cells. We analyzed the recently released genome of F. hepatica looking for a gene coding a granulin-like growth factor, a protein secreted by O. viverrini (Ov-GRN-1), which is a potent stimulator of proliferation of host cells. Using computational biology tools, we identified a granulin-like molecule in F. hepatica, here termed FhGLM, which has high sequence identity level to Ov-GRN-1 and human progranulin. We found evidence of an upstream promoter compatible with the expression of FhGLM. The FhGLM architecture showed to have five granulin domains, one of them, the domain 3, was homologue to Ov-GRN-1 and human GRNC. The structure of the FhGLM granulin domain 3 resulted to have the overall folding of its homologue the human GRNC. Our findings show the presence of a homologue of a potent modulator of cell growth in F. hepatica that might have, as other granulins, a proliferative action on host cells during fascioliasis. Future experimental assays to demonstrate the presence of FhGLM in F. hepatica are needed to confirm our hypothesis.

  8. The pancreatic beta cell surface proteome

    OpenAIRE

    Stützer, I.; Esterházy, D.; Stoffel, M.

    2012-01-01

    The pancreatic beta cell is responsible for maintaining normoglycaemia by secreting an appropriate amount of insulin according to blood glucose levels. The accurate sensing of the beta cell extracellular environment is therefore crucial to this endocrine function and is transmitted via its cell surface proteome. Various surface proteins that mediate or affect beta cell endocrine function have been identified, including growth factor and cytokine receptors, transporters, ion channels and prote...

  9. Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

    Directory of Open Access Journals (Sweden)

    Christopher Vollmers

    Full Text Available Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

  10. Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

    Science.gov (United States)

    Vollmers, Christopher; Penland, Lolita; Kanbar, Jad N.; Quake, Stephen R.

    2015-01-01

    Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain. PMID:25611855

  11. Target based screening of small molecule library identifies pregnelonene, a Nrf2 agonist, as a potential radioprotector in zebrafish

    International Nuclear Information System (INIS)

    Reactive oxygen species, cellular oxidative stress, tissue inflammation and cell death are the downstream consequences of radiation exposures which ultimately could lead to organism death. Present study aims at identifying potential targets and screening of small molecule compound library for identifying novel and effective radioprotectors. In-silco analysis of known radioprotectors revealed three main function, antioxidant, anti-inflammation and antiapoptosis. In this study, a collection of small molecules (John Hopkins Clinical Compound Library, JHCCL) were screened for these different functions using the biological activity database of NCBI with the help of in-house developed python script. Further, filtering of the JHCCL was done by searching for molecules which are known to be active against target of radiobiological significance, Nrf-2. Close observation of potential hits identified, pregnenolone, as an Nrf-2 agonist which was further evaluated for radioprotection in zebrafish model. Pregnenolone rendered significant protection (at 40 μM; added 1 hour prior to 20 Gy gamma radiation) in terms of damage manifestations (pericardial edema, microcephaly, micropthalmia, yolk sac resorption, curvature of spine, blood flow, body length, heart-beat, blood clot, roughness of skin) and survival advantage (60%) when compared to irradiated control. Further, the ability of pregnenolone to act as a neuroprotectant was also carried out using in-house developed software for assessing neuromotor functions. In comparison to radiation alone group, pregnenolone was found to possess significant neuroactive functions and diminished radiation induced neuronal impairment. Over all these results suggests that pregnenolone is an effective radioprotector which warrants further investigation for validation of its radioprotective action in higher vertebrates. Apart from that the utility of approach to screen out bioactivity data base of various chemical compound libraries for possible

  12. Human lung tumor-associated antigen identified as an extracellular matrix adhesion molecule

    OpenAIRE

    1991-01-01

    A single chain glycoprotein with an estimated molecular mass of 160 kD (gp160) was previously identified as a human lung tumor-associated antigen. This tumor marker is shown here to be associated noncovalently with a second 130-kD protein. Sequential immunoprecipitation studies of surface iodinated lung tumor cell lysates reveal that this heterodimeric complex is indistinguishable serologically and structurally from the integrin VLA-2, found originally on activated T lymphocytes and platelets...

  13. Messina: a novel analysis tool to identify biologically relevant molecules in disease.

    Directory of Open Access Journals (Sweden)

    Mark Pinese

    Full Text Available BACKGROUND: Morphologically similar cancers display heterogeneous patterns of molecular aberrations and follow substantially different clinical courses. This diversity has become the basis for the definition of molecular phenotypes, with significant implications for therapy. Microarray or proteomic expression profiling is conventionally employed to identify disease-associated genes, however, traditional approaches for the analysis of profiling experiments may miss molecular aberrations which define biologically relevant subtypes. METHODOLOGY/PRINCIPAL FINDINGS: Here we present Messina, a method that can identify those genes that only sometimes show aberrant expression in cancer. We demonstrate with simulated data that Messina is highly sensitive and specific when used to identify genes which are aberrantly expressed in only a proportion of cancers, and compare Messina to contemporary analysis techniques. We illustrate Messina by using it to detect the aberrant expression of a gene that may play an important role in pancreatic cancer. CONCLUSIONS/SIGNIFICANCE: Messina allows the detection of genes with profiles typical of markers of molecular subtype, and complements existing methods to assist the identification of such markers. Messina is applicable to any global expression profiling data, and to allow its easy application has been packaged into a freely-available stand-alone software package.

  14. The effect of antibodies against cell-surface adhesion molecules (LFA-1{alpha} and ICAM-1) on the migration and localization of {sup 99m}Tc-labeled leukocytes in acute infection

    Energy Technology Data Exchange (ETDEWEB)

    Amartey, J.K.; Parhar, R.S.; Al-Sedairy, S.T

    1997-08-01

    The deficiency of adhesion molecules on leukocytes could severely impair their ability to migrate and perform effective immunological functions leading to clinical situations such as LAD (leukocyte adhesion deficiency) syndrome. We investigated the effects of blocking anti-LFA-1{alpha} and ICAM-1 antibody-treated {sup 99m}Tc-labeled leukocytes on the migration and localization to the site of E. coli-induced acute infection in CBA/J mice. A significant inhibition of migration and localization of antibody-treated leukocytes to the site of infection was observed, reaffirming the vital role of these adhesion molecules, especially during scintigraphic examination of patients for deep infections or abscess using labeled leukocytes.

  15. Machine learning models identify molecules active against the Ebola virus in vitro [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Sean Ekins

    2016-01-01

    Full Text Available The search for small molecule inhibitors of Ebola virus (EBOV has led to several high throughput screens over the past 3 years. These have identified a range of FDA-approved active pharmaceutical ingredients (APIs with anti-EBOV activity in vitro and several of which are also active in a mouse infection model. There are millions of additional commercially-available molecules that could be screened for potential activities as anti-EBOV compounds. One way to prioritize compounds for testing is to generate computational models based on the high throughput screening data and then virtually screen compound libraries. In the current study, we have generated Bayesian machine learning models with viral pseudotype entry assay and the EBOV replication assay data. We have validated the models internally and externally. We have also used these models to computationally score the MicroSource library of drugs to select those likely to be potential inhibitors. Three of the highest scoring molecules that were not in the model training sets, quinacrine, pyronaridine and tilorone, were tested in vitro and had EC50 values of 350, 420 and 230 nM, respectively. Pyronaridine is a component of a combination therapy for malaria that was recently approved by the European Medicines Agency, which may make it more readily accessible for clinical testing. Like other known antimalarial drugs active against EBOV, it shares the 4-aminoquinoline scaffold. Tilorone, is an investigational antiviral agent that has shown a broad array of biological activities including cell growth inhibition in cancer cells, antifibrotic properties, α7 nicotinic receptor agonist activity, radioprotective activity and activation of hypoxia inducible factor-1. Quinacrine is an antimalarial but also has use as an anthelmintic. Our results suggest data sets with less than 1,000 molecules can produce validated machine learning models that can in turn be utilized to identify novel EBOV inhibitors in

  16. Machine learning models identify molecules active against the Ebola virus in vitro [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Sean Ekins

    2015-10-01

    Full Text Available The search for small molecule inhibitors of Ebola virus (EBOV has led to several high throughput screens over the past 3 years. These have identified a range of FDA-approved active pharmaceutical ingredients (APIs with anti-EBOV activity in vitro and several of which are also active in a mouse infection model. There are millions of additional commercially-available molecules that could be screened for potential activities as anti-EBOV compounds. One way to prioritize compounds for testing is to generate computational models based on the high throughput screening data and then virtually screen compound libraries. In the current study, we have generated Bayesian machine learning models with viral pseudotype entry assay and the EBOV replication assay data. We have validated the models internally and externally. We have also used these models to computationally score the MicroSource library of drugs to select those likely to be potential inhibitors. Three of the highest scoring molecules that were not in the model training sets, quinacrine, pyronaridine and tilorone, were tested in vitro and had EC50 values of 350, 420 and 230 nM, respectively. Pyronaridine is a component of a combination therapy for malaria that was recently approved by the European Medicines Agency, which may make it more readily accessible for clinical testing. Like other known antimalarial drugs active against EBOV, it shares the 4-aminoquinoline scaffold. Tilorone, is an investigational antiviral agent that has shown a broad array of biological activities including cell growth inhibition in cancer cells, antifibrotic properties, α7 nicotinic receptor agonist activity, radioprotective activity and activation of hypoxia inducible factor-1. Quinacrine is an antimalarial but also has use as an anthelmintic. Our results suggest data sets with less than 1,000 molecules can produce validated machine learning models that can in turn be utilized to identify novel EBOV inhibitors in

  17. Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing sarcoma.

    Directory of Open Access Journals (Sweden)

    Kimberly Stegmaier

    2007-04-01

    Full Text Available BACKGROUND: The presence of tumor-specific mutations in the cancer genome represents a potential opportunity for pharmacologic intervention to therapeutic benefit. Unfortunately, many classes of oncoproteins (e.g., transcription factors are not amenable to conventional small-molecule screening. Despite the identification of tumor-specific somatic mutations, most cancer therapy still utilizes nonspecific, cytotoxic drugs. One illustrative example is the treatment of Ewing sarcoma. Although the EWS/FLI oncoprotein, present in the vast majority of Ewing tumors, was characterized over ten years ago, it has never been exploited as a target of therapy. Previously, this target has been intractable to modulation with traditional small-molecule library screening approaches. Here we describe a gene expression-based approach to identify compounds that induce a signature of EWS/FLI attenuation. We hypothesize that screening small-molecule libraries highly enriched for FDA-approved drugs will provide a more rapid path to clinical application. METHODS AND FINDINGS: A gene expression signature for the EWS/FLI off state was determined with microarray expression profiling of Ewing sarcoma cell lines with EWS/FLI-directed RNA interference. A small-molecule library enriched for FDA-approved drugs was screened with a high-throughput, ligation-mediated amplification assay with a fluorescent, bead-based detection. Screening identified cytosine arabinoside (ARA-C as a modulator of EWS/FLI. ARA-C reduced EWS/FLI protein abundance and accordingly diminished cell viability and transformation and abrogated tumor growth in a xenograft model. Given the poor outcomes of many patients with Ewing sarcoma and the well-established ARA-C safety profile, clinical trials testing ARA-C are warranted. CONCLUSIONS: We demonstrate that a gene expression-based approach to small-molecule library screening can identify, for rapid clinical testing, candidate drugs that modulate previously

  18. Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.

    Science.gov (United States)

    Asanov, Alexander; Sampieri, Alicia; Moreno, Claudia; Pacheco, Jonathan; Salgado, Alfonso; Sherry, Ryan; Vaca, Luis

    2015-01-01

    Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

  19. An efficient delivery of DAMPs on the cell surface by the unconventional secretion pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Haiyan; Wang, Lan; Ruan, Yuanyuan; Zhou, Lei; Zhang, Dongmei [Department of Biochemistry and Molecular Biology, Shanghai Medical Collage, Fudan University, Shanghai 200032 (China); Min, Zhihui [Biomedical Research Center, Zhongshan Hospital, Fudan University, Shanghai 200032 (China); Xie, Jianhui [Department of Biochemistry and Molecular Biology, Shanghai Medical Collage, Fudan University, Shanghai 200032 (China); Yu, Min, E-mail: minyu@shmu.edu.cn [Department of Biochemistry and Molecular Biology, Shanghai Medical Collage, Fudan University, Shanghai 200032 (China); Gu, Jianxin [Department of Biochemistry and Molecular Biology, Shanghai Medical Collage, Fudan University, Shanghai 200032 (China)

    2011-01-21

    Research highlights: {yields} Hsp60 transported to cell surface through the classical secretory pathway was modified with N-glycosylation. {yields} HSAPB-N18 could efficiently deliver Hsp60 to the cell surface via the unconventional secretory pathway. {yields} Cell surface Hsp60 delivered by HASPB-N18 has a proper conformation. {yields} HASPB-N18 is an efficient delivery signal for other DAMP molecules such as Hsp70 and HMGB1. -- Abstract: Damage-associated molecular patterns (DAMPs) are signals released from dying cells evoking the immune system response in several inflammatory disorders. In normal situations, many of DAMPs are nuclear or cytosolic proteins with defined intracellular function, but they could be found on the cell surface following tissue injury. The biological function of the translocated DAMPs is still not well known and an efficient delivery of these molecules on the cell surface is required to clarify their biological effects. In this study, we demonstrated that an unclassical secretory signal peptide, N-terminal 18 amino acids of HASPB (HASPB-N18), could efficiently deliver Hsp60, Hsp70, and HMGB1 on the cell surface. Furthermore, the delivery of these molecules on the cell surface by HASPB-N18 is not limited to a special cell line because several cell lines could use this delivery signal to deliver these molecules on the cell surface. Moreover, we demonstrated that Hsp60 on the cell surface delivered by HASPB-N18 could be recognized by a soluble form of LOX-1, which implies that DAMPs on the cell surface delivered by HASPB-N18 have a proper conformation during transport. Therefore, delivery of DAMPs by HASPB-N18 is a reliable model to further understand the biological significance of DAMPs on the cell surface.

  20. Microplate-based assay for identifying small molecules that bind a specific intersubunit interface within the assembled HIV-1 capsid.

    Science.gov (United States)

    Halambage, Upul D; Wong, Jason P; Melancon, Bruce J; Lindsley, Craig W; Aiken, Christopher

    2015-09-01

    Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity. PMID:26077250

  1. Soluble and cell surface receptors for tumor necrosis factor

    DEFF Research Database (Denmark)

    Wallach, D; Engelmann, H; Nophar, Y;

    1991-01-01

    Tumor necrosis factor (TNF) initiates its multiple effects on cell function by binding at a high affinity to specific cell surface receptors. Two different molecular species of these receptors, which are expressed differentially in different cells, have been identified. The cDNAs of both receptor...... have recently been cloned. Antibodies to one of these receptor species (the p55, type I receptor) can trigger a variety of TNF like effects by cross-linking of the receptor molecules. Thus, it is not TNF itself but its receptors that provide the signal for the response to this cytokine....... The intracellular domains of the two receptors differ in structure, suggesting that they mediate different activities. Their extracellular domains, however, are structurally related. Both contain cysteine-rich repeats which are homologous to repeated structures found in the extracellular domains of the nerve growth...... factor receptor and the CDw40 protein. Truncated soluble forms of the two receptors, corresponding to these cysteine-rich repeated structures, have been detected in human urine and were later found to be present also in the serum. The serum levels of those soluble TNF receptors increase dramatically...

  2. Modulators of hepatic lipoprotein metabolism identified in a search for small-molecule inducers of tribbles pseudokinase 1 expression.

    Directory of Open Access Journals (Sweden)

    Marek M Nagiec

    Full Text Available Recent genome wide association studies have linked tribbles pseudokinase 1 (TRIB1 to the risk of coronary artery disease (CAD. Based on the observations that increased expression of TRIB1 reduces secretion of VLDL and is associated with lower plasma levels of LDL cholesterol and triglycerides, higher plasma levels of HDL cholesterol and reduced risk for myocardial infarction, we carried out a high throughput phenotypic screen based on quantitative RT-PCR assay to identify compounds that induce TRIB1 expression in human HepG2 hepatoma cells. In a screen of a collection of diversity-oriented synthesis (DOS-derived compounds, we identified a series of benzofuran-based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 cDNA overexpression, as they inhibit triglyceride synthesis and apoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, key components of the lipoprotein assembly pathway. However, CRISPR-Cas9 induced chromosomal disruption of the TRIB1 locus in HepG2 cells, while confirming its regulatory role in lipoprotein metabolism, demonstrated that the effects of benzofurans persist in TRIB1-null cells indicating that TRIB1 is sufficient but not necessary to transmit the effects of the drug. Remarkably, active benzofurans, as well as natural products capable of TRIB1 upregulation, also modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR transcript and LDL receptor protein, while reducing the levels of PCSK9 transcript and secreted PCSK9 protein and stimulating LDL uptake. The effects of benzofurans are not masked by cholesterol depletion and are independent of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel class of chemically tractable small-molecule modulators that shift cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging.

  3. Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

    Science.gov (United States)

    Kumar, Anand; Drozd, Mary; Pina-Mimbela, Ruby; Xu, Xiulan; Helmy, Yosra A.; Antwi, Janet; Fuchs, James R.; Nislow, Corey; Templeton, Jillian; Blackall, Patrick J.; Rajashekara, Gireesh

    2016-01-01

    Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 μM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 μM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide, and pyridazinone. Exploitation of analogs of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine. PMID:27092106

  4. Use of a Machine Learning-Based High Content Analysis Approach to Identify Photoreceptor Neurite Promoting Molecules.

    Science.gov (United States)

    Fuller, John A; Berlinicke, Cynthia A; Inglese, James; Zack, Donald J

    2016-01-01

    High content analysis (HCA) has become a leading methodology in phenotypic drug discovery efforts. Typical HCA workflows include imaging cells using an automated microscope and analyzing the data using algorithms designed to quantify one or more specific phenotypes of interest. Due to the richness of high content data, unappreciated phenotypic changes may be discovered in existing image sets using interactive machine-learning based software systems. Primary postnatal day four retinal cells from the photoreceptor (PR) labeled QRX-EGFP reporter mice were isolated, seeded, treated with a set of 234 profiled kinase inhibitors and then cultured for 1 week. The cells were imaged with an Acumen plate-based laser cytometer to determine the number and intensity of GFP-expressing, i.e. PR, cells. Wells displaying intensities and counts above threshold values of interest were re-imaged at a higher resolution with an INCell2000 automated microscope. The images were analyzed with an open source HCA analysis tool, PhenoRipper (Rajaram et al., Nat Methods 9:635-637, 2012), to identify the high GFP-inducing treatments that additionally resulted in diverse phenotypes compared to the vehicle control samples. The pyrimidinopyrimidone kinase inhibitor CHEMBL-1766490, a pan kinase inhibitor whose major known targets are p38α and the Src family member lck, was identified as an inducer of photoreceptor neuritogenesis by using the open-source HCA program PhenoRipper. This finding was corroborated using a cell-based method of image analysis that measures quantitative differences in the mean neurite length in GFP expressing cells. Interacting with data using machine learning algorithms may complement traditional HCA approaches by leading to the discovery of small molecule-induced cellular phenotypes in addition to those upon which the investigator is initially focusing.

  5. Identification of biological markers of liver X receptor (LXR activation at the cell surface of human monocytes.

    Directory of Open Access Journals (Sweden)

    Cédric Rébé

    Full Text Available BACKGROUND: Liver X receptor (LXR α and LXR β (NR1H3 and NR1H2 are oxysterol-activated nuclear receptors involved in the control of major metabolic pathways such as cholesterol homeostasis, lipogenesis, inflammation and innate immunity. Synthetic LXR agonists are currently under development and could find applications in various fields such as cardiovascular diseases, cancer, diabetes and neurodegenerative diseases. The clinical development of LXR agonists requires the identification of biological markers for pharmacodynamic studies. In this context, monocytes represent an attractive target to monitor LXR activation. They are easily accessible cells present in peripheral blood; they express LXR α and β and respond to LXR agonist stimulation in vitro. The aim of our study was to identify cell surface markers of LXR agonists on monocytes. For this, we focused on clusters of differentiation (CD markers because they are well characterized and accessible cell surface molecules allowing easy immuno-phenotyping. METHODOLOGY/PRINCIPAL FINDINGS: By using microarray analysis of monocytes treated or not with an LXR agonist in vitro, we selected three CD, i.e. CD82, CD226, CD244 for further analysis by real time PCR and flow cytometry. The three CD were up-regulated by LXR agonist treatment in vitro in a time- and dose- dependent manner and this induction was LXR specific as assessed by a SiRNA or LXR antagonist strategy. By using flow cytometry, we could demonstrate that the expression of these molecules at the cell surface of monocytes was significantly increased after LXR agonist treatment. CONCLUSIONS/SIGNIFICANCE: We have identified three new cell surface markers that could be useful to monitor LXR activation. Future studies will be required to confirm the biological and diagnostic significance of the markers.

  6. The cell surface of Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Wanderley de Souza

    1984-01-01

    Full Text Available The cell surface of trypanosomatids is formed by the plasma membrane and a layer of sub-pellicular microtubules which are connected to the plasma membrane. The plasma membrane is composed by proteins, lipids and carbohydrates which form the glycocalix. In this paper we will review briefly aspects related to the organization of the cell surface of Trypanosoma cruzi.

  7. Monte Carlo calculations of the free energy of binary sII hydrogen clathrate hydrates for identifying efficient promoter molecules.

    Science.gov (United States)

    Atamas, Alexander A; Cuppen, Herma M; Koudriachova, Marina V; de Leeuw, Simon W

    2013-01-31

    The thermodynamics of binary sII hydrogen clathrates with secondary guest molecules is studied with Monte Carlo simulations. The small cages of the sII unit cell are occupied by one H(2) guest molecule. Different promoter molecules entrapped in the large cages are considered. Simulations are conducted at a pressure of 1000 atm in a temperature range of 233-293 K. To determine the stabilizing effect of different promoter molecules on the clathrate, the Gibbs free energy of fully and partially occupied sII hydrogen clathrates are calculated. Our aim is to predict what would be an efficient promoter molecule using properties such as size, dipole moment, and hydrogen bonding capability. The gas clathrate configurational and free energies are compared. The entropy makes a considerable contribution to the free energy and should be taken into account in determining stability conditions of binary sII hydrogen clathrates.

  8. Distinct roles for dystroglycan, beta1 integrin and perlecan in cell surface laminin organization

    DEFF Research Database (Denmark)

    Henry, M D; Satz, J S; Brakebusch, C;

    2001-01-01

    integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells......, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential......Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface...

  9. Characterization of pellicle inhibition in Gluconacetobacter xylinus 53582 by a small molecule, pellicin, identified by a chemical genetics screen.

    Directory of Open Access Journals (Sweden)

    Janice L Strap

    Full Text Available Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6-benzodioxocin-8-yl]prop-2-en-1-one was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus.

  10. Hexameric Capsules Studied by Magic Angle Spinning Solid-State NMR Spectroscopy: Identifying Solvent Molecules in Pyrogallol[4]arene Capsules.

    Science.gov (United States)

    Avram, Liat; Goldbourt, Amir; Cohen, Yoram

    2016-01-18

    Powders of pyrogallol[4]arene hexamers were produced by evaporation from organic solvents and were studied, for the first time, by magic angle spinning solid-state NMR (MAS ssNMR). Evaporation selectively removed non-encapsulated solvent molecules leaving stable hexameric capsules encapsulating solvent molecules. After exposure of the powder to solvent vapors, (1)H/(13)C heteronuclear correlation MAS ssNMR experiments were used to assign the signals of the external and encapsulated solvent molecules. The formed capsules were stable for months and the process of solvent encapsulation was reversible. According to the ssNMR experiments, the encapsulated solvent molecules occupy different sites and those sites differ in their mobility. The presented approach paves the way for studying guest exchange, guest affinity, and gas storage in hexamers of this type in the solid state.

  11. Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions

    DEFF Research Database (Denmark)

    Tumova, S; Woods, A; Couchman, J R

    2000-01-01

    Heparan sulfate proteoglycans are complex molecules composed of a core protein with covalently attached glycosaminoglycan chains. While the protein part determines localization of the proteoglycan on the cell surfaces or in the extracellular matrix, the glycosaminoglycan component, heparan sulfate...... and wound repair. This review concentrates on biological roles of cell surface heparan sulfate proteoglycans, namely syndecans and glypicans, and outlines the progress achieved during the last decade in unraveling the molecular interactions behind proteoglycan functions....

  12. Cell-surface remodelling during mammalian erythropoiesis.

    Science.gov (United States)

    Wraith, D C; Chesterton, C J

    1982-10-15

    Current evidence suggests that the major cell-surface modification occurring during mammalian erythropoiesis could be generated by two separate mechanisms: either selective loss of membrane proteins during enucleation or endocytosis at the subsequent reticulocyte and erythrocyte stages. The former idea was tested by collecting developing rabbit erythroid cells before and after the enucleation step and comparing their cell-surface protein composition via radiolabelling and electrophoresis. Few changes were observed. Our data thus lend support to the endocytosis mechanism.

  13. Distribution, Arrangement and Interconnectedness of Cell Surface Receptor sites in the body of an Organism

    Directory of Open Access Journals (Sweden)

    Utoh-Nedosa

    2011-01-01

    Full Text Available Cell surface receptors have been identified as the sites of disease infectivity in living organisms in a previous study. Drugs used for the treatment or cure of infections have to eliminate infections through attacking infective organisms at the cell surface receptors to which the infective organisms are attached. Problem statement: The present study examines a wide sample of living things to get more information on the relationship of one cell surface receptor to other cell surface receptors in the body of an organism. Approach: The arrangement of cell surface receptors on the external covering of a few samples of fruits, leaves, stems, dry wood of a plant; wall gecko and some parts of the human body, were examined and photographed. Transverse and/or Longitudinal sections of soursop fruit and sycamore fruit were also examined and photographed. The five different coverings of the fleshy part of a coconut were also photographed. The photographs were studied to note the relationship of disease infection attached to cell surface receptors on the external surface of an organ to disease infection on the innermost covering of the same organ. Results: The results of the study showed that all living things had ubiquitous distribution of cell surface receptors which are usually observable with the unaided eye as dots or spots on the external covering of an organ, tissue or cell. The dots or receptor sites of cell surface receptors in the study are arranged in lines which were perpendicular, oblique, transverse or arranged in any other lineal geometrical form. The lineally arranged cell surface receptors were noted to be connected by grooves, channels or pipes which joined other receptor channels or intersected with them. Smaller cell surface receptor channels emptied into bigger channels or continued as small sized channels that ran side by side in a connective tissue bundle. These connective tissue bundles that carried many independent small-sized cell

  14. Engineering novel cell surface chemistry for selective tumor cell targeting

    Energy Technology Data Exchange (ETDEWEB)

    Bertozzi, C.R. [Univ. of California, Berkeley, CA (United States)]|[Lawrence Berkeley National Lab., CA (United States)

    1997-12-31

    A common feature of many different cancers is the high expression level of the two monosaccharides sialic acid and fucose within the context of cell-surface associated glycoconjugates. A correlation has been made between hypersialylation and/or hyperfucosylation and the highly metastatic phenotype. Thus, a targeting strategy based on sialic acid or fucose expression would be a powerful tool for the development of new cancer cell-selective therapies and diagnostic agents. We have discovered that ketone groups can be incorporated metabolically into cell-surface associated sialic acids. The ketone is can be covalently ligated with hydrazide functionalized proteins or small molecules under physiological conditions. Thus, we have discovered a mechanism to selectively target hydrazide conjugates to highly sialylated cells such as cancer cells. Applications of this technology to the generation of novel cancer cell-selective toxins and MRI contrast reagents will be discussed, in addition to progress towards the use of cell surface fucose residues as vehicles for ketone expression.

  15. Collisional relaxation of apocarotenals: identifying the S* state with vibrationally excited molecules in the ground electronic state S(0)*.

    Science.gov (United States)

    Ehlers, Florian; Scholz, Mirko; Schimpfhauser, Jens; Bienert, Jürgen; Oum, Kawon; Lenzer, Thomas

    2015-04-28

    In recent work, we demonstrated that the S* signal of β-carotene observed in transient pump-supercontinuum probe absorption experiments agrees well with the independently measured steady-state difference absorption spectrum of vibrationally hot ground state molecules S0* in solution, recorded at elevated temperatures (Oum et al., Phys. Chem. Chem. Phys., 2010, 12, 8832). Here, we extend our support for this "vibrationally hot ground state model" of S* by experiments for the three terminally aldehyde-substituted carotenes β-apo-12'-carotenal, β-apo-4'-carotenal and 3',4'-didehydro-β,ψ-caroten-16'-al ("torularhodinaldehyde") which were investigated by ultrafast pump-supercontinuum probe spectroscopy in the range 350-770 nm. The apocarotenals feature an increasing conjugation length, resulting in a systematically shorter S1 lifetime of 192, 4.9 and 1.2 ps, respectively, in the solvent n-hexane. Consequently, for torularhodinaldehyde a large population of highly vibrationally excited molecules in the ground electronic state is quickly generated by internal conversion (IC) from S1 already within the first picosecond of relaxation. As a result, a clear S* signal is visible which exhibits the same spectral characteristics as in the aforementioned study of β-carotene: a pronounced S0 → S2 red-edge absorption and a "finger-type" structure in the S0 → S2 bleach region. The cooling process is described in a simplified way by assuming an initially formed vibrationally very hot species S0** which subsequently decays with a time constant of 3.4 ps to form a still hot S0* species which relaxes with a time constant of 10.5 ps to form S0 molecules at 298 K. β-Apo-4'-carotenal behaves in a quite similar way. Here, a single vibrationally hot S0* species is sufficient in the kinetic modeling procedure. S0* relaxes with a time constant of 12.1 ps to form cold S0. Finally, no S0* features are visible for β-apo-12'-carotenal. In that case, the S1 → S0 IC process is expected

  16. Targeting of MAPK-associated molecules identifies SON as a prime target to attenuate the proliferation and tumorigenicity of pancreatic cancer cells

    Directory of Open Access Journals (Sweden)

    Furukawa Toru

    2012-12-01

    Full Text Available Abstract Background Pancreatic cancer is characterized by constitutive activation of mitogen-activated protein kinase (MAPK. Activation of MAPK is associated with the upregulation of genes implicated in the proliferation and survival of pancreatic cancer cells. We hypothesized that knockdown of these MAPK-associated molecules could produce notable anticancer phenotypes. Methods A RNA interference-mediated knockdown screening of 78 MAPK-associated molecules previously identified was performed to find molecules specifically associated with proliferation of pancreatic cancer cells in vitro. Expression of an identified molecule in pancreatic cancer tissues was examined by immunohistochemistry. In vivo tumorigenicity of cancer cells with stable knockdown of the molecule was assayed by using xenograft models. Flow cytometry and live cell imaging were employed to assess an association of the molecule with cell cycle. Results The knockdown screening revealed that knockdown of SON, the gene encoding SON, which is a large serine/arginine-rich protein involved in RNA processing, substantially suppressed pancreatic cancer cell proliferation and survival in vitro and tumorigenicity in vivo. SON expression was higher in ductal adenocarcinomas than in cells of normal ducts and precursor lesions in pancreatic cancer tissues. Knockdown of SON induced G2/M arrest and apoptosis in cultured cancer cells. The suppressive effect of SON knockdown on proliferation was less pronounced in cultured normal duct epithelial cells. SON formed nuclear speckles in the interphase of the cell cycle and dispersed in the cytoplasm during mitosis. Live cell imaging showed that SON diffusely dispersed in the early mitotic phase, accumulated in some foci in the cytoplasm in the late mitotic phase, and gradually reassembled into speckles after mitosis. Conclusion These results indicate that SON plays a critical role in the proliferation, survival, and tumorigenicity of pancreatic

  17. The yeast three-hybrid system as an experimental platform to identify proteins interacting with small signaling molecules in plant cells: Potential and limitations

    Directory of Open Access Journals (Sweden)

    Stéphanie eCottier

    2011-12-01

    Full Text Available Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx. In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA binding domain (encoded in the yeast strain, and the bioactive molecule part binding to its potential protein target fused to a DNA activating domain (encoded on a cDNA expression vector. During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discussed the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules.

  18. Identifying site-dependent effects of an extra Co atom on electronic states of single Co-phthalocyanine molecule

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jingcheng; Wang, Yu [Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China); Li, Bin, E-mail: libin@mail.ustc.edu.cn, E-mail: bwang@ustc.edu.cn; Zhao, Aidi; Wang, Bing, E-mail: libin@mail.ustc.edu.cn, E-mail: bwang@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China); Synergetic Innovation Center of Quantum Information and Quantum Physics, University of Science and Technology of China, Hefei, Anhui 230026 (China)

    2015-07-21

    We investigate the modification of electronic properties of single cobalt phthalocyanine (CoPc) molecule by an extra Co atom co-adsorbed on Au (111) surface using scanning tunneling microscopy (STM), joint with density functional theory (DFT) calculations. By manipulating CoPc molecules using the STM tip to contact individually adsorbed Co atom, two types of relatively stable complexes can be formed, denoted as CoPc-Co(I) and CoPc-Co(II). In CoPc-Co(I), the Co atom is at an intramolecular site close to aza-N atom of CoPc, which induces significant modifications of the electronic states of CoPc, such as energy shifts and splitting of nonlocal molecular orbitals. However, in CoPc-Co(II) where the Co atom is underneath a benzene lobe of CoPc, it only slightly modifies the electronic states of CoPc, and mainly local characteristics of specific molecular orbitals are affected, even though CoPc-Co(II) is more stable than CoPc-Co(I). Our DFT calculations give consistent results with the experiments, and related analyses based on the molecular orbital theory reveal mechanism behind the experimental observations.

  19. Identifying site-dependent effects of an extra Co atom on electronic states of single Co-phthalocyanine molecule.

    Science.gov (United States)

    Li, Jingcheng; Li, Bin; Wang, Yu; Zhao, Aidi; Wang, Bing

    2015-07-21

    We investigate the modification of electronic properties of single cobalt phthalocyanine (CoPc) molecule by an extra Co atom co-adsorbed on Au (111) surface using scanning tunneling microscopy (STM), joint with density functional theory (DFT) calculations. By manipulating CoPc molecules using the STM tip to contact individually adsorbed Co atom, two types of relatively stable complexes can be formed, denoted as CoPc-Co(I) and CoPc-Co(II). In CoPc-Co(I), the Co atom is at an intramolecular site close to aza-N atom of CoPc, which induces significant modifications of the electronic states of CoPc, such as energy shifts and splitting of nonlocal molecular orbitals. However, in CoPc-Co(II) where the Co atom is underneath a benzene lobe of CoPc, it only slightly modifies the electronic states of CoPc, and mainly local characteristics of specific molecular orbitals are affected, even though CoPc-Co(II) is more stable than CoPc-Co(I). Our DFT calculations give consistent results with the experiments, and related analyses based on the molecular orbital theory reveal mechanism behind the experimental observations. PMID:26203036

  20. Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

    DEFF Research Database (Denmark)

    Couchman, J R; Austria, R; Woods, A;

    1988-01-01

    sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from......In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...

  1. NSC114792, a novel small molecule identified through structure-based computational database screening, selectively inhibits JAK3

    OpenAIRE

    Kitamura Daisuke; Jayabose Somasundaram; Sandoval Claudio; Yin Chang-Hong; Jee Jun-Goo; Kim Byung-Hak; Bach Erika A; Baeg Gyeong-Hun

    2010-01-01

    Abstract Background Human or animals lacking either JAK3 or the common gamma chain (γc) expression display severe combined immunodeficiency disease, indicating the crucial role of JAK3 in T-cell development and the homeostasis of the immune system. JAK3 has also been suggested to contribute to the pathogenesis of tumorigenesis. Recent studies identified activating JAK3 mutations in patients with various hematopoietic malignancies, including acute megakaryoblastic leukemia. Importantly, functi...

  2. Implementation of a high-throughput screen for identifying small molecules to activate the Keap1-Nrf2-ARE pathway.

    Directory of Open Access Journals (Sweden)

    Kai Connie Wu

    Full Text Available Nuclear factor erythroid 2-related factor 2 (Nrf2 is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01-30 µM. Of these 238 compounds, 231 (96% increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones. In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.

  3. The AstroBiology Explorer (ABE) MIDEX Mission Concept: Using Infrared Spectroscopy to Identify Organic Molecules in Space

    Science.gov (United States)

    Sandford, Scott A.; Ennico, Kimberly; Allamandola, Louis; Bregman, Jesse; Greene, Thomas; Hudgins, Douglas

    2002-01-01

    One of the principal means by which organic compounds are detected and identified in space is by infrared spectroscopy. Past IR telescopic and laboratory studies have shown that much of the carbon in the interstellar medium (ISM) is in complex organic species but the distribution, abundance and evolutionary relationships of these materials are not well understood. The Astrobiology Explorer (ABE) is a MIDEX mission concept designed to conduct IR spectroscopic observations to detect and identify these materials and address outstanding problems in astrobiology, astrochemistry, and astrophysics. ABE's core science program includes observations of planetary nebulae and stellar outflows, protostellar objects, Solar System objects, and galaxies, and lines of sight through dense molecular clouds and the diffuse ISM. ABE is a cryogenically-cooled 60 cm diameter space telescope equipped with 3 cross-dispersed R-2000 spectrometers that share a single common slit. Each spectrometer measures one spectral octave and together cover the entire 2.5-20 micron region simultaneously. The spectrometers use state-of-the-art InSb and Si:As 1024x1024 pixel detectors. ABE would operate in a heliocentric, Earth drift-away orbit and have a core science mission lasting approximately 1.5 years. ABE is currently under study at NASA's Ames Research Center in collaboration with Ball Aerospace and Technologies Corp.

  4. Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design.

    Directory of Open Access Journals (Sweden)

    Thomas S Peat

    Full Text Available A fragment-based screen against human immunodeficiency virus type 1 (HIV integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.

  5. Analysis of endogenous peptides bound by soluble MHC class I molecules: a novel approach for identifying tumor-specific antigens.

    Science.gov (United States)

    Barnea, Eilon; Beer, Ilan; Patoka, Renana; Ziv, Tamar; Kessler, Ofra; Tzehoval, Esther; Eisenbach, Lea; Zavazava, Nicholas; Admon, Arie

    2002-01-01

    The Human MHC Project aims at comprehensive cataloging of peptides presented within the context of different human leukocyte antigens (HLA) expressed by cells of various tissue origins, both in health and in disease. Of major interest are peptides presented on cancer cells, which include peptides derived from tumor antigens that are of interest for immunotherapy. Here, HLA-restricted tumor-specific antigens were identified by transfecting human breast, ovarian and prostate tumor cell lines with truncated genes of HLA-A2 and HLA-B7. Soluble HLA secreted by these cell lines were purified by affinity chromatography and analyzed by nano-capillary electrospray ionization-tandem mass spectrometry. Typically, a large peptide pool was recovered and sequenced including peptides derived from MAGE-B2 and mucin and other new tumor-derived antigens that may serve as potential candidates for immunotherapy. PMID:11782012

  6. FINDSITE(X): a structure-based, small molecule virtual screening approach with application to all identified human GPCRs.

    Science.gov (United States)

    Zhou, Hongyi; Skolnick, Jeffrey

    2012-06-01

    We have developed FINDSITE(X), an extension of FINDSITE, a protein threading based algorithm for the inference of protein binding sites, biochemical function and virtual ligand screening, that removes the limitation that holo protein structures (those containing bound ligands) of a sufficiently large set of distant evolutionarily related proteins to the target be solved; rather, predicted protein structures and experimental ligand binding information are employed. To provide the predicted protein structures, a fast and accurate version of our recently developed TASSER(VMT), TASSER(VMT)-lite, for template-based protein structural modeling applicable up to 1000 residues is developed and tested, with comparable performance to the top CASP9 servers. Then, a hybrid approach that combines structure alignments with an evolutionary similarity score for identifying functional relationships between target and proteins with binding data has been developed. By way of illustration, FINDSITE(X) is applied to 998 identified human G-protein coupled receptors (GPCRs). First, TASSER(VMT)-lite provides updates of all human GPCR structures previously modeled in our lab. We then use these structures and the new function similarity detection algorithm to screen all human GPCRs against the ZINC8 nonredundant (TC identity > 30% to the target from the binding data library) on a 168 human GPCR set with known binding data, the average enrichment factor in the top 1% of the compound library (EF(0.01)) is 22.7, whereas EF(0.01) by FINDSITE is 7.1. For virtual screening when just the target and its native ligands are excluded, the average EF(0.01) reaches 41.4. We also analyze off-target interactions for the 168 protein test set. All predicted structures, virtual screening data and off-target interactions for the 998 human GPCRs are available at http://cssb.biology.gatech.edu/skolnick/webservice/gpcr/index.html .

  7. Two cell surface proteins bind the sponge Microciona prolifera aggregation factor.

    Science.gov (United States)

    Varner, J A; Burger, M M; Kaufman, J F

    1988-06-15

    Two extracellular matrix cell surface proteins which bind the proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) and which may function as physiological receptors for MAF were identified and characterized for the first time. By probing nitrocellulose blots of nonreducing sodium dodecyl sulfate gels containing whole sponge cell protein with iodinated MAF, a 210- and a 68-kDa protein, which have native molecular masses of approximately 200-400 and 70 kDa, were identified. MAF binding to blots is species-specific. It is also sensitive to reduction and is completely abolished by pretreatment of live cells with proteases, as was cellular aggregation, indicating that the 210- and 68-kDa proteins may be located on the cell surface. The additional observations that the 68 kDa is an endoglycosidase F-sensitive glycoprotein and that antisera against whole sponge cells or membranes can immunoprecipitate the 210 kDa when prebound to intact cells are consistent with a cell surface location. Both proteins can be isolated from sponge cell membranes and from the sponge skeleton (insoluble extracellular matrix), but the 210-kDa MAF-binding protein can also be found in the soluble extracellular matrix (buffer washes of cells and skeleton) as well. A third MAF-binding protein of molecular mass 95 kDa was also found in the sponge extracellular matrix but rarely on cells. Both of the cell-associated 210- and 68-kDa proteins are nonintegral membrane proteins, based on Triton X-114 phase separation, flotation of liposomes containing sponge membrane lysates, and their extraction from membranes by buffer washes. Both proteins bind MAF affinity resins, indicating that they each exhibit a moderate affinity for MAF under native conditions. They can also be separated from each other and from the bulk of the protein in an octylpolyoxyethylene extract of membranes by fast protein liquid chromatography Mono Q anion exchange chromatography, as assessed by native

  8. Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase

    OpenAIRE

    Swartz, Kenneth; ZHANG, YIGUAN; Valeriote, Frederick; Chen, Ben; SHAW, JIAJIU

    2013-01-01

    UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25 °C. At predetermined time points, individual ...

  9. Nanotomography of Cell Surfaces with Evanescent Fields

    Directory of Open Access Journals (Sweden)

    Michael Wagner

    2008-01-01

    Full Text Available The technique of variable-angle total internal reflection fluorescence microscopy (TIRFM and its application to nanotomography of cell surfaces are described. Present applications include (1 3D imaging of chromosomes in their metaphase to demonstrate axial resolution in the nanometre range, (2 measurements of cell-substrate topology, which upon cholesterol depletion shows some loosening of cell-substrate contacts, and (3 measurements of cell topology upon photodynamic therapy (PDT, which demonstrate cell swelling and maintenance of focal contacts. The potential of the method for in vitro diagnostics, but also some requirements and limitations are discussed.

  10. A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling.

    Directory of Open Access Journals (Sweden)

    Evelien Gebruers

    Full Text Available Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen - Jasminum gilgianum, an Oleaceae species native to Papua New Guinea - induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125 phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME's mechanism of action will help determine this compound's pharmacological utility.

  11. FolR1: a novel cell surface marker for isolating midbrain dopamine neural progenitors and nascent dopamine neurons

    Science.gov (United States)

    Gennet, Nicole; Tamburini, Claudia; Nan, Xinsheng; Li, Meng

    2016-01-01

    Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson’s disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1), a GPI-anchored cell surface molecule, specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a, a bona-fide mesDA lineage marker, during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development, as well as in ESC-derived mesDA lineage. FolR1+ neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3, whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons. PMID:27580818

  12. Cytomegalovirus m154 hinders CD48 cell-surface expression and promotes viral escape from host natural killer cell control.

    Directory of Open Access Journals (Sweden)

    Angela Zarama

    2014-03-01

    Full Text Available Receptors of the signalling lymphocyte-activation molecules (SLAM family are involved in the functional regulation of a variety of immune cells upon engagement through homotypic or heterotypic interactions amongst them. Here we show that murine cytomegalovirus (MCMV dampens the surface expression of several SLAM receptors during the course of the infection of macrophages. By screening a panel of MCMV deletion mutants, we identified m154 as an immunoevasin that effectively reduces the cell-surface expression of the SLAM family member CD48, a high-affinity ligand for natural killer (NK and cytotoxic T cell receptor CD244. m154 is a mucin-like protein, expressed with early kinetics, which can be found at the cell surface of the infected cell. During infection, m154 leads to proteolytic degradation of CD48. This viral protein interferes with the NK cell cytotoxicity triggered by MCMV-infected macrophages. In addition, we demonstrate that an MCMV mutant virus lacking m154 expression results in an attenuated phenotype in vivo, which can be substantially restored after NK cell depletion in mice. This is the first description of a viral gene capable of downregulating CD48. Our novel findings define m154 as an important player in MCMV innate immune regulation.

  13. FolR1: a novel cell surface marker for isolating midbrain dopamine neural progenitors and nascent dopamine neurons.

    Science.gov (United States)

    Gennet, Nicole; Tamburini, Claudia; Nan, Xinsheng; Li, Meng

    2016-01-01

    Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson's disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1), a GPI-anchored cell surface molecule, specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a, a bona-fide mesDA lineage marker, during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development, as well as in ESC-derived mesDA lineage. FolR1(+) neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3, whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons. PMID:27580818

  14. Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

    Science.gov (United States)

    Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

    2015-01-13

    The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy.

  15. Highly Sensitive Detection of Target Biomolecules on Cell Surface Using Gold Nanoparticle Conjugated with Aptamer Probe

    Science.gov (United States)

    Kim, Hyonchol; Terazono, Hideyuki; Hayashi, Masahito; Takei, Hiroyuki; Yasuda, Kenji

    2012-06-01

    A method of gold nanoparticle (Au NP) labeling with backscattered electron (BE) imaging of field emission scanning electron microscopy (FE-SEM) was applied for specific detection of target biomolecules on a cell surface. A single-stranded DNA aptamer, which specifically binds to the target molecule on a human acute lymphoblastic leukemia cell, was conjugated with a 20 nm Au NP and used as a probe to label its target molecule on the cell. The Au NP probe was incubated with the cell, and the interaction was confirmed using BE imaging of FE-SEM through direct counting of the number of Au NPs attached on the target cell surface. Specific Au NP-aptamer probes were observed on a single cell surface and their spatial distributions including submicron-order localizations were also clearly visualized, whereas the nonspecific aptamer probes were not observed on it. The aptamer probe can be potentially dislodged from the cell surface with treatment of nucleases, indicating that Au NP-conjugated aptamer probes can be used as sensitive and reversible probes to label target biomolecules on cells.

  16. Novel eukaryotic enzymes modifying cell-surface biopolymers

    Directory of Open Access Journals (Sweden)

    Aravind L

    2010-01-01

    Full Text Available Abstract Background Eukaryotic extracellular matrices such as proteoglycans, sclerotinized structures, mucus, external tests, capsules, cell walls and waxes contain highly modified proteins, glycans and other composite biopolymers. Using comparative genomics and sequence profile analysis we identify several novel enzymes that could be potentially involved in the modification of cell-surface glycans or glycoproteins. Results Using sequence analysis and conservation we define the acyltransferase domain prototyped by the fungal Cas1p proteins, identify its active site residues and unify them to the superfamily of classical 10TM acyltransferases (e.g. oatA. We also identify a novel family of esterases (prototyped by the previously uncharacterized N-terminal domain of Cas1p that have a similar fold as the SGNH/GDSL esterases but differ from them in their conservation pattern. Conclusions We posit that the combined action of the acyltransferase and esterase domain plays an important role in controlling the acylation levels of glycans and thereby regulates their physico-chemical properties such as hygroscopicity, resistance to enzymatic hydrolysis and physical strength. We present evidence that the action of these novel enzymes on glycans might play an important role in host-pathogen interaction of plants, fungi and metazoans. We present evidence that in plants (e.g. PMR5 and ESK1 the regulation of carbohydrate acylation by these acylesterases might also play an important role in regulation of transpiration and stress resistance. We also identify a subfamily of these esterases in metazoans (e.g. C7orf58, which are fused to an ATP-grasp amino acid ligase domain that is predicted to catalyze, in certain animals, modification of cell surface polymers by amino acid or peptides. Reviewers This article was reviewed by Gaspar Jekely and Frank Eisenhaber

  17. Novel eukaryotic enzymes modifying cell-surface biopolymers

    Science.gov (United States)

    2010-01-01

    Background Eukaryotic extracellular matrices such as proteoglycans, sclerotinized structures, mucus, external tests, capsules, cell walls and waxes contain highly modified proteins, glycans and other composite biopolymers. Using comparative genomics and sequence profile analysis we identify several novel enzymes that could be potentially involved in the modification of cell-surface glycans or glycoproteins. Results Using sequence analysis and conservation we define the acyltransferase domain prototyped by the fungal Cas1p proteins, identify its active site residues and unify them to the superfamily of classical 10TM acyltransferases (e.g. oatA). We also identify a novel family of esterases (prototyped by the previously uncharacterized N-terminal domain of Cas1p) that have a similar fold as the SGNH/GDSL esterases but differ from them in their conservation pattern. Conclusions We posit that the combined action of the acyltransferase and esterase domain plays an important role in controlling the acylation levels of glycans and thereby regulates their physico-chemical properties such as hygroscopicity, resistance to enzymatic hydrolysis and physical strength. We present evidence that the action of these novel enzymes on glycans might play an important role in host-pathogen interaction of plants, fungi and metazoans. We present evidence that in plants (e.g. PMR5 and ESK1) the regulation of carbohydrate acylation by these acylesterases might also play an important role in regulation of transpiration and stress resistance. We also identify a subfamily of these esterases in metazoans (e.g. C7orf58), which are fused to an ATP-grasp amino acid ligase domain that is predicted to catalyze, in certain animals, modification of cell surface polymers by amino acid or peptides. Reviewers This article was reviewed by Gaspar Jekely and Frank Eisenhaber PMID:20056006

  18. MHC-like molecules in some nonmammalian vertebrates can be detected by some cross-reactive xenoantisera

    DEFF Research Database (Denmark)

    Kaufman, J; Skjoedt, K; Salomonsen, J;

    1990-01-01

    in the contact regions between the chains. These latter antibodies recognized biosynthetic intermediates and also a variety of unusual cell surface MHC-like molecules present in reptile and amphibian, but absent in the mammal and chicken cells tested. These included E homodimers whose relationship to chicken B......-G molecules is unknown. 5) MHC-like molecules were identified in a bird, three reptiles, and two amphibians, but no molecules with the expected properties were found with these reagents in any of the fish tested. Udgivelsesdato: 1990-Mar-15...

  19. Heterogeneity of cell adhesion molecules in the developing nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Williams, R.K.

    1985-01-01

    Cell-surface molecules, especially glycoproteins, are believed to mediate interactions between developing neurons and their environment. These interactions include pathfinding by growing processes, recognition of appropriate targets, and formation of synaptic structures. In order to identify neuronal cell-surface molecules, monoclonal antibodies (Mab's) were prepared against synaptic fractions from adult rat brain. From this group three monoclonal antibodies, designated 3C5.59, 3G5.34, and 3G6.41, that react with cell-surface antigens of embryonic neurons were selected for further study. In immunofluoresence experiments each of these antibodies strongly reacted with the processes of cultured granule cell neurons, the major class of small cerebellar neurons, cultured from developing rat cerebellum. Mab's 3C5.59 and 3G5.34 reacted only with neurons in the cerebellar cultures. Mab 3G6.41, however, also reacted with cultured brain astrocytes. On frozen sections Mab's 3G5.34 and 3G6.41 also strongly stained the molecular layer, the site of active granule cell axon growth, in the developing cerebellum. Monoclonal and polyclonal antibodies specific for the neural cell adhesion molecule (N-CAM) were used to compare the two glycoproteins recognized by Mab 3G6.41 with N-CAM. Band 1, another large neuronal cell-surface glycoprotein was originally identified in mouse N18 neuroblastoma cells. In this study /sup 125/I-labeled N18-derived band 1 was tested for binding to 9 plant lectins and Limulus polyphemus agglutinin coupled to agarose beads. Band 1 solubilized from brain also specifically bound to LCA-agarose, indicating that mannose containing sugar moieties are present on band 1 from brain.

  20. Novel cell-based in vitro screen to identify small-molecule inhibitors against intracellular replication of Cryptococcus neoformans in macrophages.

    Science.gov (United States)

    Samantaray, Sweta; Correia, Joao N; Garelnabi, Mariam; Voelz, Kerstin; May, Robin C; Hall, Rebecca A

    2016-07-01

    The fungal pathogen Cryptococcus neoformans poses a major threat to immunocompromised patients and is a leading killer of human immunodeficiency virus (HIV)-infected patients worldwide. Cryptococci are known to manipulate host macrophages and can either remain latent or proliferate intracellularly within the host phagocyte, a favourable niche that also renders them relatively insensitive to antifungal agents. Here we report an attempt to address this limitation by using a fluorescence-based drug screening method to identify potential inhibitors of intracellular proliferation of C. neoformans. The Prestwick Chemical Library(®) of FDA-approved small molecules was screened for compounds that limit the intracellular replication of a fluorescently-tagged C. neoformans reference strain (H99-GFP) in macrophages. Preliminary screening revealed 19 of 1200 compounds that could significantly reduce intracellular growth of the pathogen. Secondary screening and host cell cytotoxicity assays highlighted fendiline hydrochloride as a potential drug candidate for the development of future anticryptococcal therapies. Live cell imaging demonstrated that this Ca(2+) channel blocker strongly enhanced phagosome maturation in macrophages leading to improved fungal killing and reduced intracellular replication. Whilst the relatively high dose of fendiline hydrochloride required renders it unfit for clinical deployment against cryptococcosis, this study highlights a novel approach for identifying new lead compounds and unravels a pharmacologically promising scaffold towards the development of novel antifungal therapies for this neglected disease. PMID:27289450

  1. Interference with ubiquitination in CFTR modifies stability of core glycosylated and cell surface pools.

    Science.gov (United States)

    Lee, Seakwoo; Henderson, Mark J; Schiffhauer, Eric; Despanie, Jordan; Henry, Katherine; Kang, Po Wei; Walker, Douglas; McClure, Michelle L; Wilson, Landon; Sorscher, Eric J; Zeitlin, Pamela L

    2014-07-01

    It is recognized that both wild-type and mutant CFTR proteins undergo ubiquitination at multiple lysines in the proteins and in one or more subcellular locations. We hypothesized that ubiquitin is added to specific sites in wild-type CFTR to stabilize it and at other sites to signal for proteolysis. Mass spectrometric analysis of wild-type CFTR identified ubiquitinated lysines 68, 710, 716, 1041, and 1080. We demonstrate that the ubiquitinated K710, K716, and K1041 residues stabilize wild-type CFTR, protecting it from proteolysis. The polyubiquitin linkage is predominantly K63. N-tail mutants, K14R and K68R, lead to increased mature band CCFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. The amount of CFTR in the K1041R mutant was drastically reduced and consisted of bands A/B, suggesting that the site in transmembrane 10 (TM10) was critical to further processing beyond the proteasome. The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition. Thus, ubiquitination at residue 1218 may destabilize wild-type CFTR in both the endoplasmic reticulum (ER) and recycling pools. Small molecules targeting the region of residue 1218 to block ubiquitination or to preserving structure at residues 710 to 716 might be protein sparing for some forms of cystic fibrosis.

  2. HIV-1 Nef and Vpu Are Functionally Redundant Broad-Spectrum Modulators of Cell Surface Receptors, Including Tetraspanins

    Science.gov (United States)

    Haller, Claudia; Müller, Birthe; Fritz, Joëlle V.; Lamas-Murua, Miguel; Stolp, Bettina; Pujol, François M.; Keppler, Oliver T.

    2014-01-01

    ABSTRACT HIV-1 Nef and Vpu are thought to optimize virus replication in the infected host, at least in part via their ability to interfere with vesicular host cell trafficking. Despite the use of distinct molecular mechanisms, Nef and Vpu share specificity for some molecules such as CD4 and major histocompatibility complex class I (MHC-I), while disruption of intracellular transport of the host cell restriction factor CD317/tetherin represents a specialized activity of Vpu not exerted by HIV-1 Nef. To establish a profile of host cell receptors whose intracellular transport is affected by Nef, Vpu, or both, we comprehensively analyzed the effect of these accessory viral proteins on cell surface receptor levels on A3.01 T lymphocytes. Thirty-six out of 105 detectable receptors were significantly downregulated by HIV-1 Nef, revealing a previously unappreciated scope with which HIV-1 Nef remodels the cell surface of infected cells. Remarkably, the effects of HIV-1 Vpu on host cell receptor exposure largely matched those of HIV-1 Nef in breadth and specificity (32 of 105, all also targeted by Nef), even though the magnitude was generally less pronounced. Of particular note, cell surface exposure of all members of the tetraspanin (TSPAN) protein family analyzed was reduced by both Nef and Vpu, and the viral proteins triggered the enrichment of TSPANs in a perinuclear area of the cell. While Vpu displayed significant colocalization and physical association with TSPANs, interactions of Nef with TSPANs were less robust. TSPANs thus emerge as a major target of deregulation in host cell vesicular transport by HIV-1 Nef and Vpu. The conservation of this activity in two independent accessory proteins suggests its importance for the spread of HIV-1 in the infected host. IMPORTANCE In this paper, we define that HIV-1 Nef and Vpu display a surprising functional overlap and affect the cell surface exposure of a previously unexpected breadth of cellular receptors. Our analyses

  3. A high throughput live transparent animal bioassay to identify non-toxic small molecules or genes that regulate vertebrate fat metabolism for obesity drug development

    Directory of Open Access Journals (Sweden)

    Woollett Laura A

    2008-08-01

    Full Text Available Abstract Background The alarming rise in the obesity epidemic and growing concern for the pathologic consequences of the metabolic syndrome warrant great need for development of obesity-related pharmacotherapeutics. The search for such therapeutics is severely limited by the slow throughput of animal models of obesity. Amenable to placement into a 96 well plate, zebrafish larvae have emerged as one of the highest throughput vertebrate model organisms for performing small molecule screens. A method for visually identifying non-toxic molecular effectors of fat metabolism using a live transparent vertebrate was developed. Given that increased levels of nicotinamide adenine dinucleotide (NAD via deletion of CD38 have been shown to prevent high fat diet induced obesity in mice in a SIRT-1 dependent fashion we explored the possibility of directly applying NAD to zebrafish. Methods Zebrafish larvae were incubated with daily refreshing of nile red containing media starting from a developmental stage of equivalent fat content among siblings (3 days post-fertilization, dpf and continuing with daily refreshing until 7 dpf. Results PPAR activators, beta-adrenergic agonists, SIRT-1 activators, and nicotinic acid treatment all caused predicted changes in fat, cholesterol, and gene expression consistent with a high degree of evolutionary conservation of fat metabolism signal transduction extending from man to zebrafish larvae. All changes in fat content were visually quantifiable in a relative fashion using live zebrafish larvae nile red fluorescence microscopy. Resveratrol treatment caused the greatest and most consistent loss of fat content. The resveratrol tetramer Vaticanol B caused loss of fat equivalent in potency to resveratrol alone. Significantly, the direct administration of NAD decreased fat content in zebrafish. Results from knockdown of a zebrafish G-PCR ortholog previously determined to decrease fat content in C. elegans support that future GPR

  4. The Cell Surface Proteome of Human Mesenchymal Stromal Cells

    OpenAIRE

    Christian Niehage; Charlotte Steenblock; Theresia Pursche; Martin Bornhäuser; Denis Corbeil; Bernard Hoflack

    2011-01-01

    BACKGROUND: Multipotent human mesenchymal stromal cells (hMSCs) are considered as promising biological tools for regenerative medicine. Their antibody-based isolation relies on the identification of reliable cell surface markers. METHODOLOGY/PRINCIPAL FINDINGS: To obtain a comprehensive view of the cell surface proteome of bone marrow-derived hMSCs, we have developed an analytical pipeline relying on cell surface biotinylation of intact cells using cell impermeable, cleavable sulfo-NHS-SS-bio...

  5. Subtractive genomics approach to identify putative drug targets and identification of drug-like molecules for beta subunit of DNA polymerase III in Streptococcus species.

    Science.gov (United States)

    Georrge, John J; Umrania, V V

    2012-07-01

    The prolonged use of the antibiotics over the years has transformed many organisms resistant to multiple drugs. This has made the field of drug discovery of vital importance in curing various infections and diseases. The drugs act by binding to a specific target protein of prime importance for the cell's survival. Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes are the few gram positive organisms that have developed resistance to drugs. It causes pneumonia, meningitis, pharyngitis, otitis media, sinusitis, bacteremia, pericarditis, and arthritis infections. The present study was carried out to identify potential drug targets and inhibitors for beta subunit of DNA polymerase III in these three Streptococcus species that might facilitate the discovery of novel drugs in near future. Various steps were adopted to find out novel drug targets. And finally 3D structure of DNA polymerase III subunit beta was modeled. The ligand library was generated from various databases to find the most suitable ligands. All the ligands were docked using Molegro Virtual Docker and the lead molecules were investigated for ADME and toxicity. PMID:22415782

  6. Fundamental studies of matrix-assisted laser desorption/ionization, using time-of-flight mass spectrometry to identify biological molecules

    Energy Technology Data Exchange (ETDEWEB)

    Eades, D.; Wruck, D.; Gregg, H.

    1996-11-11

    MALDI MS was developed as a way of getting molecular weight information on small quantities (picomole to femtomole levels) of high-mass, thermally labile macromolecules. While most other analytical MS ionization techniques cause fragmentation, decomposition, or multiple charging, MALDI efficiently places intact macromolecules into the gas phase with little fragmentation or rearrangement. This project had 3 objectives: establish the MALDI capability at LLNL, perform fundamental studies of analyte-matrix interactions, and apply the technique for biochemical research. A retired time-of-flight instrument was adapted for MALDI analyses, relevant parameters influencing the MALDI process were identified for further study (matrix molar absorptivity, sample crystal preparation), and collaborations were established with research groups in the Biology and Biotechnology Research Program at LLNL. In MALDI, the macromolecule of interest is mixed with a high-molar excess (1:100 to 1:10,000) of an organic matrix which readily absorbs energy at the wavelength corresponding to a UV laser. Upon laser irradiation, the matrix absorbs the majority of the energy, causing it to desorb from the surface and gently release the macromolecule into the gas phase with little or no fragmentation. Once in the gas phase, ion-molecule reactions between excited matrix and neutral macromolecules generated ionized analyte species which then can be focused into a MS for detection.

  7. Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Madsen, Søren M; Glenting, Jacob;

    2009-01-01

    In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel...

  8. Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

    Science.gov (United States)

    Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

    2016-01-01

    The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

  9. A Dual Receptor and Reporter for Multi-Modal Cell Surface Engineering.

    Science.gov (United States)

    Luo, Wei; Westcott, Nathan; Dutta, Debjit; Pulsipher, Abigail; Rogozhnikov, Dmitry; Chen, Jean; Yousaf, Muhammad N

    2015-10-16

    The rapid development of new small molecule drugs, nanomaterials, and genetic tools to modulate cellular function through cell surface manipulation has revolutionized the diagnosis, study, and treatment of disorders in human health. Since the cell membrane is a selective gateway barrier that serves as the first line of defense/offense and communication to its environment, new approaches that molecularly engineer or tailor cell membrane surfaces would allow for a new era in therapeutic design, therapeutic delivery, complex coculture tissue construction, and in situ imaging probe tracking technologies. In order to develop the next generation of multimodal therapies, cell behavior studies, and biotechnologies that focus on cell membrane biology, new tools that intersect the fields of chemistry, biology, and engineering are required. Herein, we develop a liposome fusion and delivery strategy to present a novel dual receptor and reporter system at cell surfaces without the use of molecular biology or metabolic biosynthesis. The cell surface receptor is based on bio-orthogonal functional groups that can conjugate a range of ligands while simultaneously reporting the conjugation through the emission of fluorescence. We demonstrate this dual receptor and reporter system by conjugating and tracking various cell surface ligands for temporal control of cell fluorescent signaling, cell-cell interaction, and tissue assembly construction. PMID:26204094

  10. Curcumin induced nanoscale CD44 molecular redistribution and antigen-antibody interaction on HepG2 cell surface

    Energy Technology Data Exchange (ETDEWEB)

    Wang Mu [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Ruan Yuxia [Department of Ophthalmology, The First Affiliated Hospital, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Xing Xiaobo; Chen Qian; Peng, Yuan [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Cai Jiye, E-mail: tjycai@jnu.edu.cn [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China)

    2011-07-04

    Graphical abstract: Highlights: > In this study, we investigate the changes of CD44 expression and distribution on HepG2 cells after curcumin treatment. > We find curcumin is able to change the morphology and ultrastructure of HepG2 cells. > Curcumin can reduce the expression of CD44 molecules and induce the nanoscale molecular redistribution on cell surface. > The binding force between CD44-modified AFM tip and the HepG2 cell surface decreases after curcumin-treatment. - Abstract: The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 {+-} 4.62 nm to 129.70 {+-} 43.72 nm) and the expression of CD44 decreased (99.79 {+-} 0.16% to 75.14 {+-} 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 {mu}M curcumin-treated) and 50-120 pN (20 {mu}M curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

  11. Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria

    OpenAIRE

    Muscariello Lidia; Boekhorst Jos; Siezen Roland; Molenaar Douwe; Renckens Bernadet; Kleerebezem Michiel

    2006-01-01

    Abstract Background Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components. Results A novel gene cluster encoding exclusively cell-surface proteins was identified, which is ...

  12. Oxidation state of chromium associated with cell surfaces of Shewanella oneidensis during chromate reduction

    Energy Technology Data Exchange (ETDEWEB)

    Neal, Andrew L.; Lowe, Kristine; Daulton, Tyrone L.; Jones-Meehan, Joanne; Little, Brenda J

    2002-12-30

    Employing electron energy loss spectroscopy (EELS) and X-ray photoelectron spectroscopy (XPS), we demonstrate that in both aerobic and anaerobic culture Shewanella oneidensis cells are capable of chromate reduction. No Cr(VI) or Cr(V) species were identified at the cell surfaces in Cr 2p{sub 3/}ore photoelectron spectra. More chromium was associated with cell surfaces recovered from anaerobic medium than aerobic. Multiplet-splitting models derived for Cr(III) and Cr(IV) were employed to determine contributions from each ion to Cr 2p{sub 3/2} photopeaks collected from the various cell treatments. Whilst in all cases Cr(III) was the major ion associated with cell surfaces, a significant contribution was identified due to Cr(IV) in anaerobically grown cells. The Cr(IV) contribution was far less when cells were grown aerobically. Moreover, when anaerobically grown cells were exposed to oxygen very little re-oxidation of Cr-precipitates occurred, the precipitates were again identified as a mixture of Cr(III) and Cr(IV). A positive relationship was observed between amounts of chromium and phosphorous associated with cell surfaces resulting from the various treatments, suggesting the precipitates included Cr(III)-phosphate. The fact that Cr(IV) remained associated with precipitates following re-oxidation suggests that under anaerobic conditions the intermediate ion is afforded sufficient stability to be incorporated within the precipitate matrix and thus conferred a degree of protection from oxidation.

  13. A high-content small molecule screen identifies sensitivity of glioblastoma stem cells to inhibition of polo-like kinase 1.

    Directory of Open Access Journals (Sweden)

    Davide Danovi

    Full Text Available Glioblastoma multiforme (GBM is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS cells and genetically normal neural stem (NS cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101 as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1 (phospho T210, with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364 phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF(-/-, or p53(-/-, as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.

  14. Cell surface proteome of the marine planctomycete Rhodopirellula baltica.

    Science.gov (United States)

    Voigt, Birgit; Hieu, Cao Xuan; Hempel, Kristina; Becher, Dörte; Schlüter, Rabea; Teeling, Hanno; Glöckner, Frank Oliver; Amann, Rudolf; Hecker, Michael; Schweder, Thomas

    2012-06-01

    The surface proteome (surfaceome) of the marine planctomycete Rhodopirellula baltica SH1(T) was studied using a biotinylation and a proteinase K approach combined with SDS-PAGE and mass spectrometry. 52 of the proteins identified in both approaches could be assigned to the group of potential surface proteins. Among them are some high molecular weight proteins, potentially involved in cell-cell attachment, that contain domains shown before to be typical for surface proteins like cadherin/dockerin domains, a bacterial adhesion domain or the fasciclin domain. The identification of proteins with enzymatic functions in the R. baltica surfaceome provides further clues for the suggestion that some degradative enzymes may be anchored onto the cell surface. YTV proteins, which have been earlier supposed to be components of the proteinaceous cell wall of R. baltica, were detected in the surface proteome. Additionally, 8 proteins with a novel protein structure combining a conserved type IV pilin/N-methylation domain and a planctomycete-typical DUF1559 domain were identified. PMID:22623273

  15. Targeting pancreatic progenitor cells in human embryonic stem cell differentiation for the identification of novel cell surface markers.

    Science.gov (United States)

    Fishman, Bettina; Segev, Hanna; Kopper, Oded; Nissenbaum, Jonathan; Schulman, Margarita; Benvenisty, Nissim; Itskovitz-Eldor, Joseph; Kitsberg, Danny

    2012-09-01

    New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.

  16. Calreticulin: Roles in Cell-Surface Protein Expression

    Directory of Open Access Journals (Sweden)

    Yue Jiang

    2014-09-01

    Full Text Available In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

  17. Cell surface carbohydrates as prognostic markers in human carcinomas

    DEFF Research Database (Denmark)

    Dabelsteen, Erik

    1996-01-01

    Tumour development is usually associated with changes in cell surface carbohydrates. These are often divided into changes related to terminal carbohydrate structures, which include incomplete synthesis and modification of normally existing carbohydrates, and changes in the carbohydrate core struc...

  18. Cadherin-9 is a novel cell surface marker for the heterogeneous pool of renal fibroblasts.

    Directory of Open Access Journals (Sweden)

    Cornelia Thedieck

    Full Text Available BACKGROUND: Interstitial fibroblasts are a minor, but nevertheless very important, component of the kidney. They secrete and remodel extracellular matrix and they produce active compounds such as erythropoietin. However, studying human renal fibroblasts has been hampered by the lack of appropriate surface markers. METHODS AND FINDINGS: The expression of cadherin-9 in various human renal cell lines and tissues was studied on the mRNA level by RT-PCR and on the protein level with the help of newly generated cadherin-9 antibodies. The classical type II cadherin-9, so far only described in the neural system, was identified as a reliable surface marker for renal fibroblasts. Compared to FSP1, a widely-used cytosolic renal fibroblast marker, cadherin-9 showed a more restricted expression pattern in human kidney. Under pathological conditions, cadherin-9 was expressed in the stroma of renal cell carcinoma, but not in the tumor cells themselves, and in renal fibrosis the percentage of cadherin-9-positive cells was clearly elevated 3 to 5 times compared to healthy kidney tissue. Induction of epithelial mesenchymal transition in renal epithelial cells with cyclosporin-A, which causes renal fibrosis as a side effect, induced cadherin-9 expression. Functional studies following siRNA-mediated knockdown of cadherin-9 revealed that it acts in the kidney like a typical classical cadherin. It was found to be associated with catenins and to mediate homophilic but not heterophilic cell interactions. CONCLUSIONS: Cadherin-9 represents a novel and reliable cell surface marker for fibroblasts in healthy and diseased kidneys. Together with the established marker molecules FSP1, CD45 and alpha smooth muscle actin, cadherin-9 can now be used to differentiate the heterogenic pool of renal fibroblasts into resident and activated fibroblasts, immigrated bone marrow derived fibroblast precursors and cells in different stages of epithelial mesenchymal transition.

  19. The cell surface proteome of Staphylococcus aureus

    NARCIS (Netherlands)

    Dreisbach, Annette; van Dijl, Jan Maarten; Buist, Girbe

    2011-01-01

    The Gram-positive bacterium Staphylococcus aureus is a wide spread opportunistic pathogen that can cause a range of life-threatening diseases. To obtain a better understanding of the global mechanisms for pathogenesis and to identify novel targets for therapeutic interventions, the S. aureus proteom

  20. EXAFS Study of Uranyl Complexation at Pseudomonas fluorescens Cell Surfaces

    Science.gov (United States)

    Bencheikh, R.; Bargar, J. R.; Tebo, B. M.

    2002-12-01

    Little is known about the roles of microbial biomass as a sink and source for uranium in contaminated aquifers, nor of the impact of bacterial biochemistry on uranium speciation in the subsurface. A significant role is implied by the high affinities of both Gram positive and Gram negative cells for binding uranyl (UO2{ 2+}). In the present study, Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy was used to identify membrane functional groups involved in uranyl binding to the Gram negative bacterium Pseudomonas fluorescens from pH 3 to pH 8. Throughout this pH-range, EXAFS spectra can be described primarily in terms of coordination of carboxylic groups to uranyl. U-C distances characteristic of 4-, 5- and 8- membered rings were observed, as well as the possibility of phosphato groups. Both shell-by-shell fits and principle component analyses indicate that the functional groups involved in binding of uranyl to the cell surface do not vary systematically across the pH range investigated. This result contrasts with EXAFS results of uranyl sorbed to Gram positive bacteria, and suggests an important role for long-chain carboxylate-terminated membrane functional groups in binding uranyl.

  1. RPE cell surface proteins in normal and dystrophic rats

    International Nuclear Information System (INIS)

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE

  2. RPE cell surface proteins in normal and dystrophic rats

    Energy Technology Data Exchange (ETDEWEB)

    Clark, V.M.; Hall, M.O.

    1986-02-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.

  3. A mass spectrometric-derived cell surface protein atlas.

    Directory of Open Access Journals (Sweden)

    Damaris Bausch-Fluck

    Full Text Available Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa. The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

  4. A mass spectrometric-derived cell surface protein atlas.

    Science.gov (United States)

    Bausch-Fluck, Damaris; Hofmann, Andreas; Bock, Thomas; Frei, Andreas P; Cerciello, Ferdinando; Jacobs, Andrea; Moest, Hansjoerg; Omasits, Ulrich; Gundry, Rebekah L; Yoon, Charles; Schiess, Ralph; Schmidt, Alexander; Mirkowska, Paulina; Härtlová, Anetta; Van Eyk, Jennifer E; Bourquin, Jean-Pierre; Aebersold, Ruedi; Boheler, Kenneth R; Zandstra, Peter; Wollscheid, Bernd

    2015-01-01

    Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments. PMID:25894527

  5. Scanning the cell surface proteome of cancer cells and identification of metastasis-associated proteins using a subtractive immunization strategy

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik J

    2009-01-01

    and technologically challenging, and no ideal method is currently available. Here, we describe a strategy that allows scanning of the entire cell surface and identification of molecules that exhibit altered expression between two cell types. Concurrently, this method gives rise to valuable reagents for further...... characterization of the identified proteins. The strategy is based on subtractive immunization of mice, and we used the two isogenic cell lines, NM-2C5 and M-4A4, derived from the MDA-MB-435 cancer cell line, as a model system. Although the two cell lines are equally tumorigenic, only M-4A4 has metastatic...... exhibiting no or very weak reactivity with normal tissues. mAb 15C7 stained a variety of cancers as well as some normal lymphoid organs and was subsequently identified to react with HLA-DR-beta. A third mAb, 31D7, that also specifically recognized HLA-DR-beta was capable of inhibiting the growth of MZ2...

  6. Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

    DEFF Research Database (Denmark)

    Lund, Rikke; Leth-Larsen, Rikke; Jensen, Ole N;

    2009-01-01

    Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface...... membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane...... proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic...

  7. Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells.

    Science.gov (United States)

    Nowatzki, Jenifer; de Sene, Reginaldo Vieira; Paludo, Katia Sabrina; Veiga, Silvio Sanches; Oliver, Constance; Jamur, Maria Célia; Nader, Helena Bonciani; Trindade, Edvaldo S; Franco, Célia Regina C

    2010-09-15

    Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.

  8. Small-Molecule Scaffolds for CYP51 Inhibitors Identified by High-Throughput Screening and Defined by X-Ray Crystallography▿

    OpenAIRE

    Larissa M. Podust; von Kries, Jens P.; Eddine, Ali Nasser; Kim, Youngchang; Yermalitskaya, Liudmila V.; Kuehne, Ronald; Ouellet, Hugues; Warrier, Thulasi; Alteköster, Markus; Lee, Jong-Seok; Rademann, Jörg; Oschkinat, Hartmut; Stefan H. E. Kaufmann; Waterman, Michael R.

    2007-01-01

    Sterol 14α-demethylase (CYP51), a major checkpoint in membrane sterol biosynthesis, is a key target for fungal antibiotic therapy. We sought small organic molecules for lead candidate CYP51 inhibitors. The changes in CYP51 spectral properties following ligand binding make CYP51 a convenient target for high-throughput screening technologies. These changes are characteristic of either substrate binding (type I) or inhibitor binding (type II) in the active site. We screened a library of 20,000 o...

  9. Melittin interaction with sulfated cell surface sugars.

    Science.gov (United States)

    Klocek, Gabriela; Seelig, Joachim

    2008-03-01

    Melittin is a 26-residue cationic peptide with cytolytic and antimicrobial properties. Studies on the action mechanism of melittin have focused almost exclusively on the membrane-perturbing properties of this peptide, investigating in detail the melittin-lipid interaction. Here, we report physical-chemical studies on an alternative mechanism by which melittin could interact with the cell membrane. As the outer surface of many cells is decorated with anionic (sulfated) glycosaminoglycans (GAGs), a strong Coulombic interaction between the two oppositely charged molecules can be envisaged. Indeed, the present study using isothermal titration calorimetry reveals a high affinity of melittin for several GAGs, that is, heparan sulfate (HS), dermatan sulfate, and heparin. The microscopic binding constant of melittin for HS is 2.4 x 10 (5) M (-1), the reaction enthalpy is Delta H melittin (0) = -1.50 kcal/mol, and the peptide-to-HS stoichiometry is approximately 11 at 10 mM Tris, 100 mM NaCl at pH 7.4 and 28 degrees C. Delta H melittin (0) is characterized by a molar heat capacity of Delta C P (0) = -227 cal mol (-1) K (-1). The large negative heat capacity change indicates that hydrophobic interactions must also be involved in the binding of melittin to HS. Circular dichroism spectroscopy demonstrates that the binding of the peptide to HS induces a conformational change to a predominantly alpha-helical structure. A model for the melittin-HS complex is presented. Melittin binding was compared with that of magainin 2 and nisin Z to HS. Magainin 2 is known for its antimicrobial properties, but it does not cause lysis of the eukaryotic cells. Nisin Z shows activity against various Gram-positive bacteria. Isothermal titration calorimetry demonstrates that magainin 2 and nisin Z do not bind to HS (5-50 degrees C, 10 mM Tris, and 100 mM NaCl at pH 7.4). PMID:18220363

  10. Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

    OpenAIRE

    Shin Soojung; Jones Karen; Lyons Ian; Mitalipova Maisam; Venable Alison; Pierce Michael; Stice Steven

    2005-01-01

    Abstract Background Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC s...

  11. Techniques to Study Specific Cell-Surface Receptor-Mediated Cellular Vitamin A Uptake

    OpenAIRE

    KAWAGUCHI, RIKI; Sun, Hui

    2010-01-01

    STRA6 is a multitransmembrane domain protein that was recently identified as the cell-surface receptor for plasma retinol binding protein (RBP), the vitamin A carrier protein in the blood. STRA6 binds to RBP with high affinity and mediates cellular uptake of vitamin A from RBP. It is not homologous to any known receptors, transporters, and channels, and it represents a new class of membrane transport protein. Consistent with the diverse physiological functions of vitamin A, STRA6 is widely ex...

  12. Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer

    Directory of Open Access Journals (Sweden)

    Richardson Andrea L

    2011-10-01

    Full Text Available Abstract Background Na+/I- symporter (NIS-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Methods Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. Results and Discussion NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Conclusions Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

  13. Pigment epithelium-derived factor binds to cell-surface F(1)-ATP synthase.

    Science.gov (United States)

    Notari, Luigi; Arakaki, Naokatu; Mueller, David; Meier, Scott; Amaral, Juan; Becerra, S P

    2010-05-01

    Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a approximately 60 kDa PEDF-binding protein in bovine retina with Bos taurus F(1)-ATP synthase beta-subunit, and that F(1)F(o)-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F(1). Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F(1). Real-time binding as determined by surface plasmon resonance demonstrated that yeast F(1) interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F(1) and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F(1). Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of approximately 60 kDa. Antibodies to F(1)beta-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F(1), these results show that PEDF is a ligand for endothelial cell-surface F(1)F(o)-ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity. PMID:20412062

  14. Bovine neonatal pancytopenia - Comparative proteomic characterization of two BVD vaccines and the producer cell surface proteome (MDBK

    Directory of Open Access Journals (Sweden)

    Euler Kerstin N

    2013-01-01

    Full Text Available Abstract Background Bovine neonatal pancytopenia (BNP is a disease syndrome in newborn calves of up to four weeks of age, first observed in southern Germany in 2006. By now, cases have been reported in several countries around the globe. Many affected calves die within days due to multiple haemorrhages, thrombocytopenia, leukocytopenia and bone marrow depletion. A certain vaccine directed against Bovine Virus Diarrhoea Virus (BVDV was recently shown to be associated with BNP pathogenesis. Immunized cows develop alloantibodies that are transferred to newborn calves via colostrum intake. In order to further elucidate BNP pathogenesis, the purpose of this study was to characterize and compare the protein composition of the associated vaccine to another vaccine directed against BVDV not related to BNP and the cell surface proteome of MDBK (Madin-Darby Bovine Kidney cells, the cell line used for production of the associated vaccine. Results By SDS-PAGE and mass spectrometry, we were able to detect several coagulation-related and immune modulatory proteins, as well as cellular and serum derived molecules being shared between the associated vaccine and MDBK cells. Furthermore, the number of proteins identified in the BNP related vaccine was almost as high as the number of surface proteins detected on MDBK cells and exceeded the amount of proteins identified in the non-BNP related vaccine over 3.5 fold. The great amount of shared cellular and serum derived proteins confirm that the BNP associated vaccine contained many molecules originating from MDBK cells and vaccine production. Conclusions The respective vaccine was not purified enough to prevent the development of alloantibodies. To narrow down possible candidate proteins, those most likely to represent a trigger for BNP pathogenesis are presented in this study, giving a fundament for further analysis in future research.

  15. Expanding the diversity of unnatural cell surface sialic acids

    Energy Technology Data Exchange (ETDEWEB)

    Luchansky, Sarah J.; Goon, Scarlett; Bertozzi, Carolyn R.

    2003-10-30

    Novel chemical reactivity can be introduced onto cell surfaces through metabolic oligosaccharide engineering. This technique exploits the substrate promiscuity of cellular biosynthetic enzymes to deliver unnatural monosaccharides bearing bioorthogonal functional groups into cellular glycans. For example, derivatives of N-acetylmannosamine (ManNAc) are converted by the cellular biosynthetic machinery into the corresponding sialic acids and subsequently delivered to the cell surface in the form of sialoglycoconjugates. Analogs of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) are also metabolized and incorporated into cell surface glycans, likely through the sialic acid and GalNAc salvage pathways, respectively. Furthermore, GlcNAc analogs can be incorporated into nucleocytoplasmic proteins in place of {beta}-O-GlcNAc residues. These pathways have been exploited to integrate unique electrophiles such as ketones and azides into the target glycoconjugate class. These functional groups can be further elaborated in a chemoselective fashion by condensation with hydrazides and by Staudinger ligation, respectively, thereby introducing detectable probes onto the cell. In conclusion, sialic acid derivatives are efficient vehicles for delivery of bulky functional groups to cell surfaces and masking of their hydroxyl groups improves their cellular uptake and utilization. Furthermore, the successful introduction of photoactivatable aryl azides into cell surface glycans opens up new avenues for studying sialic acid-binding proteins and elucidating the role of sialic acid in essential processes such as signaling and cell adhesion.

  16. Interaction of KSHV with Host Cell Surface Receptors and Cell Entry

    Directory of Open Access Journals (Sweden)

    Mohanan Valiya Veettil

    2014-10-01

    Full Text Available Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry.

  17. Recent Insights into Cell Surface Heparan Sulphate Proteoglycans and Cancer.

    Science.gov (United States)

    Couchman, John R; Multhaupt, Hinke; Sanderson, Ralph D

    2016-01-01

    A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal and regulate important cell processes, such as adhesion, migration, proliferation, and differentiation. Since many protein ligands, such as growth factors, morphogens, and cytokines, are also implicated in tumour progression, it is increasingly apparent that cell surface proteoglycans impact tumour cell behaviour. Here, we review some recent advances, emphasising that many tumour-related functions of proteoglycans are revealed only after their modification in processes subsequent to synthesis and export to the cell surface. These include enzymes that modify heparan sulphate structure, recycling of whole or fragmented proteoglycans into exosomes that can be paracrine effectors or biomarkers, and lateral interactions between some proteoglycans and calcium channels that impact the actin cytoskeleton. PMID:27408707

  18. Cell surface engineering of industrial microorganisms for biorefining applications.

    Science.gov (United States)

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-11-15

    In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coli and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed.

  19. Cell surface engineering of industrial microorganisms for biorefining applications.

    Science.gov (United States)

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-11-15

    In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coli and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed. PMID:26070720

  20. Recent Insights into Cell Surface Heparan Sulphate Proteoglycans and Cancer

    Science.gov (United States)

    Couchman, John R; Multhaupt, Hinke; Sanderson, Ralph D.

    2016-01-01

    A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal and regulate important cell processes, such as adhesion, migration, proliferation, and differentiation. Since many protein ligands, such as growth factors, morphogens, and cytokines, are also implicated in tumour progression, it is increasingly apparent that cell surface proteoglycans impact tumour cell behaviour. Here, we review some recent advances, emphasising that many tumour-related functions of proteoglycans are revealed only after their modification in processes subsequent to synthesis and export to the cell surface. These include enzymes that modify heparan sulphate structure, recycling of whole or fragmented proteoglycans into exosomes that can be paracrine effectors or biomarkers, and lateral interactions between some proteoglycans and calcium channels that impact the actin cytoskeleton. PMID:27408707

  1. Chemistry and material science at the cell surface

    Directory of Open Access Journals (Sweden)

    Weian Zhao

    2010-04-01

    Full Text Available Cell surfaces are fertile ground for chemists and material scientists to manipulate or augment cell functions and phenotypes. This not only helps to answer basic biology questions but also has diagnostic and therapeutic applications. In this review, we summarize the most recent advances in the engineering of the cell surface. In particular, we focus on the potential applications of surface engineered cells for 1 targeting cells to desirable sites in cell therapy, 2 programming assembly of cells for tissue engineering, 3 bioimaging and sensing, and ultimately 4 manipulating cell biology.

  2. Curcumin induced nanoscale CD44 molecular redistribution and antigen-antibody interaction on HepG2 cell surface

    International Nuclear Information System (INIS)

    Graphical abstract: Highlights: → In this study, we investigate the changes of CD44 expression and distribution on HepG2 cells after curcumin treatment. → We find curcumin is able to change the morphology and ultrastructure of HepG2 cells. → Curcumin can reduce the expression of CD44 molecules and induce the nanoscale molecular redistribution on cell surface. → The binding force between CD44-modified AFM tip and the HepG2 cell surface decreases after curcumin-treatment. - Abstract: The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 ± 4.62 nm to 129.70 ± 43.72 nm) and the expression of CD44 decreased (99.79 ± 0.16% to 75.14 ± 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 μM curcumin-treated) and 50-120 pN (20 μM curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

  3. Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

    Directory of Open Access Journals (Sweden)

    Craig A Gedye

    Full Text Available Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell

  4. Immunogold labels: cell-surface markers in atomic force microscopy

    NARCIS (Netherlands)

    Putman, Constant A.J.; Grooth, de Bart G.; Hansma, Paul K.; Hulst, van Niek F.; Greve, Jan

    1993-01-01

    The feasibility of using immunogold labels as cell-surface markers in atomic force microscopy is shown in this paper. The atomic force microscope (AFM) was used to image the surface of immunogold-labeled human lymphocytes. The lymphocytes were isolated from whole blood and labeled by an indirect imm

  5. Bacterial Cell Surface Damage Due to Centrifugal Compaction

    NARCIS (Netherlands)

    Peterson, Brandon W.; Sharma, Prashant K.; van der Mei, Henny C.; Busscher, Henk J.

    2012-01-01

    Centrifugal damage has been known to alter bacterial cell surface properties and interior structures, including DNA. Very few studies exist on bacterial damage caused by centrifugation because of the difficulty in relating centrifugation speed and container geometry to the damage caused. Here, we pr

  6. Genomic-Based High Throughput Screening Identifies Small Molecules That Differentially Inhibit the Antiviral and Immunomodulatory Effects of IFN-α

    OpenAIRE

    Chen, Bo; Zong, Qin; Cibotti, Ricardo; Morris, Chad; Castaneda, Juana; Naiman, Brian; Liu, Derong; Glodek, Anna; Sims, Gary P.; Herbst, Ronald; Horrigan, Stephen K.; Kiener, Peter A; Soppet, Dan; Coyle, Anthony J.; Audoly, Laurent

    2008-01-01

    Multiple lines of evidence suggest that inhibition of Type I Interferons, including IFN-α, may provide a therapeutic benefit for autoimmune diseases. Using a chemical genomics approach integrated with cellular and in vivo assays, we screened a small compound library to identify modulators of IFN-α biological effects. A genomic fingerprint was developed from both ex vivo patient genomic information and in vitro gene modulation from IFN-α cell-based stimulation. A high throughput genomic-based ...

  7. Small-molecule screening of PC3 prostate cancer cells identifies tilorone dihydrochloride to selectively inhibit cell growth based on cyclin-dependent kinase 5 expression.

    Science.gov (United States)

    Wissing, Michel D; Dadon, Tikva; Kim, Eunice; Piontek, Klaus B; Shim, Joong S; Kaelber, Nadine S; Liu, Jun O; Kachhap, Sushant K; Nelkin, Barry D

    2014-07-01

    Cyclin-dependent kinase 5 (CDK5) is a potential target for prostate cancer treatment, the enzyme being essential for prostate tumor growth and formation of metastases. In the present study, we identified agents that target prostate cancer cells based on CDK5 expression. CDK5 activity was suppressed by transfection of PC3 prostate cancer cells with a dominant-negative construct (PC3 CDK5dn). PC3 CDK5dn and PC3 control cells were screened for compounds that selectively target cells based on CDK5 expression, utilizing the Johns Hopkins Drug Library. MTS proliferation, clonogenic and 3D growth assays were performed to validate the selected hits. Screening of 3,360 compounds identified rutilantin, ethacridine lactate and cetalkonium chloride as compounds that selectively target PC3 control cells and a tilorone analog as a selective inhibitor of PC3 CDK5dn cells. A PubMed literature study indicated that tilorone may have clinical use in patients. Validation experiments confirmed that tilorone treatment resulted in decreased PC3 cell growth and invasion; PC3 cells with inactive CDK5 were inhibited more effectively. Future studies are needed to unravel the mechanism of action of tilorone in CDK5 deficient prostate cancer cells and to test combination therapies with tilorone and a CDK5 inhibitor for its potential use in clinical practice. PMID:24841903

  8. Identification of cell surface targets for HIV-1 therapeutics using genetic screens

    International Nuclear Information System (INIS)

    Human immunodeficiency virus (HIV) drugs designed to interfere with obligatory utilization of certain host cell factors by virus are less likely to encounter development of resistant strains than drugs directed against viral components. Several cellular genes required for productive infection by HIV were identified by the use of genetic suppressor element (GSE) technology as potential targets for anti-HIV drug development. Fragmented cDNA libraries from various pools of human peripheral blood mononuclear cells (PBMC) were expressed in vitro in human immunodeficiency virus type 1 (HIV-1)-susceptible cell lines and subjected to genetic screens to identify GSEs that interfered with viral replication. After three rounds of selection, more than 15 000 GSEs were sequenced, and the cognate genes were identified. The GSEs that inhibited the virus were derived from a diverse set of genes including cell surface receptors, cytokines, signaling proteins, transcription factors, as well as genes with unknown function. Approximately 2.5% of the identified genes were previously shown to play a role in the HIV-1 life cycle; this finding supports the biological relevance of the assay. GSEs were derived from the following 12 cell surface proteins: CXCR4, CCR4, CCR7, CD11C, CD44, CD47, CD68, CD69, CD74, CSF3R, GABBR1, and TNFR2. Requirement of some of these genes for viral infection was also investigated by using RNA interference (RNAi) technology; accordingly, 10 genes were implicated in early events of the viral life cycle, before viral DNA synthesis. Thus, these cell surface proteins represent novel targets for the development of therapeutics against HIV-1 infection and AIDS

  9. Association of cell surface heparan sulfate proteoglycans of Schwann cells with extracellular matrix proteins.

    Science.gov (United States)

    Carey, D J; Crumbling, D M; Stahl, R C; Evans, D M

    1990-11-25

    The terminal differentiation of Schwann cells is dependent on contact with basement membrane. The present study was undertaken to investigate the role of cell surface heparan sulfate proteoglycans (HSPGs) in mediating Schwann cell responses to extracellular matrix contact. Phosphatidylinositol-specific phospholipase C-releasable cell surface HSPGs purified from cultures of neonatal rat Schwann cells were subjected to affinity chromatography on immobilized laminin and fibronectin. Binding of the HSPG to both affinity matrices was observed. The strength of the association, however, was sensitive to the ionic strength of the buffer. In 0.1 M Tris-HCl, HSPG binding was essentially irreversible whereas in physiological ionic strength buffer (e.g. 0.142 M NaCl, 10 mM Tris), weaker binding was detected as a delay in elution of the HSPG from the affinity columns. Further studies of HSPG-laminin binding suggested that the binding was mediated by the glycosaminoglycan chains of the proteoglycans. Results of equilibrium gel filtration chromatography provided additional evidence for a reversible association of the HSPG and laminin with a Kd of approximately 1 x 10(-6) M. When Schwann cells were plated on plastic dishes coated with laminin, the cells attached and extended long slender processes. Inclusion of heparin, but not chondroitin sulfate, in the assay medium resulted in partial inhibition of process extension, but at concentrations of heparin which were higher than that needed to disrupt laminin-HSPG association in vitro. Addition of anti-integrin receptor antibodies resulted in more extensive inhibition of laminin-dependent process extension. Anti-integrin antibodies plus heparin essentially totally inhibited laminin-dependent process extension. These results demonstrate that cell surface HSPGs are capable of reversible association with extracellular matrix molecules and suggest that HSPG-laminin interactions play a role in laminin-dependent Schwann cell spreading. PMID

  10. Cell surface glycan alterations in epithelial mesenchymal transition process of Huh7 hepatocellular carcinoma cell.

    Directory of Open Access Journals (Sweden)

    Shan Li

    Full Text Available BACKGROUND AND OBJECTIVE: Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC is high. It is well known that the epithelial mesenchymal transition (EMT and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model. METHODOLOGY: HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR. RESULTS: After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase. CONCLUSIONS: The findings of this study systematically clarify the alterations of cell surface

  11. Gene expression profiling identifies FYN as an important molecule in tamoxifen resistance and a predictor of early recurrence in patients treated with endocrine therapy

    DEFF Research Database (Denmark)

    Elias, D; (Hansen) Vever, Henriette; Lænkholm, A-V;

    2015-01-01

    To elucidate the molecular mechanisms of tamoxifen resistance in breast cancer, we performed gene array analyses and identified 366 genes with altered expression in four unique tamoxifen-resistant (TamR) cell lines vs the parental tamoxifen-sensitive MCF-7/S0.5 cell line. Most of these genes were......-7/S0.5 cells. Moreover, overexpression of FYN in parental tamoxifen-sensitive MCF-7/S0.5 cells resulted in reduced sensitivity to tamoxifen treatment, whereas knockdown of FYN in the FYN-overexpressing MCF-7/S0.5 cells restored sensitivity to tamoxifen, demonstrating growth- and survival......-promoting function of FYN in MCF-7 cells. FYN knockdown in TamR cells led to reduced phosphorylation of 14-3-3 and Cdc25A, suggesting that FYN, by activation of important cell cycle-associated proteins, may overcome the anti-proliferative effects of tamoxifen. Evaluation of the subcellular localization of FYN...

  12. Infection Dynamics Vary between Symbiodinium Types and Cell Surface Treatments during Establishment of Endosymbiosis with Coral Larvae

    Directory of Open Access Journals (Sweden)

    Bette Lynn Willis

    2011-07-01

    Full Text Available Symbioses between microbes and higher organisms underpin high diversity in many ecosystems, including coral reefs, however mechanisms underlying the early establishment of symbioses remain unclear. Here we examine the roles of Symbiodinium type and cell surface recognition in the establishment of algal endosymbiosis in the reef-building coral, Acropora tenuis. We found 20–70% higher infection success (proportion of larvae infected and five-fold higher Symbiodinium abundance in larvae exposed to ITS-1 type C1 compared to ITS-1 type D in the first 96 h following exposure. The highest abundance of Symbiodinium within larvae occurred when C1-type cells were treated with enzymes that modified the 40–100 kD glycome, including glycoproteins and long chain starch residues. Our finding of declining densities of Symbiodinium C1 through time in the presence of intact cell surface molecules supports a role for cell surface recognition molecules in controlling post-phagocytosis processes, leading to rejection of some Symbiodinium types in early ontogeny. Reductions in the densities of unmodified C1 symbionts after 96 h, in contrast to increases in D symbionts may suggest the early initiation of a winnowing process contributing to the establishment of Symbiodinium D as the dominant type in one-month old juveniles of A. tenuis.

  13. Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

    Science.gov (United States)

    Hengel, Sarah R; Malacaria, Eva; Folly da Silva Constantino, Laura; Bain, Fletcher E; Diaz, Andrea; Koch, Brandon G; Yu, Liping; Wu, Meng; Pichierri, Pietro; Spies, M Ashley; Spies, Maria

    2016-01-01

    The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing. DOI: http://dx.doi.org/10.7554/eLife.14740.001 PMID:27434671

  14. Transcriptome analysis of stem wood of Nothapodytes nimmoniana (Graham) Mabb. identifies genes associated with biosynthesis of camptothecin, an anti-carcinogenic molecule

    Indian Academy of Sciences (India)

    BL Manjunatha; HR Singh; G Ravikanth; Karaba N Nataraja; Ravi Shankar; Sanjay Kumar; R Uma Shaanker

    2016-03-01

    Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of DNA topoisomerase I and has applications in treating ovarian, small lung and refractory ovarian cancers. Stem wood tissue of Nothapodytes nimmoniana (Graham) Mabb. (family Icacinaceae) is one of the richest sources of CPT. Since there is no genomic or transcriptome data available for the species, the present work sequenced and analysed transcriptome of stem wood tissue on an Illumina platform. From a total of 77,55,978 reads, 9,187 transcripts were assembled with an average length of 255 bp. Functional annotation and categorization of these assembled transcripts unraveled the transcriptome architecture and also a total of 13 genes associated with CPT biosynthetic pathway were identified in the stem wood tissue. Four genes of the pathway were cloned to full length by RACE to validate the transcriptome data. Expression analysis of 13 genes associated with CPT biosynthetic pathway in 11 different tissues vis-a-vis CPT content analysis suggested an important role of NnPG10H, NnPSLS and NnPSTR genes in the biosynthesis of CPT. These results indicated that CPT might be synthesized in the leaves and then perhaps exported to stem wood tissue for storage.

  15. Growth condition-dependent cell surface proteome analysis of Enterococcus faecium.

    Science.gov (United States)

    Sinnige, Jan C; de Been, Mark; Zhou, Miaomiao; Bonten, Marc J M; Willems, Rob J L; Top, Janetta

    2015-11-01

    The last 30 years Enterococcus faecium has become an important nosocomial pathogen in hospitals worldwide. The aim of this study was to obtain insight in the cell surface proteome of E. faecium when grown in laboratory and clinically relevant conditions. Enterococcus faecium E1162, a clinical blood stream isolate, was grown until mid-log phase in brain heart infusion medium (BHI) with, or without 0.02% bile salts, Tryptic Soy Broth with 1% glucose (TSBg) and urine, and its cell surface was "shaved" using immobilized trypsin. Peptides were identified using MS/MS. Mapping against the translated E1162 whole genome sequence identified 67 proteins that were differentially detected in different conditions. In urine, 14 proteins were significantly more and nine proteins less abundant relative to the other conditions. Growth in BHI-bile and TSBg, revealed four and six proteins, respectively, which were uniquely present in these conditions while two proteins were uniquely present in both conditions. Thus, proteolytic shaving of E. faecium cells identified differentially surface exposed proteins in different growth conditions. These proteins are of special interest as they provide more insight in the adaptive mechanisms and may serve as targets for the development of novel therapeutics against this multi-resistant emerging pathogen. All MS data have been deposited in the ProteomeXchange with identifier PXD002497 (http://proteomecentral.proteomexchange.org/dataset/PXD002497).

  16. Identification of Cell Surface Targets through Meta-analysis of Microarray Data

    Directory of Open Access Journals (Sweden)

    Henry Haeberle

    2012-07-01

    Full Text Available High-resolution image guidance for resection of residual tumor cells would enable more precise and complete excision for more effective treatment of cancers, such as medulloblastoma, the most common pediatric brain cancer. Numerous studies have shown that brain tumor patient outcomes correlate with the precision of resection. To enable guided resection with molecular specificity and cellular resolution, molecular probes that effectively delineate brain tumor boundaries are essential. Therefore, we developed a bioinformatics approach to analyze micro-array datasets for the identification of transcripts that encode candidate cell surface biomarkers that are highly enriched in medulloblastoma. The results identified 380 genes with greater than a two-fold increase in the expression in the medulloblastoma compared with that in the normal cerebellum. To enrich for targets with accessibility for extracellular molecular probes, we further refined this list by filtering it with gene ontology to identify genes with protein localization on, or within, the plasma membrane. To validate this meta-analysis, the top 10 candidates were evaluated with immunohistochemistry. We identified two targets, fibrillin 2 and EphA3, which specifically stain medulloblastoma. These results demonstrate a novel bioinformatics approach that successfully identified cell surface and extracellular candidate markers enriched in medulloblastoma versus adjacent cerebellum. These two proteins are high-value targets for the development of tumor-specific probes in medulloblastoma. This bioinformatics method has broad utility for the identification of accessible molecular targets in a variety of cancers and will enable probe development for guided resection.

  17. Recent Insights into Cell Surface Heparan Sulphate Proteoglycans and Cancer

    DEFF Research Database (Denmark)

    Couchman, John R; Multhaupt, Hinke; Sanderson, Ralph D

    2016-01-01

    A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal and re...... or fragmented proteoglycans into exosomes that can be paracrine effectors or biomarkers, and lateral interactions between some proteoglycans and calcium channels that impact the actin cytoskeleton....

  18. Immunogold labels: cell-surface markers in atomic force microscopy

    OpenAIRE

    Putman, Constant A.J.; Grooth, de, B.G.; Hansma, Paul K.; Hulst, van der, R.W.M.; Greve, Jan

    1993-01-01

    The feasibility of using immunogold labels as cell-surface markers in atomic force microscopy is shown in this paper. The atomic force microscope (AFM) was used to image the surface of immunogold-labeled human lymphocytes. The lymphocytes were isolated from whole blood and labeled by an indirect immunolabeling method using the monoclonal antibody anti-CD3 and a secondary antibody (Goat-anti-Mouse) linked to 30 nm colloidal gold particles. Some of the samples were enhanced by silver deposition...

  19. Establishment of cell surface engineering and its development.

    Science.gov (United States)

    Ueda, Mitsuyoshi

    2016-07-01

    Cell surface display of proteins/peptides has been established based on mechanisms of localizing proteins to the cell surface. In contrast to conventional intracellular and extracellular (secretion) expression systems, this method, generally called an arming technology, is particularly effective when using yeasts as a host, because the control of protein folding that is often required for the preparation of proteins can be natural. This technology can be employed for basic and applied research purposes. In this review, I describe various strategies for the construction of engineered yeasts and provide an outline of the diverse applications of this technology to industrial processes such as the production of biofuels and chemicals, as well as bioremediation and health-related processes. Furthermore, this technology is suitable for novel protein engineering and directed evolution through high-throughput screening, because proteins/peptides displayed on the cell surface can be directly analyzed using intact cells without concentration and purification. Functional proteins/peptides with improved or novel functions can be created using this beneficial, powerful, and promising technique. PMID:27305282

  20. Ancestral vascular lumen formation via basal cell surfaces.

    Directory of Open Access Journals (Sweden)

    Tomás Kucera

    Full Text Available The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

  1. Pigment Epithelium-derived Factor (PEDF) Binds to Cell-surface F1-ATP Synthase

    OpenAIRE

    NOTARI, LUIGI; Arakaki, Naokatu; Mueller, David; Meier, Scott; Amaral, Juan; Becerra, S. Patricia

    2010-01-01

    Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to a yet unknown protein on the surface of endothelial cells. Given that protein fingerprinting suggested a match of a ~60-kDa PEDF-binding protein in bovine retina to Bos taurus F1-ATP synthase β-subunit, and that F1F0-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding ...

  2. Multidimensional profiling of cell surface proteins and nuclear markers

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

    2009-01-30

    Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

  3. EMA: a developmentally regulated cell-surface glycoprotein of CNS neurons that is concentrated at the leading edge of growth cones.

    Science.gov (United States)

    Baumrind, N L; Parkinson, D; Wayne, D B; Heuser, J E; Pearlman, A L

    1992-08-01

    To identify cell-surface molecules that mediate interactions between neurons and their environment during neural development, we used monoclonal antibody techniques to define a developmentally regulated antigen in the central nervous system of the mouse. The antibody we produced (2A1) immunolabels cells throughout the central nervous system; we analyzed its distribution in the developing cerebral cortex, where it is expressed on cells very soon after they complete mitosis and leave the periventricular proliferative zone. Expression continues into adult life. The antibody also labels the epithelium of the choroid plexus and the renal proximal tubules, but does not label neurons of the peripheral nervous system in the dorsal root ganglia. In dissociated cell culture of embryonic cerebral cortex, 2A1 labels the surface of neurons but not glia. Immunolabeling of neurons in tissue culture is particularly prominent on the edge of growth cones, including filopodia and the leading edge of lamellipodia, when observed with either immunofluorescence or freeze-etch immunoelectron microscopy. Immunopurification with 2A1 of a CHAPS-extracted membrane preparation from brains of neonatal mice produces a broad (32-36 kD) electrophoretic band and a less prominent 70 kD band that are sensitive to N-glycosidase but not endoglycosidase H. Thus the 2A1 antibody recognizes a developmentally regulated, neuronal cell surface glycoprotein (or glycoproteins) with complex N-linked oligosaccharide side chains. We have termed the glycoprotein antigen EMA because of its prominence on the edge membrane of growth cones. EMA is similar to the M6 antigen (Lagenaur et al: J. Neurobiol. 23:71-88, 1992) in apparent molecular weight, distribution in tissue sections, and immunoreactivity on Western blots, suggesting that the two antigens are similar or identical. Expression of EMA is a very early manifestation of neuronal differentiation; its distribution on growth cones suggests a role in mediating the

  4. Molecule nanoweaver

    Science.gov (United States)

    Gerald, II; Rex E.; Klingler, Robert J.; Rathke, Jerome W.; Diaz, Rocio; Vukovic, Lela

    2009-03-10

    A method, apparatus, and system for constructing uniform macroscopic films with tailored geometric assemblies of molecules on the nanometer scale. The method, apparatus, and system include providing starting molecules of selected character, applying one or more force fields to the molecules to cause them to order and condense with NMR spectra and images being used to monitor progress in creating the desired geometrical assembly and functionality of molecules that comprise the films.

  5. Evaluation of Relative Yeast Cell Surface Hydrophobicity Measured by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Lisa Colling

    2005-01-01

    Full Text Available Objective: To develop an efficient method for evaluating cell surface hydrophobicity and to apply the method to demonstrate the effects of fungal growth conditions on cell surface properties.

  6. Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

    Science.gov (United States)

    Smeekens, Johanna M.; Chen, Weixuan; Wu, Ronghu

    2015-04-01

    Cell surface N-glycoproteins play extraordinarily important roles in cell-cell communication, cell-matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide- N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.

  7. Remote Control of Tissue Interactions via Engineered Photo-switchable Cell Surfaces

    OpenAIRE

    Wei Luo; Abigail Pulsipher; Debjit Dutta; Lamb, Brian M.; Yousaf, Muhammad N.

    2014-01-01

    We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture ...

  8. One Step Quick Detection of Cancer Cell Surface Marker by Integrated NiFe-based Magnetic Biosensing Cell Cultural Chip

    Institute of Scientific and Technical Information of China (English)

    Chenchen Bao; Lei Chen; Tao Wang; Chong Lei; Furong Tian; Daxiang Cui; Yong Zhou

    2013-01-01

    RGD peptides has been used to detect cell surface integrin and direct clinical effective therapeutic drug selection. Herein we report that a quick one step detection of cell surface marker that was realized by a specially designed NiFe-based magnetic biosensing cell chip combined with functionalized magnetic nanoparti-cles. Magnetic nanoparticles with 20-30 nm in diameter were prepared by coprecipitation and modified with RGD-4C, and the resultant RGD-functionalized magnetic nanoparticles were used for targeting cancer cells cul-tured on the NiFe-based magnetic biosensing chip and distinguish the amount of cell surface receptor-integrin. Cell lines such as Calu3, Hela, A549, CaFbr, HEK293 and HUVEC exhibiting different integrin expression were chosen as test samples. Calu3, Hela, HEK293 and HUVEC cells were successfully identified. This approach has advantages in the qualitative screening test. Compared with traditional method, it is fast, sensitive, low cost, easy-operative, and needs very little human intervention. The novel method has great potential in applications such as fast clinical cell surface marker detection, and diagnosis of early cancer, and can be easily extended to other biomedical applications based on molecular recognition.

  9. Cell surface hydrophobicity is conveyed by S-layer proteins - A study in recombinant lactobacilli

    NARCIS (Netherlands)

    Mei, H.C. van der; Belt-Gritter, B. van de; Pouwels, P.H.; Martinez, B.; Busscher, H.J.

    2003-01-01

    Cell surface hydrophobicity is one of the most important factors controlling adhesion of microorganisms to surfaces. In this paper, cell surface properties of lactobacilli and recombinant lactobacilli with and without a surface layer protein (SLP) associated with cell surface hydrophobicity were det

  10. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients

    Science.gov (United States)

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-01-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. PMID:26126624

  11. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

    Science.gov (United States)

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-08-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. PMID:26126624

  12. Bacterial Cell Surface Adsorption of Rare Earth Elements

    Science.gov (United States)

    Jiao, Y.; Park, D.; Reed, D.; Fujita, Y.; Yung, M.; Anderko, A.; Eslamimanesh, A.

    2015-12-01

    Rare earth elements (REE) play a critical role in many emerging clean energy technologies, including high-power magnets, wind turbines, solar panels, hybrid/electric vehicle batteries and lamp phosphors. In order to sustain demand for such technologies given current domestic REE shortages, there is a need to develop new approaches for ore processing/refining and recycling of REE-containing materials. To this end, we have developed a microbially-mediated bioadsorption strategy with application towards enrichment of REE from complex mixtures. Specifically, the bacterium Caulobacter crescentus was genetically engineered to display lanthanide binding tags (LBTs), short peptides that possess high affinity and specificity for rare earth elements, on its cell surface S-layer protein. Under optimal conditions, LBT-displayed cells adsorbed greater than 5-fold more REE than control cells lacking LBTs. Competition binding experiments with a selection of REEs demonstrated that our engineered cells could facilitate separation of light- from heavy- REE. Importantly, binding of REE onto our engineered strains was much more favorable compared to non-REE metals. Finally, REE bound to the cell surface could be stripped off using citrate, providing an effective and non-toxic REE recovery method. Together, this data highlights the potential of our approach for selective REE enrichment from REE containing mixtures.

  13. Heavy Exotic Molecules

    CERN Document Server

    Liu, Yizhuang

    2016-01-01

    We briefly review the formation of pion-mediated heavy-light exotic molecules with both charm and bottom, under the general strictures of chiral and heavy quark symmetries. The charm isosinglet exotic molecules with $J^{PC}=1^{++}$ binds, which we identify as the reported neutral $X(3872)$. The bottom isotriplet exotic with $J^{PC}=1^{+-}$ binds, and is identified as a mixed state of the reported charged exotics $Z^+_b(10610)$ and $Z^+_b(10650)$. The bound bottom isosinglet molecule with $J^{PC}=1^{++}$ is a possible neutral $X_b(10532)$ to be observed.

  14. The neural cell adhesion molecule

    DEFF Research Database (Denmark)

    Berezin, V; Bock, E; Poulsen, F M

    2000-01-01

    During the past year, the understanding of the structure and function of neural cell adhesion has advanced considerably. The three-dimensional structures of several of the individual modules of the neural cell adhesion molecule (NCAM) have been determined, as well as the structure of the complex...... between two identical fragments of the NCAM. Also during the past year, a link between homophilic cell adhesion and several signal transduction pathways has been proposed, connecting the event of cell surface adhesion to cellular responses such as neurite outgrowth. Finally, the stimulation of neurite...

  15. Longitudinal microarray analysis of cell surface antigens on peripheral blood mononuclear cells from HIV+ individuals on highly active antiretroviral therapy

    Directory of Open Access Journals (Sweden)

    Wang Bin

    2008-03-01

    Full Text Available Abstract Background The efficacy of highly active antiretroviral therapy (HAART determined by simultaneous monitoring over 100 cell-surface antigens overtime has not been attempted. We used an antibody microarray to analyze changes in the expression of 135 different cell-surface antigens overtime on PBMC from HIV+ patients on HAART. Two groups were chosen, one (n = 6 achieved sustainable response by maintaining below detectable plasma viremia and the other (n = 6 responded intermittently. Blood samples were collected over an average of 3 years and 5–8 time points were selected for microarray assay and statistical analysis. Results Significant trends over time were observed for the expression of 7 cell surface antigens (CD2, CD3epsilon, CD5, CD95, CD36, CD27 and CD28 for combined patient groups. Between groups, expression levels of 10 cell surface antigens (CD11a, CD29, CD38, CD45RO, CD52, CD56, CD57, CD62E, CD64 and CD33 were found to be differential. Expression levels of CD9, CD11a, CD27, CD28 and CD52, CD44, CD49d, CD49e, CD11c strongly correlated with CD4+ and CD8+ T cell counts, respectively. Conclusion Our findings not only detected markers that may have potential prognostic/diagnostic values in evaluating HAART efficacy, but also showed how density of cell surface antigens could be efficiently exploited in an array-like manner in relation to HAART and HIV-infection. The antigens identified in this study should be further investigated by other methods such as flow cytometry for confirmation as biological analysis of these antigens may help further clarify their role during HAART and HIV infection.

  16. Cell adhesion molecules: detection with univalent second antibody

    OpenAIRE

    1980-01-01

    Identification of cell surface molecules that play a role in cell-cell adhesion (here called cell adhesion molecules) has been achieved by demonstrating the inhibitory effect of univalent antibodies that bind these molecules in an in vitro assay of cell-cell adhesion. A more convenient reagent, intact (divalent) antibody, has been avoided because it might agglutinate the cells rather than blocking cell-cell adhesion. In this report, we show that intact rabbit immunoglobulin directed against c...

  17. Research Resource: Androgen Receptor Activity Is Regulated Through the Mobilization of Cell Surface Receptor Networks.

    Science.gov (United States)

    Hsiao, Jordy J; Ng, Brandon H; Smits, Melinda M; Martinez, Harryl D; Jasavala, Rohini J; Hinkson, Izumi V; Fermin, Damian; Eng, Jimmy K; Nesvizhskii, Alexey I; Wright, Michael E

    2015-08-01

    The aberrant expression of androgen receptor (AR)-dependent transcriptional programs is a defining pathology of the development and progression of prostate cancers. Transcriptional cofactors that bind AR are critical determinants of prostate tumorigenesis. To gain a deeper understanding of the proteins linked to AR-dependent gene transcription, we performed a DNA-affinity chromatography-based proteomic screen designed to identify proteins involved in AR-mediated gene transcription in prostate tumor cells. Functional experiments validated the coregulator roles of known AR-binding proteins in AR-mediated transcription in prostate tumor cells. More importantly, novel coregulatory functions were detected in components of well-established cell surface receptor-dependent signal transduction pathways. Further experimentation demonstrated that components of the TNF, TGF-β, IL receptor, and epidermal growth factor signaling pathways modulated AR-dependent gene transcription and androgen-dependent proliferation in prostate tumor cells. Collectively, our proteomic dataset demonstrates that the cell surface receptor- and AR-dependent pathways are highly integrated, and provides a molecular framework for understanding how disparate signal-transduction pathways can influence AR-dependent transcriptional programs linked to the development and progression of human prostate cancers.

  18. Mechanotransduction Across the Cell Surface and Through the Cytoskeleton

    Science.gov (United States)

    Wang, Ning; Butler, James P.; Ingber, Donald E.

    1993-05-01

    Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin β_1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.

  19. Vaccines based on the cell surface carbohydrates of pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Jones Christopher

    2005-01-01

    Full Text Available Glycoconjugate vaccines, in which a cell surface carbohydrate from a micro-organism is covalently attached to an appropriate carrier protein are proving to be the most effective means to generate protective immune responses to prevent a wide range of diseases. The technology appears to be generic and applicable to a wide range of pathogens, as long as antibodies against surface carbohydrates help protect against infection. Three such vaccines, against Haemophilus influenzae type b, Neisseria meningitidis Group C and seven serotypes of Streptococcus pneumoniae, have already been licensed and many others are in development. This article discusses the rationale for the development and use of glycoconjugate vaccines, the mechanisms by which they elicit T cell-dependent immune responses and the implications of this for vaccine development, the role of physicochemical methods in the characterisation and quality control of these vaccines, and the novel products which are under development.

  20. The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway.

    Science.gov (United States)

    Janßen, Linda; Ramnarayan, Venkat Raman; Aboelmagd, Mohamed; Iliopoulou, Maro; Hein, Zeynep; Majoul, Irina; Fritzsche, Susanne; Halenius, Anne; Springer, Sebastian

    2016-01-01

    In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.

  1. Myosin II controls cellular branching morphogenesis and migration in three dimensions by minimizing cell-surface curvature.

    Science.gov (United States)

    Elliott, Hunter; Fischer, Robert S; Myers, Kenneth A; Desai, Ravi A; Gao, Lin; Chen, Christopher S; Adelstein, Robert S; Waterman, Clare M; Danuser, Gaudenz

    2015-02-01

    In many cases, cell function is intimately linked to cell shape control. We used endothelial cell branching morphogenesis as a model to understand the role of myosin II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell-surface curvature. We find that Rho/ROCK-stimulated myosin II contractility minimizes cell-scale branching by recognizing and minimizing local cell-surface curvature. Using microfabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin II cortical association, where it acts to maintain minimal curvature. The feedback between regulation of myosin II by curvature and control of curvature by myosin II drives cycles of localized cortical myosin II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration.

  2. Detection of the plant parasite Cuscuta reflexa by a tomato cell surface receptor.

    Science.gov (United States)

    Hegenauer, Volker; Fürst, Ursula; Kaiser, Bettina; Smoker, Matthew; Zipfel, Cyril; Felix, Georg; Stahl, Mark; Albert, Markus

    2016-07-29

    Parasitic plants are a constraint on agriculture worldwide. Cuscuta reflexa is a stem holoparasite that infests most dicotyledonous plants. One exception is tomato, which is resistant to C. reflexa We discovered that tomato responds to a small peptide factor occurring in Cuscuta spp. with immune responses typically activated after perception of microbe-associated molecular patterns. We identified the cell surface receptor-like protein CUSCUTA RECEPTOR 1 (CuRe1) as essential for the perception of this parasite-associated molecular pattern. CuRe1 is sufficient to confer responsiveness to the Cuscuta factor and increased resistance to parasitic C. reflexa when heterologously expressed in otherwise susceptible host plants. Our findings reveal that plants recognize parasitic plants in a manner similar to perception of microbial pathogens.

  3. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

    2014-01-01

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

  4. Enumerating molecules.

    Energy Technology Data Exchange (ETDEWEB)

    Visco, Donald Patrick, Jr. (, . Tennessee Technological University, Cookeville, TN); Faulon, Jean-Loup Michel; Roe, Diana C.

    2004-04-01

    This report is a comprehensive review of the field of molecular enumeration from early isomer counting theories to evolutionary algorithms that design molecules in silico. The core of the review is a detail account on how molecules are counted, enumerated, and sampled. The practical applications of molecular enumeration are also reviewed for chemical information, structure elucidation, molecular design, and combinatorial library design purposes. This review is to appear as a chapter in Reviews in Computational Chemistry volume 21 edited by Kenny B. Lipkowitz.

  5. Cell-surface acceleration of urokinase-catalyzed receptor cleavage

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Ploug, M; Behrendt, N;

    1997-01-01

    The urokinase-type plasminogen activator (uPA) binds to a specific cell-surface receptor, uPAR. On several cell types uPAR is present both in the full-length form and a cleaved form, uPAR(2+3), which is devoid of binding activity. The formation of uPAR(2+3) on cultured U937 cells is either directly...... by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U......937 cell lysates, had the same amino termini as uPAR(2+3), generated by uPA in a purified system. In both cases cleavage had occurred at two positions in the hinge region connecting domain 1 and 2, between Arg83-Ala84 and Arg89-Ser90, respectively. The uPA-catalyzed cleavage of uPAR is a new negative...

  6. The effect of the state of differentiation on labeling of epidermal cell surface glycoproteins

    International Nuclear Information System (INIS)

    Epidermal cells were grown in a medium in which the Ca++ concentration controlled the stage of differentiation. Cell surface molecules of differentiated and undifferentiated cells were compared by lactoperoxidase-catalyzed iodination, by the interaction with 125I-lectins, and by the metabolic incorporation of L-(3H)-fucose. Molecular weights of the labeled components were determined by SDS-PAGE and autoradiography. After lactoperoxidase iodination, most of the radioactivity was found in polypeptide bands of 79,000, 65,000 and 56,000 daltons. The 79,000 band is the most intense for undifferentiated cells but disappears as differentiation proceeds. The 56,000 band is present in normal cells at all stages of differentiation but is absent from neoplastic cells. Glycoproteins reacted with 125I-lectins were found at 180,000, 130,000 and 85,000 daltons. The 130,000 band was the most prominent for differentiated cells labeled with wheat germ agglutinin but was essentially absent from the undifferentiated cells. With Ricinus communis agglutinin, this band was weaker for undifferentiated than for differentiated cells but was the most intense for both. After metabolic incorporation of tritiated fucose, radioactive glycoproteins were found at 130,000 and 85,000 daltons, with comparable intensities for differentiated and undifferentiated cells

  7. Oligonucleotide delivery with cell surface binding and cell penetrating Peptide amphiphile nanospheres.

    Science.gov (United States)

    Mumcuoglu, Didem; Sardan, Melis; Tekinay, Turgay; Guler, Mustafa O; Tekinay, Ayse B

    2015-05-01

    A drug delivery system designed specifically for oligonucleotide therapeutics can ameliorate the problems associated with the in vivo delivery of these molecules. The internalization of free oligonucleotides is challenging, and cytotoxicity is the main obstacle for current transfection vehicles. To develop nontoxic delivery vehicles for efficient transfection of oligonucleotides, we designed a self-assembling peptide amphiphile (PA) nanosphere delivery system decorated with cell penetrating peptides (CPPs) containing multiple arginine residues (R4 and R8), and a cell surface binding peptide (KRSR), and report the efficiency of this system in delivering G-3129, a Bcl-2 antisense oligonucleotide (AON). PA/AON (peptide amphiphile/antisense oligonucleotide) complexes were characterized with regards to their size and secondary structure, and their cellular internalization efficiencies were evaluated. The effect of the number of arginine residues on the cellular internalization was investigated by both flow cytometry and confocal imaging, and the results revealed that uptake efficiency improved as the number of arginines in the sequence increased. The combined effect of cell penetration and surface binding property on the cellular internalization and its uptake mechanism was also evaluated by mixing R8-PA and KRSR-PA. R8 and R8/KRSR decorated PAs were found to drastically increase the internalization of AONs compared to nonbioactive PA control. Overall, the KRSR-decorated self-assembled PA nanospheres were demonstrated to be noncytotoxic delivery vectors with high transfection rates and may serve as a promising delivery system for AONs. PMID:25828697

  8. Nanoscale biophysical properties of the cell surface galactosaminogalactan from the fungal pathogen Aspergillus fumigatus.

    Science.gov (United States)

    Beaussart, Audrey; El-Kirat-Chatel, Sofiane; Fontaine, Thierry; Latgé, Jean-Paul; Dufrêne, Yves F

    2015-09-28

    Many fungal pathogens produce cell surface polysaccharides that play essential roles in host-pathogen interactions. In Aspergillus fumigatus, the newly discovered polysaccharide galactosaminogalactan (GAG) mediates adherence to a variety of substrates through molecular mechanisms that are poorly understood. Here we use atomic force microscopy to unravel the localization and adhesion of GAG on living fungal cells. Using single-molecule imaging with tips bearing anti-GAG antibodies, we found that GAG is massively exposed on wild-type (WT) germ tubes, consistent with the notion that this glycopolymer is secreted by the mycelium of A. fumigatus, while it is lacking on WT resting conidia and on germ tubes from a mutant (Δuge3) deficient in GAG. Imaging germ tubes with tips bearing anti-β-glucan antibodies shows that exposure of β-glucan is strongly increased in the Δuge3 mutant, indicating that this polysaccharide is masked by GAG during hyphal growth. Single-cell force measurements show that expression of GAG on germ tubes promotes specific adhesion to pneumocytes and non-specific adhesion to hydrophobic substrates. These results provide a molecular foundation for the multifunctional adhesion properties of GAG, thus suggesting it could be used as a potential target in anti-adhesion therapy and immunotherapy. Our methodology represents a powerful approach for characterizing the nanoscale organization and adhesion of cell wall polysaccharides during fungal morphogenesis, thereby contributing to increase our understanding of their role in biofilm formation and immune responses.

  9. Hemagglutinin of influenza A virus binds specifically to cell surface nucleolin and plays a role in virus internalization.

    Science.gov (United States)

    Chan, Che-Man; Chu, Hin; Zhang, Anna Jinxia; Leung, Lai-Han; Sze, Kong-Hung; Kao, Richard Yi-Tsun; Chik, Kenn Ka-Heng; To, Kelvin Kai-Wang; Chan, Jasper Fuk-Woo; Chen, Honglin; Jin, Dong-Yan; Liu, Liang; Yuen, Kwok-Yung

    2016-07-01

    The hemagglutinin (HA) protein of influenza A virus initiates cell entry by binding to sialic acids on target cells. In the current study, we demonstrated that in addition to sialic acids, influenza A/Puerto Rico/8/34 H1N1 (PR8) virus HA specifically binds to cell surface nucleolin (NCL). The interaction between HA and NCL was initially revealed with virus overlay protein binding assay (VOPBA) and subsequently verified with co-immunoprecipitation. Importantly, inhibiting cell surface NCL with NCL antibody, blocking PR8 viruses with purified NCL protein, or depleting endogenous NCL with siRNA all substantially reduced influenza virus internalization. We further demonstrated that NCL was a conserved cellular factor required for the entry of multiple influenza A viruses, including H1N1, H3N2, H5N1, and H7N9. Overall, our findings identified a novel role of NCL in influenza virus life cycle and established NCL as one of the host cell surface proteins for the entry of influenza A virus. PMID:27085069

  10. Aquatic flower-inspired cell culture platform with simplified medium exchange process for facilitating cell-surface interaction studies.

    Science.gov (United States)

    Hong, Hyeonjun; Park, Sung Jea; Han, Seon Jin; Lim, Jiwon; Kim, Dong Sung

    2016-02-01

    Establishing fundamentals for regulating cell behavior with engineered physical environments, such as topography and stiffness, requires a large number of cell culture experiments. However, cell culture experiments in cell-surface interaction studies are generally labor-intensive and time-consuming due to many experimental tasks, such as multiple fabrication processes in sample preparation and repetitive medium exchange in cell culture. In this work, a novel aquatic flower-inspired cell culture platform (AFIP) is presented. AFIP aims to facilitate the experiments on the cell-surface interaction studies, especially the medium exchange process. AFIP was devised to capture and dispense cell culture medium based on interactions between an elastic polymer substrate and a liquid medium. Thus, the medium exchange can be performed easily and without the need of other instruments, such as a vacuum suction and pipette. An appropriate design window of AFIP, based on scaling analysis, was identified to provide a criterion for achieving stability in medium exchange as well as various surface characteristics of the petal substrates. The developed AFIP, with physically engineered petal substrates, was also verified to exchange medium reliably and repeatedly. A closed structure capturing the medium was sustained stably during cell culture experiments. NIH3T3 proliferation results also demonstrated that AFIP can be applied to the cell-surface interaction studies as an alternative to the conventional method. PMID:26683462

  11. M135R is a novel cell surface virulence factor of myxoma virus.

    Science.gov (United States)

    Barrett, John W; Sypula, Joanna; Wang, Fuan; Alston, Lindsay R; Shao, Zhuhong; Gao, Xiujuan; Irvine, Timothy S; McFadden, Grant

    2007-01-01

    Myxoma virus (MV) encodes a cell surface protein (M135R) that is predicted to mimic the host alpha/beta interferon receptor (IFN-alpha/beta-R) and thus prevent IFN-alpha/beta from triggering a host antiviral response. This prediction is based on sequence similarity to B18R, the viral IFN-alpha/beta-R from vaccinia virus (VV), which has been demonstrated to bind and inhibit type I interferons. However, M135R is only half the size of VV B18R. All other poxvirus-encoded IFN-alpha/beta-R homologs align only to the amino-terminal half of M135R. Peptide antibodies raised against M135R were used for immunoblotting and immunofluorescence and indicate that M135R is expressed as an early gene and that the product is a cell surface N-linked glycoprotein that is not secreted. In contrast to the predicted properties of M135R as an inhibitor of type I interferon, all binding and inhibition assays designed to demonstrate whether M135R can interact with IFN-alpha/beta have been negative. However, pathogenesis studies with a targeted M135-knockout MV construct (vMyx135KO) indicate that the deletion of M135R severely attenuates MV pathogenesis in the European rabbit. We propose that M135R is an important immunomodulatory virulence factor for myxomatosis but that the target immune ligand is not from the predicted type I interferon family and remains to be identified. PMID:17065210

  12. Signal transduction by the major histocompatibility complex class I molecule

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Skov, S; Bregenholt, S;

    1999-01-01

    Ligation of cell surface major histocompatibility class I (MHC-I) proteins by antibodies, or by their native counter receptor, the CD8 molecule, mediates transduction of signals into the cells. MHC-I-mediated signaling can lead to both increased and decreased activity of the MHC-I-expressing cell...

  13. The 37/67kDa laminin receptor (LR) inhibitor, NSC47924, affects 37/67kDa LR cell surface localization and interaction with the cellular prion protein

    Science.gov (United States)

    Sarnataro, Daniela; Pepe, Anna; Altamura, Gennaro; De Simone, Imma; Pesapane, Ada; Nitsch, Lucio; Montuori, Nunzia; Lavecchia, Antonio; Zurzolo, Chiara

    2016-01-01

    The 37/67 kDa laminin receptor (LR) is a non-integrin protein, which binds both laminin-1 of the extracellular matrix and prion proteins, that hold a central role in prion diseases. The 37/67 kDa LR has been identified as interactor for the prion protein (PrPC) and to be required for pathological PrP (PrPSc) propagation in scrapie-infected neuronal cells, leading to the possibility that 37/67 kDa LR-PrPC interaction is related to the pathogenesis of prion diseases. A relationship between 37/67 kDa LR and PrPC in the presence of specific LR inhibitor compounds has not been investigated yet. We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrPC. Here, we show that the treatment with the 37/67 kDa LR inhibitor, NSC47924, affects both the direct 37/67 kDa LR-PrPC interaction in vitro and the formation of the immunocomplex in live cells, inducing a progressive internalization of 37/67 kDa LR and stabilization of PrPC on the cell surface. These data reveal NSC47924 as a useful tool to regulate PrPC and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases. PMID:27071549

  14. A reference guide to microbial cell surface hydrophobicity based on contact angles

    NARCIS (Netherlands)

    van der Mei, HC; Busscher, HJ; Bos, R.R.M.

    1998-01-01

    Acid-base interactions form the origin of the hydrophobicity of microbial cell-surfaces and can be quantitated from contact angle measurements on microbial lawns with water, formamide, methyleneiodide and/or alpha-bromonaphthalene. This review provides a reference guide to microbial cell surface hyd

  15. A simple approach to cancer therapy afforded by multivalent pseudopeptides that target cell-surface nucleoproteins

    NARCIS (Netherlands)

    Destouches, D.; Page, N.; Hamma-Kourbali, Y.; Machi, V.; Chaloin, O.; Frechault, S.; Birmpas, C.; Katsoris, P.; Beyrath, J.D.; Albanese, P.; Maurer, M.; Carpentier, G.; Strub, J.M.; Dorsselaer, A. van; Muller, S.; Bagnard, D.; Briand, J.P.; Courty, J.

    2011-01-01

    Recent studies have implicated the involvement of cell surface forms of nucleolin in tumor growth. In this study, we investigated whether a synthetic ligand of cell-surface nucleolin known as N6L could exert antitumor activity. We found that N6L inhibits the anchorage-dependent and independent growt

  16. X-ray photoelectron spectroscopy for the study of microbial cell surfaces

    NARCIS (Netherlands)

    van der Mei, Henderina C; de Vries, Jacob; Busscher, Hendrik J

    2000-01-01

    X-ray photoelectron spectroscopy (XPS) is well known for the characterisation of material surfaces, but at first glance, is an unexpected technique to study the composition of microbial cell surfaces. Despite the fact that intimate contact between materials and microbial cell surfaces occurs in many

  17. Coupling Binding to Catalysis – Using Yeast Cell Surface Display to Select Enzymatic Activities

    OpenAIRE

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence activated cell sorting.

  18. Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

    Science.gov (United States)

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting. PMID:26060080

  19. Altered Traffic of Cardiolipin during Apoptosis: Exposure on the Cell Surface as a Trigger for “Antiphospholipid Antibodies”

    Directory of Open Access Journals (Sweden)

    Valeria Manganelli

    2015-01-01

    Full Text Available Apoptosis has been reported to induce changes in the remodelling of membrane lipids; after death receptor engagement, specific changes of lipid composition occur not only at the plasma membrane, but also in intracellular membranes. This paper focuses on one important aspect of apoptotic changes in cellular lipids, namely, the redistribution of the mitochondria-specific phospholipid, cardiolipin (CL. CL predominantly resides in the inner mitochondrial membrane, even if the rapid remodelling of its acyl chains and the subsequent degradation occur in other membrane organelles. After death receptor stimulation, CL appears to concentrate into mitochondrial “raft-like” microdomains at contact sites between inner and outer mitochondrial membranes, leading to local oligomerization of proapoptotic proteins, including Bid. Clustering of Bid in CL-enriched contacts sites is interconnected with pathways of CL remodelling that intersect membrane traffic routes dependent upon actin. In addition, CL association with cytoskeleton protein vimentin was observed. Such novel association also indicated that CL molecules may be expressed at the cell surface following apoptotic stimuli. This observation adds a novel implication of biomedical relevance. The association of CL with vimentin at the cell surface may represent a “new” target antigen in the context of the apoptotic origin of anti-vimentin/CL autoantibodies in Antiphospholipid Syndrome.

  20. Label-free and dynamic evaluation of cell-surface epidermal growth factor receptor expression via an electrochemiluminescence cytosensor.

    Science.gov (United States)

    Qiu, Youyi; Wen, Qingqing; Zhang, Lin; Yang, Peihui

    2016-04-01

    A label-free electrochemiluminescence (ECL) cytosensor was developed for dynamically evaluating of epidermal growth factor receptor (EGFR) expression on MCF-7 cancer cells based on the specific recognition of epidermal growth factor (EGF) with its receptor (EGFR). EGF-cytosensor was fabricated by in-situ electro-polymerization of polyaniline as substrate, using CdS quantum dots (CdS QDs) as ECL probe and gold nanoparticles (AuNPs) as a carrier for loading of EGF. AuNPs and CdS QDs were jointly attached on polyaniline surface to provide a sensitive and stable sensing interface, as well as a simple and label-free mode for ECL assay. Electron microscopy, atomic force microscopy (AFM) and electrochemical methods were employed to characterize the multilayer construction process of the sensing interface. The proposed EGF-cytosensor exhibited excellent analytical performance for MCF-7 cancer cells, ranging from 12 to 1.2 × 10(6) cells mL(-1), with a low detection limit of 12 cells mL(-1). Also, it was successfully applied in evaluating EGFR expression of cells surface, which was stimulated by some inhibitors or activator, and the results were confirmed by using flow cytometry and laser scanning confocal microscopy analysis. The proposed ECL cytosensor has potential applications in monitoring the dynamic variation of receptor molecules expression on cell surfaces in response to external stimulation by drugs and screening anti-cancer therapeutic agents. PMID:26838410

  1. The Role of Cell Surface Architecture of Lactobacilli in Host-Microbe Interactions in the Gastrointestinal Tract

    Directory of Open Access Journals (Sweden)

    Ranjita Sengupta

    2013-01-01

    Full Text Available Lactobacillus species can exert health promoting effects in the gastrointestinal tract (GIT through many mechanisms, which include pathogen inhibition, maintenance of microbial balance, immunomodulation, and enhancement of the epithelial barrier function. Different species of the genus Lactobacillus can evoke different responses in the host, and not all strains of the same species can be considered beneficial. Strain variations may be related to diversity of the cell surface architecture of lactobacilli and the bacteria's ability to express certain surface components or secrete specific compounds in response to the host environment. Lactobacilli are known to modify their surface structures in response to stress factors such as bile and low pH, and these adaptations may help their survival in the face of harsh environmental conditions encountered in the GIT. In recent years, multiple cell surface-associated molecules have been implicated in the adherence of lactobacilli to the GIT lining, immunomodulation, and protective effects on intestinal epithelial barrier function. Identification of the relevant bacterial ligands and their host receptors is imperative for a better understanding of the mechanisms through which lactobacilli exert their beneficial effects on human health.

  2. Heme Transfer from Streptococcal Cell Surface Protein Shp to HtsA of Transporter HtsABC

    OpenAIRE

    Liu, Mengyao; Lei, Benfang

    2005-01-01

    Human pathogen group A streptococcus (GAS) can take up heme from host heme-containing proteins as a source of iron. Little is known about the heme acquisition mechanism in GAS. We recently identified a streptococcal cell surface protein (designated Shp) and the lipoprotein component (designated HtsA) of an ATP-binding cassette (ABC) transporter made by GAS as heme-binding proteins. In an effort to delineate the molecular mechanism involved in heme acquisition by GAS, heme-free Shp (apo-Shp) a...

  3. (A structural assessment of the role of the cell surface carbohydrates of Rhizobium in the Rhizobium/legume symbiosis)

    Energy Technology Data Exchange (ETDEWEB)

    Hollingsworth, R.I.

    1991-01-01

    Research continued on the study of cell surface carbohydrates of Rhizobium. Objectives include: To characterize, at a structural level, the differences between the lipopolysaccharides of a representative number of strains from different Rhizobium species to determine which features of LPS structure are species-specific and might, therefore, be determinants of host specificity. Determine the effect(s) of nod gene induction on the structure of Rhizobium lipopolysaccharides and determine whether synthesis of a modified LPS molecule or a new surface glycoconjugate is initiated by nod gene induction. Develop a non-chemical means for rapidly screening large numbers of bacterial strains in order to determine which glycoconjugate structural features are conserved between strains of the same species. Provide the necessary structural information which, when coupled with developments in the rapidly expanding field of Rhizobium genetics, should lead to a clear understanding of the role of Rhizobium surface glycoconjugates in host/symbiont interactions. Progress is discussed.

  4. Facile Discovery of Cell-Surface Protein Targets of Cancer Cell Aptamers.

    Science.gov (United States)

    Bing, Tao; Shangguan, Dihua; Wang, Yinsheng

    2015-10-01

    Cancer biomarker discovery constitutes a frontier in cancer research. In recent years, cell-binding aptamers have become useful molecular probes for biomarker discovery. However, there are few successful examples, and the critical barrier resides in the identification of the cell-surface protein targets for the aptamers, where only a limited number of aptamer targets have been identified so far. Herein, we developed a universal SILAC-based quantitative proteomic method for target discovery of cell-binding aptamers. The method allowed for distinguishing specific aptamer-binding proteins from nonspecific proteins based on abundance ratios of proteins bound to aptamer-carrying bait and control bait. In addition, we employed fluorescently labeled aptamers for monitoring and optimizing the binding conditions. We were able to identify and validate selectin L and integrin α4 as the protein targets for two previously reported aptamers, Sgc-3b and Sgc-4e, respectively. This strategy should be generally applicable for the discovery of protein targets for other cell-binding aptamers, which will promote the applications of these aptamers.

  5. Heparanase facilitates cell adhesion and spreading by clustering of cell surface heparan sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Flonia Levy-Adam

    Full Text Available Heparanase is a heparan sulfate (HS degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys(158-Asp(171, termed KKDC was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity.

  6. Ketone isosteres of 2-N-acetamidosugars as substrates for metabolic cell surface engineering

    Energy Technology Data Exchange (ETDEWEB)

    Hang, Howard C.; Bertozzi, Carolyn R.

    2000-08-22

    Novel chemical reactivity can be engendered on cell surfaces by the metabolic incorporation of unnatural sugars into cell surface glycoconjuagtes. 2-N-Acetamido sugars such as GalNAc and GlcNAc are abundant components of cell surface glycoconjugates, and hence attractive targets for metabolic cell surface engineering. Here we report (1) the synthesis of isosteric analogs bearing a ketone group in place of the N-acetamido group, and (2) evaluation of their metabolic incorporation into mammalian cell surface glycans. A ketone isostere of GalNAc was metabolized by CHO cells through the salvage pathway and delivered to O-linked glycoproteins on the cell surface. Its residence at the core position of O-linked glycans is suggested by studies with a-benzyl GalNAc, an inhibitor of O-linked oligosaccharide extension. A mutant CHO cell line lacking endogenous UDP-GalNAc demonstrated enhanced metabolism of the GalNAc analog, suggesting that competition with native intermediates might limits enzymatic transformation in mammalian cells. A ketone isostere of GlcNAc could not be detected on CHO or human cell surfaces after incubation. Thus, the enzymes in the GlcNAc salvage pathway might be less permissive of unnatural substrates than those comprising the GalNAc salvage pathway. Alternatively, high levels of endogenous GlcNAc derivatives might compete with the ketone isostere and prevent its incorporation into oligosaccharides.

  7. Comprehensive proteomic analysis of Trypanosoma cruzi epimastigote cell surface proteins by two complementary methods

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sébastien; Motta, Flávia N;

    2013-01-01

    Trypanosoma cruzi is a protozoan that causes Chagas' disease, a neglected infectious illness that affects millions of people, mostly in Latin America. Here, the cell surface subproteome of the T. cruzi epimastigote life form was characterized. In order to prepare samples enriched in epimastigote...... plasma membrane protein, two distinct methodologies were optimized and evaluated. The first methodology was based on cell surface trypsinization (Shave) of intact living cells while the second approach used biotinylation of cell surface proteins followed by streptavidin affinity chromatography isolation...

  8. Sorption behavior of europium(III) and curium(III) on the cell surfaces of microorganisms

    Energy Technology Data Exchange (ETDEWEB)

    Ozaki, T.; Kimura, T.; Ohnuki, T.; Yoshida, Z. [Advanced Science Research Center, Japan Atomic Energy Research Inst., Ibaraki (Japan); Gillow, J.B.; Francis, A.J. [Environmental Sciences Dept., Brookhaven National Lab., Upton, NY (United States)

    2004-07-01

    We investigated the association of europium(III) and curium(III) with the microorganisms Chlorella vulgaris, Bacillus subtilis, Pseudomonas fluorescens, Halomonas sp., Halobacterium salinarum, and Halobacterium halobium. We determined the kinetics and distribution coefficients (K{sub d}) for Eu(III) and Cm(III) sorption at pH 3-5 by batch experiments, and evaluated the number of water molecules in the inner-sphere (N{sub H{sub 2}O}) and the degree of strength of ligand field (R{sub E/M}) for Eu(III) by time-resolved laser-induced fluorescence spectroscopy (TRLFS). Exudates from C. vulgaris, Halomonas sp., and H. halobium had an affinity for Eu(III) and Cm(III). The log K{sub d} of Eu(III) and Cm(III) showed that their sorption was not fully due to the exchange with three protons on the functional groups on cell surfaces. The halophilic microorganisms (Halomonas sp., Halobacterium salinarum, H. halobium) showed almost no pH dependence in log K{sub d}, indicating that an exchange with Na{sup +} on the functional groups was involved in their sorption. The {delta}N{sub H{sub 2}O} (= 9 - N{sub H{sub 2}O}) for Eu(III) on C. vulgaris was 1-3, while that for the other microorganisms was over 3, demonstrating that the coordination of Eu(III) with C. vulgaris was predominantly an outer-spherical process. The R{sub E/M} for Eu(III) on halophilic microorganisms was 2.5-5, while that for non-halophilic ones was 1-2.5. This finding suggests that the coordination environment of Eu(III) on the halophilic microorganisms is more complicated than that on the other three non-halophilic ones. (orig.)

  9. Cell surface receptors for signal transduction and ligand transport: a design principles study.

    Directory of Open Access Journals (Sweden)

    Harish Shankaran

    2007-06-01

    Full Text Available Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor-ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation.

  10. Determination of cellular lipids bound to human CD1d molecules.

    Directory of Open Access Journals (Sweden)

    Daryl Cox

    Full Text Available CD1 molecules are glycoproteins that present lipid antigens at the cell surface for immunological recognition by specialized populations of T lymphocytes. Prior experimental data suggest a wide variety of lipid species can bind to CD1 molecules, but little is known about the characteristics of cellular ligands that are selected for presentation. Here we have molecularly characterized lipids bound to the human CD1d isoform. Ligands were eluted from secreted CD1d molecules and separated by normal phase HPLC, then characterized by mass spectroscopy. A total of 177 lipid species were molecularly identified, comprising glycerophospholipids and sphingolipids. The glycerophospholipids included common diacylglycerol species, reduced forms known as plasmalogens, lyso-phospholipids (monoacyl species, and cardiolipins (tetraacyl species. The sphingolipids included sphingomyelins and glycosylated forms, such as the ganglioside GM3. These results demonstrate that human CD1d molecules bind a surprising diversity of lipid structures within the secretory pathway, including compounds that have been reported to play roles in cancer, autoimmune diseases, lipid signaling, and cell death.

  11. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

    DEFF Research Database (Denmark)

    Sundberg, Christina; Thodeti, Charles Kumar; Kveiborg, Marie;

    2004-01-01

    constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell......The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin......-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell...

  12. Hadron Molecules

    CERN Document Server

    Gutsche, Thomas; Faessler, Amand; Lee, Ian Woo; Lyubovitskij, Valery E

    2010-01-01

    We discuss a possible interpretation of the open charm mesons $D_{s0}^*(2317)$, $D_{s1}(2460)$ and the hidden charm mesons X(3872), Y(3940) and Y(4140) as hadron molecules. Using a phenomenological Lagrangian approach we review the strong and radiative decays of the $D_{s0}^* (2317)$ and $D_{s1}(2460)$ states. The X(3872) is assumed to consist dominantly of molecular hadronic components with an additional small admixture of a charmonium configuration. Determing the radiative ($\\gamma J/\\psi$ and $\\gamma \\psi(2s)$) and strong ($J/\\psi 2\\pi $ and $ J/\\psi 3\\pi$) decay modes we show that present experimental observation is consistent with the molecular structure assumption of the X(3872). Finally we give evidence for molecular interpretations of the Y(3940) and Y(4140) related to the observed strong decay modes $J/\\psi + \\omega$ or $J/\\psi + \\phi$, respectively.

  13. Cell Surface Proteins in S. Pneumoniae, S. Mitis and S. Oralis

    Directory of Open Access Journals (Sweden)

    R Hakenbeck

    2011-06-01

    Full Text Available Background and objectives: Streptococcus pneumoniae, a major human pathogen, is closely related to the commensal species S. mitis and S. oralis. S. pneumoniae surface proteins are implicated in virulence and host interaction of this species, but many of them have recently been detected in S. mitis B6 in silico. We tested for the presence of such genes usinga set of eight S. mitis and eleven S. oralis strains from different geographic locations.Materials and Methods: An oligonucleotide microarray was designed based on the genomes of S. pneumoniae R6 and TIGR4 as well as S. mitis B6 to include 63 cell surface proteins. The S. pneumoniae genes encoding neuraminidases, hyaluronidase and pneumolysin were also included. In addition to comparative genomic hybridization experiments, homologues were identified in silico in the genome of S. oralis Uo5.Results and Conclusions: The results document that many S. pneumoniae related surface proteins are ubiquitously present among the Mitis group of streptococci. All 19 samples hybridized with the pavA probe representing a gene important for adherence and invasion of S. pneumoniae. Only eight genes were not recognized in any strain, including the S. pneumoniae PcpC gene as the only virulence gene of the S. pneumoniae core genome.The fact that only 12 out of 26 genes present in the S. oralis Uo5 genome could be detected by microarray analysis confirms the sequence variation of surface components.

  14. Variability in expression of cell surface antigens of Candida albicans during morphogenesis.

    OpenAIRE

    Brawner, D L; Cutler, J. E.

    1986-01-01

    The location and expression of two different cell surface antigens on germinating and nongerminating Candida albicans cells was examined by using transmission electron microscopy after labeling with monoclonal antibodies (H9 or C6) and immunocolloidal gold. Immunodeterminant expression of the two carbohydrate antigens was followed from early germination events through 20 h of development. The determinant detected by H9 antibody, which was initially lost from the mother cell surface and prefer...

  15. The Na+/H+ Exchanger Regulatory Factor Stabilizes Epidermal Growth Factor Receptors at the Cell Surface

    OpenAIRE

    Lazar, Cheri S.; Cresson, Catherine M.; Lauffenburger, Douglas A.; Gill, Gordon N.

    2004-01-01

    Ligand binding to cell surface receptors initiates both signal transduction and endocytosis. Although signaling may continue within the endocytic compartment, down-regulation is the major mechanism that controls the concentration of cell surface receptors, their ability to receive environmental signals, and the ultimate strength of biological signaling. Internalization, recycling, and trafficking of receptor tyrosine kinases (RTKs) within the endosome compartment are each regulated to control...

  16. Exocellular esterase and emulsan release from the cell surface of Acinetobacter calcoaceticus.

    OpenAIRE

    Shabtai, Y; Gutnick, D L

    1985-01-01

    An esterase activity has been found, both in the cell-free growth medium and on the cell surface of the hydrocarbon-degrading Acinetobacter calcoaceticus RAG-1. The enzyme catalyzed the hydrolysis of acetyl and other acyl groups from triglycerides and aryl and alkyl esters. Emulsan, the extracellular heteropolysaccharide bioemulsifier produced by strain RAG-1, was also a substrate for the enzyme. Gel filtration showed that the cell-free enzyme was released from the cell surface either emulsan...

  17. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    DEFF Research Database (Denmark)

    Roldan, A L; Cubellis, M V; Masucci, M T;

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human...

  18. DMPD: Innate immune sensing of pathogens and danger signals by cell surface Toll-likereceptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17275324 Innate immune sensing of pathogens and danger signals by cell surface Toll... Show Innate immune sensing of pathogens and danger signals by cell surface Toll-likereceptors. PubmedID 172...75324 Title Innate immune sensing of pathogens and danger signals by cell surface

  19. MHC class I molecules are enriched in caveolae but do not enter with simian virus 40.

    Science.gov (United States)

    Anderson, H A; Chen, Y; Norkin, L C

    1998-06-01

    Simian virus 40 (SV40) binds to MHC class I molecules anywhere on the cell surface and then enters through caveolae. The fate of class I molecules after SV40 binding is not known. Sensitivity of 125I-surface-labelled class I molecules to papain cleavage was used to distinguish internalized class I molecules from class I molecules remaining at the cell surface. Whereas the caveolae-enriched membrane microdomain was found to also be enriched for class I molecules, no internalized papain-resistant 125I-surface-labelled class I molecules could be detected at any time in either control cells or in cells preadsorbed with saturating amounts of SV40. Instead, 125I-surface-labelled class I molecules, as well as preadsorbed 125I-labelled anti-class I antibodies, accumulated in the medium, coincident with the turnover of class I molecules at the cell surface. The class I heavy chains that accumulated in the medium were truncated and their release was specifically prevented by the metalloprotease inhibitor 1,10-phenanthroline. Thus, whereas class I molecules mediate SV40 binding, they do not appear to mediate SV40 entry.

  20. Us3 kinase encoded by herpes simplex virus 1 mediates downregulation of cell surface major histocompatibility complex class I and evasion of CD8+ T cells.

    Directory of Open Access Journals (Sweden)

    Takahiko Imai

    Full Text Available Detection and elimination of virus-infected cells by CD8(+ cytotoxic T lymphocytes (CTLs depends on recognition of virus-derived peptides presented by major histocompatibility complex class I (MHC-I molecules on the surface of infected cells. In the present study, we showed that inactivation of the activity of viral kinase Us3 encoded by herpes simplex virus 1 (HSV-1, the etiologic agent of several human diseases and a member of the alphaherpesvirinae, significantly increased cell surface expression of MHC-I, thereby augmenting CTL recognition of infected cells in vitro. Overexpression of Us3 by itself had no effect on cell surface expression of MHC-I and Us3 was not able to phosphorylate MHC-I in vitro, suggesting that Us3 indirectly downregulated cell surface expression of MHC-I in infected cells. We also showed that inactivation of Us3 kinase activity induced significantly more HSV-1-specific CD8(+ T cells in mice. Interestingly, depletion of CD8(+ T cells in mice significantly increased replication of a recombinant virus encoding a kinase-dead mutant of Us3, but had no effect on replication of a recombinant virus in which the kinase-dead mutation was repaired. These results indicated that Us3 kinase activity is required for efficient downregulation of cell surface expression of MHC-I and mediates evasion of HSV-1-specific CD8(+ T cells. Our results also raised the possibility that evasion of HSV-1-specific CD8(+ T cells by HSV-1 Us3-mediated inhibition of MHC-I antigen presentation might in part contribute to viral replication in vivo.

  1. Alteration in cell surface properties of Burkholderia spp. during surfactant-aided biodegradation of petroleum hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Mohanty, Sagarika; Mukherji, Suparna [Indian Institute of Technology Bombay, Mumbai (India). Centre for Environmental Science and Engineering (CESE)

    2012-04-15

    Chemical surfactants may impact microbial cell surface properties, i.e., cell surface hydrophobicity (CSH) and cell surface charge, and may thus affect the uptake of components from non-aqueous phase liquids (NAPLs). This work explored the impact of Triton X-100, Igepal CA 630, and Tween 80 (at twice the critical micelle concentration, CMC) on the cell surface characteristics of Burkholderia cultures, Burkholderia cepacia (ES1, aliphatic degrader) and Burkholderia multivorans (NG1, aromatic degrader), when grown on a six-component model NAPL. In the presence of Triton X-100, NAPL biodegradation was enhanced from 21% to 60% in B. cepacia and from 18% to 53% in B. multivorans. CSH based on water contact angle (50-52 ) was in the same range for both strains while zeta potential at neutral pH was -38 and -31 mV for B. cepacia and B. multivorans, respectively. In the presence of Triton X-100, their CSH increased to greater than 75 and the zeta potential decreased. This induced a change in the mode of uptake and initiated aliphatic hydrocarbon degradation by B. multivorans and increased the rate of aliphatic hydrocarbon degradation in B. cepacia. Igepal CA 630 and Tween 80 also altered the cell surface properties. For B. cepacia grown in the presence of Triton X-100 at two and five times its CMC, CSH increased significantly in the log growth phase. Growth in the presence of the chemical surfactants also affected the abundance of chemical functional groups on the cell surface. Cell surface changes had maximum impact on NAPL degradation in the presence of emulsifying surfactants, Triton X-100 and Igepal CA630.

  2. The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.

    Science.gov (United States)

    Knopf, Julia D; Tholen, Stefan; Koczorowska, Maria M; De Wever, Olivier; Biniossek, Martin L; Schilling, Oliver

    2015-10-01

    Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.

  3. BST2/CD317 counteracts human coronavirus 229E productive infection by tethering virions at the cell surface

    International Nuclear Information System (INIS)

    Bone marrow stromal antigen 2 (BST2), an interferon-inducible antiviral factor, has been shown to block the release of various enveloped viruses from cells. It has also been identified as an innate immune system component. Most enveloped viruses subject to BST2 restriction bud at the plasma membrane. Here we report our findings that (a) the production of human coronavirus 229E (HCoV-229E) progeny viruses, whose budding occurs at the ER-Golgi intermediate compartment (ERGIC), markedly decreases in the presence of BST2; and (b) BST2 knockdown expression results in enhanced HCoV-229E virion production. Electron microscopy analyses indicate that HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. Our results suggest that BST2 exerts a broad blocking effect against enveloped virus release, regardless of whether budding occurs at the plasma membrane or intracellular compartments. - Highlights: • BST2 knockdown expression results in enhanced HCoV-229E egress. • HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. • HCoV-229E infection at high MOI can significantly downregulate HeLa BST2 and rescue HIV-1 egress

  4. BST2/CD317 counteracts human coronavirus 229E productive infection by tethering virions at the cell surface

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shiu-Mei [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Huang, Kuo-Jung [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Wang, Chin-Tien, E-mail: chintien@ym.edu.tw [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan (China)

    2014-01-20

    Bone marrow stromal antigen 2 (BST2), an interferon-inducible antiviral factor, has been shown to block the release of various enveloped viruses from cells. It has also been identified as an innate immune system component. Most enveloped viruses subject to BST2 restriction bud at the plasma membrane. Here we report our findings that (a) the production of human coronavirus 229E (HCoV-229E) progeny viruses, whose budding occurs at the ER-Golgi intermediate compartment (ERGIC), markedly decreases in the presence of BST2; and (b) BST2 knockdown expression results in enhanced HCoV-229E virion production. Electron microscopy analyses indicate that HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. Our results suggest that BST2 exerts a broad blocking effect against enveloped virus release, regardless of whether budding occurs at the plasma membrane or intracellular compartments. - Highlights: • BST2 knockdown expression results in enhanced HCoV-229E egress. • HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. • HCoV-229E infection at high MOI can significantly downregulate HeLa BST2 and rescue HIV-1 egress.

  5. Anomalous cell surface structure of sickle cell anemia erythrocytes as demonstrated by cell surface labeling and endo-beta-galactosidase treatment

    Energy Technology Data Exchange (ETDEWEB)

    Fukuda, M.; Fukuda, M.N.; Hakomori, S.; Papayannopoulou, T.

    1981-01-01

    Erythrocyte surface glycoproteins from patients with various types of sickle cell anemia have been analyzed and compared with those from normal individuals. By hemagglutination with various anti-carbohydrate antibodies, sickle cells showed profound increase of i antigens and moderate increase of GlcNAc beta 1 leads to 3Gal beta 1 leads to 3 Glc structure, whereas antigenicity toward globosidic structure was unchanged. In parallel to these findings, erythrocytes of sickle cell patients have additional sialylated lactosaminoglycan in Band 3. Thus, it can be concluded that erythrocytes of sickle cell patients are characterized by an altered cell surface structure which does not appear to be due to topographical changes of cell surface membrane. It is possible that the anemia or the ''stress'' hematopoiesis in these patients is responsible for these changes.

  6. Emergence of an Apical Epithelial Cell Surface In Vivo.

    Science.gov (United States)

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

  7. Remote Control of Tissue Interactions via Engineered Photo-switchable Cell Surfaces

    Science.gov (United States)

    Luo, Wei; Pulsipher, Abigail; Dutta, Debjit; Lamb, Brian M.; Yousaf, Muhammad N.

    2014-09-01

    We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand. Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation. Remote control of cell interactions via cell surface engineering that allows for real-time manipulation of tissue dynamics may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies.

  8. Characterization of fucosyltransferase activity during mouse spermatogenesis: Evidence for a cell surface fucosyltransferase

    International Nuclear Information System (INIS)

    Fucosyltransferase activity was quantified in mouse germ cells at different stages of spermatogenesis. Specifically, fucosyltransferase activities of pachytene spermatocytes, round spermatids, and cauda epididymal sperm were compared. Fucosyltranferase activity of mixed germ cells displayed an apparent Vmax of 17 pmol (mg of protein)-1 min-1 and an apparent Km of approximately 13 μM for GDP-L-[14C]fucose in the presence of saturating amounts of asialofetuin at 33 degree C. Under these conditions, cellular fucosyltransferase activity was found to increase during spermatogenesis. In agreement with assays of intact cells, examination of subcellular fractions indicated that a large fraction of fucosyltransferase activity was associated with the cell surface. The fraction of fucosyltransferase activity that was associated with the cell surface progressively increased throughout spermatogenesis and epididymal maturation so that nearly all of the fucosyltransferase in epididymal sperm was on the cell surface. Specifically, by comparison of activities in the presence and absence of the detergent NP-40, the fraction of fucosyltransferase activity that was associated with the cell surface in pachytene spermatocytes, round spermatids, and epididymal sperm was 0.36, 0.5, and 0.85, respectively. These results suggest that a cell surface fucosyltransferase may be important during differentiation of spermatogenic cells in the testis as well as during epididymal maturation and fertilization

  9. OMG: Open Molecule Generator

    Directory of Open Access Journals (Sweden)

    Peironcely Julio E

    2012-09-01

    Full Text Available Abstract Computer Assisted Structure Elucidation has been used for decades to discover the chemical structure of unknown compounds. In this work we introduce the first open source structure generator, Open Molecule Generator (OMG, which for a given elemental composition produces all non-isomorphic chemical structures that match that elemental composition. Furthermore, this structure generator can accept as additional input one or multiple non-overlapping prescribed substructures to drastically reduce the number of possible chemical structures. Being open source allows for customization and future extension of its functionality. OMG relies on a modified version of the Canonical Augmentation Path, which grows intermediate chemical structures by adding bonds and checks that at each step only unique molecules are produced. In order to benchmark the tool, we generated chemical structures for the elemental formulas and substructures of different metabolites and compared the results with a commercially available structure generator. The results obtained, i.e. the number of molecules generated, were identical for elemental compositions having only C, O and H. For elemental compositions containing C, O, H, N, P and S, OMG produces all the chemically valid molecules while the other generator produces more, yet chemically impossible, molecules. The chemical completeness of the OMG results comes at the expense of being slower than the commercial generator. In addition to being open source, OMG clearly showed the added value of constraining the solution space by using multiple prescribed substructures as input. We expect this structure generator to be useful in many fields, but to be especially of great importance for metabolomics, where identifying unknown metabolites is still a major bottleneck.

  10. OMG: Open Molecule Generator.

    Science.gov (United States)

    Peironcely, Julio E; Rojas-Chertó, Miguel; Fichera, Davide; Reijmers, Theo; Coulier, Leon; Faulon, Jean-Loup; Hankemeier, Thomas

    2012-01-01

    Computer Assisted Structure Elucidation has been used for decades to discover the chemical structure of unknown compounds. In this work we introduce the first open source structure generator, Open Molecule Generator (OMG), which for a given elemental composition produces all non-isomorphic chemical structures that match that elemental composition. Furthermore, this structure generator can accept as additional input one or multiple non-overlapping prescribed substructures to drastically reduce the number of possible chemical structures. Being open source allows for customization and future extension of its functionality. OMG relies on a modified version of the Canonical Augmentation Path, which grows intermediate chemical structures by adding bonds and checks that at each step only unique molecules are produced. In order to benchmark the tool, we generated chemical structures for the elemental formulas and substructures of different metabolites and compared the results with a commercially available structure generator. The results obtained, i.e. the number of molecules generated, were identical for elemental compositions having only C, O and H. For elemental compositions containing C, O, H, N, P and S, OMG produces all the chemically valid molecules while the other generator produces more, yet chemically impossible, molecules. The chemical completeness of the OMG results comes at the expense of being slower than the commercial generator. In addition to being open source, OMG clearly showed the added value of constraining the solution space by using multiple prescribed substructures as input. We expect this structure generator to be useful in many fields, but to be especially of great importance for metabolomics, where identifying unknown metabolites is still a major bottleneck. PMID:22985496

  11. Novel Properties for Endoglucanase Acquired by Cell-Surface Display Technique.

    Science.gov (United States)

    Shi, Baosheng; Ke, Xiaojing; Yu, Hongwei; Xie, Jing; Jia, Yingmin; Guo, Runfang

    2015-11-01

    In order to improve the stability of endoglucanase under thermal and acidic conditions, the endoglucanase gene was fused to the N-terminus of the Saccharomyces cerevisiae pir gene, encoding the cell wall protein PIR. The fusion gene was transformed into Pichia pastoris GS115 for expression. A resulting strain with high expression and high activity was identified by examining resistance to Geneticin 418, Congo red staining, and quantitative analysis of enzyme activity. SDS-PAGE analysis revealed that the endoglucanase was successfully displayed on the yeast cell surface. The displayed endoglucanase (DEG) showed maximum activity towards sodium carboxyl methyl cellulose at approximately 275 IU/g cell dry weight. DEG exhibited greater than 60% residual activity in the pH range 2.5-8.5, higher than free endoglucanase (FEG), which had 40% residual activity at the same pH range. The highest tolerated temperature for DEG was 70°C, much higher than that of FEG, which was approximately 50°C. Moreover, DEG showed 91.1% activity at 65°C for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65°C, compared with only 150 min for FEG. DEG could be used repeatedly at least three times. These results suggest that the DEG has broad applications as a yeast whole-cell biocatalyst, due to its novel properties of high catalytic efficiency, acid-thermal stabilities, and reusability. PMID:26198121

  12. Recent Insights into Cell Surface Heparan Sulphate Proteoglycans and Cancer [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    John R Couchman

    2016-06-01

    Full Text Available A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal and regulate important cell processes, such as adhesion, migration, proliferation, and differentiation. Since many protein ligands, such as growth factors, morphogens, and cytokines, are also implicated in tumour progression, it is increasingly apparent that cell surface proteoglycans impact tumour cell behaviour. Here, we review some recent advances, emphasising that many tumour-related functions of proteoglycans are revealed only after their modification in processes subsequent to synthesis and export to the cell surface. These include enzymes that modify heparan sulphate structure, recycling of whole or fragmented proteoglycans into exosomes that can be paracrine effectors or biomarkers, and lateral interactions between some proteoglycans and calcium channels that impact the actin cytoskeleton.

  13. Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers.

    Science.gov (United States)

    González-Robles, Arturo; Castañón, Guadalupe; Cristóbal-Ramos, Ana Ruth; Hernández-Ramírez, Verónica Ivonne; Omaña-Molina, Maritza; Martínez-Palomo, Adolfo

    2007-12-01

    Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.

  14. Recent advances in yeast cell-surface display technologies for waste biorefineries.

    Science.gov (United States)

    Liu, Zhuo; Ho, Shih-Hsin; Hasunuma, Tomohisa; Chang, Jo-Shu; Ren, Nan-Qi; Kondo, Akihiko

    2016-09-01

    Waste biorefinery aims to maximize the output of value-added products from various artificial/agricultural wastes by using integrated bioprocesses. To make waste biorefinery economically feasible, it is thus necessary to develop a low-cost, environment-friendly technique to perform simultaneous biodegradation and bioconversion of waste materials. Cell-surface display engineering is a novel, cost-effective technique that can auto-immobilize proteins on the cell exterior of microorganisms, and has been applied for use with waste biofinery. Through tethering different enzymes (e.g., cellulase, lipase, and protease) or metal-binding peptides on cell surfaces, various yeast strains can effectively produce biofuels and biochemicals from sugar/protein-rich waste materials, catalyze waste oils into biodiesels, or retrieve heavy metals from wastewater. This review critically summarizes recent applications of yeast cell-surface display on various types of waste biorefineries, highlighting its potential and future challenges with regard to commercializing this technology. PMID:27039354

  15. A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yanjuan; Zhang, Shaolian; Wen, Qingqing; Huang, Hongxing; Yang, Peihui, E-mail: typh@jnu.edu.cn

    2015-06-30

    Highlights: • EGF-cytosensor was used for evaluating EGFR expression level on cell surfaces. • CdSQDs and EGF were coated on magnetic beads (MBs) for ECL-probe. • Good sensitivity was achieved due to the signal amplification of ECL-probe. - Abstract: A sensitive electrochemiluminescence (ECL) strategy for evaluating the epidermal growth factor receptor (EGFR) expression level on cell surfaces was designed by integrating the specific recognition of EGFR expressed on MCF-7 cell surfaces with an epidermal growth factor (EGF)-funtionalized CdS quantum dots (CdSQDs)-capped magnetic bead (MB) probe. The high sensitivity of ECL probe of EGF-funtionalized CdSQD-capped-MB was used for competitive recognition with EGFR expressed on cell surfaces with recombinant EGFR protein. The changes of ECL intensity depended on both the cell number and the expression level of EGFR receptor on cell surfaces. A wide linear response to cells ranging from 80 to 4 × 10{sup 6} cells mL{sup −1} with a detection limit of 40 cells mL{sup −1} was obtained. The EGF-cytosensor was used to evaluate EGFR expression levels on MCF-7 cells, and the average number of EGFR receptor on single MCF-7 cells was 1.35 × 10{sup 5} with the relative standard deviation of 4.3%. This strategy was further used for in-situ and real-time evaluating EGFR receptor expressed on cell surfaces in response to drugs stimulation at different concentration and incubation time. The proposed method provided potential applications in the detection of receptors on cancer cells and anticancer drugs screening.

  16. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

    Science.gov (United States)

    Mallanna, Sunil K; Cayo, Max A; Twaroski, Kirk; Gundry, Rebekah L; Duncan, Stephen A

    2016-09-13

    When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  17. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes

    Directory of Open Access Journals (Sweden)

    Sunil K. Mallanna

    2016-09-01

    Full Text Available When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS. Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  18. Development of a screening method to identify regulators of MICA shedding.

    Science.gov (United States)

    Kishikawa, Takahiro; Otsuka, Motoyuki; Ohno, Motoko; Yoshikawa, Takeshi; Sato, Masaya; Koike, Kazuhiko

    2015-10-01

    Immune cells, such as natural killer (NK) cells, recognize virally infected and transformed cells, and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells. Shedding of NKG2D ligands is thought to be a type of counter-mechanism employed by pathogenic cells to evade from NKG2D-mediated immune surveillance. MHC class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. We previously reported that, in soluble form, MICA expression levels are significantly associated with hepatitis virus-induced hepatocellular carcinoma. Here, we report a MICA shedding assay that utilizes membrane-bound MICA tagged at its N-terminus with a nano-luciferase reporter to quantify MICA shedding into culture media. Using this method, we screened a compound library and identified putative regulators of MICA shedding that have the potential to enhance the immune reaction by simultaneously increasing cell surface MICA levels and decreasing soluble MICA levels. This shedding assay may be useful for screening regulators of cell surface molecule shedding.

  19. A highly conserved motif at the COOH terminus dictates endoplasmic reticulum exit and cell surface expression of NKCC2.

    Science.gov (United States)

    Zaarour, Nancy; Demaretz, Sylvie; Defontaine, Nadia; Mordasini, David; Laghmani, Kamel

    2009-08-01

    Mutations in the apically located Na(+)-K(+)-2Cl(-) co-transporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder, yet the mechanisms underlying the regulation of mutated NKCC2 proteins in renal cells have not been investigated. Here, we identified a trihydrophobic motif in the distal COOH terminus of NKCC2 that was required for endoplasmic reticulum (ER) exit and surface expression of the co-transporter. Indeed, microscopic confocal imaging showed that a naturally occurring mutation depriving NKCC2 of its distal COOH-terminal region results in the absence of cell surface expression. Biotinylation assays revealed that lack of cell surface expression was associated with abolition of mature complex-glycosylated NKCC2. Pulse-chase analysis demonstrated that the absence of mature protein was not caused by reduced synthesis or increased rates of degradation of mutant co-transporters. Co-immunolocalization experiments revealed that these mutants co-localized with the ER marker protein-disulfide isomerase, demonstrating that they are retained in the ER. Cell treatment with proteasome or lysosome inhibitors failed to restore the loss of complex-glycosylated NKCC2, further eliminating the possibility that mutant co-transporters were processed by the Golgi apparatus. Serial truncation of the NKCC2 COOH terminus, followed by site-directed mutagenesis, identified hydrophobic residues (1081)LLV(1083) as an ER exit signal necessary for maturation of NKCC2. Mutation of (1081)LLV(1083) to AAA within the context of the full-length protein prevented NKCC2 ER exit independently of the expression system. This trihydrophobic motif is highly conserved in the COOH-terminal tails of all members of the cation-chloride co-transporter family, and thus may function as a common motif mediating their transport from the ER to the cell surface. Taken together, these data are consistent with a model whereby naturally occurring premature terminations that interfere with

  20. Cell wall trapping of autocrine peptides for human G-protein-coupled receptors on the yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Jun Ishii

    Full Text Available G-protein-coupled receptors (GPCRs regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP strategy. In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

  1. Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria

    Directory of Open Access Journals (Sweden)

    Muscariello Lidia

    2006-05-01

    Full Text Available Abstract Background Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components. Results A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters. Conclusion We propose that the CscA, CscB, CscC and Csc

  2. Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

    Directory of Open Access Journals (Sweden)

    Shin Soojung

    2005-07-01

    Full Text Available Abstract Background Pluripotent human embryonic stem cells (hESCs have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4, to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomatoesculetum lectin (TL, Ricinus communis agglutinin (RCA, and Concanavalin A (Con A bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA and Lotus tetragonolobus lectin (LTL did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L, Vicia villosa agglutinin (VVA, Ulex europaeus agglutinin (UEA, Phaseolus vulgaris erythro-agglutinin (PHA-E, and Maackia amurensis agglutinin (MAA bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the

  3. Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing.

    Science.gov (United States)

    2015-01-01

    The author's email has been corrected in the publication of Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to: j.zhou@uws.edu.au from: jzho7551@mail.usyd.edu.au. PMID:26167960

  4. The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1985-01-01

    Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities (Kd between 15 and 450 nM) and different off-rates of the cAMP-receptor complex (t½ between 0.7 and 150 s). The association of cAMP to the receptor and the

  5. Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

    DEFF Research Database (Denmark)

    López-Villar, Elena; Monteoliva, Lucía; Larsen, Martin Røssel;

    2006-01-01

    Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that...

  6. Growth condition-dependent cell surface proteome analysis of Enterococcus faecium

    NARCIS (Netherlands)

    Sinnige, Jan C; de Been, Mark; Zhou, Miaomiao; Bonten, Marc J M; Willems, Rob J L; Top, Janetta

    2015-01-01

    The last 30 years Enterococcus faecium has become an important nosocomial pathogen in hospitals worldwide. The aim of this study was to obtain insight in the cell surface proteome of E. faecium when grown in laboratory and clinically relevant conditions. Enterococcus faecium E1162, a clinical blood

  7. Altered expression of epithelial cell surface glycoconjugates and intermediate filaments at the margins of mucosal wounds

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Grøn, B; Mandel, U;

    1998-01-01

    Alterations in cell to cell adhesion are necessary to enable the type of cell movements that are associated with epithelial wound healing and malignant invasion. Several studies of transformed cells have related epithelial cell movement to changes in the cell surface expression of the carbohydrat...

  8. Extraction of cell surface-associated proteins from living yeast cells.

    NARCIS (Netherlands)

    F.M. Klis; M. de Jong; S. Brul; P.W.J. de Groot

    2007-01-01

    To extract cell surface-associated proteins from living fungal cells, reducing agents such as beta-mercaptoethanol and dithiothreitol are often used. We show here that both compounds are moderately lipophilic and may perturb the plasma membrane, thus causing the release of cytosolic proteins, especi

  9. Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM

    Energy Technology Data Exchange (ETDEWEB)

    Beckmann, M.A. [UT-ORNL Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN 37996-0840 (United States); Venkataraman, S. [Department of Electrical and Computer Engineering, University of Tennessee, Knoxville, TN 37996-0840 (United States); Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6123 (United States); Doktycz, M.J. [UT-ORNL Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN 37996-0840 (United States); Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6123 (United States); Nataro, J.P. [Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Sullivan, C.J. [UT-ORNL Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN 37996-0840 (United States); Morrell-Falvey, J.L. [Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6123 (United States); Allison, D.P. [UT-ORNL Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN 37996-0840 (United States) and Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996-0840 (United States) and Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6123 (United States) and Molecular Imaging Inc. Tempe, AZ 85282 (United States)]. E-mail: allisond@utk.edu

    2006-06-15

    Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria.

  10. Incorporation of Nasutitermes takasagoensis endoglucanase into cell surface-displayed minicellulosomes in Pichia pastoris X33.

    Science.gov (United States)

    Ou, Jingshen; Cao, Yicheng

    2014-09-01

    In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications. PMID:24851815

  11. Cell Surface Enzymatic Engineering-Based Approaches to Improve Cellular Therapies

    KAUST Repository

    Abuelela, Ayman F.

    2014-06-06

    The cell surface represents the interface between the cell and its environment. As such, the cell surface controls cell–cell interactions and functions such as adhesion and migration, and will transfer external cues to regulate processes such as survival, death, and differentiation. Redefining the cell surface by temporarily (or permanently) modifying the molecular landscape of the plasma membrane affects the way in which the cell interacts with its environment and influences the information that is relayed into the cell along downstream signaling pathways. This chapter outlines the role of key enzymes, the glycosyltransferases, in posttranslationally modifying proteins and lipids to fine-tune cells, ability to migrate. These enzymes are critical in controlling the formation of a platform structure, sialyl Lewis x (sLex), on circulating cells that plays a central role in the recognition and recruitment by selectin counter receptors on endothelial cells that line blood vessels of tissues throughout the body. By developing methods to manipulate the activity of these enzymes and hence the cell surface structures that result, treatments can be envisioned that direct the migration of therapeutic cells to specific locations throughout the body and also to inhibit metastasis of detrimental cells such as circulating tumor cells.

  12. Evidence of cell surface iron speciation of acidophilic iron-oxidizing microorganisms in indirect bioleaching process.

    Science.gov (United States)

    Nie, Zhen-yuan; Liu, Hong-chang; Xia, Jin-lan; Yang, Yi; Zhen, Xiang-jun; Zhang, Li-Juan; Qiu, Guan-zhou

    2016-02-01

    While indirect model has been widely accepted in bioleaching, but the evidence of cell surface iron speciation has not been reported. In the present work the iron speciation on the cell surfaces of four typically acidophilic iron-oxidizing microorganism (mesophilic Acidithiobacillus ferrooxidans ATCC 23270, moderately thermophilic Leptospirillum ferriphilum YSK and Sulfobacillus thermosulfidooxidans St, and extremely thermophilic Acidianus manzaensis YN25) grown on different energy substrates (chalcopyrite, pyrite, ferrous sulfate and elemental sulfur (S(0))) were studied in situ firstly by using synchrotron-based micro- X-ray fluorescence analysis and X-ray absorption near-edge structure spectroscopy. Results showed that the cells grown on iron-containing substrates had apparently higher surface iron content than the cells grown on S(0). Both ferrous iron and ferric iron were detected on the cell surface of all tested AIOMs, and the Fe(II)/Fe(III) ratios of the same microorganism were affected by different energy substrates. The iron distribution and bonding state of single cell of A. manzaensis were then studied in situ by scanning transmission soft X-ray microscopy based on dual-energy contrast analysis and stack analysis. Results showed that the iron species distributed evenly on the cell surface and bonded with amino, carboxyl and hydroxyl groups. PMID:26645388

  13. Cell surface physico chemistry alters biofilm development of Pseudomonas aeruginosa lipopolysaccharide mutants

    NARCIS (Netherlands)

    Flemming, CA; Palmer, RJ; Arrage, AA; Van der Mei, HC; White, DC

    1999-01-01

    The hydrophobic and electrostatic characteristics of bacterial cell surfaces were compared with attachment proclivity and biomass accumulation over time between wildtype Pseudomonas aeruginosa serotype O6 (possesses A and B band LPS), and three LPS-deficient mutants, vi;. A28 (A(+)B(-)), R5 (A(+)B(-

  14. HPLC analysis and cell surface receptor binding activities of the crude aqueous and methanolic extract of Sesamum indicum

    Institute of Scientific and Technical Information of China (English)

    Repon Kumer Saha; Md Abu Monsur Dinar; Kausain Akther Nabila; Priyanka Roy

    2014-01-01

    Objective: To identify the possible functional molecules for therapeutic uses by screening the crude aqueous and methanolic extracts derived from sesame seeds (Sesamum indicum) in vitro. Methods: High performance liquid chromatography was used to scan the functional molecules present in the extracts.Results:or its derivatives like molecules. On the other hand, the crude methanolic extract may contain Loratadine or its derivatives like molecules. Both type of extracts showed hemagglutination inhibition activities in all types of human blood samples tested. However, they showed stronger binding with AB+ blood group than those of A+ and B+ blood. The crude aqueous extracts showed the possibilities to present caffeine and cetirizine Conclusions: Sesame seeds may be considered as a functional food.

  15. Selective labelling of cell-surface proteins using CyDye DIGE Fluor minimal dyes.

    Science.gov (United States)

    Hagner-McWhirter, Asa; Winkvist, Maria; Bourin, Stephanie; Marouga, Rita

    2008-01-01

    Surface proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan DIGE technology and analysis of protein expression changes using DeCyder 2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

  16. A novel small molecule inhibitor of hepatitis C virus entry.

    Directory of Open Access Journals (Sweden)

    Carl J Baldick

    Full Text Available Small molecule inhibitors of hepatitis C virus (HCV are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc, blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

  17. Serological survey of normal humans for natural antibody to cell surface antigens of melanoma.

    Science.gov (United States)

    Houghton, A N; Taormina, M C; Ikeda, H; Watanabe, T; Oettgen, H F; Old, L J

    1980-01-01

    Sera of 106 normal adult men were tested for antibodies reacting with cell surface antigens of three established lines of cultured malignant melanoma. Positive reactions with a protein A assay for IgG antibodies were extremely rare (1-2%). The frequency of positive reactions with assays for IgM antibodies was higher: 5-15% in immune adherence assays and 55-82% in anti-C3 mixed hemadsorption assays. After low-titered sera and sera reacting with fetal calf serum components, conventional alloantigens, and widely distributed class 3 antigens were excluded, sera from seven individuals (one with IgG antibody and six with IgM antibodies) were selected for detailed analysis. The serum containing the IgG antibody came from a healthy 65-year-old Caucasian man; titers of antibody in his serum ranged from < 1/10 to 1/40,000 in tests with different melanoma cell lines. This IgG antibody identifies a differentiation antigen of melanocytes, provisionally designated Mel 1, that distinguishes two classes of melanomas: 22 melanoma cell lines typed Mel 1+ and 17 types Mel 1-. Mel 1 is expressed by fetal fibroblasts but not adult fibroblasts and can be found on a proportion of cultured epithelial cancer cell lines (5 out of 23) but not on glioma or B-cell lines. The melanoma antigens detected by the naturally occurring IgM antibodies are serologically unrelated to Mel 1 but, like Mel 1, appear to be differentiation antigens that distinguish subsets of melanoma. These IgM antibodies detect antigens that are identical or closely related to the AH antigen, a melanoma surface antigen that was initially defined by autologous antibody in a patient with melanoma. In view of the immunogenicity of both Mel 1 and the AH antigens in humans and their occurrence on more than 50% of melanomas, it remains to be seen whether antibody to these antigens can be elicited by specific vaccination of seronegative melanoma patients and whether this will have an influence on the clinical course of the disease

  18. Characterization of structural features controlling the receptiveness of empty class II MHC molecules

    DEFF Research Database (Denmark)

    Rupp, Bernd; Günther, Sebastian; Makhmoor, Talat;

    2011-01-01

    MHC class II molecules (MHC II) play a pivotal role in the cell-surface presentation of antigens for surveillance by T cells. Antigen loading takes place inside the cell in endosomal compartments and loss of the peptide ligand rapidly leads to the formation of a non-receptive state of the MHC mol...

  19. Ficolins and FIBCD1: Soluble and membrane bound pattern recognition molecules with acetyl group selectivity

    DEFF Research Database (Denmark)

    Thomsen, Theresa; Schlosser, Anders; Holmskov, Uffe;

    2011-01-01

    as pattern recognition molecules. Ficolins are soluble oligomeric proteins composed of trimeric collagen-like regions linked to fibrinogen-related domains (FReDs) that have the ability to sense molecular patterns on both pathogens and apoptotic cell surfaces and activate the complement system. The ficolins...

  20. Cell Surface Receptor Theory of Disease Infectivity; Body's Defence and Normal Body Functioning in Living Things

    Directory of Open Access Journals (Sweden)

    Utoh-Nedosa

    2011-01-01

    Full Text Available Problem statement: A study of the pattern of Candida spp. infection of the human body and the mode and pattern of reaction of the human body to this infection showed that disease infectivity and self healing by plants followed the same procedures and patterns. Approach: A comparism of these procedures and patterns of natural self- healing of disease infection by the human body and plants/plant parts with the cutaneous Candida spp. killing and elimination procedures and patterns of Vernonia amygdalina leaf extract, showed that cell surface receptors are the sites through which disease infects the body and also the sites at which the body is defended. They are also the sites where activities which result in normal body functioning are carried out. The mode and patterns of Cutaneous Candida infection in a human subject and its containment by the body was examined and photographed. The disease infection and self healing procedures and patterns of plants were also examined in comparism with those of their healthy counterparts and photographed. The findings from the observations on disease infectivity and natural body’s defence patterns and procedures of the plant parts studied and those of the human body in reaction to Candida spp. infection were compared with those of the Candida spp. killing procedures and patterns of aqueous and Arachis hypogeal oil extract of Vernonia amygdalina leaf. Results: The findings of this study also showed that disease-infective organisms gain access to the body of a host through attachment to the cell surface receptors of that host which are placed linearly and are interconnected by channels. The results of the study also indicated that living organisms have a main endogenous substance that mediates both their body’s defence and their normal physiological functioning which is therefore the owner of the cell surface receptor. Other endogenous substances which participate in normal body functioning/body’s defence or in

  1. Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics.

    Science.gov (United States)

    Ilmer, Matthias; Mazurek, Nachman; Byrd, James C; Ramirez, Karen; Hafley, Margarete; Alt, Eckhard; Vykoukal, Jody; Bresalier, Robert S

    2016-01-01

    Recurrence of gastrointestinal adenocarcinomas after surgery and chemotherapy may be attributed, in part, to the presence of a small population of tumor-initiating cancer stem cells (CSC). The expression of galectin-3 (Gal3), a multifunctional oncolectin, has been associated with biological behaviors associated with CSC. We examined the ability of Gal3 to characterize the CSC phenotype, and to identify a clinically important gastrointestinal cancer CSC population. Human colorectal and pancreatic cancer cell lines were sorted to identify subpopulations expressing commonly used CSC markers, and Gal3-positive CSC subpopulations. The association of Gal3 with the stem cell properties and alterations of these phenotypes by manipulation of Gal3 expression was examined. Gastrointestinal cancer cell lines contain both Gal3-positive and Gal3-negative subpopulations. Gal3-positive CSCs are characterized by high ALDH activity, enhanced self-renewal ability in vitro (sphere formation) and tumor forming ability in vivo, and resistance to chemotherapeutic agents and death-receptor-mediated apoptosis compared to Gal3-negative CSCs. Silencing Gal3 modifies this behavior. Cell surface Gal3 expression identifies a subset of CSCs in gastrointestinal cancers with high levels of stem cell characteristics, including chemoresistance. This may provide a platform for developing treatment strategies that target CSC. PMID:27512958

  2. Cell surface display of Yarrowia lipolytica lipase Lip2p using the cell wall protein YlPir1p, its characterization, and application as a whole-cell biocatalyst.

    Science.gov (United States)

    Yuzbasheva, Evgeniya Y; Yuzbashev, Tigran V; Perkovskaya, Natalia I; Mostova, Elizaveta B; Vybornaya, Tatiana V; Sukhozhenko, Aleksei V; Toropygin, Ilya Y; Sineoky, Sergey P

    2015-04-01

    The Yarrowia lipolytica lipase Lip2p was displayed on the yeast cell surface via N-terminal fusion variant using cell wall protein YlPir1p. The hydrolytic activity of the lipase displayed on Y. lipolytica cells reached 11,900 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. The calculated number of recombinant enzyme displayed on the cell surface corresponds to approximately 6 × 10(5) molecules per cell, which is close to the theoretical maximum (2 × 10(6) molecules/cell). Furthermore, the leaking enzyme was presented as three N-glycosylated proteins, one of which corresponds to the whole hybrid protein. Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, the surface-displayed lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. Cell-bound lipase retained 74 % of its original activity at 60 °C for 5 min of incubation, and 83 % of original activity after incubation at 50 °C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1 and 71.0 % methyl esters after 33- and 45-h reactions, respectively. PMID:25773979

  3. Cell surface polypeptides of murine T-cell clones expressing cytolytic or amplifier activity.

    OpenAIRE

    Sarmiento, M.; Glasebrook, A L; Fitch, F. W.

    1980-01-01

    Murine cytolytic T-cell and amplifier T-cell clones derived from secondary unidirectional mixed leukocyte cultures were labeled with 125I by the lactoperoxidase method and their polypeptide profiles were analyzed by NaDodSO4/polyacrylamide gel electrophoresis. All cytolytic T-cell clones derived from the same mouse strain yeilded similar cell surface polypeptide profiles. However, profiles obtained with three amplifier T-cell clones were strikingly different from each other as well as from th...

  4. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

    Science.gov (United States)

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S; Ambudkar, Suresh V

    2015-10-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

  5. A cell surface receptor complex for collagen type I recognizes the Arg- Gly-Asp sequence

    OpenAIRE

    1987-01-01

    To isolate collagen-binding cell surface proteins, detergent extracts of surface-iodinated MG-63 human osteosarcoma cells were chromatographed on affinity matrices of either type I collagen- Sepharose or Sepharose carrying a collagen-like triple-helical peptide. The peptide was designed to be triple helical and to contain the sequence Arg-Gly-Asp, which has been implicated as the cell attachment site of fibronectin, vitronectin, fibrinogen, and von Willebrand factor, and is also present in ty...

  6. Cell surface thiol isomerases may explain the platelet-selective action of S-nitrosoglutathione

    OpenAIRE

    Xiao, Fang; Gordge, Michael P

    2011-01-01

    S-nitrosoglutathione (GSNO) at low concentration inhibits platelet aggregation without causing vasodilation, suggesting platelet-selective nitric oxide delivery. The mechanism of this selectivity is unknown, but may involve cell surface thiol isomerases, in particular protein disulphide isomerase (csPDI) (EC 5.3.4.1). We have now compared csPDI expression and activity on platelets, endothelial cells and vascular smooth muscle cells, and the dependence on thiol reductase activity of these cell...

  7. Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study

    Directory of Open Access Journals (Sweden)

    Mamgain Hitesh

    2009-10-01

    Full Text Available Abstract Background Imaging tools such as scanning electron microscope (SEM and atomic force microscope (AFM can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. Methods We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK 293, human breast cancer (MCF-7 and mouse melanoma (B16F1 cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM and scanning electron microscopy (SEM. The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. Results Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. Conclusion Tumor suppressor

  8. Ultrastructural and biochemical studies of two dynamically expressed cell surface determinants on Candida albicans.

    OpenAIRE

    Brawner, D L; Cutler, J E

    1986-01-01

    Variability in the expression of two different cell surface carbohydrate determinants was examined with two agglutinating immunoglobulin M monoclonal antibodies (H9 and C6) and immunoelectron microscopy during growth of three strains of Candida albicans. A single strain of Candida parapsilosis did not express either antigen at any time during growth. Antigens were detected on the surface of C. albicans by agglutination tests with either H9 or C6 over a 48-h growth period. The difference in sp...

  9. Simian virus 40 infection via MHC class I molecules and caveolae.

    Science.gov (United States)

    Norkin, L C

    1999-04-01

    MHC class I molecules are a necessary component of the cell surface receptor for simian virus 40 (SV40). After binding to class I molecules, SV40 enters cells via a unique endocytic pathway that involves caveolae, rather than clathrin-coated pits. This pathway is dependent on a transmembrane signal that SV40 transmits from the cell surface. Furthermore, it delivers SV40 to the endoplasmic reticulum, rather than to the endosomal/lysosomal compartment, which is the usual target for endocytic traffic. The glycosphingolipid and cholesterol-enriched plasma membrane domains that contain caveolae are also enriched for class I molecules, relative to whole plasma membrane. Nevertheless, although class I molecules bind SV40, they do not enter with SV40, nor do they enter spontaneously into uninfected SV40 host cells. Instead, they are shed from the cell surface by the activity of a metalloprotease. These results imply the existence of a putative secondary receptor for SV40 that might mediate SV40 entry. It is not yet clear whether class I molecules are active in transmitting the SV40 signal. Monoclonal antibodies against class I molecules also induce a signal in the SV40 host cells. However, the antibody-induced signal is mediated by mitogen-activated protein kinase (MAP kinase), whereas the SV40 signal is independent of MAP kinase.

  10. Measurement of interaction force between RGD-peptide and Hela cell surface by optical tweezers

    Institute of Scientific and Technical Information of China (English)

    Mincheng Zhong; Guosheng Xue; Jinhua Zhou; Ziqiang Wang; Yinmei Li

    2012-01-01

    Since RGD peptides (R:arginine; G:glycine; D:aspartic acid) are found to promote cell adhesion,they are modified at numerous materials surface for medical applications such as drug delivery and regenerative medicine.Peptide-cell surface interactions play a key role in the above applications.In this letter,we study the adhesion force between the RGD-coated bead and Hela cell surface by optical tweezes.The adhesion is dominated by the binding of α5β1 and RGD-peptide with higher adhesion probability and stronger adhesion strength compared with the adhesion of bare bead and cell surface.The binding force for a single α5β1-GRGDSP pair is determined to be 16.8 pN at a loading rate of 1.5 nN/s.The unstressed off-rate is 1.65 × 10-2 s-1 and the distance of transition state for the rigid binding model is 3.0 nm.

  11. Yeast cell surface display for lipase whole cell catalyst and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

    2014-08-01

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

  12. Oxygen Modulates Human Decidual Natural Killer Cell Surface Receptor Expression and Interactions with Trophoblasts1

    Science.gov (United States)

    Wallace, Alison E.; Goulwara, Sonu S.; Whitley, Guy S.; Cartwright, Judith E.

    2014-01-01

    Decidual natural killer (dNK) cells have been shown to both promote and inhibit trophoblast behavior important for decidual remodeling in pregnancy and have a distinct phenotype compared to peripheral blood NK cells. We investigated whether different levels of oxygen tension, mimicking the physiological conditions of the decidua in early pregnancy, altered cell surface receptor expression and activity of dNK cells and their interactions with trophoblast. dNK cells were isolated from terminated first-trimester pregnancies and cultured in oxygen tensions of 3%, 10%, and 21% for 24 h. Cell surface receptor expression was examined by flow cytometry, and the effects of secreted factors in conditioned medium (CM) on the trophoblast cell line SGHPL-4 were assessed in vitro. SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 10% were significantly more invasive (P oxygen tensions of 3% or 21%. After 24 h, a lower percentage of dNK cells expressed CD56 at 21% oxygen (P oxygen (P oxygen tensions, with large patient variation. This study demonstrates dNK cell phenotype and secreted factors are modulated by oxygen tension, which induces changes in trophoblast invasion and endovascular-like differentiation. Alterations in dNK cell surface receptor expression and secreted factors at different oxygen tensions may represent regulation of function within the decidua during the first trimester of pregnancy. PMID:25232021

  13. Selective radiolabeling of cell surface proteins to a high specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, J.A.; Lau, A.L.; Cunningham, D.D.

    1987-02-10

    A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with /sup 125/I-(hydroxyphenyl)propionyl groups via /sup 125/I-sulfosuccinimidyl (hydroxyphenyl)propionate (/sup 125/II-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-(/sup 125/I)iodide labeling technique revealed that /sup 125/I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with /sup 125/I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spacial orientations on erythroctyes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane.

  14. Role of TI-VAMP and CD82 in EGFR cell-surface dynamics and signaling.

    Science.gov (United States)

    Danglot, Lydia; Chaineau, Mathilde; Dahan, Maxime; Gendron, Marie-Claude; Boggetto, Nicole; Perez, Franck; Galli, Thierry

    2010-03-01

    The v-SNARE TI-VAMP (VAMP7) mediates exocytosis during neuritogenesis, phagocytosis and lysosomal secretion. It localizes to endosomes and lysosomes but also to the trans-Golgi network. Here we show that depletion of TI-VAMP enhances the endocytosis of activated EGF receptor (EGFR) without affecting constitutive endocytosis of EGFR, or transferrin uptake. This increased EGFR internalization is mainly clathrin dependent. Searching for defects in EGFR regulators, we found that TI-VAMP depletion reduces the cell surface amount of CD82, a tetraspanin known to control EGFR localization in microdomains. We further show that TI-VAMP is required for secretion from the Golgi apparatus to the cell surface, and that TI-VAMP-positive vesicles transport CD82. Quantum dots video-microscopy indicates that depletion of TI-VAMP, or its cargo CD82, restrains EGFR diffusion and the area explored by EGFR at the cell surface. Both depletions also impair MAPK signaling and enhance endocytosis of activated EGFR by increased recruitment of AP-2. These results highlight the role of TI-VAMP in the secretory pathway of a tetraspanin, and support a model in which CD82 allows EGFR entry in microdomains that control its clathrin-dependent endocytosis and signaling.

  15. [A structural assessment of the role of the cell surface carbohydrates of Rhizobium in the Rhizobium/legume symbiosis]. Progress report, June 1989--June 1991

    Energy Technology Data Exchange (ETDEWEB)

    Hollingsworth, R.I.

    1991-12-31

    Research continued on the study of cell surface carbohydrates of Rhizobium. Objectives include: To characterize, at a structural level, the differences between the lipopolysaccharides of a representative number of strains from different Rhizobium species to determine which features of LPS structure are species-specific and might, therefore, be determinants of host specificity. Determine the effect(s) of nod gene induction on the structure of Rhizobium lipopolysaccharides and determine whether synthesis of a modified LPS molecule or a new surface glycoconjugate is initiated by nod gene induction. Develop a non-chemical means for rapidly screening large numbers of bacterial strains in order to determine which glycoconjugate structural features are conserved between strains of the same species. Provide the necessary structural information which, when coupled with developments in the rapidly expanding field of Rhizobium genetics, should lead to a clear understanding of the role of Rhizobium surface glycoconjugates in host/symbiont interactions. Progress is discussed.

  16. No major role for insulin-degrading enzyme in antigen presentation by MHC molecules.

    Directory of Open Access Journals (Sweden)

    Slobodan Culina

    Full Text Available Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands.

  17. No major role for insulin-degrading enzyme in antigen presentation by MHC molecules.

    Science.gov (United States)

    Culina, Slobodan; Mauvais, François-Xavier; Hsu, Hsiang-Ting; Burgevin, Anne; Guénette, Suzanne; Moser, Anna; van Endert, Peter

    2014-01-01

    Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE) was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands.

  18. Expression of the cell-surface heparan sulfate proteoglycan syndecan-2 in developing rat anterior pituitary gland.

    Science.gov (United States)

    Horiguchi, Kotaro; Syaidah, Rahimi; Fujiwara, Ken; Tsukada, Takehiro; Ramadhani, Dini; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

    2013-09-01

    In the anterior pituitary gland, folliculo-stellate cells and five types of hormone-producing cells are surrounded by an extracellular matrix (ECM) essential for these cells to perform their respective roles. Syndecans-type I transmembrane cell-surface heparan sulfate proteoglycans act as major ECM coreceptors via their respective heparan sulfate chains and efficiently transduce intracellular signals through the convergent action of their transmembrane and cytoplasmic domains. The syndecans comprise four family members in vertebrates: syndecan-1, -2, -3 and -4. However, whether syndecans are produced in the pituitary gland or whether they have a role as a coreceptor is not known. We therefore used (1) reverse transcription plus the polymerase chain reaction to analyze the expression of syndecan genes and (2) immunohistochemical techniques to identify the cells that produce the syndecans in the anterior pituitary gland of adult rat. Syndecan-2 mRNA expression was clearly detected in the corticotropes of the anterior pituitary gland. Moreover, the expression of syndecan-2 in the developing pituitary gland had a distinct temporospatial pattern. To identify the cells expressing syndecan-2 in the developing pituitary gland, we used double-immunohistochemistry for syndecan-2 and the cell markers E-cadherin (immature cells) and Ki-67 (proliferating cells). Some E-cadherin- and Ki-67-immunopositive cells expressed syndecan-2. Therefore, syndecan-2 expression occurs in developmentally regulated patterns and syndecan-2 probably has different roles in adult and developing anterior pituitary glands.

  19. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors.

    Directory of Open Access Journals (Sweden)

    Adiba Isa

    Full Text Available HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced a 9-42 fold increase of all six HLA-A,-B,-C gene transcripts. Interestingly, prior to stimulation, gene transcripts for all but two alleles were present in similar amounts suggesting that post-transcriptional mechanisms regulate the constitutive expression of HLA-A,-B, and -C. Locus-restricted expression of HLA-A, -B and -C challenges our current understanding of the function of these molecules as regulators of CD8(+ T-cell and NK-cell function and should lead to further inquiries into their expression on other cell types.

  20. Bacterial invasion reconstructed molecule by molecule

    Energy Technology Data Exchange (ETDEWEB)

    Werner, James H [Los Alamos National Laboratory

    2009-01-01

    We propose to visualize the initial stages of bacterial infection of a human host cell with unmatched spatial and temporal resolution. This work will develop a new capability for the laboratory (super-resolution optical imaging), will test unresolved scientific hypotheses regarding host-pathogen interaction dynamics, and leverages state of the art 3D molecular tracking instrumentation developed recently by our group. There is much to be gained by applying new single molecule tools to the important and familiar problem of pathogen entry into a host cell. For example, conventional fluorescence microscopy has identified key host receptors, such as CD44 and {alpha}5{beta}1 integrin, that aggregate near the site of Salmonella typhimurium infection of human cells. However, due to the small size of the bacteria ({approx} 2 {micro}m) and the diffraction of the emitted light, one just sees a fluorescent 'blob' of host receptors that aggregate at the site of attachment, making it difficult to determine the exact number of receptors present or whether there is any particular spatial arrangement of the receptors that facilitates bacterial adhesion/entry. Using newly developed single molecule based super-resolution imaging methods, we will visualize how host receptors are directed to the site of pathogen adhesion and whether host receptors adopt a specific spatial arrangement for successful infection. Furthermore, we will employ our 3D molecular tracking methods to follow the injection of virulence proteins, or effectors, into the host cell by the pathogen Type III secretion system (TTSS). We expect these studies to provide mechanistic insights into the early events of pathogen infection that have here-to-fore been technically beyond our reach. Our Research Goals are: Goal 1--Construct a super-resolution fluorescence microscope and use this new capability to image the spatial distribution of different host receptors (e.g. CD44, as {alpha}5{beta}1 integrin) at the

  1. High Cell Surface Death Receptor Expression Determines Type I Versus Type II Signaling*

    Science.gov (United States)

    Meng, Xue Wei; Peterson, Kevin L.; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D.; Gores, Gregory J.; Kaufmann, Scott H.

    2011-01-01

    Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression. PMID:21865165

  2. Streptococcus salivarius strains carry either fibrils or fimbriae on the cell surface.

    Science.gov (United States)

    Handley, P S; Carter, P L; Fielding, J

    1984-01-01

    Strains of Streptococcus salivarius were screened by negative staining for the presence of surface structures. Two structural subgroups were found, carrying either fibrils or fimbriae, projecting from the cell surface. Eight strains carried a very dense peritrichous array of fibrils of two distinct lengths. Long fibrils had an average length of 175 nm, and short fibrils had an average length of 95 nm. Two strains carried only long fibrils, one strain carried only short fibrils, and another strain carried a lateral tuft of very prominent fibrils of two lengths, with a fibrillar fuzz covering the remainder of the cell surface. In all the strains in which they were present, the long fibrils were unaffected by protease or trypsin treatment. In contrast, the short fibrils were completely digested by protease and partially removed by trypsin. Neither long nor short fibrils were affected structurally by mild pepsin digestion or by lipase. The Lancefield extraction procedure removed both long and short fibrils. These twelve fibrillar strains were therefore divisible into four structural subgroups. Extracts of all the fibrillar strains reacted with group K antiserum. The second main structural subgroup consisted of nine strains of S. salivarius, all of which carried morphologically identical, flexible fimbriae arranged peritrichously over the cell surface. The fimbriae were structurally distinct from fibrils and measured 0.5 to 1.0 micron long and 3 to 4 nm wide, with an irregular outline and no obvious substructure. There was no obvious reduction in the number of fimbriae after protease or trypsin treatment. Extracts of the fimbriated strains did not react with the group K antiserum. The two serological and structural subgroups could also be distinguished by colony morphology. Images PMID:6197404

  3. Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

    International Nuclear Information System (INIS)

    The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-[2-3H]mannose or L-[5,6-3H]fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with [2-3H]mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with [2-3H]mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-[1,6-3H]glucosamine and L-[1-14C]fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced 3H-labeled N-acetylglucosamine and N-acetylgalactosamine

  4. Aged mice have increased inflammatory monocyte concentration and altered expression of cell-surface functional receptors

    Indian Academy of Sciences (India)

    Kelley Strohacker; Whitney L Breslin; Katie C Carpenter; Brian K McFarlin

    2012-03-01

    The expression of monocyte cell-surface receptors represents one index of immune dysfunction, which is common with aging. Although mouse models of aging are prevalent, monocyte subset assessment is rare. Our purpose was to compare cell receptor expression on classic (CD115+/Gr-1high) and non-classic (CD115+/Gr-1low) monocytes from 80- or 20-week-old CD-1 mice. Three-colour flow cytometry was used to determine the concentration of monocyte subsets and their respective cell-surface expression of TLR2, TLR4, CD80, CD86, MHC II and CD54. These receptors were selected because they have been previously associated with altered monocyte function. Data were analysed with independent -tests; significance was set at < 0.05. Old mice had a greater concentration of both classic (258%, =0.003) and non-classic (70%, =0.026) monocytes. The classic : non-classic monocyte ratio doubled in old as compared with that in young mice (=0.006), indicating a pro-inflammatory shift. TLR4 ($\\downarrow$27%, =0.001) and CD80 ($\\downarrow$37%, =0.004) were decreased on classic monocytes from old as compared with those from young mice. TLR2 ($\\uparrow$24%, =0.002) and MHCII ($\\downarrow$21%, =0.026) were altered on non-classic monocytes from old as compared with those from young mice. The increased classic : non-classic monocyte ratio combined with changes in the cell-surface receptor expression on both monocyte subsets is indicative of immune dysfunction, which may increase age-associated disease risk.

  5. Short-chain ceramides depress integrin cell surface expression and function in colorectal cancer cells.

    Science.gov (United States)

    Morad, Samy A F; Bridges, Lance C; Almeida Larrea, Alex D; Mayen, Anthony L; MacDougall, Matthew R; Davis, Traci S; Kester, Mark; Cabot, Myles C

    2016-07-01

    Colorectal cancer (CRC) is highly metastatic, significantly so to liver, a characteristic that embodies one of the most challenging aspects of treatment. The integrin family of cell-cell and cell-matrix adhesion receptors plays a central role in migration and invasion, functions that underlie metastatic potential. In the present work we sought to determine the impact of ceramide, which plays a key modulatory role in cancer suppression, on integrin cell surface expression and function in CRC cells in order to reveal possible ceramide-centric effects on tumor cell motility. Human CRC cells LoVo, HT-29, and HCT-116 were employed, which represent lines established from primary and metastatic sites. A cell-permeable, short-chain analog, C6-ceramide, was used as ceramide mimic. Exposure of cells to C6-ceramide (24 h) promoted a dose-dependent (2.5-10 µM) decrease in the expression of cell surface β1 and β4 integrin subunits in all cell lines; at 10 µM C6-ceramide, the decreases ranged from 30 to 50% of the control. Expression of cell surface αVβ6 integrin, which is associated with advanced invasion in CRC, was also suppressed by C6-ceramide. Decreases in integrin expression translated to diminished cellular adhesion, 50% of the control at 5 µM C6-ceramide, and markedly reduced cellular migration, approximately 30-40% of the control in all cell lines. Physicochemical examination revealed potent efficacy of nano-formulated C6-ceramide, but inferior activity of dihydro-C6-ceramide and L-C6-ceramide, compared to the unsaturated counterpart and the natural d-enantiomer, respectively. These studies demonstrate novel actions of ceramides that may have application in suppression of tumor metastasis, in addition to their known tumor suppressor effects. PMID:27045476

  6. Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

    Science.gov (United States)

    Betz, N. A.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

  7. Overexpression of Cell Surface Cytokeratin 8 in Multidrug-Resistant MCF-7/MX Cells Enhances Cell Adhesion to the Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Fang Liu

    2008-11-01

    Full Text Available Accumulating evidence suggests that multiple complex mechanisms may be involved, simultaneously or complementarily, in the emergence and development of multidrug resistance (MDR in various cancers. Cell adhesion-mediated MDR is one such mechanism. In the present study, we initially observed increased cell adhesion to extracellular matrix proteins by the MDR human breast tumor cell line MCF-7/MX compared to its parental cells. We then used a strategy that combined antibody-based screening technique and mass spectrometry-based proteomics to identify membrane proteins that contribute to the enhanced adhesion of MCF-7/MX cells. Using MCF-7/MX cells as immunogen, we isolated a mouse monoclonal antibody, 9C6, that preferentially reacts with MCF-7/MX cells over the parental MCF-7 cells. The molecular target of 9C6 was identified as cytokeratin 8 (CK8, which was found to be overexpressed on the cell surface of MCF-7/MX cells. We further observed that down-regulation of cell surface levels of CK8 through siRNA transfection significantly inhibited MCF-7/MX cell adhesion to fibronectin and vitronectin. In addition, anti-CK8 siRNA partially reversed the MDR phenotype of MCF-7/MX cells. Taken together, our results suggest that alterations in the expression level and cellular localization of CK8 may play a significant role in enhancing the cellular adhesion of MDR MCF-7/MX cells.

  8. STUDY ON GLYCOCONJUGATE CHANGES ON CELL SURFACE IN PROGRESSIVE DEVELOPMENT OF PULMONARY TUMOR

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-mei; SHAN Jun; CHEN Zhuo-huai

    2002-01-01

    Aim: To investigate glycoconjugate changes on the cell surface of proliferative lesions and neoplasms of mice lungs at various stages of tumorigenesis, the relation between progressive development of mouse pulmonary tumors and expression of cell surface saccharide. Materials and methods: Thirty - one male A/J strain mice at 5 weeks of age were treated intraperitoneally with a single injection of 20 - methylcholanthrene (20 - MC), 292 pulmonary lesions including 31 hyperplasias, 145 alveolar adenomas, 61 papillary adenomas, 55 papillary adenocarcinomas and their combined type were obtained. The binding affinities of cells in normal respiratory epithelia and in proliferative lesions to four peroxidases - conjugated lectins, Maclura pomifera agglutinin (MPA), Arachis hypogea agglutinin (PNA), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) were examined. Results: Cells of hyperplasia and alveolar adenoma showed fairly strong affinity to all the four lectins. However, part of papillary adenoma cells and greater part of papillary adenocarcinoma cells lost their binding affinity to MPA, PNA, and RCA, but not to WGA. The bindings of MPA, PNA and RNA were detected predominently on the luminal surfaces of benign tumors but not on the luminal surfaces of malignant tumors. WGA might bind to varied types of benign and malignant tumors. Pretreated with neuraminidase, the lesions enhanced the staining intensity for the four lectins, the binding sites of WGA to malignant tumor cells were numerous. A distinct difference in lectin binding affinity between hyperplasia / alveolar adenoma/papillary adenoma and papillary adenocarcinoma was clearly shown( x2 = 46.89, P < 0.01, x2 = 36.77, P < 0.01 and x2 = 52.87, P < 0.01 ) in this experiment. The complex glycoconjugates on the cell surface of malignant and benign lesions during the development of pulmonary tumor were changed,malignant tumor cells differed from the surface of benign tumor cells, the levels of

  9. Glycoprotein mucin molecular brush on cancer cell surface acting as mechanical barrier against drug delivery

    Science.gov (United States)

    Wang, Xin; Shah, Aalok A.; Campbell, Robert B.; Wan, Kai-tak

    2010-12-01

    Uptake of cytotoxic drugs by typical tumor cells is limited by the dense dendritic network of oligosaccharide mucin chains that forms a mechanical barrier. Atomic force microscopy is used to directly measure the force needed to pierce the mucin layer to reach the cell surface. Measurements are analyzed by de Gennes' steric reptation theory. Multidrug resistant ovarian tumor cells shows significantly larger penetration load compared to the wide type. A pool of pancreatic, lung, colorectal, and breast cells are also characterized. The chemotherapeutic agent, benzyl-α-GalNac, for inhibiting glycosylation is shown to be effective in reducing the mechanical barrier.

  10. Role of a cell surface-associated protein in adherence and dental caries.

    OpenAIRE

    Bowen, W. H.; Schilling, K.; Giertsen, E; Pearson, S.; Lee, S. F.; Bleiweis, A; Beeman, D

    1991-01-01

    Insertional inactivation of the Streptococcus mutans spaP gene was used to construct an isogenic mutant (834) of strain NG8 (serotype c) which lacked the major cell surface-associated protein referred to as P1 (15). Results of several studies suggest that P1 is involved in the adherence of S. mutans to saliva-coated apatite surfaces. With an in vitro model system of hydroxyapatite (HA) beads coated with parotid saliva (PS) and additional HA surfaces coated with PS and in situ-formed glucan, i...

  11. M135R Is a Novel Cell Surface Virulence Factor of Myxoma Virus▿

    OpenAIRE

    Barrett, John W.; Sypula, Joanna; Wang, Fuan; Alston, Lindsay R.; Shao, Zhuhong; Gao, Xiujuan; Irvine, Timothy S.; McFadden, Grant

    2006-01-01

    Myxoma virus (MV) encodes a cell surface protein (M135R) that is predicted to mimic the host alpha/beta interferon receptor (IFN-α/β-R) and thus prevent IFN-α/β from triggering a host antiviral response. This prediction is based on sequence similarity to B18R, the viral IFN-α/β-R from vaccinia virus (VV), which has been demonstrated to bind and inhibit type I interferons. However, M135R is only half the size of VV B18R. All other poxvirus-encoded IFN-α/β-R homologs align only to the amino-ter...

  12. Roles for glycosylation of cell surface receptors involved in cellular immune recognition.

    Science.gov (United States)

    Rudd, P M; Wormald, M R; Stanfield, R L; Huang, M; Mattsson, N; Speir, J A; DiGennaro, J A; Fetrow, J S; Dwek, R A; Wilson, I A

    1999-10-22

    The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the

  13. Keynote Paper: Cell-Surface Adhesive Interactions in Microchannels and Microvessels

    CERN Document Server

    King, M R

    2003-01-01

    Adhesive interactions between white blood cells and the interior surface of the blood vessels they contact is important in inflammation and in the progression of heart disease. Parallel-plate microchannels have been useful in characterizing the strength of these interactions, in conditions that are much simplified over the complex environment these cells experience in the body. Recent computational and experimental work by several laboratories have attempted to bridge this gap between behavior observed in flow chamber experiments, and cell-surface interactions observed in the microvessels of anesthetized animals.

  14. Effects of DNP on the cell surface properties of marine bacteria and its implication for adhesion to surfaces

    Digital Repository Service at National Institute of Oceanography (India)

    Jain, A.; Nishad, K.K.; Bhosle, N.B.

    The effect of 2, 4-dinitrophenol (DNP) on extracelluar polysaccharides (EPS), cell surface charge, and hydrophobicity of six marine bacterial cultures was studied, and its influence on attachment of these bacteria to glass and polystyrene...

  15. Glycoconjugates and Related Molecules in Human Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Norihiko Sasaki

    2013-01-01

    Full Text Available Vascular endothelial cells (ECs form the inner lining of blood vessels. They are critically involved in many physiological functions, including control of vasomotor tone, blood cell trafficking, hemostatic balance, permeability, proliferation, survival, and immunity. It is considered that impairment of EC functions leads to the development of vascular diseases. The carbohydrate antigens carried by glycoconjugates (e.g., glycoproteins, glycosphingolipids, and proteoglycans mainly present on the cell surface serve not only as marker molecules but also as functional molecules. Recent studies have revealed that the carbohydrate composition of the EC surface is critical for these cells to perform their physiological functions. In this paper, we consider the expression and functional roles of endogenous glycoconjugates and related molecules (galectins and glycan-degrading enzymes in human ECs.

  16. Cell surface localization and tissue distribution of a hepatocyte cell-cell adhesion glycoprotein (cell-CAM 105)

    OpenAIRE

    Ocklind, C; Forsum, U; Obrink, B

    1983-01-01

    We recently identified a 105,000-dalton plasma membrane glycoprotein, denoted cell-CAM 105 (CAM, cell adhesion molecule), that is involved in intercellular adhesion of reaggregating rat hepatocytes (Ocklind, C., and B. Obrink, 1982, J. Biol. Chem., 257:6788-6795). In this communication we used a monospecific rabbit antiserum against cell-CAM 105 to localize the antigen by indirect immunofluorescence on isolated rat cells and on frozen rat tissue sections. This antiserum stained the surface of...

  17. Characterization of heparin-binding site of tissue transglutaminase: its importance in cell surface targeting, matrix deposition, and cell signaling.

    Science.gov (United States)

    Wang, Zhuo; Collighan, Russell J; Pytel, Kamila; Rathbone, Daniel L; Li, Xiaoling; Griffin, Martin

    2012-04-13

    Tissue transglutaminase (TG2) is a multifunctional Ca(2+)-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 (202)KFLKNAGRDCSRRSSPVYVGR(222). We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

  18. Cell surface syndecan-1 contributes to binding and function of macrophage migration inhibitory factor (MIF) on epithelial tumor cells.

    Science.gov (United States)

    Pasqualon, Tobias; Lue, Hongqi; Groening, Sabine; Pruessmeyer, Jessica; Jahr, Holger; Denecke, Bernd; Bernhagen, Jürgen; Ludwig, Andreas

    2016-04-01

    Surface expressed proteoglycans mediate the binding of cytokines and chemokines to the cell surface and promote migration of various tumor cell types including epithelial tumor cells. We here demonstrate that binding of the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) to epithelial lung and breast tumor cell lines A549 and MDA-MB231 is sensitive to enzymatic digestion of heparan sulphate chains and competitive inhibition with heparin. Moreover, MIF interaction with heparin was confirmed by chromatography and a structural comparison indicated a possible heparin binding site. These results suggested that proteoglycans carrying heparan sulphate chains are involved in MIF binding. Using shRNA-mediated gene silencing, we identified syndecan-1 as the predominant proteoglycan required for the interaction with MIF. MIF binding was decreased by induction of proteolytic shedding of syndecan-1, which could be prevented by inhibition of the metalloproteinases involved in this process. Finally, MIF induced the chemotactic migration of A549 cells, wound closure and invasion into matrigel without affecting cell proliferation. These MIF-induced responses were abrogated by heparin or by silencing of syndecan-1. Thus, our study indicates that syndecan-1 on epithelial tumor cells promotes MIF binding and MIF-mediated cell migration. This may represent a relevant mechanism through which MIF enhances tumor cell motility and metastasis.

  19. Characterization of the osteogenic potential of mesenchymal stem cells from human periodontal ligament based on cell surface markers

    Institute of Scientific and Technical Information of China (English)

    Ruth Alvarez; Hye-Lim Lee; Cun-Yu Wang; Christine Hong

    2015-01-01

    Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations:CD51/CD140a, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24%of PDLCs were CD511/CD140a1, 0.8%were CD2711, and 2.4%were STRO-11/CD1461. Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD2711 DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.

  20. Biodegradation and surfactant-mediated biodegradation of diesel fuel by 218 microbial consortia are not correlated to cell surface hydrophobicity

    Energy Technology Data Exchange (ETDEWEB)

    Owsianiak, Mikolaj; Szulc, Alicja; Chrzanowski, Lukasz; Bogacki, Mariusz [Poznan Univ. of Technology (Poland). Inst. of Chemical Technology and Engineering; Cyplik, Pawel; Olejnik-Schmidt, Agniezka K. [Poznan Univ. of Life Sciences (Poland). Dept. of Biotechnology and Food Microbiology; Heipieper, Hermann J. [Helmholtz Centre for Environmental Research - UFZ, Leipzig (Germany). Dept. of Environmental Biotechnology

    2009-09-15

    In this study, we elucidated the role of cell surface hydrophobicity (microbial adhesion to hydrocarbons method, MATH) and the effect of anionic rhamnolipids and nonionic Triton X-100 surfactants on biodegradation of diesel fuel employing 218 microbial consortia isolated from petroleum-contaminated soils. Applied enrichment procedure with floating diesel fuel as a sole carbon source in liquid cultures resulted in consortia of varying biodegradation potential and diametrically different cell surface properties, suggesting that cell surface hydrophobicity is a conserved parameter. Surprisingly, no correlations between cell surface hydrophobicity and biodegradation of diesel fuel were found. Nevertheless, both surfactants altered cell surface hydrophobicity of the consortia in similar manner: increased for the hydrophilic and decreased for the hydrophobic cultures. In addition to this, the surfactants exhibited similar influence on diesel fuel biodegradation: Increase was observed for initially slow-degrading cultures and the opposite for fast degraders. This indicates that in the surfactant-mediated biodegradation, effectiveness of surfactants depends on the specification of microorganisms and not on the type of surfactant. In contrary to what was previously reported for pure strains, cell surface hydrophobicity, as determined by MATH, is not a good descriptor of biodegrading potential for mixed cultures. (orig.)

  1. Formation of Ultracold Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Cote, Robin [Univ. of Connecticut, Storrs, CT (United States)

    2016-01-28

    Advances in our ability to slow down and cool atoms and molecules to ultracold temperatures have paved the way to a revolution in basic research on molecules. Ultracold molecules are sensitive of very weak interactions, even when separated by large distances, which allow studies of the effect of those interactions on the behavior of molecules. In this program, we have explored ways to form ultracold molecules starting from pairs of atoms that have already reached the ultracold regime. We devised methods that enhance the efficiency of ultracold molecule production, for example by tuning external magnetic fields and using appropriate laser excitations. We also investigates the properties of those ultracold molecules, especially their de-excitation into stable molecules. We studied the possibility of creating new classes of ultra-long range molecules, named macrodimers, thousand times more extended than regular molecules. Again, such objects are possible because ultra low temperatures prevent their breakup by collision. Finally, we carried out calculations on how chemical reactions are affected and modified at ultracold temperatures. Normally, reactions become less effective as the temperature decreases, but at ultracold temperatures, they can become very effective. We studied this counter-intuitive behavior for benchmark chemical reactions involving molecular hydrogen.

  2. Influence of cell surface characteristics on adhesion of Saccharomyces cerevisiae to the biomaterial hydroxylapatite.

    Science.gov (United States)

    White, Jane S; Walker, Graeme M

    2011-02-01

    The influence of the physicochemical properties of biomaterials on microbial cell adhesion is well known, with the extent of adhesion depending on hydrophobicity, surface charge, specific functional groups and acid-base properties. Regarding yeasts, the effect of cell surfaces is often overlooked, despite the fact that generalisations may not be made between closely related strains. The current investigation compared adhesion of three industrially relevant strains of Saccharomyces cerevisiae (M-type, NCYC 1681 and ALY, strains used in production of Scotch whisky, ale and lager, respectively) to the biomaterial hydroxylapatite (HAP). Adhesion of the whisky yeast was greatest, followed by the ale strain, while adhesion of the lager strain was approximately 10-times less. According to microbial adhesion to solvents (MATS) analysis, the ale strain was hydrophobic while the whisky and lager strains were moderately hydrophilic. This contrasted with analyses of water contact angles where all strains were characterised as hydrophilic. All yeast strains were electron donating, with low electron accepting potential, as indicated by both surface energy and MATS analysis. Overall, there was a linear correlation between adhesion to HAP and the overall surface free energy of the yeasts. This is the first time that the relationship between yeast cell surface energy and adherence to a biomaterial has been described.

  3. Electrophysical characteristics of Azospirillum brasilense Sp245 during interaction with antibodies to various cell surface epitopes.

    Science.gov (United States)

    Guliy, Olga I; Matora, Larisa Y; Burygin, Gennady L; Dykman, Lev A; Ostudin, Nikolai A; Bunin, Viktor D; Ignatov, Vladimir V; Ignatov, Oleg V

    2007-11-15

    This work was undertaken to examine the electrooptical characteristics of cells of Azospirillum brasilense Sp245 during their interaction with antibodies developed to various cell surface epitopes. We used the dependences of the cell suspension optical density changes induced by electroorientation on the orienting field frequency (740, 1000, 1450, 2000, and 2800kHz). Cell interactions with homologous strain-specific antibodies to the A. brasilense Sp245 O antigen and with homologous antibodies to whole bacterial cells brought about considerable changes in the electrooptical properties of the bacterial suspension. When genus-specific antibodies to the flagellin of the Azospirillum sheathed flagellum and antibodies to the serologically distinct O antigen of A. brasilense Sp7 were included in the A. brasilense Sp245 suspension, the changes caused in the electrooptical signal were slight and had values close to those for the above changes. These findings agree well with the immunochemical characteristics of the Azospirillum O antigens and with the data on the topographical distribution of the Azospirillum major cell surface antigens. The obtained results can serve as a basis for the development of a rapid test for the intraspecies detection of microorganisms.

  4. Cell surface appearance of unexpected host MHC determinants on thymocytes from radiation bone marrow chimeras

    International Nuclear Information System (INIS)

    The phenotypic appearance of cell surface antigens on murine thymocytes from long-term radiation bone marrow chimeras was analyzed using indirect immunofluorescence and flow microfluorometry. Cells maturing in the thymi of these mice were typed for MHC (Kk, I-Ak, H-2b, Kb, and Ib) and non-MHC (Lty 1, Ly 9, and TL) determinants. All cells were of donor origin as determined by non-MHC (Ly) phenotype in P1 leads to P2, P1 x P2 leads to P1, and P1 leads to P2 radiation chimeras. In contrast, the MHC phenotypes of these thymocytes were markedly affected by the host environment. Specifically, H-2 and I-A determinants of both parental phenotypes were detected on thymocytes from P1 leads to P1 x P2 chimeras; I-A determinants of host phenotype were present, whereas I-A determinants of donor phenotype were reduced on thymocytes from P1 x P2 leads to P1 chimeras; and thymocytes from P1 leads to P2 chimeras possessed H-2 and I-A determinants of host phenotype but showed reduction of donor I-A phenotype determinants. The appearance of host cell surface H-2 and I-A determinants on thymocytes from chimeras closely parallels the functional recognition of MHC determinants by T cells from chimeric mice and thus may be significantly related to the development of the self-recognition repertoire by maturing T cells

  5. Distribution of anionic groups at the cell surface of different Sporothrix schenckii cell types.

    Science.gov (United States)

    Benchimol, M; de Souza, W; Travassos, L R

    1979-06-01

    The distribution of anionic groups at the cell surface of yeastlike forms, hyphae, and conidia of Sporothrix schenckii was studied by staining with colloidal iron hydroxide and cationized ferritin. By using colloidal iron hydroxide it was shown that the external cell wall layer of one strain (strain 1099.18) could be resolved into two reactive sublayers and that these layers were present in many but not all cells of the same population. In contrast, most cells of another strain (strain 1099.12) were stained by colloidal iron hydroxide, but only one reactive layer was seen. Acidic layers of the yeastlike forms of the two strains were much thicker than those of conidia and hyphae. By the cationized ferritin staining procedure it was observed that the acidic layers of yeast forms sloughed off of cells, probably due to cell-cell or cell-medium attrition in shaken submerged cultures or to a process by which the outer layers detach from cells as they are replaced by newly synthesized ones. The colloidal iron hydroxide- and cationized ferritin-reactive cell surface layers of S. schenckii correspond to the previously described (L. R. Travassos et al., Exp. Mycol. 1:293-305, 1977) concanavalin A-reactive peptidorhamnomannan complexes, and their reactivity is probably due to the presence of acidic amino acids of low pK values rather than to glucuronic acid units.

  6. Modulation of cell surface GABA B receptors by desensitization,trafficking and regulated degradation

    Institute of Scientific and Technical Information of China (English)

    Dietmar; Benke; Khaled; Zemoura; Patrick; J; Maier

    2012-01-01

    Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.

  7. Protein Kinase C Regulates the Cell Surface Activity of Endothelin-Converting Enzyme-1.

    Science.gov (United States)

    Smith, A Ian; Lew, Rebecca A; Thomas, Walter G; Tochon-Danguy, Nathalie

    2006-09-01

    The potent vasoconstrictor endothelin is a 21 amino acid peptide whose principal physiological function is to regulate vascular tone. The generation of endothelin is crucially dependent on the local presence and activity of endothelin converting enzyme-1 (ECE-1) expressed on the surface of vascular endothelial cells. In this study, we have shown in endothelial cells that the enzyme is phosphorylated, and that phosphorylation is increased by phorbol ester stimulation of protein kinase C (PKC). Furthermore, by monitoring specific ECE-1 activity on the surface of live cells, we also show that following PKC activation, enzyme activity is significantly increased at the cell surface, where it is positioned to catalyse the generation of active endothelin. We believe this novel finding is unprecedented for a peptide processing enzyme. Indeed, this new knowledge regarding the control of endothelin production by regulating ECE-1 activity at the cell surface opens up a new area of endothelin biology and will provide novel insights into the physiology and pathophysiology of endothelin and endothelin-associated diseases. In addition, the information generated in these studies may provide valuable new insights into potential extra- and intracellular targets for the pharmacological and perhaps even therapeutic regulation of endothelin production and thus vascular tone. PMID:19617920

  8. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle

    The function of Hsp70 depends on its cellular location: When located intracellularly it exerts cytoprotective and anti-apoptotic functions, whereas it exerts immunostimulatory functions when located extracellularly. Secreted Hsp70 is for example involved in cross-presentation of cancer-derived an......The function of Hsp70 depends on its cellular location: When located intracellularly it exerts cytoprotective and anti-apoptotic functions, whereas it exerts immunostimulatory functions when located extracellularly. Secreted Hsp70 is for example involved in cross-presentation of cancer......-derived antigenic peptides, a function which is currently explored in immunotherapeutic approaches against cancer. Additionally, membrane-bound Hsp70 can stimulate antigen presenting cells to release proinflammatory cytokines and can provide a target structure for NK cell-mediated lysis. Human cancer cells...... frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 cell surface expression on cancer...

  9. SNX15 Regulates Cell Surface Recycling of APP and Aβ Generation.

    Science.gov (United States)

    Feng, Tuancheng; Niu, Mengmeng; Ji, Chengxiang; Gao, Yuehong; Wen, Jing; Bu, Guojun; Xu, Huaxi; Zhang, Yun-Wu

    2016-08-01

    Amyloid-β (Aβ) peptide plays an essential role in the pathogenesis of Alzheimer's disease (AD) and is generated from amyloid-β precursor protein (APP) through sequential proteolytic cleavages by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Trafficking dysregulation of APP, BACE1, and γ-secretase may affect Aβ generation and disease pathogenesis. Sorting nexin 15 (SNX15) is known to regulate protein trafficking. Here, we report that SNX15 is abundantly expressed in mouse neurons and astrocytes. In addition, we show that although not affecting the protein levels of APP, BACE1, and γ-secretase components and the activity of BACE1 and γ-secretase, overexpression and downregulation of SNX15 reduce and promote Aβ production, respectively. Furthermore, we find that overexpression of SNX15 increases APP protein levels in cell surface through accelerating APP recycling, whereas downregulation of SNX15 has an opposite effect. Finally, we show that exogenous expression of human SNX15 in the hippocampal dentate gyrus by adeno-associated virus (AAV) infection can significantly reduce Aβ pathology in the hippocampus and improve short-term working memory in the APPswe/PSEN1dE9 double transgenic AD model mice. Together, our results suggest that SNX15 regulates the recycling of APP to cell surface and, thus, its processing for Aβ generation. PMID:26115702

  10. The innate pattern recognition molecule Ficolin-1 is secreted by monocytes/macrophages and is circulating in human plasma

    DEFF Research Database (Denmark)

    Honoré, Christian; Rørvig, Sara; Munthe-Fog, Lea;

    2008-01-01

    Ficolin-1 (M-Ficolin) is a pattern recognition molecule of the complement system that is expressed by myeloid cells and type II alveolar epithelial cells. Ficolin-1 has been shown to localize in the secretory granules of these cells and attached to cell surfaces, but whether Ficolin-1 exists...

  11. Sequential expression of germ-layer specific molecules in the sea urchin embryo.

    Science.gov (United States)

    Wessel, G M; McClay, D R

    1985-10-01

    Described are two germ-layer specific molecules that appear coincident with the formation of two germ layer cell lineages in the sea urchin embryo. Meso1 is a molecule of 380 kDa that is first detected at the time of primary mesenchyme cell delamination from the wall of the blastula. Endo1 is a molecule of 320 kDa that appears on endoderm cells at the time of archenteron formation a few hours after Meso1 appears. Both antigens are identified by monoclonal antibodies. The appearance of these antigens is described by immunofluorescence microscopy, and quantitative data on their localization has been obtained by ultrastructural immunoelectron microscopy. The synthesis of the molecules has been followed by pulse-chase immunoprecipitation. Meso1 is first expressed in trans Golgi-like saccules, is concentrated in peripheral low electron-dense vesicles, and is found throughout the plasma membrane of the mesenchymal cells and their filopodial extensions. Newly translated Meso1 can first be immunoprecipitated upon differentiation of the mesoderm cell lineage, and pulse-chase studies suggest that the determinant is the result of a post-translational modification. [35S]Methionine pulses early in development followed by a chase to the mesenchyme blastula or prism stage show that at least a portion of the molecule is translated well in advance of the mesenchyme blastula stage. Endo1, in contrast, does not appear to be translated until the onset of gastrulation, just preceding the post-translational expression of the Endo1 determinant. Endo1 is localized to the apical and basolateral cell surfaces of the midgut and hindgut. No label is detected in foregut cells, demonstrating a heterogeneity of cell populations within the endoderm cell lineage corresponding to a difference in morphology. In addition, Endo1 is shown to be the result of new transcription by the embryonic genome. Even though the function of neither molecule is known, together they show the spatial and temporal

  12. Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells

    Science.gov (United States)

    Haga, Kei; Fujimoto, Akira; Takai-Todaka, Reiko; Miki, Motohiro; Doan, Yen Hai; Murakami, Kosuke; Yokoyama, Masaru; Murata, Kazuyoshi; Nakanishi, Akira; Katayama, Kazuhiko

    2016-01-01

    Norovirus is the leading cause of acute gastroenteritis worldwide. Since the discovery of human norovirus (HuNoV), an efficient and reproducible norovirus replication system has not been established in cultured cells. Although limited amounts of virus particles can be produced when the HuNoV genome is directly transfected into cells, the HuNoV cycle of infection has not been successfully reproduced in any currently available cell-culture system. Those results imply that the identification of a functional cell-surface receptor for norovirus might be the key to establishing a norovirus culture system. Using a genome-wide CRISPR/Cas9 guide RNA library, we identified murine CD300lf and CD300ld as functional receptors for murine norovirus (MNV). The treatment of susceptible cells with polyclonal antibody against CD300lf significantly reduced the production of viral progeny. Additionally, ectopic CD300lf expression in nonsusceptible cell lines derived from other animal species enabled MNV infection and progeny production, suggesting that CD300lf has potential for dictating MNV host tropism. Furthermore, CD300ld, which has an amino acid sequence in the N-terminal region of its extracellular domain that is highly homologous to that of CD300lf, also functions as a receptor for MNV. Our results indicate that direct interaction of MNV with two cell-surface molecules, CD300lf and CD300ld, dictates permissive noroviral infection. PMID:27681626

  13. Trapping molecules on chips

    CERN Document Server

    Santambrogio, Gabriele

    2015-01-01

    In the last years, it was demonstrated that neutral molecules can be loaded on a microchip directly from a supersonic beam. The molecules are confined in microscopic traps that can be moved smoothly over the surface of the chip. Once the molecules are trapped, they can be decelerated to a standstill, for instance, or pumped into selected quantum states by laser light or microwaves. Molecules are detected on the chip by time-resolved spatial imaging, which allows for the study of the distribution in the phase space of the molecular ensemble.

  14. [Endothelial cell adhesion molecules].

    Science.gov (United States)

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  15. In vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules

    OpenAIRE

    Yan, Jingbo; Parekh, Vrajesh V.; Mendez-Fernandez, Yanice; Olivares-Villagómez, Danyvid; Dragovic, Srdjan; Hill, Timothy; Roopenian, Derry C.; Joyce, Sebastian; Van Kaer, Luc

    2006-01-01

    Endoplasmic reticulum (ER)-associated aminopeptidase (ERAP)1 has been implicated in the final proteolytic processing of peptides presented by major histocompatibility complex (MHC) class I molecules. To evaluate the in vivo role of ERAP1, we have generated ERAP1-deficient mice. Cell surface expression of the class Ia molecules H-2Kb and H-2Db and of the class Ib molecule Qa-2 was significantly reduced in these animals. Although cells from mutant animals exhibited reduced capacity to present s...

  16. The glucose metabolite methylglyoxal inhibits expression of the glucose transporter genes by inactivating the cell surface glucose sensors Rgt2 and Snf3 in yeast.

    Science.gov (United States)

    Roy, Adhiraj; Hashmi, Salman; Li, Zerui; Dement, Angela D; Cho, Kyu Hong; Kim, Jeong-Ho

    2016-03-01

    Methylglyoxal (MG) is a cytotoxic by-product of glycolysis. MG has inhibitory effect on the growth of cells ranging from microorganisms to higher eukaryotes, but its molecular targets are largely unknown. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of expression of genes encoding glucose transporters (HXTs). Here we provide evidence that these glucose sensors are primary targets of MG in yeast. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensors. However, the glucose sensors with mutations at their putative ubiquitin-acceptor lysine residues are resistant to MG-induced degradation. These results suggest that the glucose sensors are inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. In addition, the inhibitory effect of MG on the glucose sensors is greatly enhanced in cells lacking Glo1, a key component of the MG detoxification system. Thus the stability of these glucose sensors seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell-surface glucose sensors and thus identify MG as a potential glycolytic inhibitor.

  17. Topical retinoic acid changes the epidermal cell surface glycosylation pattern towards that of a mucosal epithelium

    DEFF Research Database (Denmark)

    Griffiths, C E; Dabelsteen, Erik; Voorhees, J J

    1996-01-01

    Topical all-trans retinoic acid (RA) produces a number of epidermal changes which are indistinguishable from those observed following treatment with a local irritant, namely sodium lauryl sulphate (SLS). This observation has led to criticism that the efficacy of RA in disorders such as photoageing...... for carbohydrate synthesis, are influenced by retinoids. Thus, we investigated whether epidermal cell surface glycosylation is altered in skin treated with topical RA, and contrasted it with changes induced by topical SLS. Skin biopsies were obtained from seven normal volunteers who had been treated, on three......-treated epidermis was not significantly different from that observed after vehicle treatment. Thus, RA treatment converts normal stratified epithelium towards the phenotype of mucosal epithelium with a decrease in T antigen and a concomitant increase in Ley. These changes are not observed following treatment...

  18. Display of wasp venom allergens on the cell surface of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Borodina, Irina; Jensen, B. M.; Søndergaard, Ib;

    2010-01-01

    Background: Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast...... Saccharomyces cerevisiae preserve their native allergenic properties and whether the yeast native surface glycoproteins interfere with IgE binding. We chose to use the major allergens from the common wasp Vespula vulgaris venom: phospholipase A1, hyaluronidase and antigen 5 as the model. Results: The proteins...... their enzymatic activities. Phospholipase A1 severely inhibited the growth of the yeast cells. Antigen 5 - expressing yeast cells bound IgE antibodies from wasp venom allergic patient sera but not from control sera as demonstrated by FACS. Moreover, antigen 5 - expressing yeast cells were capable of mediating...

  19. Restoration of proper trafficking to the cell surface for membrane proteins harboring cysteine mutations.

    Science.gov (United States)

    Lopez-Rodriguez, Angelica; Holmgren, Miguel

    2012-01-01

    A common phenotype for many genetic diseases is that the cell is unable to deliver full-length membrane proteins to the cell surface. For some forms of autism, hereditary spherocytosis and color blindness, the culprits are single point mutations to cysteine. We have studied two inheritable cysteine mutants of cyclic nucleotide-gated channels that produce achromatopsia, a common form of severe color blindness. By taking advantage of the reactivity of cysteine's sulfhydryl group, we modified these mutants with chemical reagents that attach moieties with similar chemistries to the wild-type amino acids' side chains. We show that these modifications restored proper delivery to the cell membrane. Once there, the channels exhibited normal functional properties. This strategy might provide a unique opportunity to assess the chemical nature of membrane protein traffic problems. PMID:23082193

  20. Signaling at the cell surface in the circulatory and ventilatory systems

    CERN Document Server

    Thiriet, Marc

    2012-01-01

    The volumes in this authoritative series present a multidisciplinary approach to modeling and simulation of flows in the cardiovascular and ventilatory systems, especially multiscale modeling and coupled simulations. The cardiovascular and respiratory systems are tightly coupled, as their primary function is to supply oxygen to and remove carbon dioxide from the body's cells. Because physiological conduits have deformable and reactive walls, macroscopic flow behavior and prediction must be coupled to nano- and microscopic events in a corrector scheme of regulated mechanisms when the vessel lumen caliber varies markedly. Therefore, investigation of flows of blood and air in physiological conduits requires an understanding of the biology, chemistry, and physics of these systems together with the mathematical tools to describe their functioning. Volume 3 is devoted to the set of mediators of the cell surface, especially ion and molecular carriers and catalytic receptors that, once liganded and activated, initiat...

  1. Fluid dynamics and noise in bacterial cell-cell and cell-surface scattering

    CERN Document Server

    Drescher, Knut; Cisneros, Luis H; Ganguly, Sujoy; Goldstein, Raymond E; 10.1073/pnas.1019079108

    2011-01-01

    Bacterial processes ranging from gene expression to motility and biofilm formation are constantly challenged by internal and external noise. While the importance of stochastic fluctuations has been appreciated for chemotaxis, it is currently believed that deterministic long-range fluid dynamical effects govern cell-cell and cell-surface scattering - the elementary events that lead to swarming and collective swimming in active suspensions and to the formation of biofilms. Here, we report the first direct measurements of the bacterial flow field generated by individual swimming Escherichia coli both far from and near to a solid surface. These experiments allowed us to examine the relative importance of fluid dynamics and rotational diffusion for bacteria. For cell-cell interactions it is shown that thermal and intrinsic stochasticity drown the effects of long-range fluid dynamics, implying that physical interactions between bacteria are determined by steric collisions and near-field lubrication forces. This dom...

  2. Cell-surface metalloprotease ADAM12 is internalized by a clathrin- and Grb2-dependent mechanism

    DEFF Research Database (Denmark)

    Hansen, Dorte Stautz; Leyme, Anthony; Grandal, Michael Vibo;

    2012-01-01

    ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell......-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected...... in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline-rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src-homology 3 domains...

  3. Effects of sodium on cell surface and intracellular TH-naloxone binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Pollack, A.E.; Wooten, G.F.

    1987-07-27

    The binding of the opiate antagonist TH-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, TH-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the sodium effect of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites. 17 references, 2 tables.

  4. Effects of sodium on cell surface and intracellular 3H-naloxone binding sites

    International Nuclear Information System (INIS)

    The binding of the opiate antagonist 3H-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, 3H-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the sodium effect of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites. 17 references, 2 tables

  5. Cell surface proteome analysis of human-hosted Trypanosoma cruzi life stages

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sébastien; Bastos, Izabela M D;

    2014-01-01

    addressed the analysis of the plasma membrane (PM) subproteome from T. cruzi human-hosted life stages, trypomastigote and axenic amastigote, by two complementary PM protein enrichment techniques followed by identification using an LC-MS/MS approach. The results revealed an extensive repertoire of proteins...... in the PM subproteomes, including enzymes that might be suitable candidates for drug intervention. The comparison of the cell surface proteome among the life forms revealed some potentially stage-specific enzymes, although the majority was shared by both stages. Bioinformatic analysis showed that the vast......Chagas' disease is a neglected infectious illness, caused by the protozoan Trypanosoma cruzi. It remains a challenging health issue in Latin America, where it is endemic, and so far there is no immunoprophylatic vaccine or satisfactory chemotherapic treatment for its chronic stage. The present work...

  6. Molecules in galaxies

    CERN Document Server

    Omont, Alain

    2007-01-01

    The main achievements, current developments and prospects of molecular studies in external galaxies are reviewed. They are put in the context of the results of several decades of studies of molecules in local interstellar medium, their chemistry and their importance for star formation. CO observations have revealed the gross structure of molecular gas in galaxies. Together with other molecules, they are among the best tracers of star formation at galactic scales. Our knowledge about molecular abundances in various local galactic environments is progressing. They trace physical conditions and metallicity, and they are closely related to dust processes and large aromatic molecules. Major recent developments include mega-masers, and molecules in Active Galactic Nuclei; millimetre emission of molecules at very high redshift; and infrared H2 emission as tracer of warm molecular gas, shocks and photodissociation regions. The advent of sensitive giant interferometers from the centimetre to sub-millimetre range, espe...

  7. Changes in cell surface properties of shiga toxigenic Escherichia coli by Quercus infectoria G. Olivier.

    Science.gov (United States)

    Voravuthikunchai, Supayang Piyawan; Suwalak, Sakol

    2009-08-01

    The effects of Quercus infectoria (family Fagaceae) nutgalls on cell surface properties of Shiga toxigenic Escherichia coli (STEC) were investigated with an assay of microbial adhesion to hydrocarbon. The surface of bacterial cells treated with Q. infectoria exhibited a higher level of cell surface hydrophobicity (CSH) toward toluene than did the surface of untreated cells. With 50% ethanolic extract, the CSH of the three strains of STEC O157:H7 treated with 4X MIC of the extract resulted in moderate or strong hydrophobicity, whereas at 2x MIC and MIC, the CSH of only one strain of E. coli O157:H7 was significantly affected. The 95% ethanolic extract had a significant effect on CSH of all three strains at both 4X MIC and 2X MIC but not at the MIC. The effect on bacterial CSH was less pronounced with the other STEC strains. At 4X MIC, the 50% ethanolic extract increased the CSH of all non-O157 STEC strains significantly. At 2X MIC and 4X MIC, the 95% ethanolic extract affected the CSH of E. coli O26:H11 significantly but did not affect E. coli O111 :NM or E. coli O22. Electro microscopic examination revealed the loss of pili in the treated cells. The ability of Q. infectoria extract to modify hydrophobic domains enables this extract to partition the lipids of the bacterial cell membrane, rendering the membrane more permeable and allowing leakage of ions and other cell contents, which leads to cell death. Further studies are required to evaluate the effects of Q. infectoria extract in food systems or in vivo and provide support for the use of this extract as a food additive for control of these STEC pathogens. PMID:19722403

  8. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    Directory of Open Access Journals (Sweden)

    Fabio Cattaneo

    2014-10-01

    Full Text Available G protein-coupled receptors (GPCRs are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors

  9. Development of living cell force sensors for the interrogation of cell surface interactions

    Science.gov (United States)

    Brown, Scott Chang

    The measurement of cell surface interactions, or cell interaction forces, are critical for the early diagnosis and prevention of disease, the design of targeted drug and gene delivery vehicles, the development of next-generation implant materials, and much more. However, the technologies and devices that are currently available are highly limited with respect to the dynamic force range over which they can measure cell-cell or cell-substratum interactions, and with their ability to adequately mimic biologically relevant systems. Consequently, research efforts that involve cell surface interactions have been limited. In this dissertation, existing tools for research at the nanoscale (i.e., atomic force microscopy microcantilevers) are modified to develop living cell force sensors that allow for the highly sensitive measurement of cell-mediated interactions over the entire range of forces expected in biotechnology (and nano-biotechnology) research (from a single to millions of receptor-ligand bonds). Several force sensor motifs have been developed that can be used to measure interactions using single adherent cells, single suspension culture cell, and cell monolayers (tissues) over a wide range of interaction conditions (e.g., approach velocity, shear rate, contact time) using a conventional atomic force microscope. This new tool has been applied to study the pathogenesis of spontaneous pneumothorax and the interaction of cells with 14 man-made interfaces. Consequently, a new hypothesis of the interactions that manifest spontaneous pneumothorax has been developed. Additionally, these findings have the potential to lead to the development of tools for data mining materials and surfaces for unique cell interactions that could have an immense societal impact.

  10. The microbial cell surface electric field: life in an ion cloud

    Science.gov (United States)

    Yee, N.

    2005-05-01

    Electrical charge on microbial cell surfaces arises from the ionization of proton-active functional groups attached to cell wall polymers. In Gram-positive cell walls, ionizable functional groups are associated with peptidoglycan and secondary polymers such as teichoic or teichuronic acids. Carboxyl functional groups attached to the unlinked peptide crosslinks of peptidoglycan and phosphoryl groups associated with the teichoic acids can deprotonate to form negatively charged surface sites. These anionic functional groups generate charge in the cell wall which results in the formation of an electric field that surrounds the entire cell. The cell surface electric field controls the concentration and spatial distribution of ions and counterions at the cell-water interface, and strongly affects microbe-fluid and microbe-mineral interactions. Recently, we have used potentiometric titration, infrared spectroscopy, electrophoretic mobility, metal sorption experiments to characterize the surface electrical potential properties of the various Gram-positive and Gram-negative bacterial species. Potentiometric titration experiments show that the deprotonation of acidic cell wall functional groups generate surface charge density values typically ranging from 1.1 to 2.2 mol sites/g of bacteria. Spectroscopic measurements have confirmed that the dominant proton-active sites in the cell wall are carboxyl functional groups. Electrophoretic mobility experiments show that the magnitude of the electrostatic surface potential increases with increasing pH, and decreases with increasing ionic strength. Metal sorption experiments conducted with Ca(II), Sr(II) and Ba(II) exhibit strong ionic strength dependence, suggesting that high concentrations of metal ions are electrostatically bound to bacterial cell walls via outer-sphere complexation. We demonstrate that the electrostatic potential effects on ion sorption at the cell-water interface can be quantified using the Donnan model.

  11. Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

    Science.gov (United States)

    Betz, N. A.; Westhoff, B. A.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

  12. Traffic of secondary metabolites to cell surface in the red alga Laurencia dendroidea depends on a two-step transport by the cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Vanessa M Reis

    Full Text Available In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC. For chemical defence at the surface level, these metabolites are intracellularly mobilised through vesicle transport from the CC to the cell periphery for posterior exocytosis of these chemicals. The cell structures involved in this specific vesicle traffic as well as the cellular structures related to the positioning and anchoring of the CC within the cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i determine the ultrastructural apparatus involved in the vesicle traffic, ii localise cytoskeletal filaments, iii evaluate the role of different cytoskeletal filaments in the vesicle transport, iv identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the cell periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the cell surface. After that, the microtubules work in the positioning of the vesicles along the cell periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the cell periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying cellular role

  13. Traffic of secondary metabolites to cell surface in the red alga Laurencia dendroidea depends on a two-step transport by the cytoskeleton.

    Science.gov (United States)

    Reis, Vanessa M; Oliveira, Louisi S; Passos, Raoni M F; Viana, Nathan B; Mermelstein, Cláudia; Sant'anna, Celso; Pereira, Renato C; Paradas, Wladimir C; Thompson, Fabiano L; Amado-Filho, Gilberto M; Salgado, Leonardo T

    2013-01-01

    In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intracellularly mobilised through vesicle transport from the CC to the cell periphery for posterior exocytosis of these chemicals. The cell structures involved in this specific vesicle traffic as well as the cellular structures related to the positioning and anchoring of the CC within the cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the cell periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the cell surface. After that, the microtubules work in the positioning of the vesicles along the cell periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the cell periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying cellular role in the

  14. Desmosomal Molecules In and Out of Adhering Junctions: Normal and Diseased States of Epidermal, Cardiac and Mesenchymally Derived Cells

    Directory of Open Access Journals (Sweden)

    Sebastian Pieperhoff

    2010-01-01

    Full Text Available Current cell biology textbooks mention only two kinds of cell-to-cell adhering junctions coated with the cytoplasmic plaques: the desmosomes (maculae adhaerentes, anchoring intermediate-sized filaments (IFs, and the actin microfilament-anchoring adherens junctions (AJs, including both punctate (puncta adhaerentia and elongate (fasciae adhaerentes structures. In addition, however, a series of other junction types has been identified and characterized which contain desmosomal molecules but do not fit the definition of desmosomes. Of these special cell-cell junctions containing desmosomal glycoproteins or proteins we review the composite junctions (areae compositae connecting the cardiomyocytes of mature mammalian hearts and their importance in relation to human arrhythmogenic cardiomyopathies. We also emphasize the various plakophilin-2-positive plaques in AJs (coniunctiones adhaerentes connecting proliferatively active mesenchymally-derived cells, including interstitial cells of the heart and several soft tissue tumor cell types. Moreover, desmoplakin has also been recognized as a constituent of the plaques of the complexus adhaerentes connecting certain lymphatic endothelial cells. Finally, we emphasize the occurrence of the desmosomal transmembrane glycoprotein, desmoglein Dsg2, out of the context of any junction as dispersed cell surface molecules in certain types of melanoma cells and melanocytes. This broadening of our knowledge on the diversity of AJ structures indicates that it may still be too premature to close the textbook chapters on cell-cell junctions.

  15. Electron correlation in molecules

    CERN Document Server

    Wilson, S

    2007-01-01

    Electron correlation effects are of vital significance to the calculation of potential energy curves and surfaces, the study of molecular excitation processes, and in the theory of electron-molecule scattering. This text describes methods for addressing one of theoretical chemistry's central problems, the study of electron correlation effects in molecules.Although the energy associated with electron correlation is a small fraction of the total energy of an atom or molecule, it is of the same order of magnitude as most energies of chemical interest. If the solution of quantum mechanical equatio

  16. Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide.

    Science.gov (United States)

    Inokuma, Kentaro; Bamba, Takahiro; Ishii, Jun; Ito, Yoichiro; Hasunuma, Tomohisa; Kondo, Akihiko

    2016-11-01

    Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc. PMID:27183011

  17. Proteomic Analysis of Macrophages: A Potential Way to Identify Novel Proteins Associated with Activation of Macrophages for Tumor Cell Killing

    Institute of Scientific and Technical Information of China (English)

    Lingbing Zhang; Haoxuan Zhu; Yanni Lun; Dongmei Yan; Leyang Yu; Bairong Du; Xun Zhu

    2007-01-01

    One major mechanism through which macrophages effectively kill tumor cells requires cell to cell contact,indicating that certain molecules expressed on cell surface of activated macrophages may mediate the tumoricidal capability. Tumor necrosis factor (TNF) and nitric oxide (NO) are the two classical mediators of tumor cell death.However, evidence of discrepancy is accumulating indicating these known mediators do not appear to account for the broad and potent tumoricidal activity of macrophages. To obtain a full repertoire of tumoricidal activationassociated membrane proteins, we combined one-dimensional SDS-PAGE with capillary liquid chromatographytandem mass spectrometry (LC-MS/MS). Using this technique, we identified 454 activated macrophage specifically expressed proteins with extremely high confidence, including most known activation markers of macrophages,such as NO synthase (iNOS), Ym1, cyclooxygenase, etc. Membrane bound TNF-α was also identified on activated macrophages. However, it was also detected on thioglycolate elicited macrophages, indicating this molecule may not play a key role in conjugation-dependent tumor cell killing. In contrast, although NO has not been assigned as an effector molecule of conjugation-dependent tumoricidal pathway, iNOS was identified from membrane fraction of activated macrophages, suggesting NO may be involved in conjugation-dependent tumoricidal mechanism,because iNOS association with plasma membrane is ideally suited to deliver NO directly into the contacted tumor cells. This research provides not only new insights into macrophage conjugation-dependent tumoricidal mechanisms, but also a valuable data set of macrophage activation associated membrane proteins, thus providing better understanding of the functional mechanisms of macrophages in anti-tumor and other biological processes.

  18. Characterization of structural features controlling the receptiveness of empty class II MHC molecules.

    Directory of Open Access Journals (Sweden)

    Bernd Rupp

    Full Text Available MHC class II molecules (MHC II play a pivotal role in the cell-surface presentation of antigens for surveillance by T cells. Antigen loading takes place inside the cell in endosomal compartments and loss of the peptide ligand rapidly leads to the formation of a non-receptive state of the MHC molecule. Non-receptiveness hinders the efficient loading of new antigens onto the empty MHC II. However, the mechanisms driving the formation of the peptide inaccessible state are not well understood. Here, a combined approach of experimental site-directed mutagenesis and computational modeling is used to reveal structural features underlying "non-receptiveness." Molecular dynamics simulations of the human MHC II HLA-DR1 suggest a straightening of the α-helix of the β1 domain during the transition from the open to the non-receptive state. The movement is mostly confined to a hinge region conserved in all known MHC molecules. This shift causes a narrowing of the two helices flanking the binding site and results in a closure, which is further stabilized by the formation of a critical hydrogen bond between residues αQ9 and βN82. Mutagenesis experiments confirmed that replacement of either one of the two residues by alanine renders the protein highly susceptible. Notably, loading enhancement was also observed when the mutated MHC II molecules were expressed on the surface of fibroblast cells. Altogether, structural features underlying the non-receptive state of empty HLA-DR1 identified by theoretical means and experiments revealed highly conserved residues critically involved in the receptiveness of MHC II. The atomic details of rearrangements of the peptide-binding groove upon peptide loss provide insight into structure and dynamics of empty MHC II molecules and may foster rational approaches to interfere with non-receptiveness. Manipulation of peptide loading efficiency for improved peptide vaccination strategies could be one of the applications profiting

  19. Cell surface N-glycans influence the level of functional E-cadherin at the cell–cell border

    OpenAIRE

    M Kristen Hall; Douglas A Weidner; Sahil Dayal; Ruth A. Schwalbe

    2014-01-01

    E-cadherin is crucial for adhesion of cells to each other and thereby development and maintenance of tissue. While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell–cell border. Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell–cell...

  20. Alterations of electrical charge and receptors to lectins mouse lymphoma cells surface in early terms after irradiation

    International Nuclear Information System (INIS)

    Modifications of structural and functional state of OH-1 mouse lymphoma cells surface in early terms after gamma-irradiation with doses from 0.1 Gy to 10 Gy were studied. For this purpose, the methods of cell separation in a two-phase polymer system (dextran-PEG) and cell surface receptors binding with some plant lectins were used. It was revealed the decreased surface electrical charge that reached its maximum deflection 3 hours after gamma-irradiation. At the same time-dose dependent expression of irradiated cells, membrane receptors to the lectins of various specificity was observed

  1. Single-molecule dynamics at variable temperatures

    OpenAIRE

    Zondervan, Rob

    2006-01-01

    Single-molecule optics has evolved from a specialized variety of optical spectroscopy at low temperatures into a versatile tool to address questions in physics, chemistry, biology, and materials science. In this thesis, the potential of single-molecule (and ensemble) optical microscopy at variable temperatures is demonstrated: Electron transfer has been identified as a crucial step in the photodynamics of organic fluorophores, and long-term memory effects have been discovered in the relaxatio...

  2. Tunable optical absorption in silicene molecules

    KAUST Repository

    Mokkath, Junais Habeeb

    2016-07-13

    Two-dimensional materials with a tunable band gap that covers a wide range of the solar spectrum hold great promise for sunlight harvesting. For this reason, we investigate the structural, electronic, and optical properties of silicene molecules using time dependent density functional theory. We address the influence of the molecular size, buckling, and charge state as well as that of a dielectric environment. Unlike planar graphene molecules, silicene molecules prefer to form low-buckled structures with strong visible to ultraviolet optical response. We also identify molecular plasmons.

  3. Non-sequential double ionization of molecules

    CERN Document Server

    Prauzner-Bechcicki, J S; Eckhardt, B; Zakrzewski, J; Prauzner-Bechcicki, Jakub S.; Sacha, Krzysztof; Eckhardt, Bruno; Zakrzewski, Jakub

    2004-01-01

    Double ionization of diatomic molecules by short linearly polarized laser pulses is analyzed. We consider the final stage of the ionization process, that is the decay of a highly excited two electron molecule, which is formed after re-scattering. The saddles of the effective adiabatic potential energy close to which simultaneous escape of electrons takes place are identified. Numerical simulations of the ionization of molecules show that the process can be dominated by either sequential or non-sequential events. In order to increase the ratio of non-sequential to sequential ionizations very short laser pulses should be applied.

  4. Association of Intercellular Adhesion Molecule 1 (ICAM1) with Diabetes and Diabetic Nephropathy

    OpenAIRE

    Gu, Harvest F; Jun eMa; Gu, Karolin T.; Kerstin eBrismar

    2013-01-01

    Diabetes and diabetic nephropathy are complex diseases affected by genetic and environmental factors. Identification of the susceptibility genes and investigation of their roles may provide useful information for better understanding of the pathogenesis and for developing novel therapeutic approaches. Intercellular adhesion molecule 1 (ICAM1) is a cell surface glycoprotein expressed on endothelial cells and leukocytes in the immune system. The ICAM1 gene is located on chromosome 19p13 within ...

  5. Single molecules and nanotechnology

    CERN Document Server

    Vogel, Horst

    2007-01-01

    This book focuses on recent advances in the rapidly evolving field of single molecule research. These advances are of importance for the investigation of biopolymers and cellular biochemical reactions, and are essential to the development of quantitative biology. Written by leading experts in the field, the articles cover a broad range of topics, including: quantum photonics of organic dyes and inorganic nanoparticles their use in detecting properties of single molecules the monitoring of single molecule (enzymatic) reactions single protein (un)folding in nanometer-sized confined volumes the dynamics of molecular interactions in biological cells The book is written for advanced students and scientists who wish to survey the concepts, techniques and results of single molecule research and assess them for their own scientific activities.

  6. Electron-molecule collisions

    CERN Document Server

    Takayanagi, Kazuo

    1984-01-01

    Scattering phenomena play an important role in modern physics. Many significant discoveries have been made through collision experiments. Amongst diverse kinds of collision systems, this book sheds light on the collision of an electron with a molecule. The electron-molecule collision provides a basic scattering problem. It is scattering by a nonspherical, multicentered composite particle with its centers having degrees of freedom of motion. The molecule can even disintegrate, Le., dissociate or ionize into fragments, some or all of which may also be molecules. Although it is a difficult problem, the recent theoretical, experimental, and computational progress has been so significant as to warrant publication of a book that specializes in this field. The progress owes partly to technical develop­ ments in measurements and computations. No less important has been the great and continuing stimulus from such fields of application as astrophysics, the physics of the earth's upper atmosphere, laser physics, radiat...

  7. Cell surface engineering of renal cell carcinoma with glycosylphosphatidylinositol-anchored TIMP-1 blocks TGF- β 1 activation and reduces regulatory ID gene expression.

    Science.gov (United States)

    Notohamiprodjo, Susan; Djafarzadeh, Roghieh; Rieth, Nicole; Hofstetter, Monika; Jaeckel, Carsten; Nelson, Peter J

    2012-12-01

    Tissue inhibitor of metalloproteinase 1 (TIMP-1) controls matrix metalloproteinase activity through 1:1stoichiometric binding. Human TIMP-1 fused to a glycosylphosphatidylinositol(GPI) anchor (TIMP-1 - GPI) shifts the activity of TIMP-1 from the extracellular matrix to the cell surface. TIMP-1 - GPI treated renal cell carcinoma cells show increased apoptosis and reduced proliferation.Transcriptomic profiling and regulatory pathway mapping were used to identify the potential mechanisms driving these effects. Significant changes in the DNA binding inhibitors, TGF- β 1/SMAD and BMP pathways resulted from TIMP-1 - GPI treatment. These events were linked to reduced TGF- β 1 signaling mediated by inhibition of proteolytic processing of latent TGF- β 1 by TIMP-1 - GPI. PMID:23667903

  8. Optothermal Molecule Trap

    OpenAIRE

    Duhr, Stefan; Braun, Dieter

    2006-01-01

    Thermophoresis moves molecules along temperature gradients, typically from hot to cold. We superpose fluid flow with thermophoretic molecule flow under well defined microfluidic conditions, imaged by fluorescence microscopy. DNA is trapped and accumulated 16-fold in regions where both flows move in opposite directions. Strong 800-fold accumulation is expected, however with slow trapping kinetics. The experiment is equally described by a three-dimensional and one-dimensional analytical model. ...

  9. NMR detection and characterization of sialylated glycoproteins and cell surface polysaccharides

    Energy Technology Data Exchange (ETDEWEB)

    Barb, Adam W. [University of Georgia, Complex Carbohydrate Research Center (United States); Freedberg, Daron I.; Battistel, Marcos D. [Center for Biologics Evaluation and Research, Food and Drug Administration, Laboratory of Bacterial Polysaccharides (United States); Prestegard, James H., E-mail: jpresteg@ccrc.uga.edu [University of Georgia, Complex Carbohydrate Research Center (United States)

    2011-09-15

    Few solution NMR pulse sequences exist that are explicitly designed to characterize carbohydrates (glycans). This is despite the essential role carbohydrate motifs play in cell-cell communication, microbial pathogenesis, autoimmune disease progression and cancer metastasis, and despite that fact that glycans, often shed to extra-cellular fluids, can be diagnostic of disease. Here we present a suite of two dimensional coherence experiments to measure three different correlations (H3-C2, H3-C1, and C1-C2) on sialic acids, a group of nine-carbon carbohydrates found on eukaryotic cell surfaces that often play a key role in disease processes. The chemical shifts of the H3, C2, and C1 nuclei of sialic acids are sensitive to carbohydrate linkage, linkage conformation, and ionization state of the C1 carboxylate. The experiments reported include rigorous filter elements to enable detection and characterization of isotopically labeled sialic acids with high sensitivity in living cells and crude isolates with minimal interference from unwanted signals arising from the {approx}1% {sup 13}C-natural abundance of cellular metabolites. Application is illustrated with detection of sialic acids on living cells, in unpurified mixtures, and at the terminus of the N-glycan on the 55 kDa immunoglobulin G Fc.

  10. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    Science.gov (United States)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  11. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.

    Science.gov (United States)

    Katz, Adam J; Tholpady, Ashok; Tholpady, Sunil S; Shang, Hulan; Ogle, Roy C

    2005-03-01

    Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow-derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early-passage human adipose-derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of "stemness" in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.

  12. The cell surface receptor Slamf6 modulates innate immune responses during Citrobacter rodentium-induced colitis.

    Science.gov (United States)

    van Driel, Boaz; Wang, Guoxing; Liao, Gongxian; Halibozek, Peter J; Keszei, Marton; O'Keeffe, Michael S; Bhan, Atul K; Wang, Ninghai; Terhorst, Cox

    2015-09-01

    The homophilic cell surface receptors CD150 (Slamf1) and CD352 (Slamf6) are known to modulate adaptive immune responses. Although the Th17 response was enhanced in Slamf6(-/-) C57BL/6 mice upon oral infection with Citrobacter rodentium, the pathologic consequences are indistinguishable from an infection of wild-type C57BL/6 mice. Using a reporter-based binding assay, we show that Slamf6 can engage structures on the outer cell membrane of several Gram(-) bacteria. Therefore, we examined whether Slamf6, like Slamf1, is also involved in innate responses to bacteria and regulates peripheral inflammation by assessing the outcome of C. rodentium infections in Rag(-/-) mice. Surprisingly, the pathology and immune responses in the lamina propria of C. rodentium-infected Slamf6(-/-) Rag(-/-) mice were markedly reduced as compared with those of Rag(-/-) mice. Infiltration of inflammatory phagocytes into the lamina propria was consistently lower in Slamf6(-/-) Rag(-/-) mice than in Rag(-/-) animals. Concomitant with the reduced systemic translocation of the bacteria was an enhanced production of IL-22, suggesting that Slamf6 suppresses a mucosal protective program. Furthermore, administering a mAb (330) that inhibits bacterial interactions with Slamf6 to Rag(-/-) mice ameliorated the infection compared with a control antibody. We conclude that Slamf6-mediated interactions of colonic innate immune cells with specific Gram(-) bacteria reduce mucosal protection and enhance inflammation, contributing to lethal colitis that is caused by C. rodentium infections in Rag(-/-) mice.

  13. Bicistronic retroviral vectors for combining myeloprotection with cell-surface marking.

    Science.gov (United States)

    Hildinger, M; Schilz, A; Eckert, H G; Bohn, W; Fehse, B; Zander, A; Ostertag, W; Baum, C

    1999-07-01

    We have developed a retroviral vector coexpressing the multidrug-resistance 1 (MDR1) cDNA for inducing cancer drug resistance and the truncated version of the low-affinity nerve growth factor receptor (DeltaLNGFR) for cell-surface marking of transduced cells. The vector is based on the FMEV backbone which mediates high levels of gene expression in hematopoietic cells. To achieve optimal expression levels of both cDNAs, untranslated regions from MDR1 and DeltaLNGFR were removed and three different connections were tested: retroviral splice signals, an internal ribosomal entry site (IRES) from encephalomyocarditis virus, and an internal promoter from the chicken beta-actin gene. As determined by two-color flow cytometry, the best correlation of the expression of both cDNAs was obtained using the vector SF1mSdelta which utilized retroviral splice signals for co-expression. Simultaneous expression of both cDNAs at the single cell level was also shown by confocal laser microscopy. Lymphoid and hematopoietic progenitor cells, including primary human CD34+ cells, transduced with SF1mSdelta acquired dominant multidrug resistance. Transduced primary CD34+ cells could be enriched in vitro based on expression of DeltaLNGFR, avoiding exposure to cytostatic agents. Thus, monitoring the selection of chemotherapy-resistant cells and analyzing their biological properties may be alleviated, both in vitro and in vivo. PMID:10455430

  14. Membrane Vesicles as a Novel Strategy for Shedding Encrusted Cell Surfaces

    Directory of Open Access Journals (Sweden)

    Paul P. Shao

    2014-02-01

    Full Text Available Surface encrustation by minerals, which impedes cellular metabolism, is a potential hazard for microbes. The reduction of U(VI to U(IV by Shewanella oneidensis strain MR-1 leads to the precipitation of the mineral uraninite, as well as a non-crystalline U(IV product. The wild-type (WT strain can produce extracellular polymeric substances (EPS, prompting precipitation of U some distance from the cells and precluding encrustation. Using cryo-transmission electron microscopy and scanning transmission X-ray microscopy we show that, in the biofilm-deficient mutant ∆mxdA, as well as in the WT strain to a lesser extent, we observe the formation of membrane vesicles (MVs as an additional means to lessen encrustation. Additionally, under conditions in which the WT does not produce EPS, formation of MVs was the only observed mechanism to mitigate cell encrustation. Viability studies comparing U-free controls to cells exposed to U showed a decrease in the number of viable cells in conditions where MVs alone are detected, yet no loss of viability when cells produce both EPS and MVs. We conclude that MV formation is a microbial strategy to shed encrusted cell surfaces but is less effective at maintaining cell viability than the precipitation of U on EPS.

  15. Selective amine labeling of cell surface proteins guided by coiled-coil assembly.

    Science.gov (United States)

    Yano, Yoshiaki; Furukawa, Nami; Ono, Satoshi; Takeda, Yuki; Matsuzaki, Katsumi

    2016-11-01

    Covalent labeling of target proteins in living cells is useful for both fluorescence live-cell imaging and the subsequent biochemical analyses of the proteins. Here, we report an efficient method for the amine labeling of membrane proteins on the cell surface, guided by a noncovalent coiled-coil interaction. A carboxyl sulfosuccinimidyl ester introduced at the C-terminus of the coiled-coil probe reacted with target proteins under mild labeling conditions ([probe] = 150 nM, pH 7.4, 25°C) for 20 min. Various fluorescent moieties with different hydrophobicities are available for covalent labeling with high signal/background labeling ratios. Using this method, oligomeric states of glycophorin A (GpA) were compared in mammalian CHO-K1 cells and sodium dodecyl sulfate (SDS) micelles. In the cell membranes, no significant self-association of GpA was detected, whereas SDS-PAGE suggested partial dimerization of the proteins. Membrane cholesterol was found to be an important factor that suppressed the dimerization of GpA. Thus, the covalent functionality enables direct comparison of the oligomeric state of membrane proteins under various conditions. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 484-490, 2016. PMID:26285787

  16. Recruitment of Factor H to the Streptococcus suis Cell Surface is Multifactorial.

    Science.gov (United States)

    Roy, David; Grenier, Daniel; Segura, Mariela; Mathieu-Denoncourt, Annabelle; Gottschalk, Marcelo

    2016-07-07

    Streptococcus suis is an important bacterial swine pathogen and a zoonotic agent. Recently, two surface proteins of S. suis, Fhb and Fhbp, have been described for their capacity to bind factor H-a soluble complement regulatory protein that protects host cells from complement-mediated damages. Results obtained in this study showed an important role of host factor H in the adhesion of S. suis to epithelial and endothelial cells. Both Fhb and Fhbp play, to a certain extent, a role in such increased factor H-dependent adhesion. The capsular polysaccharide (CPS) of S. suis, independently of the presence of its sialic acid moiety, was also shown to be involved in the recruitment of factor H. However, a triple mutant lacking Fhb, Fhbp and CPS was still able to recruit factor H resulting in the degradation of C3b in the presence of factor I. In the presence of complement factors, the double mutant lacking Fhb and Fhbp was similarly phagocytosed by human macrophages and killed by pig blood when compared to the wild-type strain. In conclusion, this study suggests that recruitment of factor H to the S. suis cell surface is multifactorial and redundant.

  17. Modeling multivalent ligand-receptor interactions with steric constraints on configurations of cell surface receptor aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Monine, Michael [Los Alamos National Laboratory; Posner, Richard [TRANSLATION GENOMICS RESAEARCH INSTITUTE; Savage, Paul [BYU; Faeder, James [UNIV OF PITTSBURGH; Hlavacek, William S [UNM

    2008-01-01

    Signal transduction generally involves multivalent protein-protein interactions, which can produce various protein complexes and post-translational modifications. The reaction networks that characterize these interactions tend to be so large as to challenge conventional simulation procedures. To address this challenge, a kinetic Monte Carlo (KMC) method has been developed that can take advantage of a model specification in terms of reaction rules for molecular interactions. A set of rules implicitly defines the reactions that can occur as a result of the interactions represented by the rules. With the rule-based KMC method, explicit generation of the underlying chemical reaction network implied by rules is avoided. Here, we apply and extend this method to characterize the interactions of a trivalent ligand with a bivalent cell-surface receptor. This system is also studied experimentally. We consider the following kinetic models: an equivalent-site model, an extension of this model, which takes into account steric constraints on the configurations of receptor aggregates, and finally, a model that accounts for cyclic receptor aggregates. Simulation results for the equivalent-site model are consistent with an equilibrium continuum model. Using these models, we investigate the effects of steric constraints and the formation of cyclic aggregates on the kinetics and equilibria of small and large aggregate formation and the percolation phase transition that occurs in this system.

  18. Enhanced Growth Inhibition of Osteosarcoma by Cytotoxic Polymerized Liposomal Nanoparticles Targeting the Alcam Cell Surface Receptor

    Directory of Open Access Journals (Sweden)

    Noah Federman

    2012-01-01

    Full Text Available Osteosarcoma is the most common primary malignancy of bone in children, adolescents, and adults. Despite extensive surgery and adjuvant aggressive high-dose systemic chemotherapy with potentially severe bystander side effects, cure is attainable in about 70% of patients with localized disease and only 20%–30% of those patients with metastatic disease. Targeted therapies clearly are warranted in improving our treatment of this adolescent killer. However, a lack of osteosarcoma-associated/specific markers has hindered development of targeted therapeutics. We describe a novel osteosarcoma-associated cell surface antigen, ALCAM. We, then, create an engineered anti-ALCAM-hybrid polymerized liposomal nanoparticle immunoconjugate (α-AL-HPLN to specifically target osteosarcoma cells and deliver a cytotoxic chemotherapeutic agent, doxorubicin. We have demonstrated that α-AL-HPLNs have significantly enhanced cytotoxicity over untargeted HPLNs and over a conventional liposomal doxorubicin formulation. In this way, α-AL-HPLNs are a promising new strategy to specifically deliver cytotoxic agents in osteosarcoma.

  19. Study of concentric iridescent ring around the laser-induced pits on the solar cell surface

    International Nuclear Information System (INIS)

    Highlights: • We studied the laser-induced damage on solar cell surface. • Concentric iridescent ring was observed originated from the gradient distribution pattern of the thickness of the oxidized surface film. • The damaged surface film of the m-Si solar cell is SiO2, while that of the GaAs/Ge solar cell is GeO2. - Abstract: The laser-induced damage on the surface of monocrystalline silicon (m-Si) solar cells and GaAs/Gesingle heterojunction solar cells are investigated. The solar cells were irradiated by a continuous wave laser at the wavelength of 532 nm. Concentric iridescent ring appeared on the damaged surfaces when observed with an optical microscope (OM) of broad spectrum. The damaged surface film was characterized by X-ray photoelectron spectroscopy (XPS) and the Contour Meter. The laser-induced temperature in silicon was calculated. The formation mechanism of the film and the cause of the concentric iridescent ring were analyzed

  20. Cell surface thiol isomerases may explain the platelet-selective action of S-nitrosoglutathione.

    Science.gov (United States)

    Xiao, Fang; Gordge, Michael P

    2011-10-30

    S-nitrosoglutathione (GSNO) at low concentration inhibits platelet aggregation without causing vasodilation, suggesting platelet-selective nitric oxide delivery. The mechanism of this selectivity is unknown, but may involve cell surface thiol isomerases, in particular protein disulphide isomerase (csPDI) (EC 5.3.4.1). We have now compared csPDI expression and activity on platelets, endothelial cells and vascular smooth muscle cells, and the dependence on thiol reductase activity of these cell types for NO uptake from GSNO. csPDI expression was measured by flow cytometry and its reductase activity using the pseudosubstrate dieosin glutathione disulphide. This activity assay was adapted and validated for 96-well plate format. Flow cytometry revealed csPDI on all three cell types, but percentage positivity of expression was higher on platelets than on vascular cells. Consistent with this, thiol isomerase-related reductase activity was higher on platelets (Pionomycin) increased csPDI activity on both platelets and smooth muscle cells, but not on endothelium. Intracellular NO delivery from GSNO was greater in platelets than in vascular cells (Pselective actions of GSNO and help define its antithrombotic potential. PMID:21642008

  1. Regulation of NKG2D-ligand cell surface expression by intracellular calcium after HDAC-inhibitor treatment

    DEFF Research Database (Denmark)

    Jensen, Helle; Hagemann-Jensen, Michael Henrik; Lauridsen, Felicia Kathrine Bratt;

    2013-01-01

    surface expression of ULBP2, but not MICA/B, was sensitive to treatment calmidazolium and trifluoperazine, two agents known to block calcium signaling. siRNA-mediated knock-down of the calcium-regulated proteins calmodulin or calpain did however not affect NKG2D-ligand cell surface expression on Jurkat T...

  2. Effects of elaidic acid, a predominant industrial trans fatty acid, on bacterial growth and cell surface hydrophobicity of lactobacilli.

    Science.gov (United States)

    Wu, Qinglong; Shah, Nagendra P

    2014-12-01

    The consumption of trans fatty acids (TFAs) increases the risk of cardiovascular diseases and coronary heart disease in human, and there are no effective ways to remove TFAs after consumption. The aim of this study was to investigate the effects of elaidic acid on bacterial growth, cell surface hydrophobicity of lactobacilli, and metabolism of elaidic acid by lactobacilli. Lactobacilli were inoculated in MRS broth containing 0, 100, 200, and 500 mg/L of elaidic acid. Viable cell counts of lactobacilli were enumerated, concentrations of elaidic acid were determined, and cell surface hydrophobicity of lactobacilli was measured. The results showed that the growth of lactobacilli was significantly inhibited by 500 mg/L of elaidic acid, however, a cell count of 8.50 log10 CFU/mL was still reached for tested lactobacilli after 24-h incubation. In particular, a reduction of elaidic acid was found for tested lactobacilli after 24-h incubation as compared to its initial concentration of 200 mg/L. However, cell surface hydrophobicity showed no correlations with the metabolism of elaidic acid by lactobacilli. Moreover, elaidic acid was able to influence cell surface hydrophobicity, and the decrease in hydrophobicity was more obvious in Lactobacillus paracasei and Lactobacillus casei compared with that in other tested lactobacilli. This study suggests that elaidic acid could change physiochemical surface properties of lactobacilli and the lactobacilli have the potential to reduce TFAs.

  3. Proteasomal targeting and minigene repetition improve cell-surface presentation of a transfected, modified melanoma tumour antigen

    DEFF Research Database (Denmark)

    Rasmussen, A B; Zocca, M-B; Bonefeld, C M;

    2004-01-01

    on the density of specific major histocompatibility complex-peptide complexes on the surface of the antigen-presenting cell. In this study, we explored the cell-surface presentation of a substituted MART-1 peptide encoded by transfected minigenes. We investigated the potential of proteasomal targeting compared...

  4. Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy

    OpenAIRE

    1983-01-01

    Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

  5. The adhesion molecule PECAM-1 enhances the TGFβ-mediated inhibition of T cell function

    Science.gov (United States)

    Newman, Debra K.; Fu, Guoping; Adams, Tamara; Cui, Weiguo; Arumugam, Vidhyalakshmi; Bluemn, Theresa; Riese, Matthew J.

    2016-01-01

    Transforming growth factor-β (TGF-β) is an immunosuppressive cytokine that inhibits the pro-inflammatory functions of T cells, and it is a major factor in abrogating T cell activity against tumors. Canonical signaling results in the activation of Smad proteins, transcription factors that regulate target gene expression. Here, we found that the cell surface molecule platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitates non-canonical (Smad-independent) TGF-β signaling in T cells. Subcutaneously injected tumor cells dependent on TGF-β-mediated suppression of immunity grew more slowly in PECAM-1−/− mice than in their wild type counterparts. T cells isolated from PECAM-1−/− mice demonstrated relative insensitivity to the TGF-β-dependent inhibition of interferon- γ (IFN-γ) production, granzyme B synthesis and cellular proliferation. Similarly, human T cells lacking PECAM-1 demonstrated decreased sensitivity to TGF-β in a manner that was partially restored by re-expression of PECAM-1. Co-incubation of T cells with TGF-β and a T cell-activating antibody resulted in PECAM-1 phosphorylation on an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the recruitment of the inhibitory Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2). Such stimulatory conditions also induced the co-localization of PECAM-1 with the TGF-β receptor complex as identified by co-immunoprecipitation, confocal microscopy, and proximity ligation assays. These studies indicate a role for PECAM-1 in enhancing the inhibitory functions of TGF-β in T cells and suggest that therapeutic targeting of the PECAM-1-TGF-β inhibitory axis represents a means to overcome TGF-β-dependent immunosuppression within the tumor microenvironment. PMID:26956486

  6. Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans

    KAUST Repository

    Åmand, Helene L.

    2012-11-01

    Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide\\'s ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell. © 2012 Elsevier B.V.

  7. Characterization of cell surface adenosine 3',5'-monophosphate-binding proteins in Y-1 mouse adrenal tumor cells

    International Nuclear Information System (INIS)

    Adrenal cortical cells are known to export cAMP and have binding proteins and cAMP-dependent protein kinase activity associated with their plasma membranes. Because these properties suggest a function for extracellular cAMP, we have undertaken a search for specific cell surface receptors for this cyclic nucleotide. Y-1 mouse adrenal tumor cells actively export cAMP by an energy-dependent process. Analysis of Scatchard plots of the equilibrium binding of [3H]cAMP to these cells indicate the existence of two classes of cAMP binders: one with high affinity (K/sub a/ . 2.9 X 10(9) M-1) and another with low affinity (K/sub a/ . 7.0 X 10(7) M-1). The cell surface localization of these binders was established by the sensitivity of both the [3H]cAMP-binding proteins and the [32P]8-N3-cAMP photoaffinity labeled proteins of intact cells to mild trypsin digestion and by the surface distribution of a BSA-O2-monosuccinyl cAMP-gold complex revealed by electron microscopy. Analysis of radioautograms of cell surface cAMP-binding proteins from confluent monolayer tumor cells, photoaffinity labeled with [32P]8-N3-cAMP and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major 32P-labeled protein bands which were indistinguishable from the 49,000 and 55,000 mol wt regulatory subunits of the cytosolic protein kinase isoenzymes of this cell. These observations along with the demonstration of cell surface, cAMP-dependent protein kinase activity in the mouse adrenal tumor cell strongly suggest that these cAMP-binding proteins function as regulatory proteins for cell surface protein kinases

  8. Towards single molecule switches.

    Science.gov (United States)

    Zhang, Jia Lin; Zhong, Jian Qiang; Lin, Jia Dan; Hu, Wen Ping; Wu, Kai; Xu, Guo Qin; Wee, Andrew T S; Chen, Wei

    2015-05-21

    The concept of using single molecules as key building blocks for logic gates, diodes and transistors to perform basic functions of digital electronic devices at the molecular scale has been explored over the past decades. However, in addition to mimicking the basic functions of current silicon devices, molecules often possess unique properties that have no parallel in conventional materials and promise new hybrid devices with novel functions that cannot be achieved with equivalent solid-state devices. The most appealing example is the molecular switch. Over the past decade, molecular switches on surfaces have been intensely investigated. A variety of external stimuli such as light, electric field, temperature, tunneling electrons and even chemical stimulus have been used to activate these molecular switches between bistable or even multiple states by manipulating molecular conformations, dipole orientations, spin states, charge states and even chemical bond formation. The switching event can occur either on surfaces or in break junctions. The aim of this review is to highlight recent advances in molecular switches triggered by various external stimuli, as investigated by low-temperature scanning tunneling microscopy (LT-STM) and the break junction technique. We begin by presenting the molecular switches triggered by various external stimuli that do not provide single molecule selectivity, referred to as non-selective switching. Special focus is then given to selective single molecule switching realized using the LT-STM tip on surfaces. Single molecule switches operated by different mechanisms are reviewed and discussed. Finally, molecular switches embedded in self-assembled monolayers (SAMs) and single molecule junctions are addressed. PMID:25757483

  9. Multicolor Bound Soliton Molecule

    CERN Document Server

    Luo, Rui; Lin, Qiang

    2015-01-01

    We show a new class of bound soliton molecule that exists in a parametrically driven nonlinear optical cavity with appropriate dispersion characteristics. The composed solitons exhibit distinctive colors but coincide in time and share a common phase, bound together via strong inter-soliton four-wave mixing and Cherenkov radiation. The multicolor bound soliton molecule shows intriguing spectral locking characteristics and remarkable capability of spectrum management to tailor soliton frequencies, which may open up a great avenue towards versatile generation and manipulation of multi-octave spanning phase-locked Kerr frequency combs, with great potential for applications in frequency metrology, optical frequency synthesis, and spectroscopy.

  10. Topogenesis and cell surface trafficking of GPR34 are facilitated by positive-inside rule that effects through a tri-basic motif in the first intracellular loop.

    Science.gov (United States)

    Hasegawa, Haruki; Patel, Neha; Ettehadieh, Elham; Li, Peng; Lim, Ai Ching

    2016-07-01

    Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34. PMID:27086875

  11. Relationship of cell surface morphology and composition of Streptococcus salivarius K+ to adherence and hydrophobicity.

    Science.gov (United States)

    Weerkamp, A H; van der Mei, H C; Slot, J W

    1987-01-01

    The cell surfaces of a range of variants of Streptococcus salivarius HB, altered in cell wall antigen composition, were compared with those of the parent with respect to adherence, ability to adsorb to hexadecane, morphology, and exposure of lipoteichoic acid (LTA). Adherence to host surfaces was measured by using both saliva-coated hydroxyapatite beads and tissue-cultured HeLa cells, and interbacterial adherence was measured by using Veillonella alcalescens V1 cells. Progressive loss of the protease-sensitive fibril classes was generally associated with decreasing ability to adsorb to hexadecane. However, increased exposure of protein antigen C (AgC) increased the apparent hydrophobicity of the cell. This correlated with the finding that AgC was the most hydrophobic of the solubilized fibrillar cell wall antigens. Collectively, this demonstrates that adsorption to hydrophobic ligands is directly related to the density of the fibrillar layer on the cells and the properties and surface exposure of specific fibril classes. The involvement of hydrophobic interactions in AgC-associated attachment was suggested by its sensitivity to low levels of the hydrophobic bond-breaking agent tetramethyl urea, although the reduction was not to the level of adherence observed with strains lacking AgC. However, hydrophobicity was less essential to other adherence reactions. Circumstantial evidence, including immunoelectron microscopy, showing that LTA was virtually absent from the fibrillar layer, whole-cell enzyme-linked immunosorbent assay, suggesting that surface exposure of LTA related inversely to the density of the fibrillar layer, and agarose gel electrophoresis, showing that LTA was not specifically associated with protein fibrillar antigens, strongly suggested that LTA does not confer hydrophobic properties to these cells and is not involved in adherence reactions associated with the cell wall protein antigens. Images PMID:3804445

  12. Endothelial cell surface heparan sulfate (ESHS) and synthetic heparin derivatives as hemocompatible coating for biomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, M.; Huppertz, B.; Horres, R.; Baumann, H. [RWTH, Aachen (Germany). Makromolekulare Chemie und Textilchemie, Haemokompatible und Biokompatible Biomaterialien; Keller, R. [Klinische Anstalten der Stadt Koeln (Germany)

    2001-02-01

    In the present overview a coating procedure, that has been developed in our working group for medical devices e.g. implants, which are exposed to permanent blood contact and therefore have to fulfill the highest standard of hemocompatibility is described. For this purpose an endothelial cell surface heparansulfate, which belongs to the class of glycosaminoglycans is used as coating substance. This substance can be isolated from endothelial cell culture, tissue extracts or organ perfusates. Alternatively chemical regio- and stereoselective modified derivatives of the structurally related anticoagulant heparin were brought to action. These substances are anchored covalently or ionically by application of a wide spectrum of immobilization techniques on many different material surfaces. Polymer materials modified as described here have been tested for hemocompatibility in invitro and in invivo experiments with Austrian sheep. The results show, that the described method is an advanced solution for the creation of long term hemocompatible artificial material surfaces. (orig.) [German] In dem vorliegenden Uebersichtsartikel wird ein in unserer Arbeitsgruppe entwickeltes athrombogenes und plaettcheninertes Beschichtungsverfahren fuer medizinische Werkstoffoberflaechen wie z.B. Implantate, die staendigem direktem Blutkontakt ausgesetzt sind und infolge dessen ein Hoechstmass an Haemokompatibilitaet aufweisen muessen, zusammenfassend beschrieben. Hierzu wird als Beschichtungssubstanz ein aus Zellkultur, Gewebeextrakten oder Organperfusaten isolierbares zur Klasse der Glycosaminoglycane zaehlendes Endothelzelloberflaechenparansulfat (ESHS) verwendet. Alternativ werden chemisch regio- und stereoselektiv modifizierte Derivate des strukturverwandten klassischen Antikoagulanzes Heparin als Beschichtungssubstanz eingesetzt. Diese Substanzen werden unter Anwendung eines breiten Spektrums von Immobilisierungstechniken auf verschiedensten Werkstoffoberflaechen kovalent oder ionisch

  13. Proximity effect among cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface.

    Science.gov (United States)

    Bae, Jungu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.

  14. Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

    Energy Technology Data Exchange (ETDEWEB)

    Tabatabai, L.B.; Deyoe, B.L.

    1983-01-01

    Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized (/sup 125/I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis.

  15. Display of wasp venom allergens on the cell surface of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Poulsen Lars K

    2010-09-01

    Full Text Available Abstract Background Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast Saccharomyces cerevisiae preserve their native allergenic properties and whether the yeast native surface glycoproteins interfere with IgE binding. We chose to use the major allergens from the common wasp Vespula vulgaris venom: phospholipase A1, hyaluronidase and antigen 5 as the model. Results The proteins were expressed on the surface as fusions with a-agglutinin complex protein AGA2. The expression was confirmed by fluorescent cytometry (FACS after staining the cells with antibody against a C-tag attached to the C-terminal end of the allergens. Phospholipase A1 and hyaluronidase retained their enzymatic activities. Phospholipase A1 severely inhibited the growth of the yeast cells. Antigen 5 - expressing yeast cells bound IgE antibodies from wasp venom allergic patient sera but not from control sera as demonstrated by FACS. Moreover, antigen 5 - expressing yeast cells were capable of mediating allergen-specific histamine release from human basophils. Conclusions All the three major wasp venom allergens were expressed on the yeast surface. A high-level expression, which was observed only for antigen 5, was needed for detection of IgE binding by FACS and for induction of histamine release. The non-modified S. cerevisiae cells did not cause any unspecific reaction in FACS or histamine release assay despite the expression of high-mannose oligosaccharides. In perspective the yeast surface display may be used for allergen discovery from cDNA libraries and possibly for sublingual immunotherapy as the cells can serve as good adjuvant and can be produced in large amounts at a low price.

  16. Human epithelial cells exposed to functionalized multiwalled carbon nanotubes: interactions and cell surface modifications.

    Science.gov (United States)

    Fanizza, C; Casciardi, S; Incoronato, F; Cavallo, D; Ursini, C L; Ciervo, A; Maiello, R; Fresegna, A M; Marcelloni, A M; Lega, D; Alvino, A; Baiguera, S

    2015-09-01

    With the expansion of the production and applications of multiwalled carbon nanotubes (MWCNTs) in several industrial and science branches, the potential adverse effects on human health have attracted attention. Numerous studies have been conducted to evaluate how chemical functionalization may affect MWCNT effects; however, controversial data have been reported, showing either increased or reduced toxicity. In particular, the impact of carboxylation on MWCNT cytotoxicity is far from being completely understood. The aim of this work was the evaluation of the modifications induced by carboxylated-MWCNTs (MWCNTs-COOH) on cell surface and the study of cell-MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells (A549) were incubated with MWCNTs-COOH for different exposure times and concentrations (10 μg/mL for 1, 2, 4 h; 5, 10, 20 μg/mL for 24 h). At short incubation time, MWCNTs-COOH were easily observed associated with plasma membrane and in contact with microvilli. After 24 h exposure, FESEM analysis revealed that MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells: treated cells showed blebs, holes and a depletion of the microvilli density in association with structure modifications, such as widening and/or lengthening. In particular, an increase of cells showing holes and microvilli structure alterations was observed at 20 μg/mL concentration. FESEM analysis showed nanotube agglomerates, of different sizes, entering into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions, such as alveolar proteinosis. More detailed studies will be, however, necessary to

  17. Human MHC class I antigens are associated with a 90-kDa cell surface protein.

    Science.gov (United States)

    Ferm, M T; Grönberg, A

    1991-08-01

    Human MHC class I proteins are expressed on almost all nucleated cells as a heavy chain (about 45 kDa) non-covalently associated with beta 2-microglobulin (12 kDa). In this report we show that MHC class I (MHC-I) proteins can also be associated with a 90-kDa protein in the cell membrane. Surface-radiolabelled cells were treated with dithiobis succinimidyl propionate (DSP) in order to preserve multimer protein complexes during cell lysis. The lysates were immunoprecipitated and analysed by SDS-PAGE and autoradiography. Immunoprecipitation of human MHC-I proteins co-precipitated another protein of about 90 kDa in molecular weight-p90. p90 was coprecipitated from all the MHC-I expressing cells tested: U937, Raji, Molt-4 and IFN-gamma treated K562, but not from untreated, MHC-I negative K562. A 90-kDa protein was also co-precipitated with MHC-I from fresh peripheral blood mononuclear cells (PBMC). Furthermore, p90 was coprecipitated by different MoAbs to the MHC-I heavy chain or beta 2-microglobulin, but not by control antibodies. Two additional co-precipitating proteins at 34 kDa and 28 kDa were seen in MHC-I precipitates from Raji cells. Our results suggest that MHC-I proteins and the 90-kDa protein are associated in the cell membrane, probably by a close but weak, non-covalent interaction. Two additional cell surface proteins at 34 kDa and 28 kDa seem to be MHC-I associated on Raji Burkitt's lymphoma cells.

  18. Membrane secretory component is cleaved on the cell surface of rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Musil, L.S.; Baenziger, J.U.

    1986-03-05

    Transcellular transport of polymeric IgA from serum to bile by rat hepatocytes is mediated by a 105Kd membranous form of secretory component (mSC). In the presence or absence of IgA, mSC is cleaved and released into bile as a soluble 80Kd protein (fSC). They used monolayer cultures of rat hepatocytes, which synthesize mSC and efficiently cleave it to fSC, to determine the site of this conversion. (/sup 35/S)Cys-mSC accumulated in hepatocytes in the presence of leupeptin and was released as fSC when hepatocytes were placed in leupeptin-free media at 37/sup 0/. Small amounts of fSC were also produced when leupeptin was removed at 4/sup 0/, suggesting cleavage might occur on the cell membrane. Lactoperoxidase-catalyzed iodination of hepatocytes at 4/sup 0/ selectively labeled surface mSC which remained trypsin sensitive at 4/sup 0/. Hepatocytes maintained at 4/sup 0/ released significant amounts of /sup 125/I-mSC as fSC. Anti-SC antiserum reduced fSC generation at 4/sup 0/ by 70%. Following incubation at 37/sup 0/ for 10 min, /sup 125/I-mSC became resistant to degradation by trypsin and no production of fSC was seen if the cells were returned to 4/sup 0/. /sup 125/I-mSC was also cleaved to fSC following disruption by Dounce homogenization if cells were maintained at 4/sup 0/ following iodination but not if they were incubated at 37/sup 0/ for 10 min. They propose that mSC is cleaved to fSC at the plasma membrane but not intracellularly. This may reflect localization of the protease at the cell surface in a bile canalicular-like domain.

  19. Mighty Molecule Models

    Science.gov (United States)

    Brown, Tom; Rushton, Greg; Bencomo, Marie

    2008-01-01

    As part of the SMATHematics Project: The Wonder of Science, The Power of Mathematics--a collaborative partnership between Kennesaw State University and two local school districts, fifth graders had the opportunity to puzzle out chemical formulas of propane, methanol, and other important molecules. In addition, they explored properties that…

  20. Synthesis beyond the molecule

    NARCIS (Netherlands)

    Reinhoudt, D.N.; Crego-Calama, M.

    2002-01-01

    Weak, noncovalent interactions between molecules control many biological functions. In chemistry, noncovalent interactions are now exploited for the synthesis in solution of large supramolecular aggregates. The aim of these syntheses is not only the creation of a particular structure, but also the i

  1. Atoms, Molecules, and Compounds

    CERN Document Server

    Manning, Phillip

    2007-01-01

    Explores the atoms that govern chemical processes. This book shows how the interactions between simple substances such as salt and water are crucial to life on Earth and how those interactions are predestined by the atoms that make up the molecules.

  2. The extracellular matrix, p53 and estrogen compete to regulate cell-surface Fas/Apo-1 suicide receptor expression in proliferating embryonic cerebral cortical precursors, and reciprocally, Fas-ligand modifies estrogen control of cell-cycle proteins

    Directory of Open Access Journals (Sweden)

    Newman Joseph M

    2004-03-01

    Full Text Available Abstract Background Apoptosis is important for normal cerebral cortical development. We previously showed that the Fas suicide receptor was expressed within the developing cerebral cortex, and that in vitro Fas activation resulted in caspase-dependent death. Alterations in cell-surface Fas expression may significantly influence cortical development. Therefore, in the following studies, we sought to identify developmentally relevant cell biological processes that regulate cell-surface Fas expression and reciprocal consequences of Fas receptor activation. Results Flow-cytometric analyses identified two distinct neural sub-populations that expressed Fas on their cell surface at high (FasHi or moderate (FasMod levels. The anti-apoptotic protein FLIP further delineated a subset of Fas-expressing cells with potential apoptosis-resistance. FasMod precursors were mainly in G0, while FasHi precursors were largely apoptotic. However, birth-date analysis indicated that neuroblasts express the highest levels of cell-surface Fas at the end of S-phase, or after their final round of mitosis, suggesting that Fas expression is induced at cell cycle checkpoints or during interkinetic nuclear movements. FasHi expression was associated with loss of cell-matrix adhesion and anoikis. Activation of the transcription factor p53 was associated with induction of Fas expression, while the gonadal hormone estrogen antagonistically suppressed cell-surface Fas expression. Estrogen also induced entry into S-phase and decreased the number of Fas-expressing neuroblasts that were apoptotic. Concurrent exposure to estrogen and to soluble Fas-ligand (sFasL suppressed p21/waf-1 and PCNA. In contrast, estrogen and sFasL, individually and together, induced cyclin-A expression, suggesting activation of compensatory survival mechanisms. Conclusions Embryonic cortical neuronal precursors are intrinsically heterogeneous with respect to Fas suicide-sensitivity. Competing intrinsic (p53

  3. Exotic helium molecules

    International Nuclear Information System (INIS)

    We study the photo-association of an ultracold cloud of magnetically trapped helium atoms: pairs of colliding atoms interact with one or two laser fields to produce a purely long range 4He2(23S1-23P0) molecule, or a 4He2(23S1-23S1) long range molecule. Light shifts in one photon photo-association spectra are measured and studied as a function of the laser polarization and intensity, and the vibrational state of the excited molecule. They result from the light-induced coupling between the excited molecule, and bound and scattering states of the interaction between two metastable atoms. Their analysis leads to the determination of the scattering length a = (7.2 ± 0.6) ruling collisions between spin polarized atoms. The two photon photo-association spectra show evidence of the production of polarized, long-range 4He2(23S1-23S1) molecules. They are said to be exotic as they are made of two metastable atoms, each one carrying a enough energy to ionize the other. The corresponding lineshapes are calculated and decomposed in sums and products of Breit-Wigner and Fano profiles associated to one and two photon processes. The experimental spectra are fit, and an intrinsic lifetime τ = (1.4 ± 0.3) μs is deduced. It is checked whether this lifetime could be limited by spin-dipole induced Penning autoionization. This interpretation requires that there is a quasi-bound state close to the dissociation threshold in the singlet interaction potential between metastable helium atoms for the theory to match the experiment. (author)

  4. A conserved WW domain-like motif regulates invariant chain-dependent cell-surface transport of the NKG2D ligand ULBP2

    DEFF Research Database (Denmark)

    Uhlenbrock, Franziska Katharina; van Andel, Esther; Andresen, Lars;

    2015-01-01

    that the NKG2D ligand ULBP2 traffics over an invariant chain (Ii)-dependent pathway to the cell surface. This study set out to elucidate how Ii regulates ULBP2 cell-surface transport: We discovered conserved tryptophan (Trp) residues in the primary protein sequence of ULBP1-6 but not in the related MICA...

  5. Artemether Combined with shRNA Interference of Vascular Cell Adhesion Molecule-1 Significantly Inhibited the Malignant Biological Behavior of Human Glioma Cells

    OpenAIRE

    Ying-Bin Wang; Yi Hu; Zhen Li; Ping Wang; Yi-Xue Xue; Yi-Long Yao; Bo Yu; Yun-Hui Liu

    2013-01-01

    Artemether is the derivative extracted from Chinese traditional herb and originally used for malaria. Artemether also has potential therapeutic effects against tumors. Vascular cell adhesion molecule-1 (VCAM-1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we investigated the role and mechanism of artemether combined with shRNA interference of VCAM-1 (shRNA-VCAM-1) on the migration, invasion and apoptosis of glioma cells. U87 human glioma ...

  6. Cell Surface Display and Characterization of Rhizopus oryzae Lipase in Pichia pastoris Using Sed1p as an Anchor Protein.

    Science.gov (United States)

    Li, Wenqian; Shi, Hao; Ding, Huaihai; Wang, Liangliang; Zhang, Yu; Li, Xun; Wang, Fei

    2015-07-01

    It has been investigated to conduct the surface displaying of lipase from Rhizopus oryzae onto the cells of Pichia pastoris yeast using Sed1p as an anchor protein. A yeast cell surface display plasmid pPICZαA-rol-histag-sed1p was constructed by fusing rol and sed1p gene fragments into the plasmid pPICZαA, followed by introducing recombinant plasmid into P. pastoris cells. Surface display levels were monitored by Western Blot and immunofluorescence microscopy. The activity of displaying lipase obtained from recombinant mutS reached at 60 U/g-dry cell. In addition, the displaying lipase was stable in broad ranges of temperatures and pH, with the optimum temperature at 40 °C and pH 7.5. These results indicate that the P. pastoris displaying lipase may have potential in whole-cell biocatalyst. PMID:26013444

  7. A model for the biosynthesis and transport of plasma membrane-associated signaling receptors to the cell surface

    Directory of Open Access Journals (Sweden)

    Sorina Claudia Popescu

    2012-04-01

    Full Text Available Intracellular protein transport is emerging as critical in determining the outcome of receptor-activated signal transduction pathways. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating receptor synthesis and transport to the cell surface. Recent advances in this field indicate that signaling pathways and intracellular transport machinery converge and coordinate to render receptors competent for signaling at their plasma membrane activity sites. The biogenesis and transport to the cell surface of signaling receptors appears to require both general trafficking and receptor-specific factors. Several molecular determinants, residing or associated with compartments of the secretory pathway and known to influence aspects in receptor biogenesis, are discussed and integrated into a predictive cooperative model for the functional expression of signaling receptors at the plasma membrane.

  8. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C. (Scripps); (UW)

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  9. Multi-scale cell/surface interaction on modified titanium aluminum vanadium surfaces

    Science.gov (United States)

    Chen, Jianbo

    This dissertation presents a series of experimental studies of the effects of multi-scale cell/surface interactions on modified Ti-6Al-4V surfaces. These include laser-grooved surfaces; porous structures and RGD-coated laser-grooved surfaces. A nano-second DPSS UV lasers with a Gaussian pulse energy profile was used to introduce the desired micro-groove geometries onto Ti-6Al-4V surfaces. This was done without inducing micro-cracks or significant changes in surface chemistry within the heat affected zones. The desired 8-12 mum groove depths and widths were achieved by the control of pulse frequency, scan speed, and the lens focal length that controls spot size. The interactions between human osteosarcoma (HOS) cells and laser-grooved Ti-6Al-4V surfaces were investigated after 48 hours of cell culture. The cell behavior, including cell spreading, alignment and adhesion, was elucidated using scanning electronic microscopy (SEM), immuno-fluorescence staining and enzymatic detachment. Contact guidance was shown to increase as grooved spacing decreased. For the range of micro-groove geometries studied, micro-grooves with groove spacings of 20 mum provided the best combination of cell orientation and adhesion. Short-term adhesion experiments (15 mins to 1 day) also revealed that there is a positive correlation between cell orientation and cell adhesion. Contact guidance on the micro-grooved surfaces is shown to be enhanced by nano- and micro-scale asperities that provide sites for the attachment of lamellopodia during cell locomotion and spreading. Contact guidance is also promoted by the geometrical confinement provided by laser grooves. An experimental study of initial cell spreading and ingrowth into Ti-6Al-4V porous structures was also carried out on porous structures with different pore sizes and geometries. A combination of SEM, the tetrazolium salt (MTT) colorimetric assay and enzymatic detachment were used to study cell spreading and adhesion. The extent of cell

  10. Cell surface estrogen receptor alpha is upregulated during subchronic metabolic stress and inhibits neuronal cell degeneration.

    Directory of Open Access Journals (Sweden)

    Cristiana Barbati

    Full Text Available In addition to the classical nuclear estrogen receptor, the expression of non-nuclear estrogen receptors localized to the cell surface membrane (mER has recently been demonstrated. Estrogen and its receptors have been implicated in the development or progression of numerous neurodegenerative disorders. Furthermore, the pathogenesis of these diseases has been associated with disturbances of two key cellular programs: apoptosis and autophagy. An excess of apoptosis or a defect in autophagy has been implicated in neurodegeneration. The aim of this study was to clarify the role of ER in determining neuronal cell fate and the possible implication of these receptors in regulating either apoptosis or autophagy. The human neuronal cell line SH-SY5Y and mouse neuronal cells in primary culture were thus exposed to chronic minimal peroxide treatment (CMP, a form of subcytotoxic minimal chronic stress previously that mimics multiple aspects of long-term cell stress and represents a limited molecular proxy for neurodegenerative processes. We actually found that either E2 or E2-bovine serum albumin construct (E2BSA, i.e. a non-permeant form of E2 was capable of modulating intracellular cell signals and regulating cell survival and death. In particular, under CMP, the up-regulation of mERα, but not mERβ, was associated with functional signals (ERK phosphorylation and p38 dephosphorylation compatible with autophagic cytoprotection triggering and leading to cell survival. The mERα trafficking appeared to be independent of the microfilament system cytoskeletal network but was seemingly associated with microtubular apparatus network, i.e., to MAP2 molecular chaperone. Importantly, antioxidant treatments, administration of siRNA to ERα, or the presence of antagonist of ERα hindered these events. These results support that the surface expression of mERα plays a pivotal role in determining cell fate, and that ligand-induced activation of mER signalling exerts a

  11. Polymorphisms of the cell surface receptor control mouse susceptibilities to xenotropic and polytropic leukemia viruses.

    Science.gov (United States)

    Marin, M; Tailor, C S; Nouri, A; Kozak, S L; Kabat, D

    1999-11-01

    The differential susceptibilities of mouse strains to xenotropic and polytropic murine leukemia viruses (X-MLVs and P-MLVs, respectively) are poorly understood but may involve multiple mechanisms. Recent evidence has demonstrated that these viruses use a common cell surface receptor (the X-receptor) for infection of human cells. We describe the properties of X-receptor cDNAs with distinct sequences cloned from five laboratory and wild strains of mice and from hamsters and minks. Expression of these cDNAs in resistant cells conferred susceptibilities to the same viruses that naturally infect the animals from which the cDNAs were derived. Thus, a laboratory mouse (NIH Swiss) X-receptor conferred susceptibility to P-MLVs but not to X-MLVs, whereas those from humans, minks, and several wild mice (Mus dunni, SC-1 cells, and Mus spretus) mediated infections by both X-MLVs and P-MLVs. In contrast, X-receptors from the resistant mouse strain Mus castaneus and from hamsters were inactive as viral receptors. These results suggest that X-receptor polymorphisms are a primary cause of resistances of mice to members of the X-MLV/P-MLV family of retroviruses and are responsible for the xenotropism of X-MLVs in laboratory mice. By site-directed mutagenesis, we substituted sequences between the X-receptors of M. dunni and NIH Swiss mice. The NIH Swiss protein contains two key differences (K500E in presumptive extracellular loop 3 [ECL 3] and a T582 deletion in ECL 4) that are both required to block X-MLV infections. Accordingly, a single inverse mutation in the NIH Swiss protein conferred X-MLV susceptibility. Furthermore, expression of an X-MLV envelope glycoprotein in Chinese hamster ovary cells interfered efficiently with X-MLV and P-MLV infections mediated by X-receptors that contained K500 and/or T582 but had no effect on P-MLV infections mediated by X-receptors that lacked these amino acids. In contrast, moderate expression of a P-MLV (MCF247) envelope glycoprotein did not

  12. Involvement of cell surface phosphatidylinositol-anchored glycoproteins in cell-cell adhesion of chick embryo myoblasts

    OpenAIRE

    1989-01-01

    During myogenesis myoblasts fuse to form multinucleate cells that express muscle-specific proteins. A specific cell-cell adhesion process precedes lipid bilayer union during myoblast fusion (Knudsen, K. A., and A. F. Horwitz. 1977. Dev. Biol. 58:328-338) and is mediated by cell surface glycoproteins (Knudsen, K. A., 1985. J. Cell Biol. 101:891- 897). In this paper we show that myoblast adhesion and myotube formation are inhibited by treating fusion-competent myoblasts with phosphatidylinosito...

  13. Location of tumour cells in colon tissue by Texas red labelled pentosan polysulphate, an inhibitor of a cell surface protease.

    Science.gov (United States)

    Anees, M

    1996-01-01

    Pentosan polysulphate (PPS), a highly negatively charged polysaccharide, is a significant inhibitor of an isoenzymic form of a cell surface protease referred to as guanidinobenzoatase GB, associated with colonic carcinoma tissues in frozen sections and free GB in solution, in a concentration-dependent manner. However PPS failed to recognise and bind to the isoenzymic form of GB associated with normal colon epithelial cell surfaces. Texas red labelled PPS (TR-PPS) binds to the tumour cell surfaces of colonic carcinoma and colonic polyps and these cells fluoresce red, whilst the normal colon cell surfaces failed to bind the TR-PPS, and hence lacked red fluorescence. Polysulphonated suramin also selectively recognised and inhibited the colonic carcinoma GB isoenzyme. The kinetic data indicated that this inhibition was not caused by a mere polyanionic effect, since highly sulphated heparin failed to show a significant inhibition of colonic carcinoma GB, however trypan blue did show 50% inhibition. Kinetic studies have also shown that PPS is a non-competitive, reversible inhibitor of colonic carcinoma GB, with an apparent Km 6.8 x 10(-7) M. Gel analysis has shown that PPS binds to another site, distinct from the active centre, and after binding PPS changed the conformation of GB. These studies suggest that TR-PPS is a potent inhibitor of colonic carcinoma GB, and can be used as a novel fluorescent probe for the location of tumour cells in frozen sections of human colon tissues. PSS could also have potential as a vehicle for the transport of cytotoxic compounds to carcinoma cells of the colon. PMID:8835946

  14. A 160-kilodalton epithelial cell surface glycoprotein recognized by plant lectins that inhibit the adherence of Actinomyces naeslundii.

    OpenAIRE

    Brennan, M J; Cisar, J O; Sandberg, A L

    1986-01-01

    The adherence of Actinomyces naeslundii to human epithelial (KB) cells is mediated by the interaction of a fimbrial lectin on this oral bacterium with epithelial cell receptors exposed by sialidase. The D-galactose- and N-acetyl-D-galactosamine-reactive plant lectins from peanut and from Bauhinia purpurea inhibit this interaction. This report describes the partial purification and characterization of a 160-kilodalton (kDa) cell surface glycoprotein which is the principal receptor for these le...

  15. Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells.

    OpenAIRE

    Shimada, K.; Gill, P J; Silbert, J E; Douglas, W H; Fanburg, B L

    1981-01-01

    It has been postulated that lipoprotein lipase, an enzyme important in the uptake of fatty acids into tissues, is bound to the vascular endothelial cell surface and that this binding occurs through attachment to heparinlike glycosaminoglycans. Furthermore, it is thought that heparin releases the enzyme from its attachment to the endothelium into the circulation. These hypotheses have never been tested directly in cell systems in vitro. In the present study we have directly evaluated the inter...

  16. Characterization of Four Outer Membrane Proteins Involved in Binding Starch to the Cell Surface of Bacteroides thetaiotaomicron

    OpenAIRE

    Shipman, Joseph A.; Berleman, James E.; Salyers, Abigail A.

    2000-01-01

    Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. ...

  17. GABA Acts as a Ligand Chaperone in the Early Secretory Pathway to Promote Cell Surface Expression of GABAA Receptors

    OpenAIRE

    Eshaq, Randa S.; Stahl, Letha D.; Stone, Randolph; Smith, Sheryl S.; Robinson, Lucy C.; Leidenheimer, Nancy J.

    2010-01-01

    GABA (γ-aminobutyric acid) is the primary inhibitory neurotransmitter in brain. The fast inhibitory effect of GABA is mediated through the GABAA receptor, a postsynaptic ligand-gated chloride channel. We propose that GABA can act as a ligand chaperone in the early secretory pathway to facilitate GABAA receptor cell surface expression. Forty-two hrs of GABA treatment increased the surface expression of recombinant receptors expressed in HEK 293 cells, an effect accompanied by an increase in GA...

  18. Atoms, molecules & elements

    CERN Document Server

    Graybill, George

    2007-01-01

    Young scientists will be thrilled to explore the invisible world of atoms, molecules and elements. Our resource provides ready-to-use information and activities for remedial students using simplified language and vocabulary. Students will label each part of the atom, learn what compounds are, and explore the patterns in the periodic table of elements to find calcium (Ca), chlorine (Cl), and helium (He) through hands-on activities.

  19. Single Molecule Mechanochemistry

    Science.gov (United States)

    Li, Shaowei; Zhang, Yanxing; Ho, Wilson; Wu, Ruqian; Ruqian Wu, Yanxing Zhang Team; Wilson Ho, Shaowei Li Team

    Mechanical forces can be used to trigger chemical reactions through bending and stretching of chemical bonds. Using the reciprocating movement of the tip of a scanning tunneling microscope (STM), mechanical energy can be provided to a single molecule sandwiched between the tip and substrate. When the mechanical pulse center was moved to the outer ring feature of a CO molecule, the reaction rate was significantly increased compared with bare Cu surface and over Au atoms. First, DFT calculations show that the presence of CO makes the Cu cavity more attractive toward H2 Second, H2 prefers the horizontal adsorption geometry in the Cu-Cu and Au-Cu cavities and no hybridization occurs between the antibonding states of H2 and states of Cu atoms. While H2 loses electrons from its bonding state in all three cavities, the filling of its anti-bonding state only occurs in the CO-Cu cavity. Both make the CO-Cu cavity much more effectively to chop the H2 molecule. Work was supported by the National Science Foundation Center for Chemical Innovation on Chemistry at the Space-Time Limit (CaSTL) under Grant No. CHE-1414466.

  20. Photonic Molecule Lasers Revisited

    Science.gov (United States)

    Gagnon, Denis; Dumont, Joey; Déziel, Jean-Luc; Dubé, Louis J.

    2014-05-01

    Photonic molecules (PMs) formed by coupling two or more optical resonators are ideal candidates for the fabrication of integrated microlasers, photonic molecule lasers. Whereas most calculations on PM lasers have been based on cold-cavity (passive) modes, i.e. quasi-bound states, a recently formulated steady-state ab initio laser theory (SALT) offers the possibility to take into account the spectral properties of the underlying gain transition, its position and linewidth, as well as incorporating an arbitrary pump profile. We will combine two theoretical approaches to characterize the lasing properties of PM lasers: for two-dimensional systems, the generalized Lorenz-Mie theory will obtain the resonant modes of the coupled molecules in an active medium described by SALT. Not only is then the theoretical description more complete, the use of an active medium provides additional parameters to control, engineer and harness the lasing properties of PM lasers for ultra-low threshold and directional single-mode emission. We will extend our recent study and present new results for a number of promising geometries. The authors acknowledge financial support from NSERC (Canada) and the CERC in Photonic Innovations of Y. Messaddeq.

  1. Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

    Science.gov (United States)

    Poeter, Michaela; Brandherm, Ines; Rossaint, Jan; Rosso, Gonzalo; Shahin, Victor; Skryabin, Boris V.; Zarbock, Alexander; Gerke, Volker; Rescher, Ursula

    2014-04-01

    To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.

  2. Anti-cancer Lead Molecule

    KAUST Repository

    Sagar, Sunil

    2014-04-17

    Derivatives of plumbagin can be selectively cytotoxic to breast cancer cells. Derivative `A` (Acetyl Plumbagin) has emerged as a lead molecule for testing against estrogen positive breast cancer and has shown low hepatotoxicity as well as overall lower toxicity in nude mice model. The toxicity of derivative `A` was determined to be even lower than vehicle control (ALT and AST markers). The possible mechanism of action identified based on the microarray experiments and pathway mapping shows that derivative `A` could be acting by altering the cholesterol-related mechanisms. The low toxicity profile of derivative `A` highlights its possible role\\'as future anti-cancer drug and/or as an adjuvant drug to reduce the toxicity of highly toxic chemotherapeutic\\'drugs

  3. Target molecules of food phytochemicals: food science bound for the next dimension.

    Science.gov (United States)

    Murakami, Akira; Ohnishi, Kohta

    2012-05-01

    Phytochemicals are generally defined as secondary metabolites in plants that play crucial roles in their adaptation to a variety of environmental stressors. There is a great body of compelling evidence showing that these metabolites have pronounced potentials for regulating and modulating human health and disease onset, as shown by both experimental and epidemiological approaches. Concurrently, enormous efforts have been made to elucidate the mechanism of actions underlying their biological and physiological functions. For example, the pioneering work of Tachibana et al. uncovered the receptor for (-)-epigallocatechin-3-gallate (EGCg) as the 67 kDa laminin receptor, which was shown to partially mediate the functions of EGCg, such as anti-inflammatory, anti-allergic, and anti-proliferative activities. Thereafter, several protein kinases were identified as binding proteins of flavonoids, including myricetin, quercetin, and kaempferol. Isothiocyanates, sulfur-containing phytochemicals present in cruciferous plants, are well known to target Keap1 for activating the transcription factor Nrf2 for inducing self-defensive and anti-oxidative gene expression. In addition, we recently identified CD36 as a cell surface receptor for ursolic acid, a triterpenoid ubiquitously occurring in plants. Importantly, the above mentioned target proteins are indispensable for phytochemicals to exhibit, at least in part, their bioactivities. Nevertheless, it is reasonable to assume that some of the activities and potential toxicities of metabolites are exerted via their interactions with unidentified, off-target proteins. This notion may be supported by the fact that even rationally designed drugs occasionally display off-target effects and induce unexpected outcomes, including toxicity. Here we update the current status and future directions of research related to target molecules of food phytochemicals.

  4. Polysialic acid modification of the synaptic cell adhesion molecule SynCAM 1 in human embryonic stem cell-derived oligodendrocyte precursor cells

    Directory of Open Access Journals (Sweden)

    Sebastian Werneburg

    2015-05-01

    Full Text Available Oligodendrocyte precursor cells (OPCs are the progenitors of myelinating oligodendrocytes in brain development and repair. Successful myelination depends on the control of adhesiveness during OPC migration and axon contact formation. The decoration of cell surface proteins with the glycan polysialic acid (polySia is a key regulatory element of OPC interactions during development and under pathological conditions. By far the major protein carrier of polySia is the neural cell adhesion molecule NCAM, but recently, polysialylation of the synaptic cell adhesion molecule SynCAM 1 has been detected in the developing mouse brain. In mice, polySia-SynCAM 1 is associated with cells expressing NG2, a marker of a heterogeneous precursor cell population, which is the primary source for oligodendrocytes in development and myelin repair but can also give rise to astrocytes and possibly neurons. It is not yet clear if polySia-SynCAM 1 is expressed by OPCs and its occurrence in humans is elusive. By generating uniform human embryonic stem cell-derived OPC cultures, we demonstrate that polySia is present on human OPCs but down-regulated during differentiation into myelin basic protein-positive oligodendrocytes. PolySia on NCAM resides on the isoforms NCAM-180 and NCAM-140, and SynCAM 1 is identified as a novel polySia acceptor in human OPCs.

  5. Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains

    DEFF Research Database (Denmark)

    Dellgren, Christoffer; Nehlin, Jan O; Barington, Torben

    2015-01-01

    Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses....... Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly...... expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of...

  6. Identification of the essential EPE1 gene involved in retention of secreted proteins on the cell surface of Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Alexieva, K I; Klis, F; Wedler, H; Wambutt, R; Venkov, P

    1999-09-01

    Saccharomyces cerevisiae yeast cells secrete extracellularly low amounts of a few proteins. The reasons for retardation of secreted proteins on the cell surface remain obscure. We describe here a mutant able to export enhanced amount of proteins. Classical genetic methods, nucleic acids manipulations and cloning procedures were used to isolate and characterize the mutant and to clone and sequence the corresponding wild type gene. The isolated Saccharomyces cerevisiae mutant MW11, is temperature sensitive and exports on average twenty-fold more proteins at 37 degrees C than parental wild type strain (80 micrograms of proteins/1 x 10(8) mutant cells, SEM +/- 5, n22; versus 3 micrograms of proteins/1 x 10(8) parental cells, SEM +/- 1, n22). Protein overexport in the mutant requires a functional SEC1 pathway and is independent of cell lysis. Cloning and sequencing of the corresponding wild type gene identified an open reading frame of 786 bp coding for a hydrophilic protein with predicted molecular mass of 30 kDa and cytosolic localization. The newly identified gene, designated EPE1, is an essential gene. Its DNA and amino acids sequence showed no homology with other yeast genes and proteins. It is concluded that the function of unknown yet genes, such as EPE1 is needed for retention of secreted proteins on the surface of Saccharomyces cerevisiae cells.

  7. Soybean extracts increase cell surface ZIP4 abundance and cellular zinc levels: a potential novel strategy to enhance zinc absorption by ZIP4 targeting.

    Science.gov (United States)

    Hashimoto, Ayako; Ohkura, Katsuma; Takahashi, Masakazu; Kizu, Kumiko; Narita, Hiroshi; Enomoto, Shuichi; Miyamae, Yusaku; Masuda, Seiji; Nagao, Masaya; Irie, Kazuhiro; Ohigashi, Hajime; Andrews, Glen K; Kambe, Taiho

    2015-12-01

    Dietary zinc deficiency puts human health at risk, so we explored strategies for enhancing zinc absorption. In the small intestine, the zinc transporter ZIP4 functions as an essential component of zinc absorption. Overexpression of ZIP4 protein increases zinc uptake and thereby cellular zinc levels, suggesting that food components with the ability to increase ZIP4 could potentially enhance zinc absorption via the intestine. In the present study, we used mouse Hepa cells, which regulate mouse Zip4 (mZip4) in a manner indistinguishable from that in intestinal enterocytes, to screen for suitable food components that can increase the abundance of ZIP4. Using this ZIP4-targeting strategy, two such soybean extracts were identified that were specifically able to decrease mZip4 endocytosis in response to zinc. These soybean extracts also effectively increased the abundance of apically localized mZip4 in transfected polarized Caco2 and Madin-Darby canine kidney cells and, moreover, two apically localized mZip4 acrodermatitis enteropathica mutants. Soybean components were purified from one extract and soyasaponin Bb was identified as an active component that increased both mZip4 protein abundance and zinc levels in Hepa cells. Finally, we confirmed that soyasaponin Bb is capable of enhancing cell surface endogenous human ZIP4 in human cells. Our results suggest that ZIP4 targeting may represent a new strategy to improve zinc absorption in humans.

  8. Molecules in crystals

    Science.gov (United States)

    Spackman, Mark A.

    2013-04-01

    Hirshfeld surface analysis has developed from the serendipitous discovery of a novel partitioning of the crystal electron density into discrete molecular fragments, to a suite of computational tools used widely for the identification, analysis and discussion of intermolecular interactions in molecular crystals. The relationship between the Hirshfeld surface and very early ideas on the internal structure of crystals is outlined, and applications of Hirshfeld surface analysis are presented for three molecules of historical importance in the development of modern x-ray crystallography: hexamethylbenzene, hexamethylenetetramine and diketopiperazine.

  9. Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei▿ † ‡

    OpenAIRE

    Güther, Maria Lucia Sampaio; Beattie, Kenneth; Lamont, Douglas J.; James, John; Prescott, Alan R; Ferguson, Michael A. J.

    2009-01-01

    A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB3H4 labeling, that the cell surface of the TbGPI12 nul...

  10. Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false-positive control

    DEFF Research Database (Denmark)

    Solis, Nestor; Larsen, Martin Røssel; Cordwell, Stuart J

    2010-01-01

    with either trypsin or proteinase-K combined with LC-MS/MS. Trypsin-derived data were controlled using a "false-positive" strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell...... lysis and were removed from the trypsin-shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface-exposed peptides. Trypsin and proteinase-K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase-K treatment, 13 specific...

  11. Ultra-cold molecule production.

    Energy Technology Data Exchange (ETDEWEB)

    Ramirez-Serrano, Jamie; Chandler, David W.; Strecker, Kevin; Rahn, Larry A.

    2005-12-01

    The production of Ultra-cold molecules is a goal of many laboratories through out the world. Here we are pursuing a unique technique that utilizes the kinematics of atomic and molecular collisions to achieve the goal of producing substantial numbers of sub Kelvin molecules confined in a trap. Here a trap is defined as an apparatus that spatially localizes, in a known location in the laboratory, a sample of molecules whose temperature is below one degree absolute Kelvin. Further, the storage time for the molecules must be sufficient to measure and possibly further cool the molecules. We utilize a technique unique to Sandia to form cold molecules from near mass degenerate collisions between atoms and molecules. This report describes the progress we have made using this novel technique and the further progress towards trapping molecules we have cooled.

  12. Interaction of the 2,6-dimethoxysemiquinone and ascorbyl free radicals with Ehrlich ascites cells: a probe of cell-surface charge.

    OpenAIRE

    Pethig, R; Gascoyne, P R; McLaughlin, J. A.; Szent-Györgyi, A

    1984-01-01

    The rate of quenching by Ehrlich ascites cells of anionic 2,6-dimethoxy-p-semiquinone and ascorbyl free radicals is investigated as a function of cell concentration, the blocking of cell-surface sulfhydryl groups by N-ethylmaleimide, and the reduction of cell-surface charge by neuraminidase. The rate of quenching is found to be proportional to cell viability and to the number of free cell-surface sulfhydryl groups. The enzymatic action of neuraminidase results in an increase of the free radic...

  13. A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells.

    Science.gov (United States)

    Geng, Yijie; Feng, Bradley

    2016-07-01

    The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure, while cell surface marker analyses revealed a VE-cadherin(+)CD31(+)CD34(+)KDR(+)CD43(-) putative endothelial progenitor population. Furthermore, molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL(+) multi-cellular modules and a VEGFR3(+) sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation.

  14. Force measurements of TCR/pMHC recognition at T cell surface.

    Directory of Open Access Journals (Sweden)

    Pierre-Henri Puech

    Full Text Available The rupture forces and adhesion frequencies of single recognition complexes between an affinity selected peptide/MHC complex and a TCR at a murine hybridoma surface were measured using Atomic Force Microscopy. When the CD8 coreceptor is absent, the adhesion frequency depends on the nature of the peptide but the rupture force does not. When CD8 is present, no effect of the nature of the peptide is observed. CD8 is proposed to act as a time and distance lock, enabling the shorter TCR molecule to bridge the pMHC and have time to finely read the peptide. Ultimately, such experiments could help the dissection of the sequential steps by which the TCR reads the peptide/MHC complex in order to control T cell activation.

  15. Magnetic field modification of ultracold molecule-molecule collisions

    OpenAIRE

    Tscherbul, T. V.; Suleimanov, Yu. V.; Aquilanti, V.; Krems, R.V.

    2008-01-01

    We present an accurate quantum mechanical study of molecule-molecule collisions in the presence of a magnetic field. The work focusses on the analysis of elastic scattering and spin relaxation in collisions of O2(3Sigma_g) molecules at cold (~0.1 K) and ultracold (~10^{-6} K) temperatures. Our calculations show that magnetic spin relaxation in molecule-molecule collisions is extremely efficient except at magnetic fields below 1 mT. The rate constant for spin relaxation at T=0.1 K and a magnet...

  16. Single-Molecule FRET Study of DNA G-Quadruplex

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The DNA G-quadruplex formed by the human telomeric sequence is a potential target for novel anticancer drugs. We have investigated an intramolecular DNA G-quadruplex using single-molecule fluorescence resonance energy transfer and shown that individual folded quadruplexes can be identified. The mean proximity ratio measured at the single-molecule level was consistent with ensemble measurement.

  17. Passing Current through Touching Molecules

    DEFF Research Database (Denmark)

    Schull, G.; Frederiksen, Thomas; Brandbyge, Mads;

    2009-01-01

    The charge flow from a single C-60 molecule to another one has been probed. The conformation and electronic states of both molecules on the contacting electrodes have been characterized using a cryogenic scanning tunneling microscope. While the contact conductance of a single molecule between two...

  18. Mining for Molecules in the Milky Way

    Science.gov (United States)

    2008-06-01

    Scientists are using the giant Robert C. Byrd Green Bank Telescope (GBT) to go prospecting in a rich molecular cloud in our Milky Way Galaxy. They seek to discover new, complex molecules in interstellar space that may be precursors to life. The GBT and Molecules The Robert C. Byrd Green Bank Telescope and some molecules it has discovered. CREDIT: Bill Saxton, NRAO/AUI/NSF "Clouds like this one are the raw material for new stars and planets. We know that complex chemistry builds prebiotic molecules in such clouds long before the stars and planets are formed. There is a good chance that some of these interstellar molecules may find their way to the surface of young planets such as the early Earth, and provide a head start for the chemistry of life. For the first time, we now have the capability to make a very thorough and methodical search to find all the chemicals in the clouds," said Anthony Remijan, of the National Radio Astronomy Observatory (NRAO). In the past three years, Remijan and his colleagues have used the GBT to discover ten new interstellar molecules, a feat unequalled in such a short time by any other team or telescope. The scientists discovered those molecules by looking specifically for them. However, they now are changing their strategy and casting a wide net designed to find whatever molecules are present, without knowing in advance what they'll find. In addition, they are making their data available freely to other scientists, in hopes of speeding the discovery process. The research team presented its plan to the American Astronomical Society's meeting in St. Louis, MO. As molecules rotate and vibrate, they emit radio waves at specific frequencies. Each molecule has a unique pattern of such frequencies, called spectral lines, that constitutes a "fingerprint" identifying that molecule. Laboratory tests can determine the pattern of spectral lines that identifies a specific molecule. Most past discoveries came from identifying a molecule's pattern in

  19. Suppression of tumor growth and angiogenesis by a specific antagonist of the cell-surface expressed nucleolin.

    Directory of Open Access Journals (Sweden)

    Damien Destouches

    Full Text Available BACKGROUND: Emerging evidences suggest that nucleolin expressed on the cell surface is implicated in growth of tumor cells and angiogenesis. Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, here we show that the growth of tumor cells and angiogenesis are suppressed in various in vitro and in vivo experimental models. HB-19 inhibited colony formation in soft agar of tumor cell lines, impaired migration of endothelial cells and formation of capillary-like structures in collagen gel, and reduced blood vessel branching in the chick embryo chorioallantoic membrane. In athymic nude mice, HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in nude mice, and in some cases eliminated measurable tumors while displaying no toxicity to normal tissue. This potent antitumoral effect is attributed to the direct inhibitory action of HB-19 on both tumor and endothelial cells by blocking and down regulating surface nucleolin, but without any apparent effect on nucleolar nucleolin. CONCLUSION/SIGNIFICANCE: Our results illustrate the dual inhibitory action of HB-19 on the tumor development and the neovascularization process, thus validating the cell-surface expressed nucleolin as a strategic target for an effective cancer drug. Consequently, the HB-19 pseudopeptide provides a unique candidate to consider for innovative cancer therapy.

  20. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yukie; Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  1. Forces in molecules.

    Science.gov (United States)

    Hernández-Trujillo, Jesús; Cortés-Guzmán, Fernando; Fang, De-Chai; Bader, Richard F W

    2007-01-01

    Chemistry is determined by the electrostatic forces acting within a collection of nuclei and electrons. The attraction of the nuclei for the electrons is the only attractive force in a molecule and is the force responsible for the bonding between atoms. This is the attractive force acting on the electrons in the Ehrenfest force and on the nuclei in the Feynman force, one that is countered by the repulsion between the electrons in the former and by the repulsion between the nuclei in the latter. The virial theorem relates these forces to the energy changes resulting from interactions between atoms. All bonding, as signified by the presence of a bond path, has a common origin in terms of the mechanics determined by the Ehrenfest, Feynman and virial theorems. This paper is concerned in particular with the mechanics of interaction encountered in what are classically described as 'nonbonded interactions'--are atoms that 'touch' bonded or repelling one another? PMID:17328425

  2. Forces in molecules.

    Science.gov (United States)

    Hernández-Trujillo, Jesús; Cortés-Guzmán, Fernando; Fang, De-Chai; Bader, Richard F W

    2007-01-01

    Chemistry is determined by the electrostatic forces acting within a collection of nuclei and electrons. The attraction of the nuclei for the electrons is the only attractive force in a molecule and is the force responsible for the bonding between atoms. This is the attractive force acting on the electrons in the Ehrenfest force and on the nuclei in the Feynman force, one that is countered by the repulsion between the electrons in the former and by the repulsion between the nuclei in the latter. The virial theorem relates these forces to the energy changes resulting from interactions between atoms. All bonding, as signified by the presence of a bond path, has a common origin in terms of the mechanics determined by the Ehrenfest, Feynman and virial theorems. This paper is concerned in particular with the mechanics of interaction encountered in what are classically described as 'nonbonded interactions'--are atoms that 'touch' bonded or repelling one another?

  3. Lanthanide single molecule magnets

    CERN Document Server

    Tang, Jinkui

    2015-01-01

    This book begins by providing basic information on single-molecule magnets (SMMs), covering the magnetism of lanthanide, the characterization and relaxation dynamics of SMMs, and advanced means of studying lanthanide SMMs. It then systematically introduces lanthanide SMMs ranging from mononuclear and dinuclear to polynuclear complexes, classifying them and highlighting those SMMs with high barrier and blocking temperatures – an approach that provides some very valuable indicators for the structural features needed to optimize the contribution of an Ising type spin to a molecular magnet. The final chapter presents some of the newest developments in the lanthanide SMM field, such as the design of multifunctional and stimuli-responsive magnetic materials as well as the anchoring and organization of the SMMs on surfaces. In addition, the crystal structure and magnetic data are clearly presented with a wealth of illustrations in each chapter, helping newcomers and experts alike to better grasp ongoing trends and...

  4. Lanthanide single molecule magnets

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jinkui; Zhang, Peng [Chinese Academy of Sciences, Changchun (China). Changchun Inst. of Applied Chemistry

    2015-10-01

    This book begins by providing basic information on single-molecule magnets (SMMs), covering the magnetism of lanthanide, the characterization and relaxation dynamics of SMMs and advanced means of studying lanthanide SMMs. It then systematically introduces lanthanide SMMs ranging from mononuclear and dinuclear to polynuclear complexes, classifying them and highlighting those SMMs with high barrier and blocking temperatures - an approach that provides some very valuable indicators for the structural features needed to optimize the contribution of an Ising type spin to a molecular magnet. The final chapter presents some of the newest developments in the lanthanide SMM field, such as the design of multifunctional and stimuli-responsive magnetic materials as well as the anchoring and organization of the SMMs on surfaces. In addition, the crystal structure and magnetic data are clearly presented with a wealth of illustrations in each chapter, helping newcomers and experts alike to better grasp ongoing trends and explore new directions.

  5. Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin.

    OpenAIRE

    McNab, R; Holmes, A.R.; Clarke, J M; Tannock, G W; Jenkinson, H F

    1996-01-01

    Isogenic mutants of Streptococcus gordonii DL1 (Challis) in which the genes encoding high-molecular-mass cell surface polypeptides CshA and/or CshB were inactivated were deficient in binding to four strains of Actinomyces naeslundii and two strains of Streptococcus oralis. Lactose-sensitive interactions of S. gordonii with A. naeslundii ATCC 12104 and PK606 were associated with expression of cshA but not of cshB. Lactose-insensitive interactions of S. gordonii with A. naeslundii T14V and WVU6...

  6. Changes in the hydrophobic-hydrophilic cell surface character of Halomonas elongata in response to NaCl.

    OpenAIRE

    Hart, D J; Vreeland, R H

    1988-01-01

    Phase-partitioning studies of the euryhaline bacterium Halomonas elongata demonstrated that the hydrophobic-hydrophilic nature of the cell surface changed as the bacterium grew in different NaCl concentrations. Mid-log-phase cells grown in a high (3.4 M) NaCl concentration were more hydrophilic than were cells grown in a low (0.05 M) NaCl concentration. Mid-log-phase cells from defined medium containing 3.4 M NaCl normally produced a hydrophobicity reading of only 14 (hexadecane hydrophobicit...

  7. Use of enzyme label for quantitative evaluation of liposome adhesion on cell surface: studies with J774 macrophage monolayers.

    Science.gov (United States)

    Trubetskoy, V S; Dormeneva, E V; Tsibulsky, V P; Repin, V S; Torchilin, V P

    1988-07-01

    A method for quantitation of cell surface-bound liposomes utilizing J774 macrophage monolayers is developed. Surface-bound biotinyl-containing and 125I-labeled liposomes were quantified with avidin-peroxidase in an ELISA-like assay. Peroxidase substrate absorbance values were recalculated into the absolute amount of liposomal lipid using a special calibration plot. Total liposome uptake by macrophages was determined following the binding of 125I radioactivity. The approach suggested allows quantitative evaluation of the changes in the content of surface-adhered liposomes during their interaction with cells in vitro.

  8. Human antibodies targeting cell surface antigens overexpressed by the hormone refractory metastatic prostate cancer cells: ICAM-1 is a tumor antigen that mediates prostate cancer cell invasion

    OpenAIRE

    Conrad, Fraser; Zhu, Xiaodong; Zhang, Xin; Chalkley, Robert J.; Burlingame, Alma L; Marks, James D.; Liu, Bin

    2009-01-01

    Transition from hormone-sensitive to hormone-refractory metastatic tumor types poses a major challenge for prostate cancer treatment. Tumor antigens that are differentially expressed during this transition are likely to play important roles in imparting prostate cancer cells with the ability to grow in a hormone-deprived environment and to metastasize to distal sites such as the bone and thus, are likely targets for therapeutic intervention. To identify those molecules and particularly cell s...

  9. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Behrendt, N; Ploug, M; Patthy, L;

    1991-01-01

    with the internal repeats of u-PAR constitute the extracellular part of Ly-6 antigens and of the squid glycoprotein Sgp-2. Like u-PAR, these proteins are attached to the membrane by a glycosyl-phosphatidylinositol anchor. The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential...

  10. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    International Nuclear Information System (INIS)

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin

  11. A Val85Met mutation in melanocortin-1 receptor is associated with reductions in eumelanic pigmentation and cell surface expression in domestic rock pigeons (Columba livia.

    Directory of Open Access Journals (Sweden)

    Michael W Guernsey

    Full Text Available Variation in the melanocortin-1 receptor (Mc1r is associated with pigmentation diversity in wild and domesticated populations of vertebrates, including several species of birds. Among domestic bird species, pigmentation variation in the rock pigeon (Columbalivia is particularly diverse. To determine the potential contribution of Mc1r variants to pigment diversity in pigeons, we sequenced Mc1r in a wide range of pigeon breeds and identified several single nucleotide polymorphisms, including a variant that codes for an amino acid substitution (Val85Met. In contrast to the association between Val85Met and eumelanism in other avian species, this change was associated with pheomelanism in pigeons. In vitro cAMP accumulation and protein expression assays revealed that Val85Met leads to decreased receptor function and reduced cell surface expression of the mutant protein. The reduced in vitro function is consistent with the observed association with reduced eumelanic pigmentation. Comparative genetic and cellular studies provide important insights about the range of mechanisms underlying diversity among vertebrates, including different phenotypic associations with similar mutations in different species.

  12. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Wenxing; Bhatt, Avni [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States); Smith, Adam N. [University of Florida, Department of Chemistry, College of Liberal Arts and Sciences (United States); Crowley, Paula J.; Brady, L. Jeannine, E-mail: jbrady@dental.ufl.edu [University of Florida, Department of Oral Biology, College of Dentistry (United States); Long, Joanna R., E-mail: jrlong@ufl.edu [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States)

    2016-02-15

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

  13. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design

    Science.gov (United States)

    Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M.

    2016-05-01

    Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared – non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

  14. Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design.

    Science.gov (United States)

    Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M

    2016-05-05

    Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared - non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

  15. Galectin-1-mediated cell adhesion, invasion and cell death in human anaplastic large cell lymphoma: Regulatory roles of cell surface glycans

    OpenAIRE

    Suzuki, Osamu; Abe, Masafumi

    2014-01-01

    Galectin-1 is known to be one of the extracellular matrix proteins. To elucidate the biological roles of galectin-1 in cell adhesion and invasion of human anaplastic large cell lymphoma, we performed cell adhesion and invasion assays using the anaplastic large cell lymphoma cell line H-ALCL, which was previously established in our laboratory. From the cell surface lectin array, treatment with neuraminidase from Arthrobacter ureafaciens which cleaves all linkage types of cell surface sialic ac...

  16. Niemann Pick type C cells show cholesterol dependent decrease of APP expression at the cell surface its increased processing through the ?-secretase pathway

    OpenAIRE

    Malnar, Martina; KOŠIČEK, MARKO; Mitterreiter, Stefan; Omerbašić, Damir; Lichtenthaler, Stefan F.; Goate, Alison; Hećimović, Silva

    2010-01-01

    Abstract The link between cholesterol and Alzheimer's disease has recently been revealed in Niemann Pick type C disease. We found that NPC1-/- cells show decreased expression of APP at the cell surface and increased processing of APP through the ?-secretase pathway resulting in increased C99, sAPP? and intracellular A?40 levels. This effect is dependent on increased cholesterol levels, since cholesterol depletion reversed cell surface APP expression and lowered A?/C99 levels in NPC...

  17. Adhesion molecules in subjects with COPD and healthy non-smokers: a cross sectional parallel group study

    OpenAIRE

    Blidberg, Kristin; Palmberg, Lena; James, Anna; Billing, Bo; Henriksson, Elisabeth; Lantz, Ann-Sofie; Larsson, Kjell; Dahlén, Barbro

    2013-01-01

    Background The aim of the study was to investigate how the expression of adhesion molecules changes as neutrophils migrate from the circulation to the lung and if these changes differ between non-smoking subjects and smokers with and without COPD. Methods Non-smoking healthy subjects (n=22), smokers without (n=21) and with COPD (n=18) were included. Neutrophils from peripheral blood, sputum and bronchial biopsies were analysed for cell surface expression of adhesion molecules (CD11b, CD62L, C...

  18. Imaging Genetic Molecules At Atomic Resolution

    Science.gov (United States)

    Coles, L. Stephen

    1993-01-01

    Proposed method of imaging informational polymeric biological molecules at atomic resolution enables determination of sequences of component monomers about 10 to the 3rd power to 10 to the 4th power times as fast as conventional methods do. Accelerates research on genetic structures of animals and plants. Also contributes significantly to imaging processes like scanning electron microscopy (SEM), atomic-force microscopy (AFM), and scanning tunneling microscopy (STM) in cases in which necessary to locate or identify small specimens on relatively large backgrounds and subtract background images to obtain images of specimens in isolation. V-grooves on silicon wafer laid out in square pattern, intersections of which marked to identify coordinates. Specimen molecules held in grooves for reproducible positioning and scanning by AFM or STM.

  19. Specific N-glycans of Hepatocellular Carcinoma Cell Surface and the Abnormal Increase of Core-α-1, 6-fucosylated Triantennary Glycan via N-acetylglucosaminyltransferases-IVa Regulation.

    Science.gov (United States)

    Nie, Huan; Liu, Xia; Zhang, Yubao; Li, Tingting; Zhan, Chao; Huo, Wenjuan; He, Anshun; Yao, Yuanfei; Jin, Yu; Qu, Youpeng; Sun, Xue-Long; Li, Yu

    2015-11-05

    Glycosylation alterations of cell surface proteins are often observed during the progression of malignancies. The specific cell surface N-glycans were profiled in hepatocellular carcinoma (HCC) with clinical tissues (88 tumor and adjacent normal tissues) and the corresponding serum samples of HCC patients. The level of core-α-1,6-fucosylated triantennary glycan (NA3Fb) increased both on the cell surface and in the serum samples of HCC patients (p IVa (GnT-IVa), which was related to the synthesis of the NA3Fb, was substantially increased in HCC tissues. Knockdown of GnT-IVa leads to a decreased level of NA3Fb and decreased ability of invasion and migration in HCC cells. NA3Fb can be regarded as a specific cell surface N-glycan of HCC. The high expression of GnT-IVa is the cause of the abnormal increase of NA3Fb on the HCC cell surface, which regulates cell migration. This study demonstrated the specific N-glycans of the cell surface and the mechanisms of altered glycoform related with HCC. These findings lead to better understanding of the function of glycan and glycosyltransferase in the tumorigenesis, progression and metastasis of HCC.

  20. Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

    International Nuclear Information System (INIS)

    Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways

  1. Electrical transport through individual DNA molecules

    OpenAIRE

    Li, Xin-Qi; Yan, YiJing

    2001-01-01

    A theoretical study is presented to quantitatively analyze the transport experiment through individual DNA molecules reported recently by Porath {\\it et al.} [Nature {\\bf 403}, 635 (2000)]. A variety of valuable quantities are identified by contacting the theoretical model with the measured data. The partially decoherent nature on the GC pairs of DNA is elaborated in contrast to the completely incoherent hopping mechanism discussed in the context of charge transfer experiments.

  2. Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: A 600-MHz NMR study

    Energy Technology Data Exchange (ETDEWEB)

    Abeygunawardana, C.; Bush, C.A. (Univ. of Maryland, Baltimore (United States)); Cisar, J.O. (National Inst. of Dental Research, Bethesda, MD (United States))

    1991-09-03

    Specific lectin-carbohydrate interactions between certain oral streptococci and actinomyces contribute to the microbial colonization of teeth. The receptor molecules of Streptococcus oralis, 34, ATCC 10557, and Streptococcus mitis J22 for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii are antigenically distinct polysaccharides, each formed by a different phosphodiester-linked oligosaccharide repeating unit. Receptor polysaccharide was isolated form S. oralis C104 cells and was shown to contain galactose, N-acetylgalactosamine, ribitol, and phosphate with molar ratios of 4:1:1:1. The {sup 1}H NMR spectrum of the polysaccharide shows that it contains a repeating structure. The individual sugars in the repeating unit were identified by {sup 1}H coupling constants observed in E-COSY and DQF-COSY spectra. NMR methods included complete resonance assignments ({sup 1}H and {sup 13}C) by various homonuclear and heteronuclear correlation experiments that utilize scalar couplings. Sequence and linkage assignments were obtained from the heteronuclear multiple-bond correlation (HMBC) spectrum. This analysis shows that the receptor polysaccharide of S. oralis C104 is a ribitol teichoic acid polymer composed of a linear hexasaccharide repeating unit containing two residues each of galactopyranose and galactofuranose and a residue each of GalNAc and ribitol joined end to end by phosphodiester linkages.

  3. A Major Cell Surface Antigen of Coccidioides immitis Which Elicits Both Humoral and Cellular Immune Responses

    OpenAIRE

    Hung, Chiung-Yu; Ampel, Neil M.; Christian, Lara; Seshan, Kalpathi R.; Cole, Garry T.

    2000-01-01

    Multinucleate parasitic cells (spherules) of Coccidioides immitis isolates produce a membranous outer wall component (SOW) in vitro which has been reported to be reactive with antibody from patients with coccidioidal infection, elicits a potent proliferative response of murine immune T cells, and has immunoprotective capacity in a murine model of coccidioidomycosis. To identify the antigenic components of SOW, the crude wall material was first subjected to Triton X-114 extraction, and a water...

  4. CELL-SURFACE BINDING OF DEOXYNIVALENOL TO Lactobacillus paracasei subsp. tolerans ISOLATED FROM SOURDOUGH STARTER CULTURE

    OpenAIRE

    Hassan, Yousef I.; Lloyd B. Bullerman

    2013-01-01

    Deoxynivalenol (DON) and fumonisin B1 (FB1) are two contaminant-mycotoxins frequently found in food commodities produced under poor conditions. Several methods have been suggested for the detoxification of such mycotoxins. Among the proposed methods, biological detoxification seems to be the most promising and cost-efficient. This study explores the capability of one strain of lactic acid bacteria, identified as Lactobacillus paracasei subsp. tolerans, to bind both DON and FB1 in liquid cultu...

  5. The use of scFv-displaying yeast in mammalian cell surface selections.

    Science.gov (United States)

    Wang, Xin Xiang; Shusta, Eric V

    2005-09-01

    Yeast surface display has proven to be a powerful tool for the directed evolution of immunological proteins when soluble ligands are available (Cho, B.K., Kieke, M.C., Boder, E.T., Wittrup, K.D., Kranz, D.M., 1998. A yeast surface display system for the discovery of ligands that trigger cell activation. J. Immunol. Methods 220, 179; Boder, E.T., Midelfort, K.S., Wittrup, K.D., 2000. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. U. S. A. 97, 10701; Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M., Wittrup, K.D., 2000. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754; Esteban, O., Zhao, H., 2004. Directed evolution of soluble single-chain human class II MHC molecules. J. Mol. Biol. 340, 81). This investigation extends the utility of this display platform by demonstrating its capacity for use in cell panning selections. This was accomplished by employing a model single-chain antibody (scFv)-hapten system that allowed for detailed investigation of the factors governing panning success. Yeast displaying anti-fluorescein scFv (4-4-20) exhibited specific interactions with the fluoresceinated endothelial cells and could be recovered from large backgrounds of irrelevant yeast in just three rounds. Successful selections required as few as 1700 fluorescein ligands per cell, and a three-round enrichment ratio of 10(6) was possible. These results indicate that yeast surface display is a viable option for use in cell or tissue-based selections.

  6. GABAB receptor subunit GB1 at the cell surface independently activates ERK1/2 through IGF-1R transactivation.

    Directory of Open Access Journals (Sweden)

    Guillaume A Baloucoune

    Full Text Available BACKGROUND: Functional GABA(B receptor is believed to require hetero-dimerization between GABA(B1 (GB1 and GABA(B2 (GB2 subunits. The GB1 extracellular domain is required for ligand binding, and the GB2 trans-membrane domain is responsible for coupling to G proteins. Atypical GABA(B receptor responses observed in GB2-deficient mice suggested that GB1 may have activity in the absence of GB2. However the underlying mechanisms remain poorly characterized. METHODOLOGY/PRINCIPAL FINDINGS: Here, by using cells overexpressing a GB1 mutant (GB1asa with the ability to translocate to the cell surface in the absence of GB2, we show that GABA(B receptor agonists, such as GABA and Baclofen, can induce ERK1/2 phosphorylation in the absence of GB2. Furthermore, we demonstrate that GB1asa induces ERK1/2 phosphorylation through Gi/o proteins and PLC dependent IGF-1R transactivation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that GB1 may form a functional receptor at the cell surface in the absence of GB2.

  7. Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B.

    Science.gov (United States)

    Wang, Pan; He, Jie; Sun, Yufei; Reynolds, Matthew; Zhang, Li; Han, Shuangyan; Liang, Shuli; Sui, Haixin; Lin, Ying

    2016-07-01

    To modify the Pichia pastoris cell surface, two classes of hydrophobins, SC3 from Schizophyllum commune and HFBI from Trichoderma reesei, were separately displayed on the cell wall. There was an observable increase in the hydrophobicity of recombinant strains. Candida antarctica lipase B (CALB) was then co-displayed on the modified cells, generating strains GS115/SC3-61/CALB-51 and GS115/HFBI-61/CALB-51. Interestingly, the hydrolytic and synthetic activities of strain GS115/HFBI-61/CALB-51 increased by 37 and 109 %, respectively, but decreased by 26 and 43 %, respectively, in strain GS115/SC3-61/CALB-51 compared with the hydrophobin-minus recombinant strain GS115/CALB-GCW51. The amount of glycerol by-product from the transesterification reaction adsorbed on the cell surface was significantly decreased following hydrophobin modification, removing the glycerol barrier and allowing substrates to access the active sites of lipases. Electron micrographs indicated that the cell wall structures of both recombinant strains appeared altered, including changes to the inner glucan layer and outer mannan layer. These results suggest that the display of hydrophobins can change the surface structure and hydrophobic properties of P. pastoris and affect the catalytic activities of CALB displayed on the surface of P. pastoris cells. PMID:26969039

  8. Cell surface engineering of Saccharomyces cerevisiae combined with membrane separation technology for xylitol production from rice straw hydrolysate.

    Science.gov (United States)

    Guirimand, Gregory; Sasaki, Kengo; Inokuma, Kentaro; Bamba, Takahiro; Hasunuma, Tomohisa; Kondo, Akihiko

    2016-04-01

    Xylitol, a value-added polyol deriving from D-xylose, is widely used in both the food and pharmaceutical industries. Despite extensive studies aiming to streamline the production of xylitol, the manufacturing cost of this product remains high while demand is constantly growing worldwide. Biotechnological production of xylitol from lignocellulosic waste may constitute an advantageous and sustainable option to address this issue. However, to date, there have been few reports of biomass conversion to xylitol. In the present study, xylitol was directly produced from rice straw hydrolysate using a recombinant Saccharomyces cerevisiae YPH499 strain expressing cytosolic xylose reductase (XR), along with β-glucosidase (BGL), xylosidase (XYL), and xylanase (XYN) enzymes (co-)displayed on the cell surface; xylitol production by this strain did not require addition of any commercial enzymes. All of these enzymes contributed to the consolidated bioprocessing (CBP) of the lignocellulosic hydrolysate to xylitol to produce 5.8 g/L xylitol with 79.5 % of theoretical yield from xylose contained in the biomass. Furthermore, nanofiltration of the rice straw hydrolysate provided removal of fermentation inhibitors while simultaneously increasing sugar concentrations, facilitating high concentration xylitol production (37.9 g/L) in the CBP. This study is the first report (to our knowledge) of the combination of cell surface engineering approach and membrane separation technology for xylitol production, which could be extended to further industrial applications. PMID:26631184

  9. Domain-based small molecule binding site annotation

    Directory of Open Access Journals (Sweden)

    Dumontier Michel

    2006-03-01

    Full Text Available Abstract Background Accurate small molecule binding site information for a protein can facilitate studies in drug docking, drug discovery and function prediction, but small molecule binding site protein sequence annotation is sparse. The Small Molecule Interaction Database (SMID, a database of protein domain-small molecule interactions, was created using structural data from the Protein Data Bank (PDB. More importantly it provides a means to predict small molecule binding sites on proteins with a known or unknown structure and unlike prior approaches, removes large numbers of false positive hits arising from transitive alignment errors, non-biologically significant small molecules and crystallographic conditions that overpredict ion binding sites. Description Using a set of co-crystallized protein-small molecule structures as a starting point, SMID interactions were generated by identifying protein domains that bind to small molecules, using NCBI's Reverse Position Specific BLAST (RPS-BLAST algorithm. SMID records are available for viewing at http://smid.blueprint.org. The SMID-BLAST tool provides accurate transitive annotation of small-molecule binding sites for proteins not found in the PDB. Given a protein sequence, SMID-BLAST identifies domains using RPS-BLAST and then lists potential small molecule ligands based on SMID records, as well as their aligned binding sites. A heuristic ligand score is calculated based on E-value, ligand residue identity and domain entropy to assign a level of confidence to hits found. SMID-BLAST predictions were validated against a set of 793 experimental small molecule interactions from the PDB, of which 472 (60% of predicted interactions identically matched the experimental small molecule and of these, 344 had greater than 80% of the binding site residues correctly identified. Further, we estimate that 45% of predictions which were not observed in the PDB validation set may be true positives. Conclusion By

  10. Thread bonds in molecules

    CERN Document Server

    Ivlev, B

    2015-01-01

    Unusual chemical bonds are proposed. Each bond is almost covalent but is characterized by the thread of a small radius $\\sim 0.6\\times 10^{-11}$cm, between two nuclei in a molecule. The main electron density is concentrated outside the thread as in a covalent bond. The thread is formed by the electron wave function which has a tendency to be singular on it. The singularity along the thread is cut off by electron "vibrations" due to the interaction with zero point electromagnetic oscillations. The electron energy has its typical value of (1-10)eV. Due to the small tread radius the uncertainty of the electron momentum inside the thread is large resulting in a large electron kinetic energy $\\sim 1 MeV$. This energy is compensated by formation of a potential well due to the reduction of the energy of electromagnetic zero point oscillations. This is similar to formation of a negative van der Waals potential. Thread bonds are stable and cannot be created or destructed in chemical or optical processes.

  11. Tunnelling of a molecule

    International Nuclear Information System (INIS)

    A quantum-mechanical description of tunnelling is presented for a one-dimensional system with internal oscillator degrees of freedom. The 'charged diatomic molecule' is frustrated on encountering a barrier potential by its centre of charge not being coincident with its centre of mass, resulting in transitions amongst internal states. In an adiabatic limit, the tunnelling of semiclassical coherent-like oscillator states is shown to exhibit the Hartman and Bueuttiker-Landauer times tH and tBL, with the time dependence of the coherent state parameter for the tunnelled state given by α(t) = α e-iω(t+Δt) , Δt = tH - itBL. A perturbation formalism is developed, whereby the exact transfer matrix can be expanded to any desired accuracy in a suitable limit. An 'intrinsic' time, based on the oscillator transition rate during tunnelling, transmission or reflection, is introduced. In simple situations the resulting intrinsic tunnelling time is shown to vanish to lowest order. In the general case a particular (nonzero) parametrisation is inferred, and its properties discussed in comparison with the literature on tunnelling times for both wavepackets and internal clocks. Copyright (1998) CSIRO Australia

  12. Molecule-based magnets

    Indian Academy of Sciences (India)

    J V Yakhmi

    2009-06-01

    The conventional magnetic materials used in current technology, such as, Fe, Fe2O3, Cr2O3, SmCo5, Nd2Fe14B etc are all atom-based, and their preparation/processing require high temperature routes. Employing self-assembly methods, it is possible to engineer a bulk molecular material with long-range magnetic order, mainly because one can play with the weak intermolecular interactions. Since the first successful synthesis of molecular magnets in 1986, a large variety of them have been synthesized, which can be categorized on the basis of the chemical nature of the magnetic units involved: organic-, metal-based systems, heterobimetallic assemblies, or mixed organic–inorganic systems. The design of molecule-based magnets has also been extended to the design of poly-functional molecular magnets, such as those exhibiting second-order optical nonlinearity, liquid crystallinity, or chirality simultaneously with long-range magnetic order. Solubility, low density and biocompatibility are attractive features of molecular magnets. Being weakly coloured, unlike their opaque classical magnet ‘cousins’ listed above, possibilities of photomagnetic switching exist. Persistent efforts also continue to design the ever-elusive polymer magnets towards applications in industry. While providing a brief overview of the field of molecular magnetism, this article highlights some recent developments in it, with emphasis on a few studies from the author’s own lab.

  13. Strongly interacting ultracold polar molecules

    Science.gov (United States)

    Gadway, Bryce; Yan, Bo

    2016-08-01

    This paper reviews recent advances in the study of strongly interacting systems of dipolar molecules. Heteronuclear molecules feature large and tunable electric dipole moments, which give rise to long-range and anisotropic dipole–dipole interactions. Ultracold samples of dipolar molecules with long-range interactions offer a unique platform for quantum simulations and the study of correlated many-body physics. We provide an introduction to the physics of dipolar quantum gases, both electric and magnetic, and summarize the multipronged efforts to bring dipolar molecules into the quantum regime. We discuss in detail the recent experimental progress in realizing and studying strongly interacting systems of polar molecules trapped in optical lattices, with particular emphasis on the study of interacting spin systems and non-equilibrium quantum magnetism. Finally, we conclude with a brief discussion of the future prospects for studies of strongly interacting dipolar molecules.

  14. Strongly interacting ultracold polar molecules

    CERN Document Server

    Gadway, Bryce

    2016-01-01

    This paper reviews recent advances in the study of strongly interacting systems of dipolar molecules. Heteronuclear molecules feature large and tunable electric dipole moments, which give rise to long-range and anisotropic dipole-dipole interactions. Ultracold samples of dipolar molecules with long-range interactions offer a unique platform for quantum simulations and the study of correlated many-body physics. We provide an introduction to the physics of dipolar quantum gases, both electric and magnetic, and summarize the multipronged efforts to bring dipolar molecules into the quantum regime. We discuss in detail the recent experimental progress in realizing and studying strongly interacting systems of polar molecules trapped in optical lattices, with particular emphasis on the study of interacting spin systems and non-equilibrium quantum magnetism. Finally, we conclude with a brief discussion of the future prospects for studies of strongly interacting dipolar molecules.

  15. A subset of HLA-B27 molecules contains peptides much longer than nonamers.

    OpenAIRE

    Urban, R G; Chicz, R M; Lane, W S; Strominger, J.L.; Rehm, A.; Kenter, M J; Uytdehaag, F G; Ploegh, H.; Uchanska-Ziegler, B; Ziegler, A.

    1994-01-01

    An unusual monoclonal antibody (MARB4) directed against HLA-B27 that reacts with only approximately 5-20% of the cell surface HLA-B27 was used for large-scale purification of these molecules. Subsequent mass spectrometry of HLA-B27-bound peptides showed that the minor MARB4-reactive population contained peptides primarily from 900 to 4000 Da in size (approximately 8-33 amino acid residues), whereas the major HLA-B27 population contained peptides in the mass range of 900-1400...

  16. Molecules Best Paper Award 2012

    Directory of Open Access Journals (Sweden)

    Derek J. McPhee

    2012-02-01

    Full Text Available Molecules starts to institute the “Best Paper” award to recognize these outstanding papers in the area of natural products, medicinal chemistry and molecular diversity published in Molecules. We are pleased to announce the first “Molecules Best Paper Award” for 2012. Nominations were selected by the editor-in-chief and selected editorial board members from all the papers published in 2008. [...

  17. Molecules Best Paper Award 2014

    Directory of Open Access Journals (Sweden)

    Derek J. McPhee

    2014-01-01

    Full Text Available Molecules instituted some years ago a “Best Paper” award to recognize the most outstanding papers in the area of natural products, medicinal chemistry and molecular diversity published each year in Molecules. We are pleased to announce the third “Molecules Best Paper Award” for 2014. The winners were chosen by the Editor-in-Chief and selected editorial board members from among all the papers published in 2010. Reviews and research papers were evaluated separately.

  18. Molecules Best Paper Award 2013

    Directory of Open Access Journals (Sweden)

    Derek J. McPhee

    2013-02-01

    Full Text Available Molecules has started to institute a "Best Paper" award to recognize the most outstanding papers in the area of natural products, medicinal chemistry and molecular diversity published in Molecules. We are pleased to announce the second "Molecules Best Paper Award" for 2013. Candidates were chosen by the Editor-in-Chief and selected editorial board members from among all the papers published in 2009.

  19. Recoiling DNA Molecule: Simulation & Experiment

    OpenAIRE

    Neto, Jose Coelho; Dickman, Ronald; Mesquita, O. N.

    2002-01-01

    Single molecule DNA experiments often generate data from force versus extension measurements involving the tethering of a microsphere to one end of a single DNA molecule while the other is attached to a substrate. We show that the persistence length of single DNA molecules can also be measured based on the recoil dynamics of these DNA-microsphere complexes if appropriate corrections are made to the friction coefficient of the microsphere in the vicinity of the substrate. Comparison between co...

  20. Astrocytes as a source for Extracellular matrix molecules and cytokines

    Directory of Open Access Journals (Sweden)

    Stefan eWiese

    2012-06-01

    Full Text Available Research of the past 25 years has shown that astrocytes do more than participating and building up the blood brain barrier and detoxify the active synapse by reuptake of neurotransmitters and ions. Indeed, astrocytes express neurotransmitter receptors and, as a consequence, respond to stimuli. Deeper knowledge of the differentiation processes during development of the central nervous system (CNS might help explaining and even help treating neurological diseases like Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS and psychiatric disorders in which astrocytes have been shown to play a role. Astrocytes and oligodendrocytes develop from a multipotent stem cell that prior to this has produced primarily neuronal precursor cells. This switch towards the more astroglial differentiation is regulated by a change in receptor composition on the cell surface and responsiveness of the respective trophic factors Fibroblast growth factor (FGF and Epidermal growth factor (EGF. The glial precursor cell is driven into the astroglial direction by signaling molecules like Ciliary neurotrophic factor (CNTF, Bone Morphogenetic Proteins (BMPs, and EGF. However, the early astrocytes influence their environment not only by releasing and responding to diverse soluble factors but also express a wide range of extracellular matrix (ECM molecules, in particular proteoglycans of the lectican family and tenascins. Lately these ECM molecules have been shown to participate in glial development. In this regard, especially the matrix protein Tenascin C (Tnc proved to be an important regulator of astrocyte precursor cell proliferation and migration during spinal cord development. On the other hand, ECM molecules expressed by reactive astrocytes are also known to act mostly in an inhibitory fashion under pathophysiological conditions. In this regard, we further summarize recent data concerning the role of chondroitin sulfate proteoglycans and Tnc under pathological

  1. Spin squeezing an ultracold molecule

    CERN Document Server

    Bhattacharya, M

    2015-01-01

    Most research on spin squeezing thus far has focused on realizations involving either atomic or nuclear degrees of freedom. In this article we discuss a concrete proposal for spin squeezing the ultracold ground state polar paramagnetic molecule OH, a system currently under fine control in the laboratory. Starting from an experimentally relevant effective Hamiltonian, we identify a parameter regime where different combinations of static electric and magnetic fields can be used to realize the single-axis twisting Hamiltonian of Kitagawa and Ueda [M. Kitagawa and M. Ueda, Phys. Rev. A 47, 5138 (1993)], the uniform field Hamiltonian proposed by Law et al. [C. K. Law, H. T Ng and P. T. Leung, Phys. Rev. A 63, 055601 (2001)], and a model of field propagation in a Kerr medium considered by Agarwal and Puri [G. S. Agarwal and R. R. Puri, Phys. Rev. A 39, 2969 (1989)]. To support our conclusions, we provide analytical expressions as well as numerical calculations, including optimization of field strengths and accounti...

  2. Molecules Best Paper Award 2015

    Directory of Open Access Journals (Sweden)

    Derek J. McPhee

    2015-01-01

    Full Text Available Molecules instituted some years ago a “Best Paper” award to recognize the most outstanding papers in the area of organic synthesis, natural products, medicinal chemistry and molecular diversity published each year in Molecules. We are pleased to announce the third “Molecules Best Paper Award” for 2015. The winners were chosen by the Editor-in-Chief and selected editorial board members from among all the papers published in 2011. Reviews and research papers were evaluated separately. We are pleased to announce that the following eight papers have won the Molecules Best Paper Award for 2015:[...

  3. STM investigation of surfactant molecules

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Adsorption and self-organization of sodium alkyl sulfonates (STS and SHS) have been studied on HOPG by using the in situ scanning tunneling microscopy (STM). Both SHS and STS molecules adsorb on the HOPG surface and form long-range well-ordered monolayers. The neighboring molecules in different rows form a "head to head" configuration. In the high-resolution images of STS and SHS molecules, one end of the molecules shows bright spots which are attributed to the SO3- groups.

  4. Comprehensive Map of Molecules Implicated in Obesity.

    Science.gov (United States)

    Jagannadham, Jaisri; Jaiswal, Hitesh Kumar; Agrawal, Stuti; Rawal, Kamal

    2016-01-01

    Obesity is a global epidemic affecting over 1.5 billion people and is one of the risk factors for several diseases such as type 2 diabetes mellitus and hypertension. We have constructed a comprehensive map of the molecules reported to be implicated in obesity. A deep curation strategy was complemented by a novel semi-automated text mining system in order to screen 1,000 full-length research articles and over 90,000 abstracts that are relevant to obesity. We obtain a scale free network of 804 nodes and 971 edges, composed of 510 proteins, 115 genes, 62 complexes, 23 RNA molecules, 83 simple molecules, 3 phenotype and 3 drugs in "bow-tie" architecture. We classify this network into 5 modules and identify new links between the recently discovered fat mass and obesity associated FTO gene with well studied examples such as insulin and leptin. We further built an automated docking pipeline to dock orlistat as well as other drugs against the 24,000 proteins in the human structural proteome to explain the therapeutics and side effects at a network level. Based upon our experiments, we propose that therapeutic effect comes through the binding of one drug with several molecules in target network, and the binding propensity is both statistically significant and different in comparison with any other part of human structural proteome. PMID:26886906

  5. Comprehensive Map of Molecules Implicated in Obesity.

    Directory of Open Access Journals (Sweden)

    Jaisri Jagannadham

    Full Text Available Obesity is a global epidemic affecting over 1.5 billion people and is one of the risk factors for several diseases such as type 2 diabetes mellitus and hypertension. We have constructed a comprehensive map of the molecules reported to be implicated in obesity. A deep curation strategy was complemented by a novel semi-automated text mining system in order to screen 1,000 full-length research articles and over 90,000 abstracts that are relevant to obesity. We obtain a scale free network of 804 nodes and 971 edges, composed of 510 proteins, 115 genes, 62 complexes, 23 RNA molecules, 83 simple molecules, 3 phenotype and 3 drugs in "bow-tie" architecture. We classify this network into 5 modules and identify new links between the recently discovered fat mass and obesity associated FTO gene with well studied examples such as insulin and leptin. We further built an automated docking pipeline to dock orlistat as well as other drugs against the 24,000 proteins in the human structural proteome to explain the therapeutics and side effects at a network level. Based upon our experiments, we propose that therapeutic effect comes through the binding of one drug with several molecules in target network, and the binding propensity is both statistically significant and different in comparison with any other part of human structural proteome.

  6. Cell surface marker profiling of human tracheal basal cells reveals distinct subpopulations, identifies MST1/MSP as a mitogenic signal, and identifies new biomarkers for lung squamous cell carcinomas

    OpenAIRE

    Van de Laar, Emily; Clifford, Monica; Hasenoeder, Stefan; Kim, Bo Ram; Wang, Dennis; Lee, Sharon; Paterson, Josh; Vu, Nancy M; Waddell, Thomas K; Keshavjee, Shaf; Tsao, Ming-sound; Ailles, Laurie; Moghal, Nadeem

    2014-01-01

    Background The large airways of the lungs (trachea and bronchi) are lined with a pseudostratified mucociliary epithelium, which is maintained by stem cells/progenitors within the basal cell compartment. Alterations in basal cell behavior can contribute to large airway diseases including squamous cell carcinomas (SQCCs). Basal cells have traditionally been thought of as a uniform population defined by basolateral position, cuboidal cell shape, and expression of pan-basal cell lineage markers l...

  7. Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Solberg, H; Løber, D; Eriksen, J;

    1992-01-01

    -hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Danø, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand...... of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached...... by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)...

  8. CELL-SURFACE BINDING OF DEOXYNIVALENOL TO Lactobacillus paracasei subsp. tolerans ISOLATED FROM SOURDOUGH STARTER CULTURE

    Directory of Open Access Journals (Sweden)

    Yousef I. Hassan

    2013-04-01

    Full Text Available Deoxynivalenol (DON and fumonisin B1 (FB1 are two contaminant-mycotoxins frequently found in food commodities produced under poor conditions. Several methods have been suggested for the detoxification of such mycotoxins. Among the proposed methods, biological detoxification seems to be the most promising and cost-efficient. This study explores the capability of one strain of lactic acid bacteria, identified as Lactobacillus paracasei subsp. tolerans, to bind both DON and FB1 in liquid cultures. Here we report the ability of heat-inactivated cells to significantly reduce concentrations of DON in liquid cultures. Further mechanistic investigation showed that the detoxification process is a result of the physical binding of such mycotoxins to the cell wall of this bacterium.

  9. Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

    Directory of Open Access Journals (Sweden)

    Han Lanlan

    2011-10-01

    Full Text Available Abstract Background Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB. In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Results Multiple sequence alignment revealed that the C-terminal region (LcsB of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP or beta-galactosidase (Gal was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. Conclusion The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological

  10. Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Wieczorek Andrew S

    2010-09-01

    Full Text Available Abstract Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA. Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase, and were

  11. Identification of the immunoproteome of the meningococcus by cell surface immunoprecipitation and MS.

    Science.gov (United States)

    Newcombe, Jane; Mendum, Tom A; Ren, Chuan-peng; McFadden, Johnjoe

    2014-02-01

    Most healthy adults are protected from meningococcal disease by the presence of naturally acquired anti-meningococcal antibodies; however, the identity of the target antigens of this protective immunity remains unclear, particularly for protection against serogroup B disease. To identify the protein targets of natural protective immunity we developed an immunoprecipitation and proteomics approach to define the immunoproteome of the meningococcus. Sera from 10 healthy individuals showing serum bactericidal activity against both a meningococcal C strain (L91543) and the B strain MC58, together with commercially available pooled human sera, were used as probe antisera. Immunoprecipitation was performed with each serum sample and live cells from both meningococcal strains. Immunoprecipitated proteins were identified by MS. Analysis of the immunoproteome from each serum demonstrated both pan-reactive antigens that were recognized by most sera as well as subject-specific antigens. Most antigens were found in both meningococcal strains, but a few were strain-specific. Many of the immunoprecipitated proteins have been characterized previously as surface antigens, including adhesins and proteases, several of which have been recognized as vaccine candidate antigens, e.g. factor H-binding protein, NadA and neisserial heparin-binding antigen. The data demonstrate clearly the presence of meningococcal antibodies in healthy individuals with no history of meningococcal infection and a wide diversity of immune responses. The identification of the immunoreactive proteins of the meningococcus provides a basis for understanding the role of each antigen in the natural immunity associated with carriage and may help to design vaccination strategies. PMID:24275101

  12. High contrast single molecule tracking in the pericellular coat

    Science.gov (United States)

    Scrimgeour, Jan; McLane, Louis T.; Curtis, Jennifer E.

    2014-03-01

    The pericellular coat is a robust, hydrated, polymer brush-like structure that can extend several micrometers into the extracellular space around living cells. By controlling access to the cell surface, acting as a filter and storage reservoir for proteins, and actively controlling tissue-immune system interactions, the cell coat performs many important functions at scales ranging from the single cell to whole tissues. The cell coat consists of a malleable backbone - the large polysaccharide hyaluronic acid (HA) - with its structure, material properties, and ultimately its bio-functionality tuned by a diverse set of HA binding proteins. These proteins add charge, cross-links and growth factor-like ligands to the coat To probe the dynamic behavior of this soft biomaterial we have used high contrast single molecule imaging, based on highly inclined laser illumination, to observe individual fluorescently labeled HA binding proteins within the cell coat. Our work focuses on the cell coat of living chondrocyte (cartilage) cells, and in particular the effect of the large, highly charged, protein aggrecan on the properties of the coat. Through single molecule imaging we observe that aggrecan is tightly tethered to HA, and plays an important role in cell coat extension and stiffening.

  13. Characterization of a Proteasome and TAP-independent Presentation of Intracellular Epitopes by HLA-B27 Molecules

    KAUST Repository

    Magnacca, A.

    2012-07-17

    Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes.

  14. Micro-Kelvin cold molecules.

    Energy Technology Data Exchange (ETDEWEB)

    Strecker, Kevin E.; Chandler, David W.

    2009-10-01

    We have developed a novel experimental technique for direct production of cold molecules using a combination of techniques from atomic optical and molecular physics and physical chemistry. The ability to produce samples of cold molecules has application in a broad spectrum of technical fields high-resolution spectroscopy, remote sensing, quantum computing, materials simulation, and understanding fundamental chemical dynamics. Researchers around the world are currently exploring many techniques for producing samples of cold molecules, but to-date these attempts have offered only limited success achieving milli-Kelvin temperatures with low densities. This Laboratory Directed Research and Development project is to develops a new experimental technique for producing micro-Kelvin temperature molecules via collisions with laser cooled samples of trapped atoms. The technique relies on near mass degenerate collisions between the molecule of interest and a laser cooled (micro-Kelvin) atom. A subset of collisions will transfer all (nearly all) of the kinetic energy from the 'hot' molecule, cooling the molecule at the expense of heating the atom. Further collisions with the remaining laser cooled atoms will thermally equilibrate the molecules to the micro-Kelvin temperature of the laser-cooled atoms.

  15. Heavy Exotic Molecules with Charm and Bottom

    CERN Document Server

    Liu, Yizhuang

    2016-01-01

    We revisit the formation of pion-mediated heavy-light exotic molecules with both charm and bottom and their chiral partners under the general strictures of both heavy-quark and chiral symmetry. The chiral exotic partners with good parity formed using the $(0^+, 1^+)$ multiplet are about twice more bound than their primary exotic partners formed using the $(0^-,1^-)$ multiplet. The chiral couplings across the multiplets $(0^\\pm, 1^\\pm)$ cause the chiral exotic partners to unbind, and the primary exotic molecules to be about twice more bound, for $J\\leq 1$. Our multi-channel coupling results show that only the charm isosinglet exotic molecules with $J^{PC}=1^{++}$ binds, which we identify as the reported neutral $X(3872)$. Also, the bottom isotriplet exotic with $J^{PC}=1^{+-}$ binds, which we identify as a mixture of the reported charged exotics $Z^+_b(10610)$ and $Z^+_b(10650)$. The bound isosinglet with $J^{PC}=1^{++}$ is suggested as a possible neutral $X_b(10532)$ not yet reported.

  16. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

    DEFF Research Database (Denmark)

    Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad;

    2010-01-01

    HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

  17. A dimeric peptide with erythropoiesis-stimulating activity uniquely affects erythropoietin receptor ligation and cell surface expression.

    Science.gov (United States)

    Verma, Rakesh; Green, Jennifer M; Schatz, Peter J; Wojchowski, Don M

    2016-08-01

    Erythropoiesis-stimulating agents (ESAs) that exert long-acting antianemia effects have been developed recently, but their mechanisms are poorly understood. Analyses reveal unique erythropoietin receptor (EPOR)-binding properties for one such ESA, the synthetic EPOR agonist peginesatide. Compared with recombinant human EPO and darbepoietin, peginesatide exhibited a slow on rate, but sustained EPOR residency and resistant displacement. In EPO-dependent human erythroid progenitor UT7epo cells, culture in peginesatide unexpectedly upmodulated endogenous cell surface EPOR levels with parallel increases in full-length EPOR-68K levels. These unique properties are suggested to contribute to the durable activity of this (and perhaps additional) dimeric peptide hematopoietic growth factor receptor agonist. PMID:27174804

  18. Macrophage migration inhibition factor against cell-surface antigens coded by the major histocompatibility complex and other genes in mice.

    Directory of Open Access Journals (Sweden)

    Ohashi,Katsuhide

    1983-02-01

    Full Text Available We developed an indirect capillary tube method to improve reproducibility of macrophage migration inhibition (MI tests using a one-way mixed lymphocyte culture. MI response could be induced to cell-surface antigens coded by either H-2 or non-H-2 (background genes. The sensitivity was more readily induced across H-2 + background differences. The presence of only background difference did not induce the MI response to much extent. High MI activities were obtained to antigens coded by either K end or D end of the H-2 complex + background difference. Moderate activities were induced across the H-2D difference + background. These results suggest that the D region of the H-2 complex may direct a MI response when an H-2I difference is present during sensitization.

  19. Protein overexport in a Saccharomyces cerevisiae mutant is not due to facilitated release of cell-surface proteins.

    Science.gov (United States)

    Alexieva, K I; Venkov, P V

    2000-01-01

    Saccharomyces cerevisiae strain MW11 is a temperature-sensitive mutant which exports twenty times more proteins at 37 degrees C than parental or wild-type strains do. To understand the mechanism underlying the protein overexport in the mutant the possibility of an altered cell-wall structure leading to facilitated release of cell-surface proteins was studied. Data on calcofluor white and zymolyase sensitivities, resistance to killer 1 toxin and determination of exported acid phosphatase and invertase did not provide evidence for alterations in the cell-wall structure that could explain the protein overexport phenotype. The results were obtained in experiments when transcription of mutated gene was discontinued which permits the full expression of the protein overexport phenotype.

  20. Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

    Science.gov (United States)

    Babon, Aurélie; Wurceldorf, Thibault; Almunia, Christine; Pichard, Sylvain; Chenal, Alexandre; Buhot, Cécile; Beaumelle, Bruno; Gillet, Daniel

    2016-06-15

    We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes. PMID:26253725

  1. Structure-function Aspects of Extracellular Leucine-rich Repeat-containing Cell Surface Receptors in Plants

    Institute of Scientific and Technical Information of China (English)

    Zhao Zhang; Bart PHJ Thomma

    2013-01-01

    Plants exploit several types of cell surface receptors for perception of extracellular signals, of which the extracellular leucine-rich repeat (eLRR)-containing receptors form the major class. Although the function of most plant eLRR receptors remains unclear, an increasing number of these receptors are shown to play roles in innate immunity and a wide variety of developmental processes. Recent efforts using domain swaps, gene shuffling analyses, site-directed mutagenesis, interaction studies, and crystallographic analyses resulted in the current knowledge on ligand binding and the mechanism of activation of plant eLRR receptors. This review provides an overview of eLRR receptor research, specifically summarizing the recent understanding of interactions among plant eLRR receptors, their co-receptors and corresponding ligands. The functions of distinct eLRR receptor domains, and their role in structure, ligand perception and multimeric complex formation are discussed.

  2. Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Rettig, W.F.; Garin-Chesa, P.; Beresford, H.R.; Oettgen, H.F.; Melamed, M.R.; Old, L.J.

    1988-05-01

    Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and > 200 tumor specimens. Differential patterns of F19, F24, G171, G253, S5, and Thy-1 antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes.

  3. The Screw-Like Movement of a Gliding Bacterium Is Powered by Spiral Motion of Cell-Surface Adhesins.

    Science.gov (United States)

    Shrivastava, Abhishek; Roland, Thibault; Berg, Howard C

    2016-09-01

    Flavobacterium johnsoniae, a rod-shaped bacterium, glides over surfaces at speeds of ∼2 μm/s. The propulsion of a cell-surface adhesin, SprB, is known to enable gliding. We used cephalexin to generate elongated cells with irregular shapes and followed their displacement in three dimensions. These cells rolled about their long axes as they moved forward, following a right-handed trajectory. We coated gold nanoparticles with an SprB antibody and tracked them in three dimensions in an evanescent field where the nanoparticles appeared brighter when they were closer to the glass. The nanoparticles followed a right-handed spiral trajectory on the surface of the cell. Thus, if SprB were to adhere to the glass rather than to a nanoparticle, the cell would move forward along a right-handed trajectory, as observed, but in a direction opposite to that of the nanoparticle. PMID:27602728

  4. The role of a cell surface inhibitor in early signal transduction associated with the regulation of cell division and differentiation

    Science.gov (United States)

    Johnson, T. C.; Enebo, D. J.; Moos, P. J.; Fattaey, H. K.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Serum stimulation of quiescent human fibroblast cultures resulted in a hyperphosphorylation of the nuclear retinoblastoma gene susceptibility product (RB). However, serum stimulation in the presence of 9 x 10(-8) M of a purified bovine sialoglycopeptide (SGP) cell surface inhibitor abrogated the hyperphosphorylation of the RB protein and the subsequent progression of cells through the mitotic cycle. The experimental results suggest that the SGP mediated its cell cycle arrest at a site in the cell cycle that was at the time of RB phosphorylation or somewhat upstream of the modification of this regulatory protein of cell division. Both cells serum-deprived and serum stimulated in the presence of the SGP displayed only a hypophosphorylated RB protein, consistent with the SGP-mediated cell cycle arrest point being near the G1/S interface.

  5. Lectin Histochemical Study of Cell Surface Glycoconjugate in Gastric Carcinoma Using Helix Pomatia Agglutinin

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Arab

    2010-07-01

    Full Text Available "nAltered glycosylation of proteins in cancer cells is one of the main processes responsible for anaplasia, invasion and metastatic potential of neoplastic cells. Lectins are nonimmunogenetic compounds which specifically detect certain terminal sugars of glycoconjugates. The aim of the present study was to identify the N-acetylgalactosamine (GalNac containing glycoconjugates in cancer cells in all grades of gastric carcinoma. Paraffin blocks belong to 30 patients of gastric carcinoma (10 cases from each grade was collected from pathology file of Ali-Ebn-Abitaleb Hospital in Zahedan during 2005-2007. Prepared sections (5-7μm in thickness were stained by Alcian Blue, hematoxylin and eosin (H&E and helix pomatia agglutinin (HPA conjugated lectin. Lectin diluted up to 10μg/ml in PBS (0.1M, pH=6.8. Lectin reactivity was visualized by 0.03% diaminobenzidine (DAB solution. Sections were graded according to staining intensity to lectin (0-4+. Although there was some difference for lectin staining intensity between cancer cells in different grades of gastric carcinoma, statistical analysis showed that there was only a significant difference for cancer cells reactivity between histopathological grades of II and III. The pattern of reactivity to HPA lectin were also different from all histopathological grades. It seems that in cancer cells, the amount and distribution of GalNac containing glycoconjugate differ from neoplastic cells of different histopathological grades in gastric carcinoma.

  6. Lectin Histochemical Study of Cell Surface Glycoconjugate in Gastric Carcinoma Using Helix Pomatia Agglutinin

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Arab

    2010-08-01

    Full Text Available Altered glycosylation of proteins in cancer cells is one of the main processes responsible for anaplasia, invasion and metastatic potential of neoplastic cells. Lectins are nonimmunogenetic compounds which specifically detect certain terminal sugars of glycoconjugates. The aim of the present study was to identify the N-acetylgalactosamine (GalNac containing glycoconjugates in cancer cells in all grades of gastric carcinoma. Paraffin blocks belong to 30 patients of gastric carcinoma (10 cases from each grade was collected from pathology file of Ali-Ebn-Abitaleb Hospital in Zahedan during 2005-2007. Prepared sections (5-7μm in thickness were stained by Alcian Blue, hematoxylin and eosin (H&E and helix pomatia agglutinin (HPA conjugated lectin. Lectin diluted up to 10μg/ml in PBS (0.1M, pH=6.8. Lectin reactivity was visualized by 0.03% diaminobenzidine (DAB solution. Sections were graded according to staining intensity to lectin (0-4+. Although there was some difference for lectin staining intensity between cancer cells in different grades of gastric carcinoma, statistical analysis showed that there was only a significant difference for cancer cells reactivity between histopathological grades of II and III. The pattern of reactivity to HPA lectin were also different from all histopathological grades. It seems that in cancer cells, the amount and distribution of GalNac containing glycoconjugate differ from neoplastic cells of different histopathological grades in gastric carcinoma.

  7. Nanoparticles inhibit cancer cell invasion and enhance antitumor efficiency by targeted drug delivery via cell surface-related GRP78

    Directory of Open Access Journals (Sweden)

    Zhao L

    2014-12-01

    Full Text Available Liang Zhao,1,* Hongdan Li,2,* Yijie Shi,1 Guan Wang,2 Liwei Liu,1 Chang Su,3 Rongjian Su2 1School of Pharmacy, Liaoning Medical University, Jinzhou, People’s Republic of China; 2Central Laboratory of Liaoning Medical University, Jinzhou, People’s Republic of China; 3School of Veterinary Medicine, Liaoning Medical University, Jinzhou, People’s Republic of China *These authors contributed equally to this work Abstract: Nanoparticles (NPs which target specific agents could effectively recognize the target cells and increase the stability of chemical agents by encapsulation. As such, NPs have been widely used in cancer treatment research. Recently, over 90% of treatment failure cases in patients with metastatic cancer were attributed to resistance to chemotherapy. Surface-exposed glucose-regulated protein of 78 kDa (GRP78 is expressed highly on many tumor cell surfaces in many human cancers and is related to the regulation of invasion and metastasis. Herein, we report that NPs conjugated with antibody against GRP78 (mAb GRP78-NPs inhibit the adhesion, invasion, and metastasis of hepatocellular carcinoma (HCC and promote drug delivery of 5-fluorouracil into GRP78 high-expressed human hepatocellular carcinoma cells. Our new findings suggest that mAb GRP78-NPs could enhance drug accumulation by effectively transporting NPs into cell surface GRP78-overexpressed human hepatocellular carcinoma cells and then inhibit cell proliferation and viability and induce cell apoptosis by regulating caspase-3. In brief, mAb GRP78-NPs effectively inhibit cancer cell invasion and enhance antitumor efficiency by targeted drug delivery. Keywords: 5-Fu, apoptosis, HCC, caspase-3

  8. Cell surface area regulation in neurons in hippocampal slice cultures is resistant to oxygen-glucose deprivation

    Directory of Open Access Journals (Sweden)

    Natalya Shulyakova

    2010-09-01

    Full Text Available Natalya Shulyakova1,2, Jamie Fong2, Diana Diec2, Adrian Nahirny1,2, Linda R Mills1,21Department of Physiology, University of Toronto, Toronto, ON, Canada, M5T 2S8; 2Toronto Western Hospital Research Institute, University Health Network, 11-430, 399 Bathurst St, Toronto, ON, Canada, M5T 2S8Background: Neurons swell in response to a variety of insults. The capacity to recover, ie, to shrink, is critical for neuronal function and survival. Studies on dissociated neurons have shown that during swelling and shrinking, neurons reorganize their plasma membrane; as neurons swell, in response to hypo-osmotic media, the bilayer area increases. Upon restoration of normo-osmotic media, neurons shrink, forming transient invaginations of the plasma membrane known as vacuole-like dilations (VLDs, to accommodate the decrease in the bilayer.Methods: Here we used confocal microscopy to monitor neuronal swelling and shrinking in the three-dimensional (3D environment of post-natal rat hippocampal slice cultures. To label neurons, we used biolistic transfection, to introduce enhanced green fluorescent protein (eGFP targeted to the cytoplasm; and a membrane targeted GFP (lckGFP, targeted to the plasma membrane.Results: Neurons in slice cultures swelled and shrank in response to hypo-osmotic to normo-osmotic media changes. Oxygen-glucose deprivation (OGD caused sustained neuronal swelling; after reperfusion, some neurons recovered but in others, VLD recovery was stalled. OGD did not impair neuronal capacity to recover from a subsequent osmotic challenge.Conclusion: These results suggest cell surface area regulation (SAR is an intrinsic property of neurons, and that neuronal capacity for SAR may play an important role in the brain’s response to ischemic insults.Keywords: neurons, swelling, ischemia, cell surface area, hippocampal slice culture

  9. Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach

    Science.gov (United States)

    Xu, Xiaochun; Sinha, Lagnojita; Singh, Aparna; Yang, Cynthia; Xiang, Jialing; Tichauer, Kenneth M.

    2015-03-01

    Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, "untargeted" imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.

  10. The Role of Putative Phosphatidylserine-Interactive Residues of Tissue Factor on Its Coagulant Activity at the Cell Surface.

    Directory of Open Access Journals (Sweden)

    Shabbir A Ansari

    Full Text Available Exposure of phosphatidylserine (PS on the outer leaflet of the cell membrane is thought to play a critical role in tissue factor (TF decryption. Recent molecular dynamics simulation studies suggested that the TF ectodomain may directly interact with PS. To investigate the potential role of TF direct interaction with the cell surface phospholipids on basal TF activity and the enhanced TF activity following the decryption, one or all of the putative PS-interactive residues in the TF ectodomain were mutated and tested for their coagulant activity in cell systems. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A- exhibited a similar TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A, and TFK166A, was reduced substantially. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% inhibition. Mutation of all nine lipid binding residues together did not further decrease the activity of TF compared to TFG164A. A similar fold increase in TF activity was observed in wild-type TF and all TF mutants following the treatment of THP-1 cells with either calcium ionomycin or HgCl2, two agents that are commonly used to decrypt TF. Overall, our data show that a few select TF residues that are implicated in interacting with PS contribute to the TF coagulant activity at the cell surface. However, our data also indicate that TF regions outside of the putative lipid binding region may also contribute to PS-dependent decryption of TF.

  11. The Role of Putative Phosphatidylserine-Interactive Residues of Tissue Factor on Its Coagulant Activity at the Cell Surface.

    Science.gov (United States)

    Ansari, Shabbir A; Pendurthi, Usha R; Sen, Prosenjit; Rao, L Vijaya Mohan

    2016-01-01

    Exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane is thought to play a critical role in tissue factor (TF) decryption. Recent molecular dynamics simulation studies suggested that the TF ectodomain may directly interact with PS. To investigate the potential role of TF direct interaction with the cell surface phospholipids on basal TF activity and the enhanced TF activity following the decryption, one or all of the putative PS-interactive residues in the TF ectodomain were mutated and tested for their coagulant activity in cell systems. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A- exhibited a similar TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A, and TFK166A, was reduced substantially. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% inhibition. Mutation of all nine lipid binding residues together did not further decrease the activity of TF compared to TFG164A. A similar fold increase in TF activity was observed in wild-type TF and all TF mutants following the treatment of THP-1 cells with either calcium ionomycin or HgCl2, two agents that are commonly used to decrypt TF. Overall, our data show that a few select TF residues that are implicated in interacting with PS contribute to the TF coagulant activity at the cell surface. However, our data also indicate that TF regions outside of the putative lipid binding region may also contribute to PS-dependent decryption of TF. PMID:27348126

  12. Citrobacter amalonaticus phytase on the cell surface of Pichia pastoris exhibits high pH stability as a promising potential feed supplement.

    Science.gov (United States)

    Li, Cheng; Lin, Ying; Huang, Yuanyuan; Liu, Xiaoxiao; Liang, Shuli

    2014-01-01

    Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.

  13. One remarkable molecule: filaggrin.

    Science.gov (United States)

    Brown, Sara J; McLean, W H Irwin

    2012-03-01

    The discovery, in 2006, that loss-of-function mutations in the filaggrin (FLG) gene are the cause of ichthyosis vulgaris-the most common disorder of keratinization-and also a strong genetic risk factor for atopic eczema, marked a significant breakthrough in the understanding of eczema pathogenesis. Subsequent investigations of the role of FLG-null mutations have identified a series of significant associations with atopic disease phenotypes, including atopic asthma, allergic rhinitis, and peanut allergy. However, many questions remain to be answered in relation to the precise mechanisms by which deficiency of an intracellular protein expressed primarily in the differentiating epidermis may contribute to the development of cutaneous and systemic pathology. This review aims to highlight the key milestones in filaggrin research over the past 25 years, to discuss the mechanistic, clinical, and therapeutic implications, and to consider possible future directions for ongoing investigation.

  14. Identification of ATP synthase beta subunit (ATPB on the cell surface as a non-small cell lung cancer (NSCLC associated antigen

    Directory of Open Access Journals (Sweden)

    Qian Zhi

    2009-01-01

    Full Text Available Abstract Background Antibody-based immuneotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers for tumor diagnosis and therapy. Methods The membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7 was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7. Results The monoclonal antibody 4E7 (McAb4E7 specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemstry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC, but not in small cell lung cancer (SCLC. The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively. Conclusion In the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

  15. Influence of manufacturing processes on cell surface properties of probiotic strain Lactobacillus rhamnosus Lcr35®.

    Science.gov (United States)

    Nivoliez, Adrien; Veisseire, Philippe; Alaterre, Elina; Dausset, Caroline; Baptiste, Fabrice; Camarès, Olivier; Paquet-Gachinat, Marylise; Bonnet, Muriel; Forestier, Christiane; Bornes, Stéphanie

    2015-01-01

    The influence of the industrial process on the properties of probiotics, administered as complex manufactured products, has been poorly investigated. In the present study, we comparatively assessed the cell wall characteristics of the probiotic strain Lactobacillus rhamnosus Lcr35® together with three of its commercial formulations with intestinal applications. Putative secreted and transmembrane-protein-encoding genes were initially searched in silico in the genome of L. rhamnosus Lcr35®. A total of 369 candidate genes were identified which expressions were followed using a custom Lactobacillus DNA chip. Among them, 60 or 67 genes had their expression either upregulated or downregulated in the Lcr Restituo® packet or capsule formulations, compared to the native Lcr35® strain. Moreover, our data showed that the probiotic formulations (Lcr Lenio®, Lcr restituo® capsule and packet) showed a better capacity to adhere to intestinal epithelial Caco-2 cells than the native Lcr35® strain. Microbial (MATS) tests showed that the probiotic was an electron donor and that they were more hydrophilic than the native strain. The enhanced adhesion capacity of the active pharmaceutical ingredients (APIs) to epithelial Caco-2 cells and their antipathogen effect could be due to this greater surface hydrophilic character. These findings suggest that the manufacturing process influences the protein composition and the chemical properties of the cell wall. It is therefore likely that the antipathogen effect of the formulation is modulated by the industrial process. Screening of the manufactured products' properties would therefore represent an essential step in evaluating the effects of probiotic strains. PMID:25280746

  16. Multiorbital tunneling ionization of the CO molecule

    CERN Document Server

    Wu, J; Kunitski, M; Meckel, M; Voss, S; Sann, H; Kim, H; Jahnke, T; Czasch, A; Dörner, R

    2012-01-01

    We coincidently measure the molecular frame photoelectron angular distribution and the ion sum-momentum distribution of single and double ionization of CO molecules by using circularly and elliptically polarized femtosecond laser pulses, respectively. The orientation dependent ionization rates for various kinetic energy releases allow us to individually identify the ionizations of multiple orbitals, ranging from the highest occupied to the next two lower-lying molecular orbitals for various channels observed in our experiments. Not only the emission of a single electron, but also the sequential tunneling dynamics of two electrons from multiple orbitals are traced step by step. Our results confirm that the shape of the ionizing orbitals determine the strong laser field tunneling ionization in the CO molecule, whereas the linear Stark effect plays a minor role.

  17. Prebiotic molecules and interstellar grain clumps

    International Nuclear Information System (INIS)

    It is stated that interstellar molecules detected by radioastronomical techniques in galactic clouds cover a wide range of types and complexities. Amongst the heaviest recently discovered is cyanodiacetylene. There have also been earlier detections of precursors to the simplest amino-acid, glycine and probably detections of polyoxymethylene polymers and co-polymers. A possible identification of organic molecules of even greater complexity is here discussed, together with implications for the commencement of biological activity. The large departures from thermodynamic equilibrium in the interstellar medium and the co-existence of solid grains, molecules, radicals, ions, and uv photons provide conditions that are ideal for production of 'exotic' molecular species. The effect of clumping of dust grains is discussed. The possible spectral identification of highly complex organic species in the interstellar medium is also discussed and reference is made to a property common to a wide class of such molecules, that is, an absorption band centered at 2,200 A. It is tempting to identify this feature with the well-known 2,200 A band of the interstellar extinction curve. It is thought that it may be tentatively concluded that the data so far obtained could be interpreted as independent new chemical evidence of the existence of composite grain clumps in the interstellar medium and in carbonaceous chondrites, and that these grain clumps probably include a significant mass fraction of highly complex organic pre-biotic molecules that could have led to the start and dispersal of biological activity on the Earth and elsewhere in the Galaxy. Processes of natural selection probably also played an important part, particularly in the production of self-replicable peptide chains. The problem of protection of pre-biotic material against external disruptive agencies, such as u/v light, is also discussed. (U.K.)

  18. Identifiability in stochastic models

    CERN Document Server

    1992-01-01

    The problem of identifiability is basic to all statistical methods and data analysis, occurring in such diverse areas as Reliability Theory, Survival Analysis, and Econometrics, where stochastic modeling is widely used. Mathematics dealing with identifiability per se is closely related to the so-called branch of ""characterization problems"" in Probability Theory. This book brings together relevant material on identifiability as it occurs in these diverse fields.

  19. Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1F3 and C-terminal modules of fibronectin.

    Directory of Open Access Journals (Sweden)

    Jielin Xu

    Full Text Available BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7F3-(10F3. Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7F3-(10F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7F3-(10F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2F3-(14F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1F3 or the C-terminal modules to modules (2F3-(14F3 resulted in some activity, and addition of both (1F3 and the C-terminal modules resulted in a construct, (1F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1F3-C V0, (1F3-C V64, and (1F3-C Delta(V(15F3(10F1 were all able to support fibronectin assembly, suggesting that (1F3 through (11F1 and/or (12F1 were important for activity. Coatings in which the active parts of (1F3-C were present in different proteins were much less active than intact (1F3-C. CONCLUSIONS: These results suggest that (1F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

  20. Effects of fluconazole treatment of mice infected with fluconazole-susceptible and -resistant Candida tropicalis on fungal cell surface hydrophobicity, adhesion and biofilm formation

    Directory of Open Access Journals (Sweden)

    R L Kanoshiki

    2015-01-01

    Full Text Available Background : The incidence of Candida tropicalis less susceptible to fluconazole (FLC has been reported in many parts of the world. Objectives : The aim of this study was to examine the changes of putative virulence attributes of Candida tropicalis accompanying the development of resistance to FLC in vitro and in vivo. Materials and Methods : A FLC-resistant strain (FLC-R was obtained after sequential exposure of a clinical isolate FLC-sensitive (FLC-S to increasing concentrations of the antifungal. The course of infection by both strains was analyzed in BALB/c mice. Analyses of gene expression were performed by real-time polymerase chain reaction PCR. The cell surface hydrophobicity, adhesion and biofilm formation were also determined. Results : Development of resistance to FLC could be observed after 15 days of subculture in azole-containing medium. Overexpression of MDR1 and ERG11 genes were observed in FLC-R, and this strain exhibited enhanced virulence in mice, as assessed by the mortality rate. All mice challenged with the FLC-R died and FLC-treatment caused earlier death in mice infected with this strain. All animals challenged with FLC-S survived the experiment, regardless of FLC-treatment. Overall, FLC-R derivatives strains were significantly more hydrophobic than FLC-S strains and showed greater adherence and higher capacity to form biofilm on polystyrene surface. Conclusions : The expression of virulence factors was higher in FLC-R-C. tropicalis and it was enhanced after FLC-exposure. These data alert us to the importance of identifying microorganisms that show resistance to the antifungals to establish an appropriate management of candidiasis therapy.