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Sample records for sustained matrix protein

  1. Sustained-release of FGF-2 from a hybrid hydrogel of heparin-poloxamer and decellular matrix promotes the neuroprotective effects of proteins after spinal injury

    Directory of Open Access Journals (Sweden)

    Xu HL

    2018-02-01

    Full Text Available  He-Lin Xu,1,* Fu-Rong Tian,1,* Jian Xiao,1,* Pian-Pian Chen,1 Jie Xu,1 Zi-Liang Fan,1 Jing-Jing Yang,1 Cui-Tao Lu,1 Ying-Zheng Zhao1,2 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 2Hainan Medical College, Haikou, China *These authors contributed equally to this work Introduction: The short lifetime of protein-based therapies has largely limited their therapeutic efficacy in injured nervous post-spinal cord injury (post-SCI. Methods: In this study, an affinity-based hydrogel delivery system provided sustained-release of proteins, thereby extending the efficacy of such therapies. The affinity-based hydrogel was constructed using a novel polymer, heparin-poloxamer (HP, as a temperature-sensitive bulk matrix and decellular spinal cord extracellular matrix (dscECM as an affinity depot of drug. By tuning the concentration of HP in formulation, the cold ternary fibroblast growth factor-2 (FGF2-dscECM-HP solution could rapidly gelatinize into a hydrogel at body temperature. Due to the strong affinity for FGF2, hybrid FGF2-dscECM-HP hydrogel enabled sustained-release of encapsulated FGF2 over an extended period in vitro. Results: Compared to free FGF2, it was observed that both neuron functions and tissue morphology after SCI were clearly recovered in rats treated with FGF2-dscECM-HP hydrogel. Moreover, the expression of neurofilament protein and the density of axons were increased after treatment with hybrid FGF2-dscECM-HP. In addition, the neuroprotective effects of FGF2-dscECM-HP were related to inhibition of chronic endoplasmic reticulum stress-induced apoptosis.Conclusion: The results revealed that a hybrid hydrogel system may be a potential carrier to deliver macromolecular proteins to the injured site and enhance the therapeutic effects of proteins.Keywords: spinal cord injury, decellularized extracellular matrix, thermosensitive hydrogel, adsorption, basic fibroblast growth factor

  2. Formulation of Sustained-Release Diltiazem Matrix Tablets Using ...

    African Journals Online (AJOL)

    Formulation of Sustained-Release Diltiazem Matrix Tablets Using Hydrophilic Gum Blends. A Moin, H.G Shivakumar. Abstract. Purpose: To develop sustained release matrix tablets of diltiazem hydrochloride (DTZ) using karaya gum (K) alone or in combination with locust bean gum (LB) and hydroxypropyl methylcellulose ...

  3. Ubiquitination of specific mitochondrial matrix proteins

    International Nuclear Information System (INIS)

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G.; Ciechanover, Aaron

    2016-01-01

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

  4. Ubiquitination of specific mitochondrial matrix proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lehmann, Gilad [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ziv, Tamar [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Braten, Ori [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Admon, Arie [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Udasin, Ronald G. [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ciechanover, Aaron, E-mail: aaroncie@tx.technion.ac.il [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel)

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

  5. Determination of insoluble avian eggshell matrix proteins

    Czech Academy of Sciences Publication Activity Database

    Mikšík, Ivan; Sedláková, Pavla; Lacinová, Kateřina; Pataridis, Statis; Eckhardt, Adam

    2010-01-01

    Roč. 397, č. 1 (2010), s. 205-214 ISSN 1618-2642 R&D Projects: GA MŠk(CZ) 1M0510; GA ČR(CZ) GA203/09/0675; GA ČR(CZ) GA203/08/1428 Institutional research plan: CEZ:AV0Z50110509 Keywords : eggshell proteins * insoluble proteins * matrix proteins Subject RIV: CE - Biochemistry Impact factor: 3.841, year: 2010

  6. Structure and assembly of a paramyxovirus matrix protein.

    Science.gov (United States)

    Battisti, Anthony J; Meng, Geng; Winkler, Dennis C; McGinnes, Lori W; Plevka, Pavel; Steven, Alasdair C; Morrison, Trudy G; Rossmann, Michael G

    2012-08-28

    Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.

  7. Characterization of membrane association of Rinderpest virus matrix protein

    International Nuclear Information System (INIS)

    Subhashri, R.; Shaila, M.S.

    2007-01-01

    Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M protein gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein

  8. Matrix Model for Choosing Green Marketing Sustainable Strategic Alternatives

    Directory of Open Access Journals (Sweden)

    Cătălina Sitnikov

    2015-08-01

    Full Text Available Green marketing examines the symbiotic role played by marketing in ensuring sustainable business, exploring issues concerning the environment and the way strategic decisions can influence it. At present, the environmental issues concern more and more the competitive approach any organization can implement. Based on this approach, organizations can gain competitive advantage by managing environmental variables and by developing and implementing green marketing strategies. Considering the importance and impact of green marketing, by using theoretical concepts and defining a set of research directions, the paper and the research conducted were focused on creating a matrix model for choosing the optimal green marketing strategy, oriented towards competitive advantage. The model is based on the correlation that can be established among the generic strategies of competitive advantage, the variables of extended marketing mix (7Ps and the green marketing strategy matrix. There are also analyzed the implications that may be generated within a company by the adoption of a green marketing strategy and its role in promoting the environmental benefits of products.

  9. Assessing Agricultural Intensification Strategies with a Sustainable Agriculture Matrix

    Science.gov (United States)

    Zhang, X.; Davidson, E. A.

    2017-12-01

    To meet the growing global demand for food and bioenergy, agricultural production must nearly double by 2050, placing additional pressures on the environment and the society. Thus, how to efficiently use limited land, water, and nutrient resources to produce more food with low pollution (MoFoLoPo) is clearly one of the major challenges of this century. The increasingly interconnected global market provides a great opportunity for reallocating crop production to the countries and regions that use natural resources more efficiently. For example, it is estimated that optimizing the allocation of crop production around the world can mitigate 41% of nitrogen lost to the environment. However, higher efficiency in nutrients use does not necessarily lead to higher efficiency in land use or water use. In addition, the increasing share of international trade in food supply may introduce additional systemic risk and affect the resilience of global food system. Using the data/indicator from a Sustainable Agriculture Matrix and an international trade matrix, we developed a simple model to assess the trade-offs of international trade considering resource use efficiencies (including water, land, nitrogen, and phosphorus), economic costs and benefits, and the resilience of food system.

  10. Statistical Optimization of Sustained Release Venlafaxine HCI Wax Matrix Tablet.

    Science.gov (United States)

    Bhalekar, M R; Madgulkar, A R; Sheladiya, D D; Kshirsagar, S J; Wable, N D; Desale, S S

    2008-01-01

    The purpose of this research was to prepare a sustained release drug delivery system of venlafaxine hydrochloride by using a wax matrix system. The effects of bees wax and carnauba wax on drug release profile was investigated. A 3(2) full factorial design was applied to systemically optimize the drug release profile. Amounts of carnauba wax (X(1)) and bees wax (X(2)) were selected as independent variables and release after 12 h and time required for 50% (t(50)) drug release were selected as dependent variables. A mathematical model was generated for each response parameter. Both waxes retarded release after 12 h and increases the t(50) but bees wax showed significant influence. The drug release pattern for all the formulation combinations was found to be approaching Peppas kinetic model. Suitable combination of two waxes provided fairly good regulated release profile. The response surfaces and contour plots for each response parameter are presented for further interpretation of the results. The optimum formulations were chosen and their predicted results found to be in close agreement with experimental findings.

  11. Cartilage oligomeric matrix protein in patients with juvenile idiopathic arthritis

    DEFF Research Database (Denmark)

    Bjørnhart, Birgitte; Juul, Anders; Nielsen, Susan

    2009-01-01

    Cartilage oligomeric matrix protein (COMP) has been identified as a prognostic marker of progressive joint destruction in rheumatoid arthritis. In this population based study we evaluated associations between plasma concentrations of COMP, disease activity, and growth velocity in patients...

  12. In vivo extracellular matrix protein expression by human periodontal ...

    African Journals Online (AJOL)

    ONOS

    2010-08-23

    Aug 23, 2010 ... Extracellular matrix proteins (ECM) are described as molecular regulators of these events. ..... zation and adhesive interaction of cells (Yamada, 1983). .... periodontal ligament fibroblasts after simulation of orthodontic force.

  13. Polyether sulfone/hydroxyapatite mixed matrix membranes for protein purification

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Junfen, E-mail: junfensun@dhu.edu.cn [State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, North People Road 2999, Shanghai 201620 (China); Wu, Lishun [Department of Chemistry and Chemical Engineering, Heze University, Daxue Road 2269, Heze, Shandong Province 274015 (China)

    2014-07-01

    This work proposes a novel approach for protein purification from solution using mixed matrix membranes (MMMs) comprising of hydroxyapatite (HAP) inside polyether sulfone (PES) matrix. The influence of HAP particle loading on membrane morphology is studied. The MMMs are further characterized concerning permeability and adsorption capacity. The MMMs show purification of protein via both diffusion as well as adsorption, and show the potential of using MMMs for improvements in protein purification techniques. The bovine serum albumin (BSA) was used as a model protein. The properties and structures of MMMs prepared by immersion phase separation process were characterized by pure water flux, BSA adsorption and scanning electron microscopy (SEM).

  14. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space

    NARCIS (Netherlands)

    Stuart, Rosemary A.; Gruhler, Albrecht; Klei, Ida van der; Guiard, Bernard; Koll, Hans; Neupert, Walter

    1994-01-01

    The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted.

  15. In vivo extracellular matrix protein expression by human periodontal ...

    African Journals Online (AJOL)

    It is well known that the orthodontic force applied to teeth generates a series of events that remodel the periodontal ligament (PDL). Extracellular matrix proteins (ECM) are described as molecular regulators of these events. However, the exact contribution of these proteins in human PDL modeling by orthodontic force ...

  16. Protein structure estimation from NMR data by matrix completion.

    Science.gov (United States)

    Li, Zhicheng; Li, Yang; Lei, Qiang; Zhao, Qing

    2017-09-01

    Knowledge of protein structures is very important to understand their corresponding physical and chemical properties. Nuclear Magnetic Resonance (NMR) spectroscopy is one of the main methods to measure protein structure. In this paper, we propose a two-stage approach to calculate the structure of a protein from a highly incomplete distance matrix, where most data are obtained from NMR. We first randomly "guess" a small part of unobservable distances by utilizing the triangle inequality, which is crucial for the second stage. Then we use matrix completion to calculate the protein structure from the obtained incomplete distance matrix. We apply the accelerated proximal gradient algorithm to solve the corresponding optimization problem. Furthermore, the recovery error of our method is analyzed, and its efficiency is demonstrated by several practical examples.

  17. Formulation of Sustained-Release Diltiazem Matrix Tablets Using ...

    African Journals Online (AJOL)

    Erah

    surface, their drug release behavior appears simple, but ... matrix material for the formulation of ..... formulation F5 (,) and reference formulations. ( , □). 0. 50. 100. 150. 200. 250. 300. 0. 3. 6 .... Coviello T, Matricardi P, Marianecci C, Alhaique F.

  18. Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

    Directory of Open Access Journals (Sweden)

    Wei-ling Cui

    2016-01-01

    Full Text Available Vascularization of acellular nerves has been shown to contribute to nerve bridging. In this study, we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves. The rat nerve defects were treated with acellular nerve grafting (control group alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein (experimental group. As shown through two-dimensional imaging, the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation, and gradually covered the entire graft at day 21. The vascular density, vascular area, and the velocity of revascularization in the experimental group were all higher than those in the control group. These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.

  19. A major protein component of the Bacillus subtilis biofilm matrix.

    Science.gov (United States)

    Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

    2006-02-01

    Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

  20. Mixed-matrix membrane adsorbers for protein separation

    NARCIS (Netherlands)

    Avramescu, M.E.; Borneman, Z.; Wessling, M.

    2003-01-01

    The separation of two similarly sized proteins, bovine serum albumin (BSA) and bovine hemoglobin (Hb) was carried out using a new type of ion-exchange mixed-matrix adsorber membranes. The adsorber membranes were prepared by incorporation of various types of Lewatit ion-exchange resins into an

  1. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Energy Technology Data Exchange (ETDEWEB)

    Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  2. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    International Nuclear Information System (INIS)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress

  3. Dry fractionation for sustainable production of functional legume protein concentrates

    NARCIS (Netherlands)

    Schutyser, M.A.I.; Pelgrom, P.J.M.; Goot, van der A.J.; Boom, R.M.

    2015-01-01

    Plant proteins gain increasing interest as part of a sustainable diet. Because plant materials not only contain protein, they are generally isolated via an energy intensive wet fractionation. This review discusses dry fractionation as an alternative and more sustainable route for producing

  4. Conformational plasticity of the Ebola virus matrix protein.

    Science.gov (United States)

    Radzimanowski, Jens; Effantin, Gregory; Weissenhorn, Winfried

    2014-11-01

    Filoviruses are the causative agents of a severe and often fatal hemorrhagic fever with repeated outbreaks in Africa. They are negative sense single stranded enveloped viruses that can cross species barriers from its natural host bats to primates including humans. The small size of the genome poses limits to viral adaption, which may be partially overcome by conformational plasticity. Here we review the different conformational states of the Ebola virus (EBOV) matrix protein VP40 that range from monomers, to dimers, hexamers, and RNA-bound octamers. This conformational plasticity that is required for the viral life cycle poses a unique opportunity for development of VP40 specific drugs. Furthermore, we compare the structure to homologous matrix protein structures from Paramyxoviruses and Bornaviruses and we predict that they do not only share the fold but also the conformational flexibility of EBOV VP40. © 2014 The Protein Society.

  5. Domain organizations of modular extracellular matrix proteins and their evolution.

    Science.gov (United States)

    Engel, J

    1996-11-01

    Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

  6. Binding of triiodothyronine to rat liver nuclear matrix. influence of thyroid hormones on the phosphorylation of nuclear matrix proteins

    International Nuclear Information System (INIS)

    Adylova, A.T.; Atakhanova, B.A.

    1986-01-01

    The interaction of thyroid hormones with rat liver nuclear matrix proteins was investigated. It was shown that the nuclear matrix contains sites that bind triiodothyronine with high affinity (K = 1.07.10 9 M -1 ) and limited capacity (the maximum binding capacity is equal to 28 /SUP a/ .5 fmoles of triiodothyronine per 100 ug protein). Electrophoretic identification of the matrix proteins that bind triiodothyronine was performed. The molecular weight of the main triiodothyronine-binding fraction is 50,000-52,000. It was shown that the administration of triiodothyronine to thyroidectomized rats stimulates the phosphorylation of all the protein fractions of the nuclear matrix

  7. Sustainable protein technology : an evaluation on the STW Protein programme and an outlook for the future

    NARCIS (Netherlands)

    Voudouris, Panagiotis; Tamayo Tenorio, Angelica; Lesschen, Jan Peter; Kyriakopoulou, Konstantina; Sanders, Johan P.M.; Goot, van der Atze Jan; Bruins, Marieke E.

    2017-01-01

    In 2013 a new STW research programme was started on sustainable protein recovery. This STW Protein Programme consisted of five sustainable protein technology projects, which aimed at developing innovative methods to extract proteins from plant leaves, microalgae and insects to meet the increasing

  8. Cartilage oligomeric matrix protein enhances matrix assembly during chondrogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S; Chen, Faye H

    2012-04-01

    Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention.

  9. CARTILAGE OLIGOMERIC MATRIX PROTEIN ENHANCES MATRIX ASSEMBLY DURING CHONDROGENESIS OF HUMAN MESENCHYMAL STEM CELLS

    Science.gov (United States)

    Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S.; Chen, Faye H.

    2011-01-01

    Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate-hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention. PMID:22095699

  10. Improved gel electrophoresis matrix for hydrophobic protein separation and identification.

    Science.gov (United States)

    Tokarski, Caroline; Fillet, Marianne; Rolando, Christian

    2011-03-01

    We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N'-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N'-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: -0.085) than in the classical gel (average GRAVY: -0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach. 2010 Elsevier Inc. All rights reserved.

  11. Development of Sustained Release Capsules Containing “Coated Matrix Granules of Metoprolol Tartrate”

    OpenAIRE

    Siddique, Sabahuddin; Khanam, Jasmina; Bigoniya, Papiya

    2010-01-01

    The objective of this investigation was to prepare sustained release capsule containing coated matrix granules of metoprolol tartrate and to study its in vitro release and in vivo absorption. The design of dosage form was performed by choosing hydrophilic hydroxypropyl methyl cellulose (HPMC K100M) and hydrophobic ethyl cellulose (EC) polymers as matrix builders and Eudragit® RL/RS as coating polymers. Granules were prepared by composing drug with HPMC K100M, EC, dicalcium phosphate by wet gr...

  12. Development and evaluation of Ketoprofen sustained release matrix tablet using Hibiscus rosa-sinensis leaves mucilage

    OpenAIRE

    Kaleemullah, M.; Jiyauddin, K.; Thiban, E.; Rasha, S.; Al-Dhalli, S.; Budiasih, S.; Gamal, O.E.; Fadli, A.; Eddy, Y.

    2016-01-01

    Currently, the use of natural gums and mucilage is of increasing importance in pharmaceutical formulations as valuable drug excipient. Natural plant-based materials are economic, free of side effects, biocompatible and biodegradable. Therefore, Ketoprofen matrix tablets were formulated by employing Hibiscus rosa-sinensis leaves mucilage as natural polymer and HPMC (K100M) as a synthetic polymer to sustain the drug release from matrix system. Direct compression method was used to develop susta...

  13. Interaction between the enamel matrix proteins amelogenin and ameloblastin

    International Nuclear Information System (INIS)

    Ravindranath, Hanumanth H.; Chen, Li-Sha; Zeichner-David, Margaret; Ishima, Rieko; Ravindranath, Rajeswari M.H.

    2004-01-01

    Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [ 3 H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly

  14. Interaction between the enamel matrix proteins amelogenin and ameloblastin.

    Science.gov (United States)

    Ravindranath, Hanumanth H; Chen, Li-Sha; Zeichner-David, Margaret; Ishima, Rieko; Ravindranath, Rajeswari M H

    2004-10-22

    Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [(3)H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly.

  15. Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

    Science.gov (United States)

    Yachie, Nozomu; Petsalaki, Evangelia; Mellor, Joseph C; Weile, Jochen; Jacob, Yves; Verby, Marta; Ozturk, Sedide B; Li, Siyang; Cote, Atina G; Mosca, Roberto; Knapp, Jennifer J; Ko, Minjeong; Yu, Analyn; Gebbia, Marinella; Sahni, Nidhi; Yi, Song; Tyagi, Tanya; Sheykhkarimli, Dayag; Roth, Jonathan F; Wong, Cassandra; Musa, Louai; Snider, Jamie; Liu, Yi-Chun; Yu, Haiyuan; Braun, Pascal; Stagljar, Igor; Hao, Tong; Calderwood, Michael A; Pelletier, Laurence; Aloy, Patrick; Hill, David E; Vidal, Marc; Roth, Frederick P

    2016-04-22

    High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  16. Molecular events in matrix protein metabolism in the aging kidney

    Science.gov (United States)

    Sataranatarajan, Kavithalakshmi; Feliers, Denis; Mariappan, Meenalakshmi M.; Lee, Hak Joo; Lee, Myung Ja; Day, Robert T.; Yalamanchili, Hima Bindu; Choudhury, Goutam G.; Barnes, Jeffrey L.; Van Remmen, Holly; Richardson, Arlan; Kasinath, Balakuntalam S.

    2018-01-01

    Summary We explored molecular events associated with aging-induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle-aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age-phase-specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle-aged mice. These changes occurred with increment in TGFβ mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFβ-regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR-21 and miR-200c, but not miR-192, miR-200a, or miR-200b, was increased with aging. Increased miR-21 and miR-200c contents were associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Agephase-specific regulation of matrix protein synthesis occurs and involves matrix protein-specific transcriptional and post-transcriptional mechanisms. PMID:23020145

  17. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Science.gov (United States)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

  18. Distance matrix-based approach to protein structure prediction.

    Science.gov (United States)

    Kloczkowski, Andrzej; Jernigan, Robert L; Wu, Zhijun; Song, Guang; Yang, Lei; Kolinski, Andrzej; Pokarowski, Piotr

    2009-03-01

    Much structural information is encoded in the internal distances; a distance matrix-based approach can be used to predict protein structure and dynamics, and for structural refinement. Our approach is based on the square distance matrix D = [r(ij)(2)] containing all square distances between residues in proteins. This distance matrix contains more information than the contact matrix C, that has elements of either 0 or 1 depending on whether the distance r (ij) is greater or less than a cutoff value r (cutoff). We have performed spectral decomposition of the distance matrices D = sigma lambda(k)V(k)V(kT), in terms of eigenvalues lambda kappa and the corresponding eigenvectors v kappa and found that it contains at most five nonzero terms. A dominant eigenvector is proportional to r (2)--the square distance of points from the center of mass, with the next three being the principal components of the system of points. By predicting r (2) from the sequence we can approximate a distance matrix of a protein with an expected RMSD value of about 7.3 A, and by combining it with the prediction of the first principal component we can improve this approximation to 4.0 A. We can also explain the role of hydrophobic interactions for the protein structure, because r is highly correlated with the hydrophobic profile of the sequence. Moreover, r is highly correlated with several sequence profiles which are useful in protein structure prediction, such as contact number, the residue-wise contact order (RWCO) or mean square fluctuations (i.e. crystallographic temperature factors). We have also shown that the next three components are related to spatial directionality of the secondary structure elements, and they may be also predicted from the sequence, improving overall structure prediction. We have also shown that the large number of available HIV-1 protease structures provides a remarkable sampling of conformations, which can be viewed as direct structural information about the

  19. Evaluating the environmental sustainability of biomass-based energy strategy: Using an impact matrix framework

    Energy Technology Data Exchange (ETDEWEB)

    Weldu, Yemane W., E-mail: ywweldem@ucalgary.ca [Faculty of Environmental Design, University of Calgary, Calgary, Alberta 2500, University Drive NW, T2N 1N4 (Canada); Assefa, Getachew [Faculty of Environmental Design, University of Calgary, Calgary, Alberta 2500, University Drive NW, T2N 1N4 (Canada); Athena Chair in Life Cycle Assessment in Design (Canada)

    2016-09-15

    A roadmap for a more sustainable energy strategy is complex, as its development interacts critically with the economic, social, and environmental dimensions of sustainable development. This paper applied an impact matrix method to evaluate the environmental sustainability and to identify the desirable policy objectives of biomass-based energy strategy for the case of Alberta. A matrix with the sustainability domains on one axis and areas of environmental impact on the other was presented to evaluate the nexus effect of policy objectives and bioenergy production. As per to our analysis, economic diversification, technological innovation, and resource conservation came up as the desirable policy objectives of sustainable development for Alberta because they demonstrated environmental benefits in all environmental impact categories, namely climate change, human health, and ecosystem. On the other hand, human health and ecosystem impacts were identified as trade-offs when the policy objectives for sustainability were energy security, job creation, and climate change. Thus, bioenergy can mitigate climate change but may impact human health and ecosystem which then in turn can become issues of concern. Energy strategies may result in shifting of risks from one environmental impact category to another, and from one sustainable domain to another if the technical and policy-related issues are not identified.

  20. Evaluating the environmental sustainability of biomass-based energy strategy: Using an impact matrix framework

    International Nuclear Information System (INIS)

    Weldu, Yemane W.; Assefa, Getachew

    2016-01-01

    A roadmap for a more sustainable energy strategy is complex, as its development interacts critically with the economic, social, and environmental dimensions of sustainable development. This paper applied an impact matrix method to evaluate the environmental sustainability and to identify the desirable policy objectives of biomass-based energy strategy for the case of Alberta. A matrix with the sustainability domains on one axis and areas of environmental impact on the other was presented to evaluate the nexus effect of policy objectives and bioenergy production. As per to our analysis, economic diversification, technological innovation, and resource conservation came up as the desirable policy objectives of sustainable development for Alberta because they demonstrated environmental benefits in all environmental impact categories, namely climate change, human health, and ecosystem. On the other hand, human health and ecosystem impacts were identified as trade-offs when the policy objectives for sustainability were energy security, job creation, and climate change. Thus, bioenergy can mitigate climate change but may impact human health and ecosystem which then in turn can become issues of concern. Energy strategies may result in shifting of risks from one environmental impact category to another, and from one sustainable domain to another if the technical and policy-related issues are not identified.

  1. Formulation and in vitro evaluation of sustained release matrix tablets using cross-linked natural gum.

    Science.gov (United States)

    Jamil, Qurratul Ain; Masood, Muhammad Irfan; Jamil, Muhammad Nauman; Masood, Imran; Iqbal, Shahid Muhammad

    2017-03-01

    Polysaccharide gums because of their biocompatibility, biodegradability and non-immunogenic properties are considered as the best choice for preparing sustained release tablets as compared to their synthetic counterpart. The cross linking of natural gums in matrix tablets increase the sustained release property of matrix tablets. Isoniazid is a first line therapy of tuberculosis, belongs to BCS I with half-life of 3-4 hours. These characteristics make isoniazid a good candidate for sustained release dosage form. Karaya gum crossed linked with trisodium tri metaphosphate was used as release rate retardant for preparing isoniazid cross-linked matrix tablet. Total 8 sustained release formulations were prepared. Both granules and tablets were evaluated under in vitro condition against different parameters. Dissolution studies were performed with all eight formulations for 12 hours using USP apparatus I. Four formulations designated as F1, F2, F3, F4 have drug and karaya gum while other four formulations F5, F6, F7, F8 have drug and crossed linked polymer in ratios of 1:1, 1:2, 1:3 and 1:4 respectively. Dissolution data was analyzed by using different kinetic models. Best fit model for most efficient formulation was zero order while release mechanism was super case I. Formulation 8 showed sufficiently slow release kinetics and about 83% of drug was released in 10 hours, indicating that cross-linked karaya gum proved efficient in preparing sustained release tablets.

  2. Predicting protein-protein interactions from multimodal biological data sources via nonnegative matrix tri-factorization.

    Science.gov (United States)

    Wang, Hua; Huang, Heng; Ding, Chris; Nie, Feiping

    2013-04-01

    Protein interactions are central to all the biological processes and structural scaffolds in living organisms, because they orchestrate a number of cellular processes such as metabolic pathways and immunological recognition. Several high-throughput methods, for example, yeast two-hybrid system and mass spectrometry method, can help determine protein interactions, which, however, suffer from high false-positive rates. Moreover, many protein interactions predicted by one method are not supported by another. Therefore, computational methods are necessary and crucial to complete the interactome expeditiously. In this work, we formulate the problem of predicting protein interactions from a new mathematical perspective--sparse matrix completion, and propose a novel nonnegative matrix factorization (NMF)-based matrix completion approach to predict new protein interactions from existing protein interaction networks. Through using manifold regularization, we further develop our method to integrate different biological data sources, such as protein sequences, gene expressions, protein structure information, etc. Extensive experimental results on four species, Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens, and Caenorhabditis elegans, have shown that our new methods outperform related state-of-the-art protein interaction prediction methods.

  3. Collagen and related extracellular matrix proteins in atherosclerotic plaque development.

    Science.gov (United States)

    Shami, Annelie; Gonçalves, Isabel; Hultgårdh-Nilsson, Anna

    2014-10-01

    The structure, composition and turnover of the extracellular matrix (ECM) as well as cell-matrix interactions are crucial in the developing atherosclerotic plaque. There is a need for further insight into specific proteins in the ECM and their functions in the developing plaque, and during the last few years a number of publications have highlighted this very important field of research. These novel findings will be addressed in the present review. This review covers literature focused on collagen and ECM proteins interacting with collagen, and what their roles may be in plaque development. Acute myocardial infarction and stroke are common diseases that cause disability and mortality, and the underlying mechanism is often the rupture of a vulnerable atherosclerotic plaque. The vascular ECM and the tissue repair in the atherosclerotic lesion are important players in plaque progression. Understanding how specific proteins in the ECM interact with cells in the plaque and affect the fate of the plaque can lead to new treatments for cardiovascular disease.

  4. Cartilage oligomeric matrix protein specific antibodies are pathogenic

    DEFF Research Database (Denmark)

    Geng, Hui; Nandakumar, Kutty Selva; Pramhed, Anna

    2012-01-01

    -specific monoclonal antibodies (mAbs). METHODS: B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated......ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP...

  5. Thermodynamics of protein folding: a random matrix formulation.

    Science.gov (United States)

    Shukla, Pragya

    2010-10-20

    The process of protein folding from an unfolded state to a biologically active, folded conformation is governed by many parameters, e.g. the sequence of amino acids, intermolecular interactions, the solvent, temperature and chaperon molecules. Our study, based on random matrix modeling of the interactions, shows, however, that the evolution of the statistical measures, e.g. Gibbs free energy, heat capacity, and entropy, is single parametric. The information can explain the selection of specific folding pathways from an infinite number of possible ways as well as other folding characteristics observed in computer simulation studies. © 2010 IOP Publishing Ltd

  6. Membrane and inclusion body targeting of lyssavirus matrix proteins.

    Science.gov (United States)

    Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

    2013-02-01

    Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

  7. Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules

    Science.gov (United States)

    Killian, C. E.; Wilt, F. H.

    1996-01-01

    In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

  8. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

    Science.gov (United States)

    Watson, Steve P

    2009-01-01

    The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

  9. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    Science.gov (United States)

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  10. NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

    Directory of Open Access Journals (Sweden)

    Lola A. Brown

    2015-04-01

    Full Text Available Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2 is mediated by Gag’s N-terminally myristylated matrix (MA domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV, a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S. These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

  11. NMR structure of the myristylated feline immunodeficiency virus matrix protein.

    Science.gov (United States)

    Brown, Lola A; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G; Kuo, Lillian; Freed, Eric O; Summers, Michael F

    2015-04-30

    Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag's N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

  12. Influence of Natural, Synthetic Polymers and Fillers on sustained release matrix tablets of Pregabalin

    OpenAIRE

    Vijaya Durga. K; Ashok Kumar. P; Suresh V Kulkarni

    2013-01-01

    The objective of the present study was to develop sustained release matrix tablets of Pregabalin for the treatment of neuropathic pain and epilepsy. The tablets were prepared by wet granulation and formulated using drug with Hydrophilic, hydrophobic, synthetic, natural polymers and 4 different fillers were used. The effect of Polymer concentration, combination and fillers on drug release rate was analyzed for the formulations F-1 to F-17. The tablets were subjected to physicochemical studies,...

  13. Development and evaluation of Ketoprofen sustained release matrix tablet using Hibiscus rosa-sinensis leaves mucilage

    Directory of Open Access Journals (Sweden)

    M. Kaleemullah

    2017-07-01

    Full Text Available Currently, the use of natural gums and mucilage is of increasing importance in pharmaceutical formulations as valuable drug excipient. Natural plant-based materials are economic, free of side effects, biocompatible and biodegradable. Therefore, Ketoprofen matrix tablets were formulated by employing Hibiscus rosa-sinensis leaves mucilage as natural polymer and HPMC (K100M as a synthetic polymer to sustain the drug release from matrix system. Direct compression method was used to develop sustained released matrix tablets. The formulated matrix tablets were evaluated in terms of physical appearance, weight variation, thickness, diameter, hardness, friability and in vitro drug release. The difference between the natural and synthetic polymers was investigated concurrently. Matrix tablets developed from each formulation passed all standard physical evaluation tests. The dissolution studies of formulated tablets revealed sustained drug release up to 24 h compared to the reference drug Apo Keto® SR tablets. The dissolution data later were fitted into kinetic models such as zero order equation, first order equation, Higuchi equation, Hixson Crowell equation and Korsmeyer-Peppas equation to study the release of drugs from each formulation. The best formulations were selected based on the similarity factor (f2 value of 50% and more. Through the research, it is found that by increasing the polymers concentration, the rate of drug release decreased for both natural and synthetic polymers. The best formulation was found to be F3 which contained 40% Hibiscus rosa-sinensis mucilage polymer and showed comparable dissolution profile to the reference drug with f2 value of 78.03%. The release kinetics of this formulation has shown to follow non-Fickian type which involved both diffusion and erosion mechanism. Additionally, the statistical results indicated that there was no significant difference (p > 0.05 between the F3 and reference drug in terms of MDT and

  14. Development and evaluation of Ketoprofen sustained release matrix tablet using Hibiscus rosa-sinensis leaves mucilage.

    Science.gov (United States)

    Kaleemullah, M; Jiyauddin, K; Thiban, E; Rasha, S; Al-Dhalli, S; Budiasih, S; Gamal, O E; Fadli, A; Eddy, Y

    2017-07-01

    Currently, the use of natural gums and mucilage is of increasing importance in pharmaceutical formulations as valuable drug excipient. Natural plant-based materials are economic, free of side effects, biocompatible and biodegradable. Therefore, Ketoprofen matrix tablets were formulated by employing Hibiscus rosa-sinensis leaves mucilage as natural polymer and HPMC (K100M) as a synthetic polymer to sustain the drug release from matrix system. Direct compression method was used to develop sustained released matrix tablets. The formulated matrix tablets were evaluated in terms of physical appearance, weight variation, thickness, diameter, hardness, friability and in vitro drug release. The difference between the natural and synthetic polymers was investigated concurrently. Matrix tablets developed from each formulation passed all standard physical evaluation tests. The dissolution studies of formulated tablets revealed sustained drug release up to 24 h compared to the reference drug Apo Keto® SR tablets. The dissolution data later were fitted into kinetic models such as zero order equation, first order equation, Higuchi equation, Hixson Crowell equation and Korsmeyer-Peppas equation to study the release of drugs from each formulation. The best formulations were selected based on the similarity factor ( f 2 ) value of 50% and more. Through the research, it is found that by increasing the polymers concentration, the rate of drug release decreased for both natural and synthetic polymers. The best formulation was found to be F3 which contained 40% Hibiscus rosa-sinensis mucilage polymer and showed comparable dissolution profile to the reference drug with f 2 value of 78.03%. The release kinetics of this formulation has shown to follow non-Fickian type which involved both diffusion and erosion mechanism. Additionally, the statistical results indicated that there was no significant difference (p > 0.05) between the F3 and reference drug in terms of MDT and T50% with p

  15. A matrix in life cycle perspective for selecting sustainable materials for buildings in Sri Lanka

    Energy Technology Data Exchange (ETDEWEB)

    Abeysundara, U.G. Yasantha [Ministry of Education, Isurupaya, Battaramulla (Sri Lanka); Babel, Sandhya [Environmental Technology Program, School of Biochemical Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University, P.O. Box 22, Pathumthani 12121 (Thailand); Gheewala, Shabbir [The Joint Graduate School of Energy and Environment, King Mongkut' s University of Technology Thonburi, Bangkok 10140 (Thailand)

    2009-05-15

    This paper presents a matrix to select sustainable materials for buildings in Sri Lanka, taking into consideration environmental, economic and social assessments of materials in a life cycle perspective. Five building elements, viz., foundations, roofs, ceilings, doors and windows, and floors are analyzed based on materials used for these elements. Environmental burdens associated with these elements are analyzed in terms of embodied energy and environmental impacts such as global warming, acidification and nutrient enrichment. Economic analysis is based on market prices and affordability of materials. Social factors that are taken into account are thermal comfort, interior (aesthetics), ability to construct quickly, strength and durability. By compiling the results of analyses, two building types with minimum and maximum impacts are identified. These two cases along with existing buildings are compared in a matrix of environmental, economic and social scores. Analysis of the results also indicates need for higher consideration of environmental parameters in decision-making over social and economic factors, as social and economic scores do not vary much between cases. Hence, this matrix helps decision-makers to select sustainable materials for buildings, meaningfully, and thus helps to move towards a more sustainable buildings and construction sector. (author)

  16. Graphene oxide as a protein matrix: influence on protein biophysical properties.

    Science.gov (United States)

    Hernández-Cancel, Griselle; Suazo-Dávila, Dámaris; Ojeda-Cruzado, Axel J; García-Torres, Desiree; Cabrera, Carlos R; Griebenow, Kai

    2015-10-19

    This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.

  17. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

    Energy Technology Data Exchange (ETDEWEB)

    Farhat, Walid A [Department of Surgery, Division of Urology, University of Toronto and Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman [Department of Developmental and Stem Cell Biology, Research Institute, Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Sherman, Christopher [Department of Anatomic Pathology, Sunnybrook and Women' s College Health Sciences Centre, Toronto, ON (Canada); Derwin, Kathleen [Department of Biomedical Engineering, Lerner Research Institute and Orthopaedic Research Center, Cleveland Clinic Foundation, Cleveland, OH (United States)], E-mail: walid.farhat@sickkids.ca

    2008-06-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

  18. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

    International Nuclear Information System (INIS)

    Farhat, Walid A; Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman; Sherman, Christopher; Derwin, Kathleen

    2008-01-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization

  19. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties.

    Science.gov (United States)

    Farhat, Walid A; Chen, Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Sherman, Christopher; Derwin, Kathleen; Yeger, Herman

    2008-06-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

  20. A National Scale Sustainable Agriculture Matrix of Indicators to Inform Policy

    Science.gov (United States)

    Davidson, E. A.; Zhang, X.

    2017-12-01

    The ratification of Sustainable Development Goals (SDGs) by all member countries of the United Nations demonstrates the determination of the international community in moving towards a sustainable future. To enable and encourage accountability, independent and transparent measurements of national sustainability efforts are essential. Among all sectors, agriculture is fundamental to all three pillars of sustainability, namely environment, society, and economy. However, the definition of a sustainable agriculture and the feasibility of measuring it remain elusive, in part because it encompasses both biophysical and socio-economic components that are still poorly integrated. Therefore, we have been developing a Sustainable Agriculture Matrix (SAM) on a national scale in order to measure country-level performance in agriculture. First proposed by Swaminathan for agricultural research and policy in 1990s, SAM is a collection of indicators measuring sustainable agriculture from environmental, social, and economic dimensions. The environmental dimension evaluates various impacts of agricultural production on the environment, such as water consumption and nutrient pollution. The economic dimension quantifies the costs and benefits for major stakeholders involved in agricultural production, including government, industry, farmers, and consumers. The social dimension considers three major aspects: 1) social welfare (e.g., hunger and poverty rate, nutritional quality, demography of rural community); 2) equity over sectors, space, and gender (e.g., access to resources/services and opportunities, distribution of income, land ownership and tenure rights); 3) systemic risk (e.g., fragility of the global agricultural production and trade system, resilience of a farm or a country to market and natural shocks). Translating the illustrative concepts into measureable indicators will not only provide an independent and transparent measurement of national performance in the

  1. Responsibility and Sustainability in a Food Chain: A Priority Matrix Analysis

    Directory of Open Access Journals (Sweden)

    Francesco Caracciolo

    2011-12-01

    Full Text Available  This paper shows the results of empirical research conducted to assess the sustainability of a typical food supply chain, suggesting feasible solutions to satisfy inter-dimensional requisites of durable development. The analysis was conducted with reference to the supply chain of the San Marzano tomato (SMZ, a typical local food. The product is endowed with an origin certification label (PDO, meeting demand within high-value market niches. The SMZ is a flagship product in the Italian region of Campania and has benefited from several regionally funded interventions, such as genetic research and support for the application for EU certification of origin. Two key findings emerged from the research. First, the results allowed us to define a Stakeholder Priority and Responsibilities’ Matrix (SPRM, and monitor the sustainability trend of SMZ food supply chains. Second, the consistency between the adoption of quality strategy (brand of origin and sustainable development of the sector was evaluated. Despite its intrinsic characteristics and its organized, well-defined structure, the SMZ food supply chain is unable to address sustainable objectives without considerable public intervention and support. In terms of sustainability, to be able to show desirable food chain characteristics, the existence of a fully collaborative relationship between the actors has to be ascertained. Identifying shared goals is essential to assign and implement coordinated actions, pooling responsibility for product quality into social and environmental dimensions.

  2. Sustained-release of caffeine from a polymeric tablet matrix: An in vitro and pharmacokinetic study

    International Nuclear Information System (INIS)

    Tan, Donna; Zhao Bin; Moochhala, Shabbir; Yang Yiyan

    2006-01-01

    Caffeine is utilized as a stimulant to impart a desired level of alertness during certain working hours. Usually, a single dose of caffeine induces 2-3 h of alertness coupled with side effects whereas a longer effect of 8-12 h is very useful for both daily life and military action. Thus, there is a need to deliver the stimulant continuously to an individual at one time to impart an increased level of alertness for the period stated after administration. This study aimed to design a polymeric microparticle system for sustained delivery of caffeine using a polymeric matrix. Poly(ethylene oxide) (PEO) was used as the erodible matrix material and the caffeine polymeric tablets were fabricated by compression using a Graseby Specac hydraulic press. In vitro release profiles as well as the pharmacokinetics studies data were obtained. Caffeine tablets fabricated using various polymers showed a high initial burst release type profile as compared to the caffeine-PEO-tablet. The PK studies showed sustained delivery of caffeine resulted in two expected phenomena: a reduction in the initial high rate of caffeine release (burst release) as well as a reduction in the change in caffeine concentration in the systemic circulation. A simple two-component system for sustained-release caffeine formulation therefore has been achieved

  3. Sustained-release of caffeine from a polymeric tablet matrix: An in vitro and pharmacokinetic study

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Donna [Defence Medical and Environmental Research Institute, DSO National Laboratories (Kent Ridge), 27 Medical Drive, 12-00, Singapore 117597 (Singapore); Zhao Bin [Defence Medical and Environmental Research Institute, DSO National Laboratories (Kent Ridge), 27 Medical Drive, 12-00, Singapore 117597 (Singapore); Moochhala, Shabbir [Defence Medical and Environmental Research Institute, DSO National Laboratories (Kent Ridge), 27 Medical Drive, 12-00, Singapore 117597 (Singapore)]. E-mail: mshabbir@dso.org.sg; Yang Yiyan [Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, 04-01, The Nanos, Singapore 138669 (Singapore)

    2006-07-25

    Caffeine is utilized as a stimulant to impart a desired level of alertness during certain working hours. Usually, a single dose of caffeine induces 2-3 h of alertness coupled with side effects whereas a longer effect of 8-12 h is very useful for both daily life and military action. Thus, there is a need to deliver the stimulant continuously to an individual at one time to impart an increased level of alertness for the period stated after administration. This study aimed to design a polymeric microparticle system for sustained delivery of caffeine using a polymeric matrix. Poly(ethylene oxide) (PEO) was used as the erodible matrix material and the caffeine polymeric tablets were fabricated by compression using a Graseby Specac hydraulic press. In vitro release profiles as well as the pharmacokinetics studies data were obtained. Caffeine tablets fabricated using various polymers showed a high initial burst release type profile as compared to the caffeine-PEO-tablet. The PK studies showed sustained delivery of caffeine resulted in two expected phenomena: a reduction in the initial high rate of caffeine release (burst release) as well as a reduction in the change in caffeine concentration in the systemic circulation. A simple two-component system for sustained-release caffeine formulation therefore has been achieved.

  4. Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

    Science.gov (United States)

    Ito, Mikako; Ohno, Kinji

    2018-02-20

    Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression

  5. Formulation and in vivo evaluation of diclofenac sodium sustained release matrix tablet: effect of compression force.

    Science.gov (United States)

    Taha, Ehab Ibrahim; Shazly, Gamal Abdel-Ghany; Harisa, Gamaleldin Ibrahim; Barakat, Nahla Sedik; Al-Enazi, Fouza Kayem; Elbagory, Ibrahim Mostafa

    2015-03-01

    In the present study, Diclofenac Sodium (DS) matrix tablets were prepared by direct compression method under different compression forces (5, 10, 15 and 20 KN), using ethylcellulose as matrix forming material. The produced tablets were characterized on the foundation of satisfactory tablet properties such as hardness, friability, drug content, weight variations and in vitro drug release rate. Differential scanning calorimetry (DSC), Fourier Transform Infrared (FT-IR) spectroscopy and X-ray diffraction have been used to investigate any incompatibilities of the tablet's ingredients. Additionally, in vivo bioavailability has been investigated on beagle dogs. Data obtained revealed that, upon increasing compression force the in vitro drug release was sustained and the T(max) value was four hours (for formulations compressed at 15 and 20 kN) compared to the conventional voltarine(®) 50 tablets (T(max) value of 2 hours).

  6. Storage and sustained release of volatile substances from a hollow silica matrix

    Energy Technology Data Exchange (ETDEWEB)

    Wang Jiexin [Key Lab for Nanomaterials, Ministry of Education, Beijing 100029 (China); Ding Haomin [Research Center of the Ministry of Education for High Gravity Engineering and Technology, Beijing University of Chemical Technology, Beijing 100029 (China); Tao Xia [Key Lab for Nanomaterials, Ministry of Education, Beijing 100029 (China); Chen Jianfeng [Key Lab for Nanomaterials, Ministry of Education, Beijing 100029 (China)

    2007-06-20

    Porous hollow silica nanospheres (PHSNSs) prepared by adopting a nanosized CaCO{sub 3} template were utilized for the first time as a novel carrier for the storage and sustained release of volatile substances. Two types of volatile substances, Indian pipal from perfumes and peroxyacetic acid from disinfectants, were selected and then tested by one simple adsorption process with two separate comparative carriers, i.e. activated carbon and solid porous silica. It was demonstrated that a high storage capacity (9.6 ml{sub perfume}/mg{sub carrier}) of perfume could be achieved in a PHSNS matrix, which was almost 14 times as much as that of activated carbon. The perfume release profiles showed that PHSNSs exhibited sustained multi-stage release behaviour, while the constant release of activated carbon at a low level was discerned. Further, a Higuchi model study proved that the release process of perfume in both carriers followed a Fickian diffusion mechanism. For peroxyacetic acid as a disinfectant model, PHSNSs also displayed a much better delayed-delivery process than a solid porous silica system owing to the existence of unique hollow frameworks. Therefore, the aforementioned excellent sustained-release behaviours would make PHSNSs a promising carrier for storage and sustained delivery applications of volatile substances.

  7. Oligomerization and polymerization of the filovirus matrix protein VP40

    International Nuclear Information System (INIS)

    Timmins, Joanna; Schoehn, Guy; Kohlhaas, Christine; Klenk, Hans-Dieter; Ruigrok, Rob W.H.; Weissenhorn, Winfried

    2003-01-01

    The matrix protein VP40 from Ebola virus plays an important role in the assembly process of virus particles by interacting with cellular factors, cellular membranes, and the ribonuclearprotein particle complex. Here we show that the N-terminal domain of VP40 folds into a mixture of two different oligomeric states in vitro, namely hexameric and octameric ringlike structures, as detected by gel filtration chromatography, chemical cross-linking, and electron microscopy. Octamer formation depends largely on the interaction with nucleic acids, which in turn confers in vitro SDS resistance. Refolding experiments with a nucleic acid free N-terminal domain preparation reveal a mostly dimeric form of VP40, which is transformed into an SDS resistant octamer upon incubation with E. coli nucleic acids. In addition, we demonstrate that the N-terminal domain of Marburg virus VP40 also folds into ringlike structures, similar to Ebola virus VP40. Interestingly, Marburg virus VP40 rings reveal a high tendency to polymerize into rods composed of stacked rings. These results may suggest distinct roles for different oligomeric forms of VP40 in the filovirus life cycle

  8. Formulation and evaluation of sustained release matrix tablet of rabeprazole using wet granulation technique

    Directory of Open Access Journals (Sweden)

    Ruqaiyah Khan

    2014-01-01

    Full Text Available Introduction: Rabeprazole, a member of substituted benzimidazoles, inhibits the final step in gastric acid secretions. This drug claims to cause fastest acid separation (due to higher pKa, and more rapidly converts to the active species to aid gastric mucin synthesis. The most significant pharmacological action of Rabeprazole is dose dependent suppression of gastric acid secretion; without anticholinergic or H2-blocking action. It completely abolishes the hydrochloric acid secretion as it is powerful inhibitor of gastric acid. Rabeprazole is acid labile and hence commonly formulated as an enteric coated tablet. The absorption of rabeprazole occurs rapidly as soon as tablet leaves the stomach. Aim: In the present study an attempt was made to formulate and evaluate Rabeprazole sustained release matrix tablet using wet granulation technique incorporating various polymers like HPMC-E15, Carbopol934, and sodium carboxymethyl cellulose (CMC. Materials and Methods: The Formulated tablets were evaluated for different physicochemical properties like rheological properties, weight variation, thickness, hardness, % friability, in vitro release studies and drug content. Results: Studies revealed that all the physicochemical parameters comply with the official standards. The in vitro release studies exhibits the release up to 90%, over a prolonged period of time which confirms the extended release profile of formulation, having better bioavailability as well as decreased dosing frequency with reduced doses. Conclusion: The sustained release matrix tablets of rabiprazole shown better bioavailability, efficacy and potency, when compared with official standards.

  9. Preparation of mixed matrix adsorber membranes for protein recovery

    NARCIS (Netherlands)

    Avramescu, M.E.; Girones nogue, Miriam; Borneman, Zandrie; Wessling, Matthias

    2003-01-01

    This paper presents a generic technology allowing the incorporation of functional entities into a porous substrate. Various ion exchange particles were incorporated into an ethylene vinyl alcohol (EVAL) copolymer porous matrix by an immersion phase separation process and a heterogeneous matrix,

  10. Protein instability and immunogenicity: roadblocks to clinical application of injectable protein delivery systems for sustained release.

    Science.gov (United States)

    Jiskoot, Wim; Randolph, Theodore W; Volkin, David B; Middaugh, C Russell; Schöneich, Christian; Winter, Gerhard; Friess, Wolfgang; Crommelin, Daan J A; Carpenter, John F

    2012-03-01

    Protein instability and immunogenicity are two main roadblocks to the clinical success of novel protein drug delivery systems. In this commentary, we discuss the need for more extensive analytical characterization in relation to concerns about protein instability in injectable drug delivery systems for sustained release. We then will briefly address immunogenicity concerns and outline current best practices for using state-of-the-art analytical assays to monitor protein stability for both conventional and novel therapeutic protein dosage forms. Next, we provide a summary of the stresses on proteins arising during preparation of drug delivery systems and subsequent in vivo release. We note the challenges and difficulties in achieving the absolute requirement of quantitatively assessing the degradation of protein molecules in a drug delivery system. We describe the potential roles for academic research in further improving protein stability and developing new analytical technologies to detect protein degradation byproducts in novel drug delivery systems. Finally, we provide recommendations for the appropriate approaches to formulation design and assay development to ensure that stable, minimally immunogenic formulations of therapeutic proteins are created. These approaches should help to increase the probability that novel drug delivery systems for sustained protein release will become more readily available as effective therapeutic agents to treat and benefit patients. Copyright © 2011 Wiley Periodicals, Inc.

  11. Interactions of rat repetitive sequence MspI8 with nuclear matrix proteins during spermatogenesis

    International Nuclear Information System (INIS)

    Rogolinski, J.; Widlak, P.; Rzeszowska-Wolny, J.

    1996-01-01

    Using the Southwestern blot analysis we have studied the interactions between rat repetitive sequence MspI8 and the nuclear matrix proteins of rats testis cells. Starting from 2 weeks the young to adult animal showed differences in type of testis nuclear matrix proteins recognizing the MspI8 sequence. The same sets of nuclear matrix proteins were detected in some enriched in spermatocytes and spermatids and obtained after fractionation of cells of adult animal by the velocity sedimentation technique. (author). 21 refs, 5 figs

  12. Development of sustained release capsules containing "coated matrix granules of metoprolol tartrate".

    Science.gov (United States)

    Siddique, Sabahuddin; Khanam, Jasmina; Bigoniya, Papiya

    2010-09-01

    The objective of this investigation was to prepare sustained release capsule containing coated matrix granules of metoprolol tartrate and to study its in vitro release and in vivo absorption. The design of dosage form was performed by choosing hydrophilic hydroxypropyl methyl cellulose (HPMC K100M) and hydrophobic ethyl cellulose (EC) polymers as matrix builders and Eudragit® RL/RS as coating polymers. Granules were prepared by composing drug with HPMC K100M, EC, dicalcium phosphate by wet granulation method with subsequent coating. Optimized formulation of metoprolol tartrate was formed by using 30% HPMC K100M, 20% EC, and ratio of Eudragit® RS/RL as 97.5:2.5 at 25% coating level. Capsules were filled with free flowing optimized granules of uniform drug content. This extended the release period upto 12 h in vitro study. Similarity factor and mean dissolution time were also reported to compare various dissolution profiles. The network formed by HPMC and EC had been coupled satisfactorily with the controlled resistance offered by Eudragit® RS. The release mechanism of capsules followed Korsemeyer-Peppas model that indicated significant contribution of erosion effect of hydrophilic polymer. Biopharmaceutical study of this optimized dosage form in rabbit model showed 10 h prolonged drug release in vivo. A close correlation (R(2) = 0.9434) was established between the in vitro release and the in vivo absorption of drug. The results suggested that wet granulation with subsequent coating by fluidized bed technique, is a suitable method to formulate sustained release capsules of metoprolol tartrate and it can perform therapeutically better than conventional immediate release dosage form.

  13. Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen-induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures

    NARCIS (Netherlands)

    Blumbach, K.; Bastiaansen-Jenniskens, Y.M.; Groot, J. de; Paulsson, M.; Osch, G.J.V.M. van; Zaucke, F.

    2009-01-01

    Objective. Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril-associated proteins

  14. The Geocybernetic Assessment Matrix (GAM) — A new assessment tool for evaluating the level and nature of sustainability or unsustainability

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, Jason, E-mail: jp1@tiscali.co.uk

    2016-01-15

    Evaluating sustainability from EIA-based assessments has been problematic at best. This is due to the use of reductionist and qualitative approaches which is dependent upon the perspective of the assessor(s). Therefore, a more rigorous and holistic approach is required to evaluate sustainability in a more consistent way. In this paper, a matrix-based methodology in order to assess the indicated level and nature of sustainability for any project, policy, indicators, legislation, regulation, or other framework is described. The Geocybernetic Assessment Matrix (GAM) is designed to evaluate the level and nature of sustainability or unsustainability occurring in respect the fundamental and complex geocybernetic paradigms. The GAM method is described in detail in respect to the theory behind it and the methodology. The GAM is then demonstrated using an appropriate case study — Part 1 of the UK Climate Change Act (2008) concerning carbon budgets and targets. The results indicate that the Part 1 of Act may not achieve the desired goals in contributing towards sustainable development through the stated mechanisms for carbon budgets and targets. The paper then discusses the broader context of the GAM with respect to the core themes evident in the development and application of the GAM of: sustainability science; sustainability assessment; application value of the GAM; and future research and development. - Highlights: • A new assessment tool called the Geocybernetic Assessment Matrix (GAM) described. • GAM evaluates the level and nature of sustainability or unsustainability. • GAM demonstrated by application to Part 1 of the UK Climate Change Act (CCA). • Part 1 of CCA has significant flaws in achieving a sustainable pathway. • GAM offers a potentially useful tool for quantitatively evaluating sustainability.

  15. The Geocybernetic Assessment Matrix (GAM) — A new assessment tool for evaluating the level and nature of sustainability or unsustainability

    International Nuclear Information System (INIS)

    Phillips, Jason

    2016-01-01

    Evaluating sustainability from EIA-based assessments has been problematic at best. This is due to the use of reductionist and qualitative approaches which is dependent upon the perspective of the assessor(s). Therefore, a more rigorous and holistic approach is required to evaluate sustainability in a more consistent way. In this paper, a matrix-based methodology in order to assess the indicated level and nature of sustainability for any project, policy, indicators, legislation, regulation, or other framework is described. The Geocybernetic Assessment Matrix (GAM) is designed to evaluate the level and nature of sustainability or unsustainability occurring in respect the fundamental and complex geocybernetic paradigms. The GAM method is described in detail in respect to the theory behind it and the methodology. The GAM is then demonstrated using an appropriate case study — Part 1 of the UK Climate Change Act (2008) concerning carbon budgets and targets. The results indicate that the Part 1 of Act may not achieve the desired goals in contributing towards sustainable development through the stated mechanisms for carbon budgets and targets. The paper then discusses the broader context of the GAM with respect to the core themes evident in the development and application of the GAM of: sustainability science; sustainability assessment; application value of the GAM; and future research and development. - Highlights: • A new assessment tool called the Geocybernetic Assessment Matrix (GAM) described. • GAM evaluates the level and nature of sustainability or unsustainability. • GAM demonstrated by application to Part 1 of the UK Climate Change Act (CCA). • Part 1 of CCA has significant flaws in achieving a sustainable pathway. • GAM offers a potentially useful tool for quantitatively evaluating sustainability.

  16. Matrix proteins as centralized organizers of negative-sense RNA virions.

    Science.gov (United States)

    Liljeroos, Lassi; Butcher, Sarah J

    2013-01-01

    Matrix proteins are essential components of most negative-sense RNA, enveloped viruses. They serve a wide range of duties ranging from self-driven membrane budding and coordination of other viral components to modulation of viral transcription. The functional similarity between these proteins is striking, despite major differences in their structures. Whereas biochemical and structural studies have partly been hindered by the inherent aggregation properties of these proteins, their cellular functions are beginning to be understood. In this review we summarize the current knowledge on negative-sense RNA virus matrix proteins and their interactions with other viral and cellular proteins. We also discuss the similarities and differences in matrix protein functions between the different families within the negative-sense RNA viruses.

  17. Novel serological neo-epitope markers of extracellular matrix proteins for the detection of portal hypertension

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; Karsdal, M A; Byrjalsen, I

    2013-01-01

    The hepatic venous pressure gradient (HVPG) is an invasive, but important diagnostic and prognostic marker in cirrhosis with portal hypertension (PHT). During cirrhosis, remodelling of fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process generating small fragments of degrade...... extracellular matrix (ECM) proteins known as neoepitopes, which are then released into the circulation....

  18. Matrix Gla Protein polymorphism, but not concentrations, is associated with radiographic hand osteoarthritis

    Science.gov (United States)

    Objective. Factors associated with mineralization and osteophyte formation in osteoarthritis (OA) are incompletely understood. Genetic polymorphisms of matrix Gla protein (MGP), a mineralization inhibitor, have been associated clinically with conditions of abnormal calcification. We therefore evalua...

  19. Extracellular matrix proteins: a positive feedback loop in lung fibrosis?

    NARCIS (Netherlands)

    Blaauboer, M.E.; van Boeijen, F.R.; Emson, C.L.; Turner, S.M.; Zandieh-Doulabi, B.; Hanemaaijer, R.; Smit, T.H.; Stoop, R.; Everts, V.

    2014-01-01

    Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the

  20. Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

    NARCIS (Netherlands)

    Blaauboer, M.E.; Boeijen, F.R.; Emson, C.L.; Turner, S.M.; Zandieh-Doulabi, B.; Hanemaaijer, R.; Smit, T.H.; Stoop, R.; Everts, V.

    2014-01-01

    Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the

  1. Growth of thin films of low molecular weight proteins by matrix assisted pulsed laser evaporation (MAPLE)

    DEFF Research Database (Denmark)

    Matei, Andreea; Schou, Jørgen; Constantinescu, C.

    2011-01-01

    Thin films of lysozyme and myoglobin grown by matrix assisted pulsed laser evaporation (MAPLE) from a water ice matrix have been investigated. The deposition rate of these two low molecular weight proteins (lysozyme: 14307 amu and myoglobin: 17083 amu) exhibits a maximum of about 1–2 ng/cm2 per....... The results for lysozyme demonstrate that the fragmentation rate of the proteins during the MAPLE process is not influenced by the pH of the water solution prior to freezing....

  2. Mixed matrix membrane adsorbers for protein and blood purification

    NARCIS (Netherlands)

    Saiful, S.

    2007-01-01

    Biotechnology and bio-manufacturing markets are continuously growing, generating new sources of many valuable healthcare and life science products including therapeutic proteins and polysaccharides, monoclonals, vaccines, diagnostics, pharmaceutical chemicals and enzymes. These bioproducts have to

  3. Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus.

    Science.gov (United States)

    Price, Daniel L; Li, Pingdong; Chen, Chun-Hao; Wong, Danni; Yu, Zhenkun; Chen, Nanhai G; Yu, Yong A; Szalay, Aladar A; Cappello, Joseph; Fong, Yuman; Wong, Richard J

    2016-02-01

    Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral β-galactosidase expression as compared to PBS. The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes. © 2014 Wiley Periodicals, Inc.

  4. Novel Polyurethane Matrix Systems Reveal a Particular Sustained Release Behavior Studied by Imaging and Computational Modeling.

    Science.gov (United States)

    Campiñez, María Dolores; Caraballo, Isidoro; Puchkov, Maxim; Kuentz, Martin

    2017-07-01

    The aim of the present work was to better understand the drug-release mechanism from sustained release matrices prepared with two new polyurethanes, using a novel in silico formulation tool based on 3-dimensional cellular automata. For this purpose, two polymers and theophylline as model drug were used to prepare binary matrix tablets. Each formulation was simulated in silico, and its release behavior was compared to the experimental drug release profiles. Furthermore, the polymer distributions in the tablets were imaged by scanning electron microscopy (SEM) and the changes produced by the tortuosity were quantified and verified using experimental data. The obtained results showed that the polymers exhibited a surprisingly high ability for controlling drug release at low excipient concentrations (only 10% w/w of excipient controlled the release of drug during almost 8 h). The mesoscopic in silico model helped to reveal how the novel biopolymers were controlling drug release. The mechanism was found to be a special geometrical arrangement of the excipient particles, creating an almost continuous barrier surrounding the drug in a very effective way, comparable to lipid or waxy excipients but with the advantages of a much higher compactability, stability, and absence of excipient polymorphism.

  5. Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

    Science.gov (United States)

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-01-01

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329

  6. Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

    Science.gov (United States)

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-06-29

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

  7. Rhabdovirus matrix protein structures reveal a novel mode of self-association.

    Directory of Open Access Journals (Sweden)

    Stephen C Graham

    2008-12-01

    Full Text Available The matrix (M proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus and from Lagos bat virus (genus: Lyssavirus, revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.

  8. Suppression of matrix protein synthesis in endothelial cells by herpes simplex virus is not dependent on viral protein synthesis

    International Nuclear Information System (INIS)

    Kefalides, N.A.

    1986-01-01

    The synthesis of matrix proteins by human endothelial cells (EC) in vitro was studied before and at various times after infection with Herpes Simplex virus Type 1 (HSV-1) or 2 (HSV-2). Monolayers of EC were either mock-infected or infected with virus for 1 hr at a multiplicity infection (MOI) of 5 to 20 at 37 0 C. Control and infected cultures were pulse-labeled for 1 or 2 hrs with either [ 14 C]proline or [ 35 S]methionine. Synthesis of labeled matrix proteins was determined by SDS-gel electrophoresis. Suppression of synthesis of fibronectin, Type IV collagen and thrombospondin began as early as 2 hrs and became almost complete by 10 hrs post-infection. The degree of suppression varied with the protein and the virus dose. Suppression of Type IV collagen occurred first followed by that of fibronectin and then thrombospondin. Infection of EC with UV irradiated HSV-1 or HSV-2 resulted in suppression of host-cell protein synthesis as well as viral protein synthesis. Infection with intact virus in the presence of actinomycin-D resulted in suppression of both host-cell and viral protein synthesis. The data indicate that infection of EC with HSV leads to suppression of matrix protein synthesis which does not depend on viral protein synthesis

  9. Import of peroxisomal matrix proteins in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Gunkel, Katja

    2005-01-01

    Archaea, prokaryotes and eukaryotes form the three kingdoms of life. The smallest unit of life, which can exist independently, is a cell. Archaea and prokaryotes have a relatively very simple architecture. The cytoplasm (cellulars pace), containing all metabolites, proteins and genetic material

  10. Evaluation of gum mastic (Pistacia lentiscus as a microencapsulating and matrix forming material for sustained drug release

    Directory of Open Access Journals (Sweden)

    Dinesh M. Morkhade

    2017-09-01

    Full Text Available In this study, a natural gum mastic was evaluated as a microencapsulating and matrix-forming material for sustained drug release. Mastic was characterized for its physicochemical properties. Microparticles were prepared by oil-in-oil solvent evaporation method. Matrix tablets were prepared by wet and melt granulation techniques. Diclofenac sodium (DFS and diltiazem hydrochloride (DLTZ were used as model drugs. Mastic produced discrete and spherical microspheres with DLTZ and microcapsules with DFS. Particle size and drug loading of microparticles was in the range of 22–62 µm and 50–87%, respectively. Increase in mastic: drug ratio increased microparticle size, improved drug loading and decreased the drug release rate. Microparticles with gum: drug ratio of 2:1 could sustain DLTZ release up to 12 h and released 57% DFS in 12 h. Mastic produced tablets with acceptable pharmacotechnical properties. A 30% w/w of mastic in tablet could sustain DLTZ release for 5 h from wet granulation, and DFS release for 8 h and 11 h from wet and melt granulation, respectively. Results revealed that a natural gum mastic can be used successfully to formulate matrix tablets and microparticles for sustained drug release.

  11. Degenerated human intervertebral discs contain autoantibodies against extracellular matrix proteins

    Directory of Open Access Journals (Sweden)

    S Capossela

    2014-04-01

    Full Text Available Degeneration of intervertebral discs (IVDs is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.

  12. Sustainable protein from biogas. The perspective of susteine

    International Nuclear Information System (INIS)

    Oosterhuis, N.; Hooijer, H.

    2007-12-01

    Production of single-cell protein (Susteine) on the basis of biogas (methane) from manure digestion might be an interesting option to reduce the amounts of manure from cattle, pigs and poultry and also might be an option for a sustainable production of high-value protein. An earlier made investigation leaded to the conclusion that many questions had to be answered to make a better picture of this opportunity for the Dutch agricultural society. In this study we investigated the feasibility of such a process and product. It is concluded that the product will be accepted in the market as long as the HACCP-requirements which are applicable in the feed production are fulfilled. The product as such, assuming that the quality is comparable to the product as was produced by Norferm, could get an attractive position especially in the application for piglets. A quantity of 10.000 t/y could easily be sold in this market segment. Of course the product first has to be registered, which needs additional studies. Technically we foresee no major hurdles in the production of Susteine on basis of methane from biogas. The biogas has to be purified to such a quality that SCP-production is feasible, however, technologies are available. It needs a further investigation to establish the quality requirements for such a process. The scale of operation of the biogas installation creates a financial-economic problem. In order to produce 10.000 t protein / year, a quantity of 30-35 million m 3 of biogas is needed, for which 2 million ton of manure has to be digested. In case a centralized production of gas and protein is foreseen, the number of transport movements will be unacceptable. Besides the quantity of manure to be transported also approximately the same amount of digested material has to be transported. A unique opportunity in The Netherlands is the presence of a pipe network for natural gas. Such a network could serve as a transport system of locally produced, purified, biogas and a

  13. Serum protein fractionation using supported molecular matrix electrophoresis.

    Science.gov (United States)

    Dong, Weijie; Matsuno, Yu-ki; Kameyama, Akihiko

    2013-08-01

    Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Calcinosis in juvenile dermatomyositis : a possible role for the vitamin K-dependent protein matrix Gla protein

    NARCIS (Netherlands)

    Van Summeren, M. J. H.; Spliet, W. G. M.; Van Royen-Kerkhof, A.; Vermeer, C.; Lilien, M.; Kuis, W.; Schurgers, L. J.

    Objectives. The aims of the present study were to investigate whether the calcification inhibitor matrix Gla protein (MGP) is expressed in muscle biopsies of patients with juvenile dermatomyositis (JDM), and whether different forms of MGP are differentially expressed in JDM patients with and without

  15. Distribution of cytoskeletal proteins, integrins, leukocyte adhesion molecules and extracellular matrix proteins in plastic-embedded human and rat kidneys

    NARCIS (Netherlands)

    van Goor, H; Coers, W; van der Horst, MLC; Suurmeijer, AJH

    2001-01-01

    OBJECTIVE: To study the distribution of cytoskeletal proteins (actin, alpha -actinin, vinculin, beta -tubulin, keratin, vimentin, desmin), adhesion molecules for cell-matrix interations (very later antigens [VLA1-6], beta1, beta2 [CD18], vitronectin receptor [alphav beta3], CD 11b), leukocyte

  16. An Overview of the Medical Applications of Marine Skeletal Matrix Proteins

    Directory of Open Access Journals (Sweden)

    M. Azizur Rahman

    2016-09-01

    Full Text Available In recent years, the medicinal potential of marine organisms has attracted increasing attention. This is due to their immense diversity and adaptation to unique ecological niches that has led to vast physiological and biochemical diversification. Among these organisms, marine calcifiers are an abundant source of novel proteins and chemical entities that can be used for drug discovery. Studies of the skeletal organic matrix proteins of marine calcifiers have focused on biomedical applications such as the identification of growth inducing proteins that can be used for bone regeneration, for example, 2/4 bone morphogenic proteins (BMP. Although a few reports on the functions of proteins derived from marine calcifiers can be found in the literature, marine calcifiers themselves remain an untapped source of proteins for the development of innovative pharmaceuticals. Following an overview of the current knowledge of skeletal organic matrix proteins from marine calcifiers, this review will focus on various aspects of marine skeletal protein research including sources, biosynthesis, structures, and possible strategies for chemical or physical modification. Special attention will be given to potential medical applications and recent discoveries of skeletal proteins and polysaccharides with biologically appealing characteristics. In addition, I will introduce an effective protocol for sample preparation and protein purification that includes isolation technology for biopolymers (of both soluble and insoluble organic matrices from coralline algae. These algae are a widespread but poorly studied group of shallow marine calcifiers that have great potential for marine drug discovery.

  17. Sustained subconjunctival protein delivery using a thermosetting gel delivery system.

    Science.gov (United States)

    Rieke, Erin R; Amaral, Juan; Becerra, S Patricia; Lutz, Robert J

    2010-02-01

    An effective treatment modality for posterior eye diseases would provide prolonged delivery of therapeutic agents, including macromolecules, to eye tissues using a safe and minimally invasive method. The goal of this study was to assess the ability of a thermosetting gel to deliver a fluorescently labeled protein, Alexa 647 ovalbumin, to the choroid and retina of rats following a single subconjunctival injection of the gel. Additional experiments were performed to compare in vitro to in vivo ovalbumin release rates from the gel. The ovalbumin content of the eye tissues was monitored by spectrophotometric assays of tissue extracts of Alexa 647 ovalbumin from dissected sclera, choroid, and retina at time points ranging from 2 h to 14 days. At the same time points, fluorescence microscopy images of tissue samples were also obtained. Measurement of intact ovalbumin was verified by LDS-PAGE analysis of the tissue extract solutions. In vitro release of Alexa 488 ovalbumin into 37 degrees C PBS solutions from ovalbumin-loaded gel pellets was also monitored over time by spectrophotometric assay. In vivo ovalbumin release rates were determined by measurement of residual ovalbumin extracted from gel pellets removed from rat eyes at various time intervals. Our results indicate that ovalbumin concentrations can be maintained at measurable levels in the sclera, choroid, and retina of rats for up to 14 days using the thermosetting gel delivery system. The concentration of ovalbumin exhibited a gradient that decreased from sclera to choroid and to retina. The in vitro release rate profiles were similar to the in vivo release profiles. Our findings suggest that the thermosetting gel system may be a feasible method for safe and convenient sustained delivery of proteins to choroidal and retinal tissue in the posterior segments of the eye.

  18. LRPPRC is a mitochondrial matrix protein that is conserved in metazoans

    International Nuclear Information System (INIS)

    Sterky, Fredrik H.; Ruzzenente, Benedetta; Gustafsson, Claes M.; Samuelsson, Tore; Larsson, Nils-Goeran

    2010-01-01

    Research highlights: → LRPPRC orthologs are restricted to metazoans. → LRPPRC is imported to the mitochondrial matrix. → No evidence of nuclear isoform. -- Abstract: LRPPRC (also called LRP130) is an RNA-binding pentatricopeptide repeat protein. LRPPRC has been recognized as a mitochondrial protein, but has also been shown to regulate nuclear gene transcription and to bind specific RNA molecules in both the nucleus and the cytoplasm. We here present a bioinformatic analysis of the LRPPRC primary sequence, which reveals that orthologs to the LRPPRC gene are restricted to metazoan cells and that all of the corresponding proteins contain mitochondrial targeting signals. To address the subcellular localization further, we have carefully analyzed LRPPRC in mammalian cells and identified a single isoform that is exclusively localized to mitochondria. The LRPPRC protein is imported to the mitochondrial matrix and its mitochondrial targeting sequence is cleaved upon entry.

  19. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

    DEFF Research Database (Denmark)

    Gannagé, Monique; Dormann, Dorothee; Albrecht, Randy

    2009-01-01

    Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here, we...... demonstrate that influenza A virus inhibits macroautophagy, a cellular process known to be manipulated by diverse pathogens. Influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes, and one viral protein, matrix protein 2, is necessary and sufficient...... for this inhibition of autophagosome degradation. Macroautophagy inhibition by matrix protein 2 compromises survival of influenza virus-infected cells but does not influence viral replication. We propose that influenza A virus, which also encodes proapoptotic proteins, is able to determine the death of its host cell...

  20. Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

    DEFF Research Database (Denmark)

    Houen, G.; Olsen, D.T.; Hansen, P.R.

    2003-01-01

    A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated......, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates...... of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings...

  1. Sustainability.

    Science.gov (United States)

    Chang, Chein-Chi; DiGiovanni, Kimberly; Mei, Ying; Wei, Li

    2016-10-01

    This review on Sustainability covers selected 2015 publications on the focus of Sustainability. It is divided into the following sections : • Sustainable water and wastewater utilities • Sustainable water resources management • Stormwater and green infrastructure • Sustainability in wastewater treatment • Life cycle assessment (LCA) applications • Sustainability and energy in wastewater industry, • Sustainability and asset management.

  2. One-step separation of myristoylated and nonmyristoylated retroviral matrix proteins

    Czech Academy of Sciences Publication Activity Database

    Doležal, Michal; Zábranský, Aleš; Hrabal, R.; Ruml, T.; Pichová, Iva; Rumlová, Michaela

    2013-01-01

    Roč. 92, č. 1 (2013), s. 94-99 ISSN 1046-5928 R&D Projects: GA ČR GA204/09/1388 Institutional support: RVO:61388963 Keywords : matrix protein * mouse mammary tumor virus * murine leukemia virus * myristoylation * N-myristoyltransferase * retrovirus Subject RIV: CE - Biochemistry Impact factor: 1.508, year: 2013

  3. β-TCP/HA with or without enamel matrix proteins for maxillary sinus floor augmentation

    DEFF Research Database (Denmark)

    Nery, James Carlos; Pereira, Luís Antônio Violin Dias; Guimarães, George Furtado

    2017-01-01

    BACKGROUND: It is still unclear whether enamel matrix proteins (EMD) as adjunct to bone grafting enhance bone healing. This study compared histomorphometrically maxillary sinus floor augmentation (MSFA) with β-TCP/HA in combination with or without EMD in humans. METHODS: In ten systemically healthy...

  4. Topical application of amelogenin extracellular matrix protein in non-healing venous ulcers

    Directory of Open Access Journals (Sweden)

    Burçin Abud

    2014-12-01

    Full Text Available Background and Design: Treatment of chronic venous ulcers of the lower extremity is still an important difficulty. The principal treatment of these ulcers includes compression therapy, local wound care and surgery. Unresponsiveness to these standard treatments is a frequent situation with negative effects on life quality and reductions in personal productivity. Therefore, there is a need for new applications to increase the effectiveness of treatment in treatment-resistant cases. In the present study, we retrospectively evaluated the results of topical application of amelogenin extracellular matrix protein in resistant venous ulcers. Materials and Methods: We analyzed the records of patients with treatment-resistant venous ulceration who were treated with amelogenin extracellular matrix protein between June 2011 and December 2012.. Results: 26 patients (21 male and 5 female with a total number of 28 ulcers (24 patients with 1 ulcer, 2 patients with two ulcers were evaluated. The patients were treated with topically applied amelogenin extracellular matrix protein and regional four bandage compression. Bandages were changed weekly. Each cure continued for six weeks. In fourteen patients (15 ulcers, we observed a complete healing by the end of the first cure. In another twelve cases (13 ulcers, the same period resulted with a reduction in wound diameter. We continued to the second cure for these patients. By the end of the second cure, complete healing was achieved in five cases (6 ulcers. Conclusion: Topical application of amelogenin extracellular matrix protein may be considered as an effective therapeutic choice for refractory venous ulcers.

  5. The application of an enamel matrix protein derivative (Emdogain) in regenerative periodontal therapy: a review.

    NARCIS (Netherlands)

    Sculean, A.; Schwarz, F.; Becker, J.; Brecx, M.

    2007-01-01

    Regenerative periodontal therapy aims at reconstitution of the lost periodontal structures such as new formation of root cementum, periodontal ligament and alveolar bone. Findings from basic research indicate that enamel matrix protein derivative (EMD) has a key role in periodontal wound healing.

  6. Beyond the Protein Matrix : Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction

    NARCIS (Netherlands)

    Martinoli, Christian; Dudek, Hanna M.; Orru, Roberto; Edmondson, Dale E.; Fraaije, Marco W.; Mattevi, Andrea

    2013-01-01

    A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP(+) and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically modified cofactor analogues. Like

  7. Kidney stone matrix proteins ameliorate calcium oxalate monohydrate induced apoptotic injury to renal epithelial cells.

    Science.gov (United States)

    Narula, Shifa; Tandon, Simran; Singh, Shrawan Kumar; Tandon, Chanderdeep

    2016-11-01

    Kidney stone formation is a highly prevalent disease, affecting 8-10% of the human population worldwide. Proteins are the major constituents of human kidney stone's organic matrix and considered to play critical role in the pathogenesis of disease but their mechanism of modulation still needs to be explicated. Therefore, in this study we investigated the effect of human kidney stone matrix proteins on the calcium oxalate monohydrate (COM) mediated cellular injury. The renal epithelial cells (MDCK) were exposed to 200μg/ml COM crystals to induce injury. The effect of proteins isolated from human kidney stone was studied on COM injured cells. The alterations in cell-crystal interactions were examined by phase contrast, polarizing, fluorescence and scanning electron microscopy. Moreover, its effect on the extent of COM induced cell injury, was quantified by flow cytometric analysis. Our study indicated the antilithiatic potential of human kidney stone proteins on COM injured MDCK cells. Flow cytometric analysis and fluorescence imaging ascertained that matrix proteins decreased the extent of apoptotic injury caused by COM crystals on MDCK cells. Moreover, the electron microscopic studies of MDCK cells revealed that matrix proteins caused significant dissolution of COM crystals, indicating cytoprotection against the impact of calcium oxalate injury. The present study gives insights into the mechanism implied by urinary proteins to restrain the pathogenesis of kidney stone disease. This will provide a better understanding of the formation of kidney stones which can be useful for the proper management of the disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    Science.gov (United States)

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

  9. A single peroxisomal targeting signal mediates matrix protein import in diatoms.

    Directory of Open Access Journals (Sweden)

    Nicola H Gonzalez

    Full Text Available Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

  10. Three-dimensional structure of the human immunodeficiency virus type 1 matrix protein.

    Science.gov (United States)

    Massiah, M A; Starich, M R; Paschall, C; Summers, M F; Christensen, A M; Sundquist, W I

    1994-11-25

    The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. Genetic analyses have indicated that the protein performs important functions throughout the viral life-cycle, including anchoring the transmembrane envelope protein on the surface of the virus, assisting in viral penetration, transporting the proviral integration complex across the nuclear envelope, and localizing the assembling virion to the cell membrane. We now report the three-dimensional structure of recombinant HIV-1 matrix protein, determined at high resolution by nuclear magnetic resonance (NMR) methods. The HIV-1 matrix protein is the first retroviral matrix protein to be characterized structurally and only the fourth HIV-1 protein of known structure. NMR signal assignments required recently developed triple-resonance (1H, 13C, 15N) NMR methodologies because signals for 91% of 132 assigned H alpha protons and 74% of the 129 assignable backbone amide protons resonate within chemical shift ranges of 0.8 p.p.m. and 1 p.p.m., respectively. A total of 636 nuclear Overhauser effect-derived distance restraints were employed for distance geometry-based structure calculations, affording an average of 13.0 NMR-derived distance restraints per residue for the experimentally constrained amino acids. An ensemble of 25 refined distance geometry structures with penalties (sum of the squares of the distance violations) of 0.32 A2 or less and individual distance violations under 0.06 A was generated; best-fit superposition of ordered backbone heavy atoms relative to mean atom positions afforded root-mean-square deviations of 0.50 (+/- 0.08) A. The folded HIV-1 matrix protein structure is composed of five alpha-helices, a short 3(10) helical stretch, and a three-strand mixed beta-sheet. Helices I to III and the 3(10) helix pack about a central helix (IV) to form a compact globular domain that is capped by the beta-sheet. The C-terminal helix (helix V) projects away

  11. Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins.

    Science.gov (United States)

    Mizumoto, Shuji; Yamada, Shuhei; Sugahara, Kazuyuki

    2015-10-01

    Recent functional studies on chondroitin sulfate-dermatan sulfate (CS-DS) demonstrated its indispensable roles in various biological events including brain development and cancer. CS-DS proteoglycans exert their physiological activity through interactions with specific proteins including growth factors, cell surface receptors, and matrix proteins. The characterization of these interactions is essential for regulating the biological functions of CS-DS proteoglycans. Although amino acid sequences on the bioactive proteins required for these interactions have already been elucidated, the specific saccharide sequences involved in the binding of CS-DS to target proteins have not yet been sufficiently identified. In this review, recent findings are described on the interaction between CS-DS and some proteins which are especially involved in the central nervous system and cancer development/metastasis. Copyright © 2015. Published by Elsevier Ltd.

  12. The Regional-Matrix Approach to the Training of Highly Qualified Personnel for the Sustainable Development of the Mining Region

    Science.gov (United States)

    Zhernov, Evgeny; Nehoda, Evgenia

    2017-11-01

    The state, regional and industry approaches to the problem of personnel training for building an innovative knowledge economy at all levels that ensures sustainable development of the region are analyzed in the article using the cases of the Kemerovo region and the coal industry. A new regional-matrix approach to the training of highly qualified personnel is proposed, which allows to link the training systems with the regional economic matrix "natural resources - cognitive resources" developed by the author. A special feature of the new approach is the consideration of objective conditions and contradictions of regional systems of personnel training, which have formed as part of economic systems of regions differ-entiated in the matrix. The methodology of the research is based on the statement about the interconnectivity of general and local knowledge, from which the understanding of the need for a combination of regional, indus-try and state approaches to personnel training is derived. A new form of representing such a combination is the proposed approach, which is based on matrix analysis. The results of the research can be implemented in the practice of modernization of professional education of workers in the coal industry of the natural resources extractive region.

  13. Development and evaluation of sustained release losartan potassium matrix tablet using kollidon SR as release retardant

    Directory of Open Access Journals (Sweden)

    Shahid Sarwar

    2012-12-01

    Full Text Available The present study was undertaken to develop sustained release (SR matrix tablets of losartan potassium, an angiotensin-II antagonist for the treatment of hypertension. The tablets were prepared by direct compression method, along with Kollidon SR as release retardant polymer. The amount of losartan potassium remains fixed (100 mg for all the three formulations whereas the amounts of Kollidon SR were 250 mg, 225 mg, and 200 mg for F-1, F-2, and F-3 respectively. The evaluation involves three stages: the micromeritic properties evaluation of granules, physical property studies of tablets, and in-vitro release kinetics studies. The USP apparatus type II was selected to perform the dissolution test, and the dissolution medium was 900 mL phosphate buffer pH 6.8. The test was carried out at 75 rpm, and the temperature was maintained at 37 ºC ± 0.5 ºC. The release kinetics was analyzed using several kinetics models. Higher polymeric content in the matrix decreased the release rate of drug. At lower polymeric level, the rate and extent of drug release were enhanced. All the formulations followed Higuchi release kinetics where the Regression co-efficient (R² values are 0.958, 0.944, and 0.920 for F-1, F-2, and F-3 respectively, and they exhibited diffusion dominated drug release. Statistically significant (PO presente estudo foi realizado para desenvolver (SR matriz de comprimidos de liberação sustentada de losartana, um antagonista da angiotensina II, para o tratamento da hipertensão arterial. Os comprimidos foram preparados pelo método de compressão direta com Kollidon SR como polímero de liberação lenta. A quantidade de losartana potássica permanece fixa (100 mg para todas as três formulações enquanto que as quantidades de Kollidon SR foram de 250 mg, 225 mg e 200 mg para F-1, F-2 e F-3, respectivamente. A avaliação envolve três etapas- propriedades micromeríticas dos grânulos, estudo das propriedades físicas dos comprimidos e

  14. Distinct profile of vascular progenitor attachment to extracellular matrix proteins in cancer patients.

    Science.gov (United States)

    Labonté, Laura; Li, Yuhua; Addison, Christina L; Brand, Marjorie; Javidnia, Hedyeh; Corsten, Martin; Burns, Kevin; Allan, David S

    2012-04-01

    Vascular progenitor cells (VPCs) facilitate angiogenesis and initiate vascular repair by homing in on sites of damage and adhering to extracellular matrix (ECM) proteins. VPCs also contribute to tumor angiogenesis and induce angiogenic switching in sites of metastatic cancer. In this study, the binding of attaching cells in VPC clusters that form in vitro on specific ECM proteins was investigated. VPC cluster assays were performed in vitro on ECM proteins enriched in cancer cells and in remodelling tissue. Profiles of VPC clusters from patients with cancer were compared to healthy controls. The role of VEGF and integrin-specific binding of angiogenic attaching cells was addressed. VPC clusters from cancer patients were markedly increased on fibronectin relative to other ECM proteins tested, in contrast to VPC clusters from control subjects, which formed preferentially on laminin. Specific integrin-mediated binding of attaching cells in VPC clusters was matrix protein-dependent. Furthermore, cancer patients had elevated plasma VEGF levels compared to healthy controls and VEGF facilitated preferential VPC cluster formation on fibronectin. Incubating cells from healthy controls with VEGF induced a switch from the 'healthy' VPC binding profile to the profile observed in cancer patients with a marked increase in VPC cluster formation on fibronectin. The ECM proteins laminin and fibronectin support VPC cluster formation via specific integrins on attaching cells and can facilitate patterns of VPC cluster formation that are distinct in cancer patients. Larger studies, however, are needed to gain insight on how tumor angiogenesis may differ from normal repair processes.

  15. Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

    Directory of Open Access Journals (Sweden)

    Srsen Vlastimil

    2009-04-01

    Full Text Available Abstract Background Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood. Results We performed studies on mouse myoblast cell lines that were induced to differentiate in culture, to characterize the early events of centrosome protein re-localization. We demonstrate that this re-localization occurs already at the single cell stage, prior to fusion into myotubes. Centrosome proteins that accumulate at the nuclear surface form an insoluble matrix that can be reversibly disassembled if isolated nuclei are exposed to mitotic cytoplasm from Xenopus egg extract. Our microscopy data suggest that this perinuclear matrix of centrosome proteins consists of a system of interconnected fibrils. Conclusion Our data provide new insights into the reorganization of centrosome proteins during muscular differentiation, at the structural and biochemical level. Because we observe that centrosome protein re-localization occurs early during differentiation, we believe that it is of functional importance for the reorganization of the cytoskeleton in the differentiation process.

  16. Phrase Mining of Textual Data to Analyze Extracellular Matrix Protein Patterns Across Cardiovascular Disease.

    Science.gov (United States)

    Liem, David Alexandre; Murali, Sanjana; Sigdel, Dibakar; Shi, Yu; Wang, Xuan; Shen, Jiaming; Choi, Howard; Caufield, J Harry; Wang, Wei; Ping, Peipei; Han, Jiawei

    2018-05-18

    Extracellular matrix (ECM) proteins have been shown to play important roles regulating multiple biological processes in an array of organ systems, including the cardiovascular system. By using a novel bioinformatics text-mining tool, we studied six categories of cardiovascular disease (CVD), namely ischemic heart disease (IHD), cardiomyopathies (CM), cerebrovascular accident (CVA), congenital heart disease (CHD), arrhythmias (ARR), and valve disease (VD), anticipating novel ECM protein-disease and protein-protein relationships hidden within vast quantities of textual data. We conducted a phrase-mining analysis, delineating the relationships of 709 ECM proteins with the six groups of CVDs reported in 1,099,254 abstracts. The technology pipeline known as Context-aware Semantic Online Analytical Processing (CaseOLAP) was applied to semantically rank the association of proteins to each and all six CVDs, performing analyses to quantify each protein-disease relationship. We performed principal component analysis and hierarchical clustering of the data, where each protein is visualized as a six dimensional vector. We found that ECM proteins display variable degrees of association with the six CVDs; certain CVDs share groups of associated proteins whereas others have divergent protein associations. We identified 82 ECM proteins sharing associations with all six CVDs. Our bioinformatics analysis ascribed distinct ECM pathways (via Reactome) from this subset of proteins, namely insulin-like growth factor regulation and interleukin-4 and interleukin-13 signaling, suggesting their contribution to the pathogenesis of all six CVDs. Finally, we performed hierarchical clustering analysis and identified protein clusters associated with a targeted CVD; analyses revealed unexpected insights underlying ECM-pathogenesis of CVDs.

  17. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

    Science.gov (United States)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    2006-05-01

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

  18. Postsynaptic density protein 95 in the striosome and matrix compartments of the human neostriatum.

    Directory of Open Access Journals (Sweden)

    Ryoma eMorigaki

    2015-11-01

    Full Text Available The human neostriatum consists of two functional subdivisions referred to as the striosome (patch and matrix compartments. The striosome-matrix dopamine systems play a central role in cortico-thalamo-basal ganglia circuits, and their involvement is thought to underlie the genesis of multiple movement and behavioral disorders, and of drug addiction. Human neuropathology also has shown that striosomes and matrix have differential vulnerability patterns in several striatal neurodegenerative diseases. Postsynaptic density protein 95 (PSD-95, also known as DLG4, is a major scaffolding protein in the postsynaptic densities of dendritic spines. PSD-95 is now known to negatively regulate not only N-methyl-D-aspartate glutamate signaling, but also dopamine D1 signals at sites of postsynaptic transmission. Accordingly, a neuroprotective role for PSD-95 against dopamine D1 receptor (D1R-mediated neurotoxicity in striatal neurodegeneration also has been suggested. Here, we used a highly sensitive immunohistochemistry technique to show that in the human neostriatum, PSD-95 is differentially concentrated in the striosome and matrix compartments, with a higher density of PSD-95 labeling in the matrix compartment than in the striosomes. This compartment-specific distribution of PSD-95 was strikingly complementary to that of D1R. In addition to the possible involvement of PSD-95-mediated synaptic function in compartment-specific dopamine signals, we suggest that the striosomes might be more susceptible to D1R-mediated neurotoxicity than the matrix compartment. This notion may provide new insight into the compartment-specific vulnerability of MSNs in striatal neurodegeneration.

  19. Tissue specificity of the hormonal response in sex accessory tissues is associated with nuclear matrix protein patterns.

    Science.gov (United States)

    Getzenberg, R H; Coffey, D S

    1990-09-01

    The DNA of interphase nuclei have very specific three-dimensional organizations that are different in different cell types, and it is possible that this varying DNA organization is responsible for the tissue specificity of gene expression. The nuclear matrix organizes the three-dimensional structure of the DNA and is believed to be involved in the control of gene expression. This study compares the nuclear structural proteins between two sex accessory tissues in the same animal responding to the same androgen stimulation by the differential expression of major tissue-specific secretory proteins. We demonstrate here that the nuclear matrix is tissue specific in the rat ventral prostate and seminal vesicle, and undergoes characteristic alterations in its protein composition upon androgen withdrawal. Three types of nuclear matrix proteins were observed: 1) nuclear matrix proteins that are different and tissue specific in the rat ventral prostate and seminal vesicle, 2) a set of nuclear matrix proteins that either appear or disappear upon androgen withdrawal, and 3) a set of proteins that are common to both the ventral prostate and seminal vesicle and do not change with the hormonal state of the animal. Since the nuclear matrix is known to bind androgen receptors in a tissue- and steroid-specific manner, we propose that the tissue specificity of the nuclear matrix arranges the DNA in a unique conformation, which may be involved in the specific interaction of transcription factors with DNA sequences, resulting in tissue-specific patterns of secretory protein expression.

  20. Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

    Science.gov (United States)

    Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-01-01

    Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

  1. Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

    Science.gov (United States)

    Zhang, Wenting; Zheng, Wenjie; Toh, Yukimatsu; Betancourt-Solis, Miguel A; Tu, Jiagang; Fan, Yanlin; Vakharia, Vikram N; Liu, Jun; McNew, James A; Jin, Meilin; Tao, Yizhi J

    2017-08-08

    Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.

  2. Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

    Science.gov (United States)

    Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

    2018-02-28

    Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

  3. [Preparation of hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata and study on its in vitro release mechanism].

    Science.gov (United States)

    Xu, Fang-Fang; Shi, Wei; Zhang, Hui; Guo, Qing-Ming; Wang Zhen-Zhong; Bi, Yu-An; Wang, Zhi-Min; Xiao, Wei

    2015-01-01

    In this study, hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata were prepared and the in vitro release behavior were also evaluated. The optimal prescription was achieved by studying the main factor of the type and amount of hydroxypropyl methylcellulose (HPMC) using single factor test and evaluating through cumulative release of three lactones. No burst drug release from the obtained matrix tablets was observed. Drug release sustained to 14 h. The release mechanism of three lactones from A. paniculata was accessed by zero-order, first-order, Higuchi and Peppas equation. The release behavior of total lactones from A. paniculata was better agreed with Higuchi model and the drug release from the tablets was controlled by degradation of the matrix. The preparation of hydrophilic matrix sustained release tablets of total lactones from A. paniculata with good performance of drug release was simple.

  4. Alterations in proteins of bone marrow extracellular matrix in undernourished mice

    Directory of Open Access Journals (Sweden)

    C.L. Vituri

    2000-08-01

    Full Text Available The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM. Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5% and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase and laminin (4.8-fold increase when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

  5. Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques

    DEFF Research Database (Denmark)

    Woods, Alan A; Linton, Stuart M; Davies, Michael Jonathan

    2003-01-01

    Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has...... been interpreted in terms of the occurrence of two oxidative mechanisms, one involving oxygen-derived radicals catalysed by trace transition metal ions, and a second involving chlorinating species (HOCl or Cl2), generated by the haem enzyme myeloperoxidase (MPO). As MPO is released extracellularly...... for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions...

  6. Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

    Science.gov (United States)

    Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

    2018-01-01

    This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

  7. Trends in global warming and evolution of matrix protein 2 family from influenza A virus.

    Science.gov (United States)

    Yan, Shao-Min; Wu, Guang

    2009-12-01

    The global warming is an important factor affecting the biological evolution, and the influenza is an important disease that threatens humans with possible epidemics or pandemics. In this study, we attempted to analyze the trends in global warming and evolution of matrix protein 2 family from influenza A virus, because this protein is a target of anti-flu drug, and its mutation would have significant effect on the resistance to anti-flu drugs. The evolution of matrix protein 2 of influenza A virus from 1959 to 2008 was defined using the unpredictable portion of amino-acid pair predictability. Then the trend in this evolution was compared with the trend in the global temperature, the temperature in north and south hemispheres, and the temperature in influenza A virus sampling site, and species carrying influenza A virus. The results showed the similar trends in global warming and in evolution of M2 proteins although we could not correlate them at this stage of study. The study suggested the potential impact of global warming on the evolution of proteins from influenza A virus.

  8. Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants.

    Science.gov (United States)

    Echtay, Karim S; Murphy, Michael P; Smith, Robin A J; Talbot, Darren A; Brand, Martin D

    2002-12-06

    Superoxide activates nucleotide-sensitive mitochondrial proton transport through the uncoupling proteins UCP1, UCP2, and UCP3 (Echtay, K. S., et al. (2002) Nature 415, 1482-1486). Two possible mechanisms were proposed: direct activation of the UCP proton transport mechanism by superoxide or its products and a cycle of hydroperoxyl radical entry coupled to UCP-catalyzed superoxide anion export. Here we provide evidence for the first mechanism and show that superoxide activates UCP2 in rat kidney mitochondria from the matrix side of the mitochondrial inner membrane: (i) Exogenous superoxide inhibited matrix aconitase, showing that external superoxide entered the matrix. (ii) Superoxide-induced uncoupling was abolished by low concentrations of the mitochondrially targeted antioxidants 10-(6'-ubiquinonyl)decyltriphenylphosphonium (mitoQ) or 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (mitoVit E), which are ubiquinone (Q) or tocopherol derivatives targeted to the matrix by covalent attachment to triphenylphosphonium cation. However, superoxide-induced uncoupling was not affected by similar concentrations of the nontargeted antioxidants Q(o), Q(1), decylubiquinone, vitamin E, or 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid (TROLOX) or of the mitochondrially targeted but redox-inactive analogs decyltriphenylphosphonium or 4-chlorobutyltriphenylphosphonium. Thus matrix superoxide appears to be necessary for activation of UCP2 by exogenous superoxide. (iii) When the reduced to oxidized ratio of mitoQ accumulated by mitochondria was increased by inhibiting cytochrome oxidase, it induced nucleotide-sensitive uncoupling that was not inhibited by external superoxide dismutase. Under these conditions quinols are known to produce superoxide, and because mitoQ is localized within the mitochondrial matrix this suggests that production of superoxide in the matrix was sufficient to activate UCP2. Furthermore, the superoxide

  9. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

    Science.gov (United States)

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-12-29

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

  10. Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

    Science.gov (United States)

    Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

    2014-11-01

    Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people

  11. Preparation and In-Vitro Evaluation of Sustained Release Matrix Diclofenac Sodium Tablets Using HPMC KM 100 and Gums

    Directory of Open Access Journals (Sweden)

    Zafar Iqbal, Raza Khan, Fazli Nasir, Jamshaid Ali Khan, Lateef Ahmad, Abad Khan, Yaser Shah

    2010-12-01

    Full Text Available Objectives: The impact of hydroxypropylmethyl cellulose(HPMC K 100M alone and in combination with the guar gum,xanthan gum and gum tragacanth on the release of the diclofenac sodium matrix tablets were evaluated.Materials and Methods: The granules were prepared using wet granulation method and compressed into tablets using different ratio of drug and gum ratio. The physical properties of the tablets were within acceptable pharmacopeial limits.The release profiles of the matrix tablets were evaluated in vitro,using USP dissolution apparatus II (paddle method.Results: The formulations containing HPMC K 100M drug ratio1:1.3 and 1:1.6 and formulations containing HPMC, gum and drug with different ratio also sustained the release of diclofenac sodium for 12 hours. The mechanism of drug release from the matrix tablets was studied using Zero order, First order, Higuchi and Korsmeyer’s models using regression coefficient method. The stability of the selected formulations was evaluated at 40˚C and 70% RH for 6 months.Conclusions: HPMC K100M alone and in combination with natural gums as the retarding material retarded the release upto 12 hours and showed little deviation from the theoretical release pattern.

  12. Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein.

    Science.gov (United States)

    Serrière, Jennifer; Robert, Xavier; Perez, Magali; Gouet, Patrice; Guillon, Christophe

    2013-06-24

    Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins

  13. Prediction of residue-residue contact matrix for protein-protein interaction with Fisher score features and deep learning.

    Science.gov (United States)

    Du, Tianchuan; Liao, Li; Wu, Cathy H; Sun, Bilin

    2016-11-01

    Protein-protein interactions play essential roles in many biological processes. Acquiring knowledge of the residue-residue contact information of two interacting proteins is not only helpful in annotating functions for proteins, but also critical for structure-based drug design. The prediction of the protein residue-residue contact matrix of the interfacial regions is challenging. In this work, we introduced deep learning techniques (specifically, stacked autoencoders) to build deep neural network models to tackled the residue-residue contact prediction problem. In tandem with interaction profile Hidden Markov Models, which was used first to extract Fisher score features from protein sequences, stacked autoencoders were deployed to extract and learn hidden abstract features. The deep learning model showed significant improvement over the traditional machine learning model, Support Vector Machines (SVM), with the overall accuracy increased by 15% from 65.40% to 80.82%. We showed that the stacked autoencoders could extract novel features, which can be utilized by deep neural networks and other classifiers to enhance learning, out of the Fisher score features. It is further shown that deep neural networks have significant advantages over SVM in making use of the newly extracted features. Copyright © 2016. Published by Elsevier Inc.

  14. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    International Nuclear Information System (INIS)

    Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro; Yamaguchi, Kazuo; Garcia, Andres J

    2011-01-01

    The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG 7 ). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni 2+ -ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG 7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII 7-10 ) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII 7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  15. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    Energy Technology Data Exchange (ETDEWEB)

    Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro [World Premier International (WPI) Research Center Initiative, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science - NIMS (Japan); Yamaguchi, Kazuo [Department of Chemistry, Faculty of Science and Research Institute for Photofunctionalized Materials, Kanagawa University (Japan); Garcia, Andres J, E-mail: NAKANISHI.Jun@nims.go.jp [Institute for Bioengineering and Bioscience, Woodruff School of Mechanical Engineering, Georgia Institute of Technology (United States)

    2011-08-15

    The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG{sub 7}). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni{sup 2+}-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG{sub 7} underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII{sub 7-10}) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII{sub 7-10} was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  16. Simulation of Forest Environment as a Measure to sustain Protein ...

    African Journals Online (AJOL)

    It was observed that domestication and shade types under which of wild Vegetables were grown had effects on their nutritive values (the protein, vitamin A and C contents). Nevertheless, banana shades closely upheld vitamin C contents of L.curnuta and B. pilosa. Also, it did well to maintain protein contents in L. curnuta ...

  17. Dry fractionation for sustainable production of plant protein concentrates

    NARCIS (Netherlands)

    Pelgrom, P.J.M.

    2015-01-01

    The global demand for protein-rich foods is expected to double in the coming decades due to the increasing prosperity and world population. To keep up with the demand, the transition from an animal to a plant-based protein supply is desirable from long-term economic and environmental

  18. Crosslinking of a Peritrophic Matrix Protein Protects Gut Epithelia from Bacterial Exotoxins

    Science.gov (United States)

    Shibata, Toshio; Maki, Kouki; Hadano, Jinki; Fujikawa, Takumi; Kitazaki, Kazuki; Koshiba, Takumi; Kawabata, Shun-ichiro

    2015-01-01

    Transglutaminase (TG) catalyzes protein-protein crosslinking, which has important and diverse roles in vertebrates and invertebrates. Here we demonstrate that Drosophila TG crosslinks drosocrystallin, a peritrophic matrix protein, to form a stable fiber structure on the gut peritrophic matrix. RNA interference (RNAi) of the TG gene was highly lethal in flies and induced apoptosis of gut epithelial cells after oral infection with Pseudomonas entomophila. Moreover, AprA, a metalloprotease secreted by P. entomophila, digested non-crosslinked drosocrystallin fibers, but not drosocrystallin fibers crosslinked by TG. In vitro experiments using recombinant drosocrystallin and monalysin proteins demonstrated that monalysin, a pore-forming exotoxin of P. entomophila, was adsorbed on the crosslinked drosocrystallin fibers in the presence of P. entomophila culture supernatant. In addition, gut-specific TG-RNAi flies had a shorter lifespan than control flies after ingesting P. entomophila, whereas the lifespan after ingesting AprA-knockout P. entomophila was at control levels. We conclude that drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, form an important physical barrier against exotoxins of invading pathogenic microbes. PMID:26506243

  19. Crosslinking of a Peritrophic Matrix Protein Protects Gut Epithelia from Bacterial Exotoxins.

    Directory of Open Access Journals (Sweden)

    Toshio Shibata

    2015-10-01

    Full Text Available Transglutaminase (TG catalyzes protein-protein crosslinking, which has important and diverse roles in vertebrates and invertebrates. Here we demonstrate that Drosophila TG crosslinks drosocrystallin, a peritrophic matrix protein, to form a stable fiber structure on the gut peritrophic matrix. RNA interference (RNAi of the TG gene was highly lethal in flies and induced apoptosis of gut epithelial cells after oral infection with Pseudomonas entomophila. Moreover, AprA, a metalloprotease secreted by P. entomophila, digested non-crosslinked drosocrystallin fibers, but not drosocrystallin fibers crosslinked by TG. In vitro experiments using recombinant drosocrystallin and monalysin proteins demonstrated that monalysin, a pore-forming exotoxin of P. entomophila, was adsorbed on the crosslinked drosocrystallin fibers in the presence of P. entomophila culture supernatant. In addition, gut-specific TG-RNAi flies had a shorter lifespan than control flies after ingesting P. entomophila, whereas the lifespan after ingesting AprA-knockout P. entomophila was at control levels. We conclude that drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, form an important physical barrier against exotoxins of invading pathogenic microbes.

  20. Enhance and Maintain Chondrogenesis of Synovial Fibroblasts by Cartilage Extracellular Matrix Protein Matrilins

    Science.gov (United States)

    Pei, Ming; Luo, Junming; Chen, Qian

    2008-01-01

    Summary Objective Cartilage-specific extracellular matrix (ECM) proteins have been proposed to play key roles in modulating cellular phenotypes during chondrogenesis of mesenchymal stem cells. Matrilin (MATN) 1 and 3 are among the most up-regulated ECM proteins during chondrogenesis. The aim of this study was to analyze their roles in chondrogenesis of mesenchymal fibroblasts from synovium. Methods Primary synovial fibroblasts (SFBs) were purified from porcine synovium and incubated in pellet culture for 18 days. Chondrogenesis of SFB was analyzed by histological staining with safranin-O/fast green, and by quantifying glycosaminoglycans with dimethylmethylene blue assay. The mRNA levels of chondrogenic markers including collagen II, aggrecan, and Sox 9 were quantified by real-time RT-PCR, while the protein levels of Col II and matrilins were determined by western blot analysis. Results SFBs underwent chondrogenesis after incubation with TGF-β1 for three days; however, this process was attenuated during the subsequent incubation period. Expression of a MATN1 or 3 cDNA maintained and further enhanced chondrogenesis of SFBs as shown by increased cartilaginous matrix areas, elevated amount of glycosaminoglycans, and stimulated expression of chondrogenic markers. Conclusion Our findings suggest a novel function for MATN1 and 3 to maintain and enhance chondrogenesis of mesenchymal fibroblasts initiated by TGF-β. Our results also support a critical role of cartilage-specific ECM proteins to modulate cellular phenotypes in the microenvironment during chondrogenic differentiation. PMID:18282772

  1. Enhancing and maintaining chondrogenesis of synovial fibroblasts by cartilage extracellular matrix protein matrilins.

    Science.gov (United States)

    Pei, M; Luo, J; Chen, Q

    2008-09-01

    Cartilage-specific extracellular matrix (ECM) proteins have been proposed to play key roles in modulating cellular phenotypes during chondrogenesis of mesenchymal stem cells. Matrilin (MATN)1 and MATN3 are among the most up-regulated ECM proteins during chondrogenesis. The aim of this study was to analyze their roles in chondrogenesis of mesenchymal fibroblasts from synovium. Primary synovial fibroblasts (SFBs) were purified from porcine synovium and incubated in pellet culture for 18 days. Chondrogenesis of SFB was analyzed by histological staining with safranin-O/fast green, and by quantifying glycosaminoglycans (GAG) with dimethylmethylene blue assay. The mRNA levels of chondrogenic markers including collagen II, aggrecan, and Sox 9 were quantified by real-time reverse transcription polymerase chain reaction, while the protein levels of Col II and MATNs were determined by western blot analysis. SFBs underwent chondrogenesis after incubation with transforming growth factor-beta1 (TGF-beta1) for 3 days; however, this process was attenuated during the subsequent incubation period. Expression of a Matn1 or Matn3 cDNA maintained and further enhanced chondrogenesis of SFBs as shown by increased cartilaginous matrix areas, elevated amount of GAG, and stimulated expression of chondrogenic markers. Our findings suggest a novel function for MATN1 and MATN3 to maintain and enhance chondrogenesis of mesenchymal fibroblasts initiated by TGF-beta. Our results also support a critical role of cartilage-specific ECM proteins to modulate cellular phenotypes in the microenvironment during chondrogenic differentiation.

  2. Hyriopsis cumingii Hic52-A novel nacreous layer matrix protein with a collagen-like structure.

    Science.gov (United States)

    Liu, Xiaojun; Pu, Jingwen; Zeng, Shimei; Jin, Can; Dong, Shaojian; Li, Jiale

    2017-09-01

    Nacre is a product of a precisely regulated biomineralization process and a major contributor to the luster of pearls. Nacre is composed of calcium carbonate and an organic matrix of proteins that is secreted from mollusc mantle tissue and is exclusively associated with shell formation. In this study, hic52, a novel matrix protein gene from mantle of Hyriopsis cumingii, was cloned and functionally analyzed. The full-length cDNA of hic52 encoded 542 amino acids and contained a signal peptide of 18 amino acids. Excluding the signal peptide, the theoretical molecular mass of the polypeptide was 52.2kDa. The predicted isoelectric point was 10.37, indicating a basic shell protein. The amino acid sequence of hic52 featured high proportion of Gly (28.8%) and Gln (12.4%) residues. The predicted tertiary structure was characterized as having similarities to collagen I, alpha 1 and alpha 2 in the structure. The polypeptide sequence shared no homology with collagen. The hic52 expression pattern by quantitative real-time PCR and in situ hybridization exhibits at the dorsal epithelial cells of the mantle. Expression increased during the stages of pearl sac development. The data showed that hic52 is probably a framework shell protein that mediates and controls the nacreous biomineralization process. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Protein-transitions in and out of the dough matrix in wheat flour mixing.

    Science.gov (United States)

    Wang, Xiaolong; Appels, Rudi; Zhang, Xiaoke; Bekes, Ferenc; Torok, Kitti; Tomoskozi, Sandor; Diepeveen, Dean; Ma, Wujun; Islam, Shahidul

    2017-02-15

    Sequential protein behavior in the wheat dough matrix under continuous mixing and heating treatment has been studied using Mixolab-dough samples from two Australian wheat cultivars, Westonia and Wyalkatchem. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis indicated that 32min (80°C) was a critical time point in forming large protein complexes and loosing extractability of several protein groups like y-type high molecular weight glutenin subunits (HMW-GSs), gamma-gliadins, beta-amylases, serpins, and metabolic proteins with higher mass. Up to 32min (80°C) Westonia showed higher protein extractability compared to Wyalkatchem although it was in the opposite direction thereafter. Twenty differentially expressed proteins could be assigned to chromosomes 1D, 3A, 4A, 4B, 4D, 6A, 6B, 7A and 7B. The results expanded the range of proteins associated with changes in the gluten-complex during processing and provided targets for selecting new genetic variants associated with altered quality attributes of the flour. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Vitamin K Intake and Plasma Desphospho-Uncarboxylated Matrix Gla-Protein Levels in Kidney Transplant Recipients

    NARCIS (Netherlands)

    Boxma, P.Y.; Berg, van den E.; Geleijnse, J.M.; Laverman, G.D.; Schurgers, L.J.; Vermeer, C.; Kema, I.P.; Muskiet, F.A.J.; Navis, G.; Bakker, S.J.L.; Borst, de M.H.

    2012-01-01

    Vitamin K is essential for activation of ¿-carboxyglutamate (Gla)-proteins including the vascular calcification inhibitor matrix Gla-protein (MGP). Insufficient vitamin K intake leads to production of uncarboxylated, mostly inactive proteins and contributes to an increased cardiovascular risk. In

  5. Improved success of sparse matrix protein crystallization screening with heterogeneous nucleating agents.

    Directory of Open Access Journals (Sweden)

    Anil S Thakur

    2007-10-01

    Full Text Available Crystallization is a major bottleneck in the process of macromolecular structure determination by X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth generally occurs spontaneously in a supersaturated solution as a result of homogenous nucleation. However, in a typical sparse matrix screening experiment, precipitant and protein concentration are not sampled extensively, and supersaturation conditions suitable for nucleation are often missed.We tested the effect of nine potential heterogenous nucleating agents on crystallization of ten test proteins in a sparse matrix screen. Several nucleating agents induced crystal formation under conditions where no crystallization occurred in the absence of the nucleating agent. Four nucleating agents: dried seaweed; horse hair; cellulose and hydroxyapatite, had a considerable overall positive effect on crystallization success. This effect was further enhanced when these nucleating agents were used in combination with each other.Our results suggest that the addition of heterogeneous nucleating agents increases the chances of crystal formation when using sparse matrix screens.

  6. Patchwork structure-function analysis of the Sendai virus matrix protein.

    Science.gov (United States)

    Mottet-Osman, Geneviève; Miazza, Vincent; Vidalain, Pierre-Olivier; Roux, Laurent

    2014-09-01

    Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. The Role of Structural Extracellular Matrix Proteins in Urothelial Bladder Cancer

    Directory of Open Access Journals (Sweden)

    Andrea Brunner

    2007-01-01

    Full Text Available The extracellular matrix (ECM plays a key role in the modulation of cancer cell invasion. In urothelial carcinoma of the bladder (UC the role of ECM proteins has been widely studied. The mechanisms, which are involved in the development of invasion, progression and generalization, are complex, depending on the interaction of ECM proteins with each other as well as with cancer cells. The following review will focus on the pathogenetic role and prognostic value of structural proteins, such as laminins, collagens, fi bronectin (FN, tenascin (Tn-C and thrombospondin 1 (TSP1 in UC. In addition, the role of integrins mediating the interaction of ECM molecules and cancer cells will be addressed, since integrin-mediated FN, Tn-C and TSP1 interactions seem to play an important role during tumor cell invasion and angiogenesis.

  8. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    Directory of Open Access Journals (Sweden)

    Jun Nakanishi, Hidekazu Nakayama, Kazuo Yamaguchi, Andres J Garcia and Yasuhiro Horiike

    2011-01-01

    Full Text Available The development of methods for the off–on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs of three disulfide compounds containing (i a photocleavable poly(ethylene glycol (PEG, (ii nitrilotriacetic acid (NTA and (iii hepta(ethylene glycol (EG7. Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7–10 to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  9. Intergenotypic replacement of lyssavirus matrix proteins demonstrates the role of lyssavirus M proteins in intracellular virus accumulation.

    Science.gov (United States)

    Finke, Stefan; Granzow, Harald; Hurst, Jose; Pollin, Reiko; Mettenleiter, Thomas C

    2010-02-01

    Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

  10. ADAM 12 cleaves extracellular matrix proteins and correlates with cancer status and stage

    DEFF Research Database (Denmark)

    Roy, Roopali; Wewer, Ulla M; Zurakowski, David

    2004-01-01

    -Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an approximately 68-k......Da band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen...... or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme...

  11. The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

    Science.gov (United States)

    Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

    2012-07-01

    Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells.

  12. EcmPred: Prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection

    KAUST Repository

    Kandaswamy, Krishna Kumar Umar; Ganesan, Pugalenthi; Kalies, Kai Uwe; Hartmann, Enno; Martinetz, Thomas M.

    2013-01-01

    The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan

  13. Glycation of extracellular matrix proteins and its role in atherosclerosis 

    Directory of Open Access Journals (Sweden)

    Aleksandra Kuzan

    2012-10-01

    Full Text Available Glycation consists in formation of advanced glycation end-products (AGE during non-enzymatic reaction between reducing sugars and proteins, lipids or nucleic acids. This review is focused mainly on glycation of collagen and its role in acceleration of vascular disease. Collagen is an extracellular matrix protein characterized by unique structure forming fibrils with great anti-tensile and anti-breaking strength. The protein builds the connective tissue and is responsible for biomechanical properties of blood vessels. It is reported that higher content of glycated collagen correlates with lower elasticity and greater toughness of the vessel walls and, as a consequence, a faster rate of atherosclerosis development. Numerous mechanisms connected with AGE formation are involved in atherogenesis, among others: receptor-mediated production of free radicals, triggering an inflammatory process, activation of leukocytes and thrombocytes, facilitation of LDL binding, change in level of growth factors, adhesion molecules, MMP and some other proteins’ expression. The coverages allow the development of therapeutic strategies to prevent or slow down the pathological processes connected with glycation of collagen and other proteins in the artery wall. The main strategies are based on limitation of exogenous AGE, consumption of products which contain rutin, treatment with drugs which inhibit AGE formation, such aspyridoxamine, and chemicals which are able to cleave already formed AGE protein-protein crosslinks, such as ALT-711.

  14. Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading

    Directory of Open Access Journals (Sweden)

    Belinda Pingguan-Murphy

    2012-08-01

    Full Text Available OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05. The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05, indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading.

  15. Evaluation of rate of swelling and erosion of verapamil (VRP) sustained-release matrix tablets.

    Science.gov (United States)

    Khamanga, Sandile M; Walker, Roderick B

    2006-01-01

    Tablets manufactured in-house were compared to a marketed sustained-release product of verapamil to investigate the rate of hydration, erosion, and drug-release mechanism by measuring the wet and subsequent dry weights of the products. Swelling and erosion rates depended on the polymer and granulating fluid used, which ultimately pointed to their permeability characteristics. Erosion rate of the marketed product was highest, which suggests that the gel layer that formed around these tablets was weak as opposed to the robust and resistant layers of test products. Anomalous and near zero-order transport mechanisms were dominant in tests and commercial product, respectively.

  16. Expression of extracellular matrix proteins: tenascin-C, fibronectin and galectin-3 in prostatic adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Monika Ulamec

    2015-12-01

    Full Text Available Introduction: The interchanged stromal-epithelial relations and altered expression profiles of various extracellular matrix (ECM proteins creates a suitable microenvironment for cancer development and growth. We support the opinion that remodeling of the extracellular matrix (ECM plays an important role in the cancer progression. The aim of this study was to examine the expression of ECM proteins tenascin-C, fibronectin and galectin-3 in prostatic adenocarcinoma. Methods: Glands and surrounding stroma were analyzed in randomly selected specimens from 52 patients with prostate cancer and 28 patients with benign prostatic hyperplasia (BHP. To evaluate the intensity of tenascin-C, fibronectin and galectin-3 expression the percentage of positively immunostained stromal cells was examined.Results: Compared to BPH, stroma of prostatic adenocarcinoma showed statistically significant increase in tenascin-C expression (p<0.001, predominantly around neoplastic glands, while fibronectin (p=0.001 and galectin-3 (p<0.001 expression in the same area was decreased.Conclusions: Our study confirms changes in the expression of ECM proteins of prostate cancer which may have important role in the cancer development.

  17. Structure and expression of an unusually acidic matrix protein of pearl oyster shells

    International Nuclear Information System (INIS)

    Tsukamoto, Daiki; Sarashina, Isao; Endo, Kazuyoshi

    2004-01-01

    We report identification and characterization of the unusually acidic molluscan shell matrix protein Aspein, which may have important roles in calcium carbonate biomineralization. The Aspein gene (aspein) encodes a sequence of 413 amino acids, including a high proportion of Asp (60.4%), Gly (16.0%), and Ser (13.2%), and the predicted isoelectric point is 1.45; this is the most acidic of all the molluscan shell matrix proteins sequenced so far, or probably even of all known proteins on earth. The main body of Aspein is occupied by (Asp) 2-10 sequences punctuated with Ser-Gly dipeptides. RT-PCR demonstrated that the transcript of aspein is expressed at the outer edge of the mantle, corresponding to the calcitic prismatic layer, but not at the inner part of the mantle, corresponding to the aragonitic nacreous layer. Our findings and previous in vitro experiments taken together suggest that Aspein is responsible for directed formation of calcite in the shell of the pearl oyster Pinctada fucata

  18. Matrix Gla Protein is Involved in Crystal Formation in Kidney of Hyperoxaluric Rats

    Directory of Open Access Journals (Sweden)

    Xiuli Lu

    2013-02-01

    Full Text Available Background: Matrix Gla protein (MGP is a molecular determinant regulating vascular calcification of the extracellular matrix. However, it is still unclear how MGP may be invovled in crystal formation in the kidney of hyperoxaluric rats. Methods: Male Sprague-Dawley rats were divided into the hyperoxaluric group and control group. Hyperoxaluric rats were administrated by 0.75% ethylene glycol (EG for up to 8 weeks. Renal MGP expression was detected by the standard avidin-biotin complex (ABC method. Renal crystal deposition was observed by a polarizing microscope. Total RNA and protein from the rat kidney tissue were extracted. The levels of MGP mRNA and protein expression were analyzed by the real-time polymerase chain reaction (RT-PCR and Western blot. Results: Hyperoxaluria was induced successfully in rats. The MGP was polarly distributed, on the apical membrane of renal tubular epithelial cells, and was found in the ascending thick limbs of Henle's loop (cTAL and the distal convoluted tubule (DCT in hyperoxaluric rats, its expression however, was present in the medullary collecting duct (MCD in stone-forming rats. Crystals with multilaminated structure formed in the injurious renal tubules with lack of MGP expression.MGP mRNA expression was significantly upregulated by the crystals' stimulations. Conclusion: Our results suggested that the MGP was involved in crystals formation by the continuous expression, distributing it polarly in the renal tubular cells and binding directly to the crystals.

  19. Preparation and characterization of alginate microspheres for sustained protein delivery within tissue scaffolds

    International Nuclear Information System (INIS)

    Zhai Peng; Chen, X B; Schreyer, David J

    2013-01-01

    Tissue engineering scaffolds are designed not only to provide structural support for the repair of damaged tissue, but can also serve the function of bioactive protein delivery. Here we present a study on the preparation and characterization of protein-loaded microspheres, either alone or incorporated into mock tissue scaffolds, for sustained protein delivery. Alginate microspheres were prepared by a novel, small-scale water-in-oil emulsion technique and loaded with fluorescently labeled immunoglobulin G (IgG). Microsphere size appears to be influenced by the magnitude and distribution of force generated by mechanical stirring during emulsion. Protein release studies show that sustained IgG release from microspheres could be achieved and that application of a secondary coating of chitosan could further slow the rate of protein release. Preservation of bioactivity of released IgG protein was confirmed using an immunohistochemical assay. When IgG-loaded microspheres were incorporated into mock scaffolds, initial protein release was diminished and the overall time course of release was extended. The present study demonstrates that protein-loaded microspheres can be prepared with a controlled release profile and preserved biological activity, and can be incorporated into scaffolds to achieve sustained and prolonged protein delivery in a tissue engineering application. (paper)

  20. Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

    Science.gov (United States)

    Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

    2013-11-01

    Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular

  1. Myristoylation drives dimerization of matrix protein from mouse mammary tumor virus

    Czech Academy of Sciences Publication Activity Database

    Doležal, Michal; Zábranský, Aleš; Dostál, Jiří; Vaněk, O.; Brynda, Jiří; Lepšík, Martin; Hadravová, Romana; Pichová, Iva

    2016-01-01

    Roč. 13, Jan 5 (2016), č. článku 2. ISSN 1742-4690 R&D Projects: GA MŠk LO1302; GA MŠk(CZ) LO1304; GA ČR GBP208/12/G016 Institutional support: RVO:61388963 Keywords : dimerization * matrix protein * MMTV * molecular dynamics * mouse mammary tumor virus * myristoylation Subject RIV: CE - Biochemistry Impact factor: 3.867, year: 2016 http://retrovirology.biomedcentral.com/articles/10.1186/s12977-015-0235-8

  2. Efficacy of enamel matrix protein applied to spontaneous periodontal disease in two dogs.

    Science.gov (United States)

    Watanabe, Kazuhiro; Kikuchi, Masahiro; Okumura, Masahiro; Kadosawa, Tsuyoshi; Fujinaga, Toru

    2003-09-01

    Enamel matrix protein (EMP) was applied for regeneration of periodontal tissue in 2 dogs with spontaneous periodontal disease. Case 1 had bony resorption around the root and root apex of the maxillary fourth premolars. Case 2 had vertical resorption of bone between the mandibular first and second molars. A flap was formed in the buccal gingiva, and EMP was applied onto the surface of the exposed root. One or 4 months postoperatively, increased bone level and clinical attachment were recognized. EMP was therefore suggested to be effective to induce regeneration of periodontal tissues in the cases with periodontal disease.

  3. OmpL1 is an extracellular matrix- and plasminogen-interacting protein of Leptospira spp.

    Science.gov (United States)

    Fernandes, Luis G V; Vieira, Monica L; Kirchgatter, Karin; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Romero, Eliete C; Nascimento, Ana L T O

    2012-10-01

    Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical β-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with K(D) (equilibrium dissociation constant) values of 2,099.93 ± 871.03 nM and 1,239.23 ± 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a K(D) of 368.63 ± 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

  4. Protein-resistant polymer coatings obtained by matrix assisted pulsed laser evaporation

    Energy Technology Data Exchange (ETDEWEB)

    Rusen, L. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Mustaciosu, C. [Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH, Magurele, Bucharest (Romania); Mitu, B.; Filipescu, M.; Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Dinca, V., E-mail: dinali@nipne.ro [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania)

    2013-08-01

    Adsorption of proteins and polysaccharides is known to facilitate microbial attachment and subsequent formation of biofilm on surfaces that ultimately results in its biofouling. Therefore, protein repellent modified surfaces are necessary to block the irreversible attachment of microorganisms. Within this context, the feasibility of using the Poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether (PEG-block-PCL Me) copolymer as potential protein-resistant coating was explored in this work. The films were deposited using Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique that allows good control of composition, thickness and homogeneity. The chemical and morphological characteristics of the films were examined using Fourier Transform Infrared Spectroscopy (FTIR), contact angle measurements and Atomic Force Microscopy (AFM). The FTIR data demonstrates that the functional groups in the MAPLE-deposited films remain intact, especially for fluences below 0.5 J cm{sup −2}. Optical Microscopy and AFM images show that the homogeneity and the roughness of the coatings are related to both laser parameters (fluence, number of pulses) and target composition. Protein adsorption tests were performed on the PEG-block-PCL Me copolymer coated glass and on bare glass surface as a control. The results show that the presence of copolymer as coating significantly reduces the adsorption of proteins.

  5. Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways ▿

    Science.gov (United States)

    Larrous, Florence; Gholami, Alireza; Mouhamad, Shahul; Estaquier, Jérôme; Bourhy, Hervé

    2010-01-01

    The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control. PMID:20631119

  6. Two overlapping domains of a lyssavirus matrix protein that acts on different cell death pathways.

    Science.gov (United States)

    Larrous, Florence; Gholami, Alireza; Mouhamad, Shahul; Estaquier, Jérôme; Bourhy, Hervé

    2010-10-01

    The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control.

  7. [Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

    Science.gov (United States)

    Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

    2013-01-01

    After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

  8. Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

    Science.gov (United States)

    Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

    2012-12-01

    Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

  9. Self-reinforcement and protein sustained delivery of hyaluronan hydrogel by tailoring a dually cross-linked network

    International Nuclear Information System (INIS)

    Luo, Chunhong; Xu, Guoguang; Wang, Xinghui; Tu, Mei; Zeng, Rong; Rong, Jianhua; Zhao, Jianhao

    2015-01-01

    A series of self-reinforcing hyaluronan hydrogels were developed to improve mechanical properties and protein sustained delivery thanks to a dually cross-linked network. Hyaluronan gel particles (HGPs, 1–5 μm in diameter) with different cross-linking densities, i.e. HGPs-1.5, HGPs-3 and HGPs-15, were prepared in an inverse emulsion system and used as the reinforcing phase after glycidyl methacrylation, while glycidyl methacrylated hyaluronan with a substitution degree of 45.2% was synthesized as the matrix phase. These two phases were cross-linked under ultraviolet irradiation to form self-reinforcing hyaluronan hydrogels (srHAs) that showed typical cross-linked structure of HGPs connecting the matrix phase by cross-section observation. In comparison to hyaluronan bulk gels and their blends with HGPs, srHAs distinctly enhanced the mechanical properties and BSA long-term sustained delivery, especially srHA-1.5 showed the highest compressive modulus of 220 ± 15 kPa and the slowest BSA delivery (67% release at 14 d). The 3T3 fibroblast cell culture showed that all the srHAs had no cytotoxicity. - Highlights: • New self-reinforcing HA hydrogels with a dually cross-linked network were developed. • Self-reinforcing HA hydrogels greatly enhanced the mechanical properties. • Self-reinforcing HA hydrogels prolonged the sustained delivery of BSA. • The self-reinforcing mechanism and BSA diffusion mechanism were discussed. • Self-reinforcing HA hydrogels had no cytotoxicity to 3T3 fibroblast cells

  10. Self-reinforcement and protein sustained delivery of hyaluronan hydrogel by tailoring a dually cross-linked network

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Chunhong; Xu, Guoguang; Wang, Xinghui [Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Tu, Mei; Zeng, Rong; Rong, Jianhua [Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Zhao, Jianhao, E-mail: jhzhao@jnu.edu.cn [Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China)

    2015-01-01

    A series of self-reinforcing hyaluronan hydrogels were developed to improve mechanical properties and protein sustained delivery thanks to a dually cross-linked network. Hyaluronan gel particles (HGPs, 1–5 μm in diameter) with different cross-linking densities, i.e. HGPs-1.5, HGPs-3 and HGPs-15, were prepared in an inverse emulsion system and used as the reinforcing phase after glycidyl methacrylation, while glycidyl methacrylated hyaluronan with a substitution degree of 45.2% was synthesized as the matrix phase. These two phases were cross-linked under ultraviolet irradiation to form self-reinforcing hyaluronan hydrogels (srHAs) that showed typical cross-linked structure of HGPs connecting the matrix phase by cross-section observation. In comparison to hyaluronan bulk gels and their blends with HGPs, srHAs distinctly enhanced the mechanical properties and BSA long-term sustained delivery, especially srHA-1.5 showed the highest compressive modulus of 220 ± 15 kPa and the slowest BSA delivery (67% release at 14 d). The 3T3 fibroblast cell culture showed that all the srHAs had no cytotoxicity. - Highlights: • New self-reinforcing HA hydrogels with a dually cross-linked network were developed. • Self-reinforcing HA hydrogels greatly enhanced the mechanical properties. • Self-reinforcing HA hydrogels prolonged the sustained delivery of BSA. • The self-reinforcing mechanism and BSA diffusion mechanism were discussed. • Self-reinforcing HA hydrogels had no cytotoxicity to 3T3 fibroblast cells.

  11. Contribution of matrix converter in microgeneration for a sustainable energy planning; Contribuicao do conversor matricial na microgeracao para um planejamento energetico sustentavel

    Energy Technology Data Exchange (ETDEWEB)

    Romero, Javier Alexis Andrade; Romero, Jesus Franklin Andrade [Universidade Federal do ABC (UFABC), Santo Andre, SP (Brazil). Programa de Pos-Graduacao em Energia], emails: javier.romero@ufabc.edu.br, jesus.romero@ufabc.edu.br

    2010-07-01

    Once microturbine (MT) based generation systems present suitable and short term application conditions in distributed generation, a rigorous sustainability evaluation is necessary. In this work a Two Stage Matrix Converter (CM2E) that improves the energy efficiency performance is evaluated. In this sense, an analysis based on technological indicators regarding a sustainable energy planning is presented in technological, economic, social and environmental dimensions (author)

  12. Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.

    Science.gov (United States)

    Bobone, Sara; Hilsch, Malte; Storm, Julian; Dunsing, Valentin; Herrmann, Andreas; Chiantia, Salvatore

    2017-06-15

    Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still

  13. Matrix reloaded: CCN, tenascin and SIBLING group of matricellular proteins in orchestrating cancer hallmark capabilities.

    Science.gov (United States)

    Thakur, Ravi; Mishra, Durga Prasad

    2016-12-01

    Matricellular proteins (MCPs) are the non-structural extracellular matrix (ECM) proteins with various regulatory functions. MCPs are critical regulators of ECM homeostasis and are often found dysregulated in various malignancies. They interact with various proteins like ECM structural proteins, integrins, growth factor receptors and growth factors to modulate their availability and activity. Cancer-supporting MCPs are known to induce proliferation, migration and invasion of cancer cells. MCPs also support cancer stem (like) cell growth and induce a drug-resistant state. Apart from their direct effects on cancer cells, they play key roles in angiogenesis, immunomodulation, stromal cell infiltration, stromal proliferation and matrix remodeling. High expression of various MCPs belonging to the tenascin, CCN and SIBLING families is often associated with aggressive tumors and poor patient prognosis. Due to their differential expression and distinct functional role, these MCPs are perceived as attractive therapeutic targets in cancer. Studies on preclinical models have indicated that targeting tumor-supportive MCPs could be a potent avenue for developing anti-cancer therapies. The MCP receptors, like integrins and some associated growth factor receptors, are already being targeted using pharmacological inhibitors and neutralizing antibodies. Neutralizing antibodies against CCNs, tenascins and SIBLINGs have shown promising results in preclinical cancer models, suggesting an opportunity to develop anti-MCP therapies to target cancer. Peptides derived from anti-cancer MCPs could also serve as therapeutic entities. In the present review, in continuation with the expanding horizon of MCP functions and disease association, we focus on (i) their unique domain arrangement, (ii) their association with cancer hallmarks and (iii) available and possible therapeutic interventions. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    International Nuclear Information System (INIS)

    Gualdrón-López, Melisa; Michels, Paul A.M.

    2013-01-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M r of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M r of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and 35 S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed

  15. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Gualdrón-López, Melisa [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium); Michels, Paul A.M., E-mail: paul.michels@uclouvain.be [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium)

    2013-02-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

  16. Admixtures in Cement-Matrix Composites for Mechanical Reinforcement, Sustainability, and Smart Features

    Science.gov (United States)

    Bastos, Guillermo; Patiño-Barbeito, Faustino; Patiño-Cambeiro, Faustino; Armesto, Julia

    2016-01-01

    For more than a century, several inclusions have been mixed with Portland cement—nowadays the most-consumed construction material worldwide—to improve both the strength and durability required for construction. The present paper describes the different families of inclusions that can be combined with cement matrix and reviews the achievements reported to date regarding mechanical performance, as well as two other innovative functionalities of growing importance: reducing the high carbon footprint of Portland cement, and obtaining new smart features. Nanomaterials stand out in the production of such advanced features, allowing the construction of smart or multi-functional structures by means of thermal- and strain-sensing, and photocatalytic properties. The first self-cleaning concretes (photocatalytic) have reached the markets. In this sense, it is expected that smart concretes will be commercialized to address specialized needs in construction and architecture. Conversely, other inclusions that enhance strength or reduce the environmental impact remain in the research stage, in spite of the promising results reported in these issues. Despite the fact that such functionalities are especially profitable in the case of massive cement consumption, the shift from the deeply established Portland cement to green cements still has to overcome economic, institutional, and technical barriers. PMID:28774091

  17. Effects of extracellular matrix proteins on the growth of haematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Celebi, Betuel; Pineault, Nicolas; Mantovani, Diego

    2011-01-01

    Umbilical cord blood (UCB) transplantation and haematological recovery are currently limited by the amount of haematopoietic progenitor cells (HPCs) present in each unit. HPCs and haematopoietic stem cells (HSCs) normally interact with cells and extracellular matrix (ECM) proteins present within the endosteal and vascular niches. Hence, we investigated whether coating of culture surfaces with ECM proteins normally present in the marrow microenvironment could benefit the ex vivo expansion of HPCs. Towards this, collagen types I and IV (COL I and IV), laminin (LN) and fibronectin (FN) were tested individually or as component of two ECM-mix complexes. Individually, ECM proteins had both common and unique properties on the growth and differentiation of UCB CD34+ cells; some ECM proteins favoured the differentiation of some lineages over that of others (e.g. FN for erythroids), some the expansion of HPCs (e.g. LN and megakaryocyte (MK) progenitor) while others had less effects. Next, two ECM-mix complexes were tested; the first one contained all four ECM proteins (4ECMp), while the second 'basement membrane-like structure' was without COL I (3ECMp). Removal of COL I led to strong reductions in cell growth and HPCs expansion. Interestingly, the 4ECMp-mix complex reproducibly increased CD34+ (1.3-fold) and CD41+ (1.2-fold) cell expansions at day 6 (P < 0.05) versus control, and induced greater myeloid progenitor expansion (P < 0.05) than 3ECMp. In conclusion, these results suggest that optimization of BM ECM protein complexes could provide a better environment for the ex vivo expansion of haematopoietic progenitors than individual ECM protein.

  18. Effects of extracellular matrix proteins on the growth of haematopoietic progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Celebi, Betuel; Pineault, Nicolas [Hema-Quebec, Research and Development Department, Quebec City, G1V 5C3, PQ (Canada); Mantovani, Diego, E-mail: nicolas.pineault@hema-quebec.qc.ca [Laboratory for Biomaterials and Bioengineering, Department of Materials Engineering and University Hospital Research Center, Laval University, Quebec City, G1V 0A6, PQ (Canada)

    2011-10-15

    Umbilical cord blood (UCB) transplantation and haematological recovery are currently limited by the amount of haematopoietic progenitor cells (HPCs) present in each unit. HPCs and haematopoietic stem cells (HSCs) normally interact with cells and extracellular matrix (ECM) proteins present within the endosteal and vascular niches. Hence, we investigated whether coating of culture surfaces with ECM proteins normally present in the marrow microenvironment could benefit the ex vivo expansion of HPCs. Towards this, collagen types I and IV (COL I and IV), laminin (LN) and fibronectin (FN) were tested individually or as component of two ECM-mix complexes. Individually, ECM proteins had both common and unique properties on the growth and differentiation of UCB CD34+ cells; some ECM proteins favoured the differentiation of some lineages over that of others (e.g. FN for erythroids), some the expansion of HPCs (e.g. LN and megakaryocyte (MK) progenitor) while others had less effects. Next, two ECM-mix complexes were tested; the first one contained all four ECM proteins (4ECMp), while the second 'basement membrane-like structure' was without COL I (3ECMp). Removal of COL I led to strong reductions in cell growth and HPCs expansion. Interestingly, the 4ECMp-mix complex reproducibly increased CD34+ (1.3-fold) and CD41+ (1.2-fold) cell expansions at day 6 (P < 0.05) versus control, and induced greater myeloid progenitor expansion (P < 0.05) than 3ECMp. In conclusion, these results suggest that optimization of BM ECM protein complexes could provide a better environment for the ex vivo expansion of haematopoietic progenitors than individual ECM protein.

  19. Enhanced release of bone morphogenetic proteins from demineralized bone matrix by gamma irradiation

    International Nuclear Information System (INIS)

    Sung, Nak-Yun; Choi, Jong-il

    2015-01-01

    Gamma irradiation is a useful method for sterilizing demineralized bone matrix (DBM), but its effect on the osteoinductivity of DBM is still controversial. In this study, the osteoinductive activity of gamma-irradiated DBM was examined using a mouse myoblastic cell line (C2C12). DBM was extracted from adult bovine bone and was irradiated at a dose of 25 kGy using a 60 cobalt gamma-irradiator. Cell proliferation with DBM was not affected by gamma-irradiation, but alkaline phosphatase and osteocalcin productions were significantly increased in C2C12 cell groups treated with gamma-irradiated DBM. It was reasoned that bone morphogenetic proteins were more efficiently released from gamma-irradiated DBM than from the non-irradiated control. This result suggests the effectiveness of radiation sterilization of bone implants - Highlights: • Demineralized bone matrix (DBM) was gamma-irradiated for sterilization. • Irradiated DBM had higher alkaline phosphatase and osteocalcin production. • It was reasoned the more released bone morphogenetic proteins by irradiation. • This result supports the application of radiation sterilization for bone implants

  20. Periostin is an extracellular matrix protein required for eruption of incisors in mice

    International Nuclear Information System (INIS)

    Kii, Isao; Amizuka, Norio; Minqi, Li; Kitajima, Satoshi; Saga, Yumiko; Kudo, Akira

    2006-01-01

    A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin -/- mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin -/- mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone

  1. Crystal Structure of the Oligomeric Form of Lassa Virus Matrix Protein Z.

    Science.gov (United States)

    Hastie, Kathryn M; Zandonatti, Michelle; Liu, Tong; Li, Sheng; Woods, Virgil L; Saphire, Erica Ollmann

    2016-05-01

    The arenavirus matrix protein Z is highly multifunctional and occurs in both monomeric and oligomeric forms. The crystal structure of a dodecamer of Z from Lassa virus, presented here, illustrates a ring-like structure with a highly basic center. Mutagenesis demonstrates that the dimeric interface within the dodecamer and a Lys-Trp-Lys triad at the center of the ring are important for oligomerization. This structure provides an additional template to explore the many functions of Z. The arenavirus Lassa virus causes hundreds of thousands of infections each year, many of which develop into fatal hemorrhagic fever. The arenavirus matrix protein Z is multifunctional, with at least four distinct roles. Z exists in both monomeric and oligomeric forms, each of which likely serves a specific function in the viral life cycle. Here we present the dodecameric form of Lassa virus Z and demonstrate that Z forms a "wreath" with a highly basic center. This structure and that of monomeric Z now provide a pair of critical templates by which the multiple roles of Z in the viral life cycle may be interpreted. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

    Directory of Open Access Journals (Sweden)

    Karen L Posey

    2010-04-01

    Full Text Available Mutations in cartilage oligomeric matrix protein (COMP, a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

  3. New low-flux mixed matrix membranes that offer superior removal of protein-bound toxins from human plasma

    Science.gov (United States)

    Pavlenko, Denys; van Geffen, Esmée; van Steenbergen, Mies J.; Glorieux, Griet; Vanholder, Raymond; Gerritsen, Karin G. F.; Stamatialis, Dimitrios

    2016-10-01

    Hemodialysis is a widely available and well-established treatment for patients with End Stage Renal Disease (ESRD). However, although life-sustaining, patient mortality rates are very high. Several recent studies corroborated the link between dialysis patients’ outcomes and elevated levels of protein-bound uremic toxins (PBUT) that are poorly removed by conventional hemodialysis. Therefore, new treatments are needed to improve their removal. Recently, our group showed that the combination of dialysis and adsorption on one membrane, the mixed matrix membrane (MMM), can effectively remove those toxins from human plasma. However, these first MMMs were rather large in diameter and their mass transport characteristics needed improvement before application in the clinical setting. Therefore, in this study we developed a new generation of MMMs that have a smaller diameter and optimized characteristics offering superior ability in removing the PBUT indoxyl sulfate (IS) and p-cresyl sulfate (pCS) in comparison to first generation MMMs (30 and 125% respectively), as well as, a commercial dialysis membrane (more than 100% better removal).

  4. Binding of a cementum attachment protein to extracellular matrix components and to dental surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Pitaru, S; Hekmati, H [Department of Oral Biology, Goldschleger School of Dental Medicine, Tel Aviv University (Israel); Savion, N [Goldschleger Eye Institute, Sackler School of Medicine, Tel Aviv University (Israel); Olsen, S; Narayanan, S A [Department of Pathology, School of Medicine, University of Washington, Seattle, Washington (United States)

    1992-01-01

    Cementum proteins (CP) have been shown to mediate cell attachment. Among these, a 55 kDa protein was isolated. The purpose of the present study was to assess the capacity of CP to bind to non-demineralized and demineralized root surfaces and to support cell attachment to dentin. CP were prepared by sequential extraction of bovine cementum with 25 mM EDTA, 0.5 M acetic acid followed by 4 M guanidine HCl. The latter was subjected to ion exchange chromatography on a DEAE-3SW column and eluted stepwise with a 0-0.5 M NaCl gradient. CP were labelled with [sup 125]I and the capacity of [sup 125]I-CP to bind to mineralized and partially demineralized dentin, synthetic hydroxyapatite, collagen, fibronectin and fibrillar collagen-fibronectin cimplex was assessed. It was found that CP bind specifically to mineralized dentin and synthetic hydroxyapatite but not to demineralized dentin. The specific binding was 60% of the total binding. SDS-PAGE analysis of the proteins bound to dentin indicated that the main bound protein had a molecular weight of 55 kDa. CP exhibited high affinity for fibronectin (k[sub D] = 1.56 x 10[sup -10] M) and fibronectincollagen complex, but their binding to either molecular or fibrillar collagen was negligible. It is suggested that CP may play an important role in the attachment of cells of the periodontium to cementum extracellular matrix during homeostasis and regeneration. (au).

  5. In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins

    International Nuclear Information System (INIS)

    Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Soker, Shay; Khang, Gilson

    2013-01-01

    The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation. (paper)

  6. Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: powerful analytical tools in recombinant protein chemistry

    DEFF Research Database (Denmark)

    Andersen, Jens S.; Svensson, B; Roepstorff, P

    1996-01-01

    Electrospray ionization and matrix assisted laser desorption/ionization are effective ionization methods for mass spectrometry of biomolecules. Here we describe the capabilities of these methods for peptide and protein characterization in biotechnology. An integrated analytical strategy is presen......Electrospray ionization and matrix assisted laser desorption/ionization are effective ionization methods for mass spectrometry of biomolecules. Here we describe the capabilities of these methods for peptide and protein characterization in biotechnology. An integrated analytical strategy...... is presented encompassing protein characterization prior to and after cloning of the corresponding gene....

  7. Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix.

    Science.gov (United States)

    Yun, Jing-Ping; Chew, Eng Ching; Liew, Choong-Tsek; Chan, John Y H; Jin, Mei-Lin; Ding, Ming-Xiao; Fai, Yam Hin; Li, H K Richard; Liang, Xiao-Man; Wu, Qiu-Liang

    2003-12-15

    It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix. Copyright 2003 Wiley-Liss, Inc.

  8. The human metapneumovirus matrix protein stimulates the inflammatory immune response in vitro.

    Directory of Open Access Journals (Sweden)

    Audrey Bagnaud-Baule

    Full Text Available Each year, during winter months, human Metapneumovirus (hMPV is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients.

  9. The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins)

    NARCIS (Netherlands)

    Faber, Klaas Nico; Haima, Pieter; Gietl, Christine; Harder, Willem; Ab, Geert; Veenhuis, Marten

    1994-01-01

    Two main types of peroxisomal targeting signals have been identified that reside either at the extreme C terminus (PTS1) or the N terminus (PTS2) of the protein. In the methylotrophic yeast Hansenula polymorpha the majority of peroxisomal matrix proteins are of the PTS1 type. Thus far, for H.

  10. Staphylococcus aureus manganese transport protein C (MntC is an extracellular matrix- and plasminogen-binding protein.

    Directory of Open Access Journals (Sweden)

    Natália Salazar

    Full Text Available Infections caused by Staphylococcus aureus--particularly nosocomial infections--represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM. Manganese transport protein C (MntC, a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA. The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.

  11. Future Protein Supply and Demand: Strategies and Factors Influencing a Sustainable Equilibrium

    Directory of Open Access Journals (Sweden)

    Maeve Henchion

    2017-07-01

    Full Text Available A growing global population, combined with factors such as changing socio-demographics, will place increased pressure on the world’s resources to provide not only more but also different types of food. Increased demand for animal-based protein in particular is expected to have a negative environmental impact, generating greenhouse gas emissions, requiring more water and more land. Addressing this “perfect storm” will necessitate more sustainable production of existing sources of protein as well as alternative sources for direct human consumption. This paper outlines some potential demand scenarios and provides an overview of selected existing and novel protein sources in terms of their potential to sustainably deliver protein for the future, considering drivers and challenges relating to nutritional, environmental, and technological and market/consumer domains. It concludes that different factors influence the potential of existing and novel sources. Existing protein sources are primarily hindered by their negative environmental impacts with some concerns around health. However, they offer social and economic benefits, and have a high level of consumer acceptance. Furthermore, recent research emphasizes the role of livestock as part of the solution to greenhouse gas emissions, and indicates that animal-based protein has an important role as part of a sustainable diet and as a contributor to food security. Novel proteins require the development of new value chains, and attention to issues such as production costs, food safety, scalability and consumer acceptance. Furthermore, positive environmental impacts cannot be assumed with novel protein sources and care must be taken to ensure that comparisons between novel and existing protein sources are valid. Greater alignment of political forces, and the involvement of wider stakeholders in a governance role, as well as development/commercialization role, is required to address both sources of

  12. Future Protein Supply and Demand: Strategies and Factors Influencing a Sustainable Equilibrium

    Science.gov (United States)

    Henchion, Maeve; Hayes, Maria; Mullen, Anne Maria; Fenelon, Mark; Tiwari, Brijesh

    2017-01-01

    A growing global population, combined with factors such as changing socio-demographics, will place increased pressure on the world’s resources to provide not only more but also different types of food. Increased demand for animal-based protein in particular is expected to have a negative environmental impact, generating greenhouse gas emissions, requiring more water and more land. Addressing this “perfect storm” will necessitate more sustainable production of existing sources of protein as well as alternative sources for direct human consumption. This paper outlines some potential demand scenarios and provides an overview of selected existing and novel protein sources in terms of their potential to sustainably deliver protein for the future, considering drivers and challenges relating to nutritional, environmental, and technological and market/consumer domains. It concludes that different factors influence the potential of existing and novel sources. Existing protein sources are primarily hindered by their negative environmental impacts with some concerns around health. However, they offer social and economic benefits, and have a high level of consumer acceptance. Furthermore, recent research emphasizes the role of livestock as part of the solution to greenhouse gas emissions, and indicates that animal-based protein has an important role as part of a sustainable diet and as a contributor to food security. Novel proteins require the development of new value chains, and attention to issues such as production costs, food safety, scalability and consumer acceptance. Furthermore, positive environmental impacts cannot be assumed with novel protein sources and care must be taken to ensure that comparisons between novel and existing protein sources are valid. Greater alignment of political forces, and the involvement of wider stakeholders in a governance role, as well as development/commercialization role, is required to address both sources of protein and ensure

  13. Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

    International Nuclear Information System (INIS)

    Warters, R.L.; Brizgys, L.M.; Lyons, B.W.

    1987-01-01

    The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 0 C and 45 0 C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 0 C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells were incubated at 37 0 C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 0 C a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks. (author)

  14. A Novel Acidic Matrix Protein, PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite*

    Science.gov (United States)

    Pan, Cong; Fang, Dong; Xu, Guangrui; Liang, Jian; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2014-01-01

    Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium. PMID:24302723

  15. The influenza A virus matrix protein as a marker to monitor initial virus internalisation.

    Science.gov (United States)

    Eierhoff, Thorsten; Ludwig, Stephan; Ehrhardt, Christina

    2009-01-01

    The uptake of influenza A viruses (IAV) into cells represents an attractive antiviral drug target, e.g., by interfering with essential cellular or viral entry factors. So far, this process could only be studied by time-consuming microscopical methods. Thus, there is a lack of rapid and easy assay systems to monitor viral entry. Here, we describe a rapid procedure to analyse internalisation of IAV via Western blot detection of virion-associated matrix protein (M1), the most abundant protein within the viral particle. The assay is broadly applicable and detects different virus strains of various subtypes. As a proof of principle, treatment of cells with various known or presumed entry inhibitors resulted in reduced M1 levels. Removal of sialic acids, the receptors for IAV, led to a complete loss of the M1 signal, indicating that virus internalisation can be monitored already at the stage of attachment. Prevention of endosomal acidification resulted in a delayed degradation of M1 indicative of IAV particles trapped in endosomes. Thus, early detection of the virus-associated M1 protein is a rapid method to monitor different steps of influenza virus internalisation and has potential for application as a screening method for drugs that interfere with the uptake of IAV.

  16. Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

    Science.gov (United States)

    Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

    2018-05-01

    The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

  17. The YPLGVG sequence of the Nipah virus matrix protein is required for budding

    Directory of Open Access Journals (Sweden)

    Yan Lianying

    2008-11-01

    Full Text Available Abstract Background Nipah virus (NiV is a recently emerged paramyxovirus capable of causing fatal disease in a broad range of mammalian hosts, including humans. Together with Hendra virus (HeV, they comprise the genus Henipavirus in the family Paramyxoviridae. Recombinant expression systems have played a crucial role in studying the cell biology of these Biosafety Level-4 restricted viruses. Henipavirus assembly and budding occurs at the plasma membrane, although the details of this process remain poorly understood. Multivesicular body (MVB proteins have been found to play a role in the budding of several enveloped viruses, including some paramyxoviruses, and the recruitment of MVB proteins by viral proteins possessing late budding domains (L-domains has become an important concept in the viral budding process. Previously we developed a system for producing NiV virus-like particles (VLPs and demonstrated that the matrix (M protein possessed an intrinsic budding ability and played a major role in assembly. Here, we have used this system to further explore the budding process by analyzing elements within the M protein that are critical for particle release. Results Using rationally targeted site-directed mutagenesis we show that a NiV M sequence YPLGVG is required for M budding and that mutation or deletion of the sequence abrogates budding ability. Replacement of the native and overlapping Ebola VP40 L-domains with the NiV sequence failed to rescue VP40 budding; however, it did induce the cellular morphology of extensive filamentous projection consistent with wild-type VP40-expressing cells. Cells expressing wild-type NiV M also displayed this morphology, which was dependent on the YPLGVG sequence, and deletion of the sequence also resulted in nuclear localization of M. Dominant-negative VPS4 proteins had no effect on NiV M budding, suggesting that unlike other viruses such as Ebola, NiV M accomplishes budding independent of MVB cellular proteins

  18. Novel serological neo-epitope markers of extracellular matrix proteins for the detection of portal hypertension.

    Science.gov (United States)

    Leeming, D J; Karsdal, M A; Byrjalsen, I; Bendtsen, F; Trebicka, J; Nielsen, M J; Christiansen, C; Møller, S; Krag, A

    2013-11-01

    The hepatic venous pressure gradient (HVPG) is an invasive, but important diagnostic and prognostic marker in cirrhosis with portal hypertension (PHT). During cirrhosis, remodelling of fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process generating small fragments of degraded extracellular matrix (ECM) proteins known as neoepitopes, which are then released into the circulation. To investigate their potential as plasma markers for detection of PHT. Ninety-four patients with alcoholic cirrhosis and 20 liver-healthy controls were included. Clinical and laboratory data of the patients were collected. All patients received HVPG measurement with blood sampling. In these samples, the following degradation or formation markers were measured: C1M (type I-collagen), C3M and PRO-C3 (type III collagen), C4M and P4NP 7S (type IV collagen), C5M (type V collagen), C6M (type VI collagen), BGM (biglycan), ELM (elastin), CRPM (CRP). All ECM markers except for CRPM correlated significantly with HVPG. Interestingly, C4M, C5M and ELM levels were significantly higher in patients with HVPG >10 mmHg. Multiple regression analysis identified PRO-C3, C6M and ELM as significant determinants, while the models A and B including PRO-C3, ELM, C6M and model for end-stage liver disease (MELD) provided better description of PHT (r = 0.75, P models provided odds ratios of >100 for having clinical significant PHT. These novel non-invasive extracellular matrix markers reflect the degree of liver dysfunction. The different degrees of portal hypertension correlated with these circulating neoepitopes. Using a single blood sample, these neoepitopes in combination with MELD detect the level of portal hypertension. © 2013 The Authors. Alimentary Pharmacology and Therapeutics published by John Wiley & Sons Ltd.

  19. The role of Matrix Gla Protein in ossification and recovery of the avian growth plate

    Directory of Open Access Journals (Sweden)

    Harel eDan

    2012-07-01

    Full Text Available ECM mineralization is an essential physiologic process in bone, teeth, and hypertrophic cartilage. Matrix Gla Protein (MGP, an inhibitor of mineralization, is expressed by chondrocytes and vascular smooth muscle cells to inhibit calcification of those soft tissues.Tibial Dyschondroplasia (TD, a skeletal abnormality apparent as a plug of non-vascularized, non-mineralized, white opaque cartilage in the tibial growth plate of avian species can serve as a good model for studying process and genes involved in matrix mineralization and calcification. In this work, we studied the involvement of MGP in the development of TD, as well as in the processes of spontaneous and induced recovery from this syndrome. First, we found that during normal bone development, MGP is expressed in specific time and locations, starting from wide spread expression in the yet un-ossified diaphysis during embryonic development, to specific expression in hypertrophic chondrocytes adjacent to the chondro-osseous junction and the secondary ossification center just prior to calcification. In addition, we show that MGP is not expressed in the impaired TD lesion, however when the lesion begins to heal, it strongly express MGP prior to its calcification. Moreover, we show that when calcification is inhibited, a gap is formed between the expression zones of MGP and BMP2 and that this gap is closed during the healing process. To conclude, we suggest that MGP, directly or through interaction with BMP2, plays a role as ossification regulator, rather then simple inhibitor that acts prior to ossification.

  20. Cartilage oligomeric matrix protein in patients with juvenile idiopathic arthritis: relation to growth and disease activity

    DEFF Research Database (Denmark)

    Bjørnhart, Birgitte; Juul, Anders; Nielsen, Susan

    2009-01-01

    OBJECTIVE: Cartilage oligomeric matrix protein (COMP) has been identified as a prognostic marker of progressive joint destruction in rheumatoid arthritis. In this population based study we evaluated associations between plasma concentrations of COMP, disease activity, and growth velocity...... in patients with recent-onset juvenile idiopathic arthritis (JIA). COMP levels in JIA and healthy children were compared with those in healthy adults. Plasma levels of insulin-like growth factor I (IGF-1), which has been associated with COMP expression and growth velocity, were studied in parallel. METHODS......: 87 patients with JIA entered the study, including oligoarticular JIA (n = 34), enthesitis-related arthritis (n = 8), polyarticular rheumatoid factor (RF)-positive JIA (n = 2), polyarticular RF-negative JIA (n = 27), systemic JIA (n = 6), and undifferentiated JIA (n = 10). Plasma levels of COMP were...

  1. Microseed matrix screening for optimization in protein crystallization: what have we learned?

    Science.gov (United States)

    D'Arcy, Allan; Bergfors, Terese; Cowan-Jacob, Sandra W; Marsh, May

    2014-09-01

    Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time- and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.

  2. Modulating bacterial and gut mucosal interactions with engineered biofilm matrix proteins.

    Science.gov (United States)

    Duraj-Thatte, Anna M; Praveschotinunt, Pichet; Nash, Trevor R; Ward, Frederick R; Joshi, Neel S

    2018-02-22

    Extracellular appendages play a significant role in mediating communication between bacteria and their host. Curli fibers are a class of bacterial fimbria that is highly amenable to engineering. We demonstrate the use of engineered curli fibers to rationally program interactions between bacteria and components of the mucosal epithelium. Commensal E. coli strains were engineered to produce recombinant curli fibers fused to the trefoil family of human cytokines. Biofilms formed from these strains bound more mucins than those producing wild-type curli fibers, and modulated mucin rheology as well. When treated with bacteria producing the curli-trefoil fusions mammalian cells behaved identically in terms of their migration behavior as when they were treated with the corresponding soluble trefoil factors. Overall, this demonstrates the potential utility of curli fibers as a scaffold for the display of bioactive domains and an untapped approach to rationally modulating host-microbe interactions using bacterial matrix proteins.

  3. Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

    Science.gov (United States)

    Gc, Jeevan B; Gerstman, Bernard S; Chapagain, Prem P

    2017-10-01

    The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP 2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP 2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. [Morphology of basement membrane and associated matrix proteins in normal and pathological tissues].

    Science.gov (United States)

    Nerlich, A

    1995-01-01

    Basement membranes (BM) are specialized structures of the extracellular matrix. Their composition is of particular importance for the maintenance of normal morphological and functional properties of a multitude of organs and tissue systems and it is thus required for regular homeostasis of body function. Generally, they possess three main functions, i.e. participation in the maintenance of tissue structure, control of fluid and substrate exchange, and regulation of cell growth and differentiation. BMs are made up by various components which are in part specifically localized within the BM zone, or which represent ubiquitous matrix constituents with specific quantitative and/or qualitative differences in their localization. On the basis of a thorough immunohistochemical analysis of normal and diseased tissues, we provide here a concept of "functional morphology/pathomorphology" of the different BM components analyzed: 1.) The ubiquitous BM-constituent collagen IV primarily stabilizes the BM-zone and thus represents the "backbone" of the BM providing mechanical strength. Its loss leads to cystic tissue transformation as it is evidenced from the analysis of polycystic nephropathies. Thus, in other cystic tissue transformations a similar formal pathogenesis may be present. 2.) The specific localization of collagen VII as the main structural component of anchoring fibrils underlines the mechanical anchoring function of this collagenous protein. Defects in this protein lead to hereditary epidermolysis. The rapid re-occurrence of epidermal collagen VII during normal human wound healing indicates a quick reconstitution of the mechanical tensile strength of healing wounds. 3.) The BM-specific heparan sulfate proteoglycan (HSPG, Perlecan) with its highly negative anionic charge can be assumed to exert filter control. This assumption is corroborated by the localizatory findings of a preferential deposition of HSPG in endothelial and particularly in glomerular BM. Similarly

  5. The influence of γ-radiation on biosynthesis of nuclear matrix proteins of hepatic cells of pregnant rats

    International Nuclear Information System (INIS)

    Mirkhamidova, P.; Shamsutdinova, G.T.; Mirakhmedov, A.K.; Filatova, L.S.; Bul'dyaeva, T.V.; Zbarskij, I.B.

    1992-01-01

    A study was made of incorporation of 35 S-methionine into nuclear matrix proteins of hepatic cells of pregnant rats and their embryos subjected to single γ-irradiation ( 60 Co, 1 and 2 Gy, 0.0233 Gy/s) on days 3, 13 and 17 of pregrnancy and embryogenesis. On day 21 of pregnancy and embryogenesis a decrease in the rate of incorporation of 35 S-methionine into nuclear matrix proteins was shown to be a function of radiation dose and time of pregnancy and embryogenesis on the moment of exposure

  6. Peeping into human renal calcium oxalate stone matrix: characterization of novel proteins involved in the intricate mechanism of urolithiasis.

    Directory of Open Access Journals (Sweden)

    Kanu Priya Aggarwal

    Full Text Available BACKGROUND: The increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal-membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure. METHODS: Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin-Darby Canine Kidney (MDCK renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated. RESULTS: Five proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level. CONCLUSIONS: We identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone

  7. Formulation development and optimization of sustained release matrix tablet of Itopride HCl by response surface methodology and its evaluation of release kinetics.

    Science.gov (United States)

    Bose, Anirbandeep; Wong, Tin Wui; Singh, Navjot

    2013-04-01

    The objective of this present investigation was to develop and formulate sustained release (SR) matrix tablets of Itopride HCl, by using different polymer combinations and fillers, to optimize by Central Composite Design response surface methodology for different drug release variables and to evaluate drug release pattern of the optimized product. Sustained release matrix tablets of various combinations were prepared with cellulose-based polymers: hydroxy propyl methyl cellulose (HPMC) and polyvinyl pyrolidine (pvp) and lactose as fillers. Study of pre-compression and post-compression parameters facilitated the screening of a formulation with best characteristics that underwent here optimization study by response surface methodology (Central Composite Design). The optimized tablet was further subjected to scanning electron microscopy to reveal its release pattern. The in vitro study revealed that combining of HPMC K100M (24.65 MG) with pvp(20 mg)and use of LACTOSE as filler sustained the action more than 12 h. The developed sustained release matrix tablet of improved efficacy can perform therapeutically better than a conventional tablet.

  8. Patterned layers of adsorbed extracellular matrix proteins: influence on mammalian cell adhesion.

    Science.gov (United States)

    Dupont-Gillain, C C; Alaerts, J A; Dewez, J L; Rouxhet, P G

    2004-01-01

    Three patterned systems aiming at the control of mammalian cell behavior are presented. The determinant feature common to these systems is the spatial distribution of extracellular matrix (ECM) proteins (mainly collagen) on polymer substrates. This distribution differs from one system to another with respect to the scale at which it is affected, from the supracellular to the supramolecular scale, and with respect to the way it is produced. In the first system, the surface of polystyrene was oxidized selectively to form micrometer-scale patterns, using photolithography. Adsorption of ECM proteins in presence of a competitor was enhanced on the oxidized domains, allowing selective cell adhesion to be achieved. In the second system, electron beam lithography was used to engrave grooves (depth and width approximately 1 microm) on a poly(methyl methacrylate) (PMMA) substratum. No modification of the surface chemistry associated to the created topography could be detected. Cell orientation along the grooves was only observed when collagen was preadsorbed on the substratum. In the third system, collagen adsorbed on PMMA was dried in conditions ensuring the formation of a nanometer-scale pattern. Cell adhesion was enhanced on such patterned collagen layers compared to smooth collagen layers.

  9. Two-dimensional electrophoretic analysis of nuclear matrix proteins in human colon adenocarcinoma.

    Science.gov (United States)

    Toumpanaki, A; Baltatzis, G E; Gaitanarou, E; Seretis, E; Toumpanakis, C; Aroni, K; Kittas, Christos; Voloudakis-Baltatzis, I E

    2009-01-01

    The aim of the present study was to observe possible qualitative and quantitative expression differences between nuclear matrix proteins (NMPs) of human colon adenocarcinoma and their mirror biopsies, using the technique of two-dimensional gel electrophoresis, in order to identify the existence of specific NMP fingerprints for colon cancer. Colon tissues were examined ultrastructurally and NMPs were isolated biochemically, by serial extraction of lipids, soluble proteins, DNA, RNA, and intermediate filaments and were separated according to their isoelectric point (pI) and their molecular weight (MW) by high-resolution two-dimensional electrophoresis (2D). By comparing the 2D electropherograms of colon cancer tissues and mirror biopsy tissues we observed qualitative and quantitative expression differences between their NMPs but also a differentiation of NMP composition between the stages of malignancy. Moreover, despite the similarities between mirror biopsy samples, a highlight percentage of exception was observed. Electrophoretic results provided in this study demonstrated that the examined NMPs could be further investigated as potential markers for detection of colorectal cancer in an early stage, for the assessment of the disease progression, as well as useful tools for individual therapy and for preventing a possible recurrence of cancer and metastasis.

  10. [Nuclear protein matrix from giant nuclei of Chironomus plumosus determinates polythene chromosome organization].

    Science.gov (United States)

    Makarov, M S; Chentsov, Iu S

    2010-01-01

    Giant nuclei from salivary glands of Chironomus plumosus were treated in situ with detergent, 2 M NaCl and nucleases in order to reveal residual nuclear matrix proteins (NMP). It was shown, that preceding stabilization of non-histone proteins with 2 mM CuCl2 allowed to visualize the structure of polythene chromosomes at every stage of the extraction of histones and DNA. Stabilized NPM of polythene chromosomes maintains their morphology and banding patterns, which is observed by light and electron microscopy, whereas internal fibril net or residual nucleoli are not found. In stabilized NPM of polythene chromosomes, topoisomerase IIalpha and SMC1 retain their localization that is typical of untreated chromosomes. NPM of polythene chromosomes also includes sites of DNA replication, visualized with BrDU incubation, and some RNA-components. So, we can conclude that structure of NPM from giant nuclei is equal to NPM from normal interphase nuclei, and that morphological features of polythene chromosomes depend on the presence of NMP.

  11. Structure and self-assembly of the calcium binding matrix protein of human metapneumovirus.

    Science.gov (United States)

    Leyrat, Cedric; Renner, Max; Harlos, Karl; Huiskonen, Juha T; Grimes, Jonathan M

    2014-01-07

    The matrix protein (M) of paramyxoviruses plays a key role in determining virion morphology by directing viral assembly and budding. Here, we report the crystal structure of the human metapneumovirus M at 2.8 Å resolution in its native dimeric state. The structure reveals the presence of a high-affinity Ca²⁺ binding site. Molecular dynamics simulations (MDS) predict a secondary lower-affinity site that correlates well with data from fluorescence-based thermal shift assays. By combining small-angle X-ray scattering with MDS and ensemble analysis, we captured the structure and dynamics of M in solution. Our analysis reveals a large positively charged patch on the protein surface that is involved in membrane interaction. Structural analysis of DOPC-induced polymerization of M into helical filaments using electron microscopy leads to a model of M self-assembly. The conservation of the Ca²⁺ binding sites suggests a role for calcium in the replication and morphogenesis of pneumoviruses. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Interaction of glutaric aciduria type 1-related glutaryl-CoA dehydrogenase with mitochondrial matrix proteins.

    Directory of Open Access Journals (Sweden)

    Jessica Schmiesing

    Full Text Available Glutaric aciduria type 1 (GA1 is an inherited neurometabolic disorder caused by mutations in the GCDH gene encoding glutaryl-CoA dehydrogenase (GCDH, which forms homo- and heteromeric complexes in the mitochondrial matrix. GA1 patients are prone to the development of encephalopathic crises which lead to an irreversible disabling dystonic movement disorder. The clinical and biochemical manifestations of GA1 vary considerably and lack correlations to the genotype. Using an affinity chromatography approach we report here for the first time on the identification of mitochondrial proteins interacting directly with GCDH. Among others, dihydrolipoamide S-succinyltransferase (DLST involved in the formation of glutaryl-CoA, and the β-subunit of the electron transfer flavoprotein (ETFB serving as electron acceptor, were identified as GCDH binding partners. We have adapted the yellow fluorescent protein-based fragment complementation assay and visualized the oligomerization of GCDH as well as its direct interaction with DLST and ETFB in mitochondria of living cells. These data suggest that GCDH is a constituent of multimeric mitochondrial dehydrogenase complexes, and the characterization of their interrelated functions may provide new insights into the regulation of lysine oxidation and the pathophysiology of GA1.

  13. Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis.

    Science.gov (United States)

    Zhang, Zhichun; Tian, Hua; Lv, Ping; Wang, Weiping; Jia, Zhuqing; Wang, Sainan; Zhou, Chunyan; Gao, Xuejun

    2015-01-01

    Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

  14. A Novel Matrix Protein Hic31 from the Prismatic Layer of Hyriopsis Cumingii Displays a Collagen-Like Structure.

    Science.gov (United States)

    Liu, Xiaojun; Zeng, Shimei; Dong, Shaojian; Jin, Can; Li, Jiale

    2015-01-01

    In this study, we clone and characterize a novel matrix protein, hic31, from the mantle of Hyriopsis cumingii. The amino acid composition of hic31 consists of a high proportion of Glycine residues (26.67%). Tissue expression detection by RT-PCR indicates that hic31 is expressed specifically at the mantle edge. In situ hybridization results reveals strong signals from the dorsal epithelial cells of the outer fold at the mantle edge, and weak signals from inner epithelial cells of the same fold, indicating that hic31 is a prismatic-layer matrix protein. Although BLASTP results identify no shared homology with other shell-matrix proteins or any other known proteins, the hic31 tertiary structure is similar to that of collagen I, alpha 1 and alpha 2. It has been well proved that collagen forms the basic organic frameworks in way of collagen fibrils and minerals present within or outside of these fibrils. Therefore, hic31 might be a framework-matrix protein involved in the prismatic-layer biomineralization. Besides, the gene expression of hic31 increase in the early stages of pearl sac development, indicating that hic31 may play important roles in biomineralization of the pearl prismatic layer.

  15. A Novel Matrix Protein Hic31 from the Prismatic Layer of Hyriopsis Cumingii Displays a Collagen-Like Structure.

    Directory of Open Access Journals (Sweden)

    Xiaojun Liu

    Full Text Available In this study, we clone and characterize a novel matrix protein, hic31, from the mantle of Hyriopsis cumingii. The amino acid composition of hic31 consists of a high proportion of Glycine residues (26.67%. Tissue expression detection by RT-PCR indicates that hic31 is expressed specifically at the mantle edge. In situ hybridization results reveals strong signals from the dorsal epithelial cells of the outer fold at the mantle edge, and weak signals from inner epithelial cells of the same fold, indicating that hic31 is a prismatic-layer matrix protein. Although BLASTP results identify no shared homology with other shell-matrix proteins or any other known proteins, the hic31 tertiary structure is similar to that of collagen I, alpha 1 and alpha 2. It has been well proved that collagen forms the basic organic frameworks in way of collagen fibrils and minerals present within or outside of these fibrils. Therefore, hic31 might be a framework-matrix protein involved in the prismatic-layer biomineralization. Besides, the gene expression of hic31 increase in the early stages of pearl sac development, indicating that hic31 may play important roles in biomineralization of the pearl prismatic layer.

  16. Preparation and characterization of monodisperse large-porous silica microspheres as the matrix for protein separation.

    Science.gov (United States)

    Xia, Hongjun; Wan, Guangping; Zhao, Junlong; Liu, Jiawei; Bai, Quan

    2016-11-04

    High performance liquid chromatography (HPLC) is a kind of efficient separation technology and has been used widely in many fields. Micro-sized porous silica microspheres as the most popular matrix have been used for fast separation and analysis in HPLC. In this paper, the monodisperse large-porous silica microspheres with controllable size and structure were successfully synthesized with polymer microspheres as the templates and characterized. First, the poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) microspheres (P GMA-EDMA ) were functionalized with tetraethylenepentamine (TEPA) to generate amino groups which act as a catalyst in hydrolysis of tetraethyl orthosilicate (TEOS) to form Si-containing low molecular weight species. Then the low molecular weight species diffused into the functionalized P GMA-EDMA microspheres by induction force of the amino groups to form polymer/silica hybrid microspheres. Finally, the organic polymer templates were removed by calcination, and the large-porous silica microspheres were obtained. The compositions, morphology, size distribution, specific surface area and pore size distribution of the porous silica microspheres were characterized by infrared analyzer, scanning-electron microscopy, dynamic laser scattering, the mercury intrusion method and thermal gravimetric analysis, respectively. The results show that the agglomeration of the hybrid microspheres can be overcome when the templates were functionalized with TEPA as amination reagent, and the yield of 95.7% of the monodisperse large-porous silica microspheres can be achieved with high concentration of polymer templates. The resulting large-porous silica microspheres were modified with octadecyltrichlorosilane (ODS) and the chromatographic evaluation was performed by separating the proteins and the digest of BSA. The baseline separation of seven kinds of protein standards was achieved, and the column delivered a better performance when separating BSA digests

  17. Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products.

    Science.gov (United States)

    Bae, Hyun W; Zhao, Li; Kanim, Linda E A; Wong, Pamela; Delamarter, Rick B; Dawson, Edgar G

    2006-05-20

    Enzyme-linked immunosorbent assay was used to detect bone morphogenetic proteins (BMPs) 2, 4, and 7 in 9 commercially available ("off the shelf") demineralized bone matrix (DBM) product formulations using 3 different manufacturer's production lots of each DBM formulation. To evaluate and compare the quantity of BMPs among several different DBM formulations (inter-product variability), as well as examine the variability of these proteins in different production lots within the same DBM formulation (intra-product variability). DBMs are commonly used to augment available bone graft in spinal fusion procedures. Surgeons are presented with an ever-increasing variety of commercially available human DBMs from which to choose. Yet, there is limited information on a specific DBM product's osteoinductive efficacy, potency, and constancy. There were protein extracts from each DBM sample separately dialyzed 4 times against distilled water at 4 degrees C for 48 hours. The amount of BMP-2, BMP-4, and BMP-7 was determined using enzyme-linked immunosorbent assay. RESULTS.: The concentrations of detected BMP-2 and BMP-7 were low for all DBM formulations, only nanograms of BMP were extracted from each gram of DBM (20.2-120.6 ng BMP-2/g DBM product; 54.2-226.8 ng BMP-7/g DBM). The variability of BMP concentrations among different lots of the same DBM formulation, intra-product variability, was higher than the variability of concentrations among different DBM formulations, inter-product variability (coefficient of variation range BMP-2 [16.34% to 76.01%], P DBMs are low, in the order of 1 x 10(-9) g of BMP/g of DBM. There is higher variability in concentration of BMPs among 3 different lots of the same DBM formulation than among different DBM formulations. This variability questions DBM products' reliability and, possibly, efficacy in providing consistent osteoinduction.

  18. Influenza A virus infection engenders a poor antibody response against the ectodomain of matrix protein 2

    Directory of Open Access Journals (Sweden)

    Wunner William

    2006-12-01

    Full Text Available Abstract Background Matrix protein 2 (M2 is an integral tetrameric membrane protein of influenza A virus (IAV. Its ectodomain (M2e shows remarkably little diversity amongst human IAV strains. As M2e-specific antibodies (Abs have been shown to reduce the severity of infection in animals, M2e is being studied for its capability of providing protection against a broad range of IAV strains. Presently, there is little information about the concentration of M2e-specific Abs in humans. Two previous studies made use of ELISA and Western blot against M2e peptides and recombinant M2 protein as immunosorbents, respectively, and reported Ab titers to be low or undetectable. An important caveat is that these assays may not have detected all Abs capable of binding to native tetrameric M2e. Therefore, we developed an assay likely to detect all M2e tetramer-specific Abs. Results We generated a HeLa cell line that expressed full length tetrameric M2 (HeLa-M2 or empty vector (HeLa-C10 under the control of the tetracycline response element. These cell lines were then used in parallel as immunosorbents in ELISA. The assay was standardized and M2e-specific Ab titers quantified by means of purified murine or chimeric (mouse variable regions, human constant regions M2e-specific Abs in the analysis of mouse and human sera, respectively. We found that the cell-based ELISA was substantially more effective than immobilized M2e peptide in detecting M2e-specific Abs in sera of mice that had recovered from repetitive IAV infections. Still, titers remained low ( Conclusion The results provide convincing evidence that M2e-specific Ab-mediated protection is currently lacking or suboptimal in humans.

  19. Adhesion dynamics of porcine esophageal fibroblasts on extracellular matrix protein-functionalized poly(lactic acid)

    International Nuclear Information System (INIS)

    Cai Ning; Gong Yingxue; Chan, Vincent; Liao Kin; Chian, Kerm Sin

    2008-01-01

    Effective attachment of esophageal cells on biomaterials is one important requirement in designing engineered esophagus substitute for esophageal cancer treatment. In this study, poly(lactic acid) (PLA) was subjected to surface modification by coupling extracellular matrix (ECM) proteins on its surface to promote cell adhesion. Two typical ECM proteins, collagen type I (COL) and fibronectin (FN), were immobilized on the PLA surface with the aid of glutaraldehyde as a cross linker between aminolyzed PLA and ECM proteins. By using confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy, the long-term adhesion dynamics of porcine esophageal fibroblasts (PEFs) on four types of surfaces (unmodified PLA, PLA-COOH, PLA-COL and PLA-FN) was investigated during 24 h of culture. It is demonstrated by C-RICM results that PEFs form strong adhesion contact on all four types of surfaces at different stages of cell seeding. Among the four surfaces, PEFs on the PLA-FN surface reach the maximum adhesion energy (9.5 x 10 -7 J m -2 ) in the shortest time (20 min) during the initial stage of cell seeding. After adhesion energy reaches the maximum value, PEFs maintain their highly deformed geometries till they reached a steady state after 20 h of culture. F-actin immunostaining results show that the evolvement of spatial organization of F-actin is tightly correlated with the formation of adhesion contact and cell spreading. Furthermore, the cell attachment ratio of PEFs on PLA in 2 h is only 26% compared with 88% on PLA-FN, 73% on PLA-COL and 36% on PLA-COOH. All the results demonstrate the effect of surface functionalization on the biophysical responses of PEFs in cell adhesion. Fibronectin-immobilized PLA demonstrates promising potential for application as an engineered esophagus substitute

  20. Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry for the Investigation of Proteins and Peptides

    Science.gov (United States)

    Burnum, Kristin E.; Frappier, Sara L.; Caprioli, Richard M.

    2008-07-01

    Mass spectrometry (MS) is an excellent technology for molecular imaging because of its high data dimensionality. MS can monitor thousands of individual molecular data channels measured as mass-to-charge (m/z). We describe the use of matrix-assisted laser desorption/ionization (MALDI) MS for the image analysis of proteins, peptides, lipids, drugs, and metabolites in tissues. We discuss the basic instrumentation and sample preparation methods needed to produce high-resolution images and high image reproducibility. Matrix-addition protocols are briefly discussed along with normal operating procedures, and selected biological and medical applications of MALDI imaging MS are described. We give examples of both two- and three-dimensional imaging, including normal mouse embryo implantation, sperm maturation in mouse epididymis, protein distributions in brain sections, protein alterations as a result of drug administration, and protein changes in brain due to neurodegeneration and tumor formation. Advantages of this technology and future challenges for its improvement are discussed.

  1. Can microbes compete with cows for sustainable protein production - A feasibility study on high quality protein

    DEFF Research Database (Denmark)

    Vestergaard, Mike; Chan, Siu Hung Joshua; Jensen, Peter Ruhdal

    2016-01-01

    An increasing population and their increased demand for high-protein diets will require dramatic changes in the food industry, as limited resources and environmental issues will make animal derived foods and proteins, gradually more unsustainable to produce. To explore alternatives to animal...... derived proteins, an economic model was built around the genome-scale metabolic network of E. coli to study the feasibility of recombinant protein production as a food source. Using a novel model, we predicted which microbial production strategies are optimal for economic return, by capturing the tradeoff...... between the market prices of substrates, product output and the efficiency of microbial production. A case study with the food protein, Bovine Alpha Lactalbumin was made to evaluate the upstream economic feasibilities. Simulations with different substrate profiles at maximum productivity were used...

  2. KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms.

    Science.gov (United States)

    Aguilar, Claudio; Vlamakis, Hera; Guzman, Alejandra; Losick, Richard; Kolter, Roberto

    2010-05-18

    Bacillus subtilis cells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion of kinD suppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed.

  3. Leptospira interrogans induces uterine inflammatory responses and abnormal expression of extracellular matrix proteins in dogs.

    Science.gov (United States)

    Wang, Wei; Gao, Xuejiao; Guo, Mengyao; Zhang, Wenlong; Song, Xiaojing; Wang, Tiancheng; Zhang, Zecai; Jiang, Haichao; Cao, Yongguo; Zhang, Naisheng

    2014-10-01

    Leptospira interrogans (L. interrogans), a worldwide zoonosis, infect humans and animals. In dogs, four syndromes caused by leptospirosis have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups), and also it caused inflammation. Extracellular matrix (ECM) is a complex mixture of matrix molecules that is crucial to the reproduction. Both inflammatory response and ECM are closed relative to reproductive. The aim of this study was to clarify how L. interrogans affected the uterus of dogs, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans. In the present study, 27 dogs were divided into 3 groups, intrauterine infusion with L. interrogans, to make uterine infection, sterile EMJH, and normal saline as a control, respectively. The uteruses were removed by surgical operation in 10, 20, and 30 days, respectively. The methods of histopathological analysis, ELISA, Western blot and qPCR were used. The results showed that L. interrogans induced significantly inflammatory responses, which were characterized by inflammatory cellular infiltration and high expression levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in uterine tissue of these dogs. Furthermore, L. interrogans strongly down-regulated the expression of ECM (collagens (CL) IV, fibronectins (FN) and laminins (LN)) in mRNA and protein levels. These data indicated that strongly inflammatory responses, and abnormal regulation of ECM might contribute to the proliferation of dogs infected by L. interrogans. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner.

    Science.gov (United States)

    Qiao, F; Moss, A; Kupfer, G M

    2001-06-29

    Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix.

  5. Inherited complex I deficiency is associated with faster protein diffusion in the matrix of moving mitochondria

    NARCIS (Netherlands)

    Koopman, W.J.H.; Distelmaier, F.; Hink, M.A.; Verkaart, S.; Wijers, M.; Fransen, J.; Smeitink, J.A.M.; Willems, P.H.G.M.

    2008-01-01

    Mitochondria continuously change shape, position, and matrix configuration for optimal metabolite exchange. It is well established that changes in mitochondrial metabolism influence mitochondrial shape and matrix configuration. We demonstrated previously that inhibition of mitochondrial complex I

  6. Dimerization Efficiency of Canine Distemper Virus Matrix Protein Regulates Membrane-Budding Activity.

    Science.gov (United States)

    Bringolf, Fanny; Herren, Michael; Wyss, Marianne; Vidondo, Beatriz; Langedijk, Johannes P; Zurbriggen, Andreas; Plattet, Philippe

    2017-08-15

    Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher-order oligomers may facilitate membrane budding. To gain functional insights, we employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane-budding activity. Three six-alanine-block (6A-block) mutants with mutations located at strategic oligomeric positions were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the other two display amino acids located within two distal surface-exposed α-helices proposed to be involved in dimer-dimer contacts. We further focused on the core of the dimeric interface by mutating asparagine 138 (N138) to several nonconservative amino acids. Cellular localization combined with dimerization and coimmunopurification assays, performed under various denaturing conditions, revealed that all 6A-block mutants were impaired in self-assembly and cell periphery accumulation. These phenotypes correlated with deficiencies in relocating CDV nucleocapsid proteins to the cell periphery and in virus-like particle (VLP) production. Conversely, all M-N138 mutants remained capable of self-assembly, though to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that the M-N138 variants exhibiting the most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provide mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane-budding activity. IMPORTANCE Despite the availability of

  7. Regulation of complement by cartilage oligomeric matrix protein allows for a novel molecular diagnostic principle in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Happonen, Kaisa E; Saxne, Tore; Aspberg, Anders

    2010-01-01

    Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage, where it catalyzes collagen fibrillogenesis. Elevated amounts of COMP are found in serum during increased turnover of cartilage associated with active joint disease, such as rheumatoid arthritis (RA) and osteoarthr...

  8. Ingestion of Casein in a Milk Matrix Modulates Dietary Protein Digestion and Absorption Kinetics but Does Not Modulate Postprandial Muscle Protein Synthesis in Older Men.

    Science.gov (United States)

    Churchward-Venne, Tyler A; Snijders, Tim; Linkens, Armand M A; Hamer, Henrike M; van Kranenburg, Janneau; van Loon, Luc J C

    2015-07-01

    The slow digestion and amino acid absorption kinetics of isolated micellar casein have been held responsible for its relatively lower postprandial muscle protein synthetic response compared with rapidly digested proteins such as isolated whey. However, casein is normally consumed within a milk matrix. We hypothesized that protein digestion and absorption kinetics and the subsequent muscle protein synthetic response after micellar casein ingestion are modulated by the milk matrix. The aim of this study was to determine the impact of a milk matrix on casein protein digestion and absorption kinetics and postprandial muscle protein synthesis in older men. In a parallel-group design, 32 healthy older men (aged 71 ± 1 y) received a primed continuous infusion of L-[ring-(2)H5]-phenylalanine, L-[ring-3,5-(2)H2]-tyrosine, and L-[1-(13)C]-leucine, and ingested 25 g intrinsically L-[1-(13)C]-phenylalanine and L-[1-(13)C]-leucine labeled casein dissolved in bovine milk serum (Cas+Serum) or water (Cas). Plasma samples and muscle biopsies were collected in the postabsorptive state and for 300 min in the postprandial period to examine whole-body and skeletal muscle protein metabolism. Casein ingestion increased plasma leucine and phenylalanine concentrations and L-[1-(13)C]-phenylalanine enrichments, with a more rapid rise after Cas vs. Cas+Serum. Nonetheless, dietary protein-derived phenylalanine availability did not differ between Cas+Serum (47 ± 2%, mean ± SEM) and Cas (46 ± 3%) when assessed over the 300-min postprandial period (P = 0.80). The milk matrix did not modulate postprandial myofibrillar protein synthesis rates from 0 to 120 min (0.038 ± 0.005 vs. 0.031 ± 0.007%/h) or from 120 to 300 min (0.052 ± 0.004 vs. 0.067 ± 0.005%/h) after Cas+Serum vs. Cas. Similarly, no treatment differences in muscle protein-bound L-[1-(13)C]-phenylalanine enrichments were observed at 120 min (0.003 ± 0.001 vs. 0.002 ± 0.001) or 300 min (0.015 ± 0.002 vs. 0.016 ± 0.002 mole

  9. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  10. Can microbes compete with cows for sustainable protein production - A feasibility study on high quality protein.

    Science.gov (United States)

    Vestergaard, Mike; Chan, Siu Hung Joshua; Jensen, Peter Ruhdal

    2016-11-08

    An increasing population and their increased demand for high-protein diets will require dramatic changes in the food industry, as limited resources and environmental issues will make animal derived foods and proteins, gradually more unsustainable to produce. To explore alternatives to animal derived proteins, an economic model was built around the genome-scale metabolic network of E. coli to study the feasibility of recombinant protein production as a food source. Using a novel model, we predicted which microbial production strategies are optimal for economic return, by capturing the tradeoff between the market prices of substrates, product output and the efficiency of microbial production. A case study with the food protein, Bovine Alpha Lactalbumin was made to evaluate the upstream economic feasibilities. Simulations with different substrate profiles at maximum productivity were used to explore the feasibility of recombinant Bovine Alpha Lactalbumin production coupled with market prices of utilized materials. We found that recombinant protein production could be a feasible food source and an alternative to traditional sources.

  11. Can microbes compete with cows for sustainable protein production - A feasibility study on high quality protein

    Science.gov (United States)

    Vestergaard, Mike; Chan, Siu Hung Joshua; Jensen, Peter Ruhdal

    2016-11-01

    An increasing population and their increased demand for high-protein diets will require dramatic changes in the food industry, as limited resources and environmental issues will make animal derived foods and proteins, gradually more unsustainable to produce. To explore alternatives to animal derived proteins, an economic model was built around the genome-scale metabolic network of E. coli to study the feasibility of recombinant protein production as a food source. Using a novel model, we predicted which microbial production strategies are optimal for economic return, by capturing the tradeoff between the market prices of substrates, product output and the efficiency of microbial production. A case study with the food protein, Bovine Alpha Lactalbumin was made to evaluate the upstream economic feasibilities. Simulations with different substrate profiles at maximum productivity were used to explore the feasibility of recombinant Bovine Alpha Lactalbumin production coupled with market prices of utilized materials. We found that recombinant protein production could be a feasible food source and an alternative to traditional sources.

  12. Smart sustainable bottle (SSB) system for E. coli based recombinant protein production.

    Science.gov (United States)

    Li, Zhaopeng; Carstensen, Bettina; Rinas, Ursula

    2014-11-05

    Recombinant proteins are usually required in laboratories interested in the protein but not in the production process itself. Thus, technical equipment which is easy to handle and straight forward protein production procedures are of great benefit to those laboratories. Companies selling single use cultivation bags and bioreactors are trying to satisfy at least part of these needs. However, single-use systems can contribute to major costs which might be acceptable when "good manufacturing practices" are required but not acceptable for most laboratories facing tight funding. The assembly and application of a simple self-made "smart sustainable bottle" (SSB) system for E. coli based protein production is presented. The core of the SSB system is a 2-L glass bottle which is operated at constant temperature, air flow, and stirrer speed without measurement and control of pH and dissolved oxygen. Oxygen transfer capacities are in the range as in conventional bioreactors operated at intermediate aeration rates and by far exceed those found in conventional shaking flasks and disposable bioreactors. The SSB system was applied for the production of various recombinant proteins using T7-based expression systems and a defined autoinduction medium. The production performance regarding amount and solubility of proteins with robust and delicate properties was as good as in state-of-the-art stirred tank commercial bioreactors. The SSB system represents a low cost protein production device applicable for easy, effective, and reproducible recombinant protein production.

  13. Not All Inner Ears are the Same: Otolith Matrix Proteins in the Inner Ear of Sub-Adult Cichlid Fish, Oreochromis Mossambicus, Reveal Insights Into the Biomineralization Process.

    Science.gov (United States)

    Weigele, Jochen; Franz-Odendaal, Tamara A; Hilbig, Reinhard

    2016-02-01

    The fish ear stones (otoliths) consist mainly of calcium carbonate and have lower amounts of a proteinous matrix. This matrix consists of macromolecules, which directly control the biomineralization process. We analyzed the composition of this proteinous matrix by mass spectrometry in a shotgun approach. For this purpose, an enhanced protein purification technique was developed that excludes any potential contamination of proteins from body fluids. Using this method we identified eight proteins in the inner ear of Oreochromis mossambicus. These include the common otolith matrix proteins (OMP-1, otolin-1, neuroserpin, SPARC and otoconin), and three proteins (alpha tectorin, otogelin and transferrin) not previously localized to the otoliths. Moreover, we were able to exclude the occurrence of two matrix proteins (starmaker and pre-cerebellin-like protein) known from other fish species. In further analyses, we show that the absence of the OMP starmaker corresponds to calcitic otoliths and that pre-cerebellin-like protein is not present at any stage during the development of the otoliths of the inner ear. This study shows O. mossambicus does not have all of the known otolith proteins indicating that the matrix proteins in the inner ear of fish are not the same across species. Further functional studies of the novel proteins we identified during otolith development are required. © 2015 Wiley Periodicals, Inc.

  14. Peroxisomal matrix protein import - Suppression of protein import defects in Hansenula polymorpha pex mutants by overproduction of the PTS1 receptor pex5p

    NARCIS (Netherlands)

    Kiel, JAKW; Veenhuis, M

    2000-01-01

    In the past decade, much progress has been made in understanding the mechanisms that govern sorting of proteins to the peroxisomal lumen. This article summarizes the principal features of how peroxisomal matrix enzymes are thought to reach the peroxisome. In addition, it describes recent data that

  15. Expression of uncarboxylated matrix Gla protein in ankylosing spondylitis and its significance

    Directory of Open Access Journals (Sweden)

    Han-qing HUANG

    2013-07-01

    Full Text Available Objective To investigate the serum level of uncarboxylated matrix Gla protein (ucMGP in ankylosing spondylitis (AS patients, and to evaluate its diagnostic value and the relation of ucMGP to inflammation and ossification process in AS. Methods Eight-two AS patients and 76 healthy controls were enrolled in this randomized controlled study. The clinical indices (age, gender, course of disease, disease activity, changes in radiographic studies, and indices of bone metabolism or inflammation, including erythrocyte sedimentation rate (ESR, C-reactive protein (CRP, osteocalcin (OC, and bone-specific alkaline phosphatase (BALP were evaluated or measured. The disease activity was assessed by Bath Ankylosing Spondylitis Disease Activity Index (BASDAI, and changes in radiographic pictures were evaluated according to the modified Stoke AS Spine Score (mSASSS, and serum level of ucMGP was measured by a competitive ELISA. The relationship between ucMGP and clinical indexes, radiographic scoring, indices in bone metabolism or inflammation was estimated by SPSS software, and the diagnostic value of ucMGP was analyzed by receiver operator characteristic (ROC curve. Results The levels of ESR and CRP in AS patients were higher than those in healthy controls, but the serum ucMGP was lower (2958±654nmol/L compared with healthy controls (4551±1036nmol/L, P0, r=-0.715, P1, r=-0.741, P10, r=-0.776, P<0.01; mSASSS <10, r=-0.297, P=0.028. Conclusion Serum ucMGP may serve as a diagnostic biomarker of AS and progression index of ossification, especially in late stage of AS.

  16. Spatial Expression of Otolith Matrix Protein-1 and Otolin-1 in Normally and Kinetotically Swimming Fish.

    Science.gov (United States)

    Weigele, Jochen; Franz-Odendaal, Tamara A; Hilbig, Reinhard

    2015-10-01

    Kinetosis (motion sickness) has been repeatedly shown to affect some fish of a given clutch following the transition from 1g to microgravity or from hypergravity to 1g. This susceptibility to kinetosis may be correlated with irregular inner ear otolith growth. Otoliths are mainly composed of calcium carbonate and matrix proteins, which play an important role in the process of otolith mineralization. Here, we examine the morphology of otoliths and the expression pattern of the major otolith proteins OMP-1 and otolin-1 in a series of hypergravity experiments. In the utricle, OMP-1 is present in centripetal (medial) and centrifugal (lateral) regions of the meshwork area. In the saccule, OMP-1 was expressed within a dorsal and a ventral narrow band of the meshwork area opposite to the periphery of the sulcus acusticus. In normal animals, the spatial expression pattern of OMP-1 reaches more posteriorly in the centrifugal aspect and is considerably broader in the centripetal portion of the utricle compared to kinetotic animals. However, otolin-1 was not expressed in the utricule. In the saccule, no differences were observed for either gene when comparing normal and kinetotically behaving fish. The difference in the utricular OMP-1 expression pattern between normally and kinetotically swimming fish indicates a different otolith morphology and thus a different geometry of the otoliths resting on the corresponding sensory maculae. As the utricle is the endorgan responsible for sensing gravity, the aberrant morphology of the utricular otoliths, based on OMP-1 expression, likely leads to the observed kinetotic behavior. © 2015 Wiley Periodicals, Inc.

  17. Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis.

    Directory of Open Access Journals (Sweden)

    Zhichun Zhang

    Full Text Available Mutation of distal-less homeobox 3 (DLX3 is responsible for human tricho-dento-osseous syndrome (TDO with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

  18. Modulation of enamel matrix proteins on the formation and nano-assembly of hydroxyapatite in vitro

    International Nuclear Information System (INIS)

    Li Hong; Huang Weiya; Zhang Yuanming; Xue Bo; Wen Xuejun

    2012-01-01

    Natural enamel has a hierarchically nanoassembled architecture that is regulated by enamel matrix proteins (EMPs) during the formation of enamel crystals. To understand the role of EMPs on enamel mineralization, calcium phosphate (CaP) growth experiments in both the presence and absence of native rat EMPs in a single diffusion system were conducted. The morphology and organization of formed CaP crystals were examined by X-Ray Diffraction (XRD), High-Resolution Transmission Microscopy (HRTEM) and Selected Area Electron Diffraction (SAED). In the system containing the EMPs, hydroxyapatite (HAP) with hierarchical lamellar nanostructure can be formed and the aligned HAP assembly tightly bundled by 3–4 rod-like nanocrystals like an enamel prism. However, in the absence of EMPs, only a sheet-like structure of octacalcium phosphate (OCP) phase was presented. EMPs promote HAP formation and inhibit the growth of OCP on the (010) plane. It is discussed that the organized Amelogenin/Amorphous Calcium Phosphate might be the precursor to the bundled HAP crystal prism. The study benefits the understanding of biomineralization of tooth enamel. - Highlights: ► An aligned hydroxyapatite crystal bundled by rod-like nanosize crystals was obtained. ► An organized Amel/ACP would be the precursor of the bundled hydroxyapatite crystal prism. ► EMPs inhibit the growth of octacalcium phosphate in a defined plane.

  19. Matrix Gla Protein Polymorphisms are Associated with Coronary Artery Calcification in Men

    Science.gov (United States)

    Crosier, Michael D.; Booth, Sarah L.; Peter, Inga; Dawson-Hughes, Bess; Price, Paul A.; O’Donnell, Christopher J.; Hoffmann, Udo; Williamson, Matthew K.; Ordovas, Jose M.

    2009-01-01

    Summary Matrix Gla protein (MGP) is a key regulator of vascular calcification. Genetic variation at the MGP locus could modulate the development of coronary artery calcification (CAC). Our aim was to examine the cross-sectional association between MGP single nucleotide polymorphisms (SNPs) [rs1800802 (T-138C), rs1800801 (G-7A), and rs4236 (Ala102Thr)] and CAC. CAC was measured by multidetector computed tomography (MDCT), in older men and women of European descent, (n = 386; 60 to 80 y of age). Serum MGP was measured by radioimmunoassay. Linear, Tobit and Ordinal regression analyses all revealed that in men, homozygous carriers of the minor allele of rs1800802 , rs1800801 , or rs4236 (minor allele frequency: 21, 38, and 40%, respectively) were associated with a decreased quantity of CAC, relative to major allele carriers. This association was not found in women. Although genetic variation in MGP was associated with serum MGP concentrations, there were no associations between serum MGP and CAC. The results of this study suggest a role for MGP genetic variants in coronary atherosclerosis among men that is not reflected in serum MGP concentrations. PMID:19352064

  20. Hydroxychloroquine induces inhibition of collagen type II and oligomeric matrix protein COMP expression in chondrocytes

    Directory of Open Access Journals (Sweden)

    Tao Li

    2016-06-01

    Full Text Available The aim of this study was to investigate the effect of hydroxychloroquine on the level of collagen type II and oligomeric matrix protein COMP expression in chondrocytes of knee osteoarthritis. The rate of growth in cartilage cells was analyzed using MTT assay whereas the Col-2 and COMP expression levels were detected by RT-PCR and Western blotting analyses. For the determination of MMP-13 expression, ELISA test was used. The results revealed no significant change in the rate of cartilage cell proliferation in hydroxychloroquine-treated compared to untreated cells. Hydroxychloro-quine treatment exhibited concentration- and time-dependent effect on the inhibition of collagen type II and COMP expression in chondrocytes. However, its treatment caused a significant enhancement in the expression levels of MMP-13 compared to the untreated cells. Therefore, hydroxychloro-quine promotes expression of MMP-13 and reduces collagen type II and COMP expression levels in chondrocytes without any significant change in the growth of cells.

  1. Modulation of enamel matrix proteins on the formation and nano-assembly of hydroxyapatite in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Li Hong, E-mail: tlihong@jnu.edu.cn [Department of Materials Science and Engineering, Jinan University, Guangzhou, Guangdong 510630 (China); Department of Bioengineering, Clemson University, Charleston, SC 29425 (United States); Huang Weiya [Department of Chemistry, Jinan University, Guangzhou, Guangdong 510630 (China); Department of Materials Science and Engineering, Taizhou, Taizhou University, Zhejiang 317000 (China); Zhang Yuanming [Department of Chemistry, Jinan University, Guangzhou, Guangdong 510630 (China); Xue Bo [Department of Materials Science and Engineering, Jinan University, Guangzhou, Guangdong 510630 (China); Wen Xuejun [Department of Bioengineering, Clemson University, Charleston, SC 29425 (United States)

    2012-05-01

    Natural enamel has a hierarchically nanoassembled architecture that is regulated by enamel matrix proteins (EMPs) during the formation of enamel crystals. To understand the role of EMPs on enamel mineralization, calcium phosphate (CaP) growth experiments in both the presence and absence of native rat EMPs in a single diffusion system were conducted. The morphology and organization of formed CaP crystals were examined by X-Ray Diffraction (XRD), High-Resolution Transmission Microscopy (HRTEM) and Selected Area Electron Diffraction (SAED). In the system containing the EMPs, hydroxyapatite (HAP) with hierarchical lamellar nanostructure can be formed and the aligned HAP assembly tightly bundled by 3-4 rod-like nanocrystals like an enamel prism. However, in the absence of EMPs, only a sheet-like structure of octacalcium phosphate (OCP) phase was presented. EMPs promote HAP formation and inhibit the growth of OCP on the (010) plane. It is discussed that the organized Amelogenin/Amorphous Calcium Phosphate might be the precursor to the bundled HAP crystal prism. The study benefits the understanding of biomineralization of tooth enamel. - Highlights: Black-Right-Pointing-Pointer An aligned hydroxyapatite crystal bundled by rod-like nanosize crystals was obtained. Black-Right-Pointing-Pointer An organized Amel/ACP would be the precursor of the bundled hydroxyapatite crystal prism. Black-Right-Pointing-Pointer EMPs inhibit the growth of octacalcium phosphate in a defined plane.

  2. Undersulfation of proteoglycans and proteins alter C6 glioma cells proliferation, adhesion and extracellular matrix organization.

    Science.gov (United States)

    Mendes de Aguiar, Claudia B N; Garcez, Ricardo Castilho; Alvarez-Silva, Marcio; Trentin, Andréa Gonçalves

    2002-11-01

    Proteoglycans are considered to be important molecule in cell-microenvironment interactions. They are overexpressed in neoplastic cells modifying their growth and migration in hosts. In this work we verified that undersulfation of proteoglycans and other sulfated molecules, induced by sodium chlorate treatment, inhibited C6 glioma cells proliferation in a dose-dependent way. This effect was restored by the addition of exogenous heparin. We could not detect significant cell mortality in our culture condition. The treatment also impaired in a dose-dependent manner, C6 cell adhesion to extracellular matrix (ECM) proteins (collagen IV, laminin and fibronectin). In addition, sodium chlorate treatment altered C6 glioma cell morphology, from the fibroblast-like to a more rounded one. This effect was accompanied by increased synthesis of fibronectin and alterations in its extracellular network organization. However, we could not observe modifications on laminin organization and synthesis. The results suggest an important connection between sulfation degree with important tumor functions, such as proliferation and adhesion. We suggest that proteoglycans may modulate the glioma microenvironment network during tumor cell progression and invasion.

  3. [Antitumor effects of matrix protein of vesicular stomatic virus on EL4 lymphoma mice].

    Science.gov (United States)

    Lin, Shi-jia; Yu, Qin-mei; Meng, Wen-tong; Wen, Yan-jun; Chen, Li-juan; Niu, Ting

    2011-03-01

    To explore antitumor effects of plasmid pcDNA3. 1-MP encoding matrix protein of vesicular stomatitis virus (VSV) complexed with cationic liposome (DOTAP:CHOL) in mice with EL4 lymphoma. C57BL/6 mouse model with EL4 lymphoma was established. Sixty mice bearing EL4 lymphoma were divided randomly into five groups including Lip-MP, Lip-pVAX, Lip, ADM and NS groups, which were intravenously injected with liposome-pcDNA 3. 1-MP complex, liposome-pVAX complex, empty liposome, Adriamycin and normal saline respectively every three days. Tumor volumes and survival time were monitored. Microvessel density and tumor proliferative index in tumor tissues were determined by CD31, Ki-67 immunohistochemistry staining, meanwhile the tumor apoptosis index was measured by TUNEL method. From 6 days after treatments on, the tumor volume in Lip-MP group was much smaller than that in Lip-pVAX, Lip and NS group (P EL4 tumor cells in vivo (P EL4 lymphoma, which may be related to the induction of tumor cell apoptosis, inhibition of tumor angiogenesis, and suppression of tumor cell proliferation.

  4. Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

    International Nuclear Information System (INIS)

    Heremans, A.; Cassiman, J.J.; Van den Berghe, H.; David, G.

    1988-01-01

    Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. (Abstract Truncated)

  5. Airway remodelling and inflammation in asthma are dependent on the extracellular matrix protein fibulin-1c.

    Science.gov (United States)

    Liu, Gang; Cooley, Marion A; Nair, Prema M; Donovan, Chantal; Hsu, Alan C; Jarnicki, Andrew G; Haw, Tatt Jhong; Hansbro, Nicole G; Ge, Qi; Brown, Alexandra C; Tay, Hock; Foster, Paul S; Wark, Peter A; Horvat, Jay C; Bourke, Jane E; Grainge, Chris L; Argraves, W Scott; Oliver, Brian G; Knight, Darryl A; Burgess, Janette K; Hansbro, Philip M

    2017-12-01

    Asthma is a chronic inflammatory disease of the airways. It is characterized by allergic airway inflammation, airway remodelling, and airway hyperresponsiveness (AHR). Asthma patients, in particular those with chronic or severe asthma, have airway remodelling that is associated with the accumulation of extracellular matrix (ECM) proteins, such as collagens. Fibulin-1 (Fbln1) is an important ECM protein that stabilizes collagen and other ECM proteins. The level of Fbln1c, one of the four Fbln1 variants, which predominates in both humans and mice, is increased in the serum and airways fluids in asthma but its function is unclear. We show that the level of Fbln1c was increased in the lungs of mice with house dust mite (HDM)-induced chronic allergic airway disease (AAD). Genetic deletion of Fbln1c and therapeutic inhibition of Fbln1c in mice with chronic AAD reduced airway collagen deposition, and protected against AHR. Fbln1c-deficient (Fbln1c -/- ) mice had reduced mucin (MUC) 5 AC levels, but not MUC5B levels, in the airways as compared with wild-type (WT) mice. Fbln1c interacted with fibronectin and periostin that was linked to collagen deposition around the small airways. Fbln1c -/- mice with AAD also had reduced numbers of α-smooth muscle actin-positive cells around the airways and reduced airway contractility as compared with WT mice. After HDM challenge, these mice also had fewer airway inflammatory cells, reduced interleukin (IL)-5, IL-13, IL-33, tumour necrosis factor (TNF) and CXCL1 levels in the lungs, and reduced IL-5, IL-33 and TNF levels in lung-draining lymph nodes. Therapeutic targeting of Fbln1c reduced the numbers of GATA3-positive Th2 cells in the lymph nodes and lungs after chronic HDM challenge. Treatment also reduced the secretion of IL-5 and IL-13 from co-cultured dendritic cells and T cells restimulated with HDM extract. Human epithelial cells cultured with Fbln1c peptide produced more CXCL1 mRNA than medium-treated controls. Our data show

  6. EcmPred: Prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection

    KAUST Repository

    Kandaswamy, Krishna Kumar Umar

    2013-01-01

    The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan syndrome, osteogenesis imperfecta, numerous chondrodysplasias, and skin diseases. In this work, we report a random forest approach, EcmPred, for the prediction of ECM proteins from protein sequences. EcmPred was trained on a dataset containing 300 ECM and 300 non-ECM and tested on a dataset containing 145 ECM and 4187 non-ECM proteins. EcmPred achieved 83% accuracy on the training and 77% on the test dataset. EcmPred predicted 15 out of 20 experimentally verified ECM proteins. By scanning the entire human proteome, we predicted novel ECM proteins validated with gene ontology and InterPro. The dataset and standalone version of the EcmPred software is available at http://www.inb.uni-luebeck.de/tools-demos/Extracellular_matrix_proteins/EcmPred. © 2012 Elsevier Ltd.

  7. Poly(lactic-co-glycolic acid) devices: Production and applications for sustained protein delivery.

    Science.gov (United States)

    Lee, Parker W; Pokorski, Jonathan K

    2018-03-13

    Injectable or implantable poly(lactic-co-glycolic acid) (PLGA) devices for the sustained delivery of proteins have been widely studied and utilized to overcome the necessity of repeated administrations for therapeutic proteins due to poor pharmacokinetic profiles of macromolecular therapies. These devices can come in the form of microparticles, implants, or patches depending on the disease state and route of administration. Furthermore, the release rate can be tuned from weeks to months by controlling the polymer composition, geometry of the device, or introducing additives during device fabrication. Slow-release devices have become a very powerful tool for modern medicine. Production of these devices has initially focused on emulsion-based methods, relying on phase separation to encapsulate proteins within polymeric microparticles. Process parameters and the effect of additives have been thoroughly researched to ensure protein stability during device manufacturing and to control the release profile. Continuous fluidic production methods have also been utilized to create protein-laden PLGA devices through spray drying and electrospray production. Thermal processing of PLGA with solid proteins is an emerging production method that allows for continuous, high-throughput manufacturing of PLGA/protein devices. Overall, polymeric materials for protein delivery remain an emerging field of research for the creation of single administration treatments for a wide variety of disease. This review describes, in detail, methods to make PLGA devices, comparing traditional emulsion-based methods to emerging methods to fabricate protein-laden devices. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Peptide-Based Structures. © 2018 Wiley Periodicals, Inc.

  8. Proteomic analysis of the crayfish gastrolith chitinous extracellular matrix reveals putative protein complexes and a central role for GAP 65.

    Science.gov (United States)

    Glazer, Lilah; Roth, Ziv; Weil, Simy; Aflalo, Eliahu D; Khalaila, Isam; Sagi, Amir

    2015-10-14

    Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather

  9. Antinuclear Matrix Protein 2 Autoantibodies and Edema, Muscle Disease, and Malignancy Risk in Dermatomyositis Patients.

    Science.gov (United States)

    Albayda, Jemima; Pinal-Fernandez, Iago; Huang, Wilson; Parks, Cassie; Paik, Julie; Casciola-Rosen, Livia; Danoff, Sonye K; Johnson, Cheilonda; Christopher-Stine, Lisa; Mammen, Andrew L

    2017-11-01

    Dermatomyositis (DM) patients typically present with proximal weakness and autoantibodies that are associated with distinct clinical phenotypes. We observed that DM patients with autoantibodies recognizing the nuclear matrix protein NXP-2 often presented with especially severe weakness. The aim of this study was to characterize the clinical features associated with anti-NXP-2 autoantibodies. There were 235 DM patients who underwent testing for anti-NXP-2 autoantibodies. Patient characteristics, including muscle strength, were compared between those with and without these autoantibodies. The number of cancer cases observed in anti-NXP-2-positive subjects was compared with the number expected in the general population. Of the DM patients, 56 (23.8%) were anti-NXP-2-positive. There was no significant difference in the prevalence of proximal extremity weakness in patients with and without anti-NXP-2. In contrast, anti-NXP-2-positive patients had more prevalent weakness in the distal arms (35% versus 20%; P = 0.02), distal legs (25% versus 8%; P edema (36% versus 19%; P = 0.01) than anti-NXP-2-negative patients. Five anti-NXP-2-positive subjects (9%) had cancer-associated myositis, representing a 3.68-fold increased risk (95% confidence interval 1.2-8.6) compared to the expected prevalence in the general population. In DM, anti-NXP-2 autoantibodies are associated with subcutaneous edema, calcinosis, and a muscle phenotype characterized by myalgia, proximal and distal weakness, and dysphagia. As anti-NXP-2-positive patients have an increased risk of cancer, we suggest that they undergo comprehensive cancer screening. © 2017, American College of Rheumatology.

  10. Vitamin K-Dependent Carboxylation of Matrix Gla Protein Influences the Risk of Calciphylaxis.

    Science.gov (United States)

    Nigwekar, Sagar U; Bloch, Donald B; Nazarian, Rosalynn M; Vermeer, Cees; Booth, Sarah L; Xu, Dihua; Thadhani, Ravi I; Malhotra, Rajeev

    2017-06-01

    Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification. The ability of MGP to inhibit calcification requires the activity of a vitamin K-dependent enzyme, which mediates MGP carboxylation. We investigated how MGP carboxylation influences the risk of calciphylaxis in adult patients receiving dialysis and examined the effects of vitamin K deficiency on MGP carboxylation. Our study included 20 patients receiving hemodialysis with calciphylaxis (cases) and 20 patients receiving hemodialysis without calciphylaxis (controls) matched for age, sex, race, and warfarin use. Cases had higher plasma levels of uncarboxylated MGP (ucMGP) and carboxylated MGP (cMGP) than controls. However, the fraction of total MGP that was carboxylated (relative cMGP concentration = cMGP/[cMGP + uncarboxylated MGP]) was lower in cases than in controls (0.58±0.02 versus 0.69±0.03, respectively; P =0.003). In patients not taking warfarin, cases had a similarly lower relative cMGP concentration. Each 0.1 unit reduction in relative cMGP concentration associated with a more than two-fold increase in calciphylaxis risk. Vitamin K deficiency associated with lower relative cMGP concentration in multivariable adjusted analyses ( β =-8.99; P =0.04). In conclusion, vitamin K deficiency-mediated reduction in relative cMGP concentration may have a role in the pathogenesis of calciphylaxis. Whether vitamin K supplementation can prevent and/or treat calciphylaxis requires further study. Copyright © 2017 by the American Society of Nephrology.

  11. Matrix metalloproteinase-20 mediates dental enamel biomineralization by preventing protein occlusion inside apatite crystals.

    Science.gov (United States)

    Prajapati, Saumya; Tao, Jinhui; Ruan, Qichao; De Yoreo, James J; Moradian-Oldak, Janet

    2016-01-01

    Reconstruction of enamel-like materials is a central topic of research in dentistry and material sciences. The importance of precise proteolytic mechanisms in amelogenesis to form a hard tissue with more than 95% mineral content has already been reported. A mutation in the Matrix Metalloproteinase-20 (MMP-20) gene results in hypomineralized enamel that is thin, disorganized and breaks from the underlying dentin. We hypothesized that the absence of MMP-20 during amelogenesis results in the occlusion of amelogenin in the enamel hydroxyapatite crystals. We used spectroscopy and electron microscopy techniques to qualitatively and quantitatively analyze occluded proteins within the isolated enamel crystals from MMP-20 null and Wild type (WT) mice. Our results showed that the isolated enamel crystals of MMP-20 null mice had more organic macromolecules occluded inside them than enamel crystals from the WT. The crystal lattice arrangements of MMP-20 null enamel crystals analyzed by High Resolution Transmission Electron Microscopy (HRTEM) were found to be significantly different from those of the WT. Raman studies indicated that the crystallinity of the MMP-20 null enamel crystals was lower than that of the WT. In conclusion, we present a novel functional mechanism of MMP-20, specifically prevention of unwanted organic material entrapped in the forming enamel crystals, which occurs as the result of precise amelogenin cleavage. MMP-20 action guides the growth morphology of the forming hydroxyapatite crystals and enhances their crystallinity. Elucidating such molecular mechanisms can be applied in the design of novel biomaterials for future clinical applications in dental restoration or repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Identification of Proteins with Potential Osteogenic Activity Present in the Water-Soluble Matrix Proteins from Crassostrea gigas Nacre Using a Proteomic Approach

    Directory of Open Access Journals (Sweden)

    Daniel V. Oliveira

    2012-01-01

    Full Text Available Nacre, when implanted in vivo in bones of dogs, sheep, mice, and humans, induces a biological response that includes integration and osteogenic activity on the host tissue that seems to be activated by a set of proteins present in the nacre water-soluble matrix (WSM. We describe here an experimental approach that can accurately identify the proteins present in the WSM of shell mollusk nacre. Four proteins (three gigasin-2 isoforms and a cystatin A2 were for the first time identified in WSM of Crassostrea gigas nacre using 2DE and LC-MS/MS for protein identification. These proteins are thought to be involved in bone remodeling processes and could be responsible for the biocompatibility shown between bone and nacre grafts. These results represent a contribution to the study of shell biomineralization process and opens new perspectives for the development of new nacre biomaterials for orthopedic applications.

  13. Matrix-assisted laser-desorption-ionization mass spectrometry of proteins using a free-electron laser

    International Nuclear Information System (INIS)

    Cramer, R.; Hillenkamp, F.; Haglund, R.

    1995-01-01

    Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry (MS) is one of the most promising techniques for spectral fingerprinting large molecules, such as proteins, oligonucleotides and carbohydrates. In the usual implementation of this technique, the analyte molecule is dissolved in an aromatic liquid matrix material which resonantly absorbs ultraviolet laser light. Resonant absorption by π-π* transitions volatilizes the matrix and initiates subsequent charge transfer to the analyte molecules, which are detected by time-of-flight mass spectrometry. Recent MALDI-MS studies with Er:YAG (2.94 μm) and CO 2 4 (9.4-10.6 μm) lasers suggest that them is significant unexplored potential for mass spectrometry of macromolecules, including oligonucleotide, in the mid-infrared. Preliminary experiments show that it is possible to capitalize on the rich rovibronic absorption spectrum of virtually all organics to initiate resonant desorption in matrix material over the entire range of pH values. However, the mechanism of charge transfer is particularly problematic for infrared MALDI because of the low photon energy. In this paper, we report the results of MALI-MS studies on small proteins using the Vanderbilt FEL and several matrix materials. Proteins with masses up to roughly 6,000 amu were detected with high resolution in a linear time-of-flight mass spectrometer. By varying the pulse duration using a broadband Pockels cell, we have been able to compare the results of relatively long (5 μs) and short (0.1 μs) irradiation on the desorption and ionization processes. Compared to uv-MALDI spectra of identical analytes obtained with a nitrogen laser (337 nm) in the same time-of-flight spectrometer, the infrared results appear to show that the desorption and ionization process goes on over a somewhat longer time scale

  14. A Human Nuclear Shuttling Protein That Interacts with Human Immunodeficiency Virus Type 1 Matrix Is Packaged into Virions

    Science.gov (United States)

    Gupta, Kalpana; Ott, David; Hope, Thomas J.; Siliciano, Robert F.; Boeke, Jef D.

    2000-01-01

    Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells. Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55Gag and genomic RNA. Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues. Its expression is upregulated upon activation of CD4+ T cells. VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm. Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture. We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55Gag and viral genomic RNA during virion production. Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport. PMID:11090181

  15. Canine distemper virus matrix protein influences particle infectivity, particle composition, and envelope distribution in polarized epithelial cells and modulates virulence.

    Science.gov (United States)

    Dietzel, Erik; Anderson, Danielle E; Castan, Alexandre; von Messling, Veronika; Maisner, Andrea

    2011-07-01

    In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis of the closely related canine distemper virus (CDV) has not been characterized. To this end this, we generated a recombinant wild-type CDV carrying a vaccine strain M protein. The recombinant virus retained the parental growth phenotype in VerodogSLAMtag cells, but displayed an increased particle-to-infectivity ratio very similar to that of the vaccine strain, likely due to inefficient H protein incorporation. Even though infectious virus was released only from the apical surface, consistent with the release polarity of the wild-type CDV strain, envelope protein distribution in polarized epithelial cells reproduced the bipolar pattern seen in vaccine strain-infected cells. Most notably, the chimeric virus was completely attenuated in ferrets and caused only a mild and transient leukopenia, indicating that the differences in particle infectivity and envelope protein sorting mediated by the vaccine M protein contribute importantly to vaccine strain attenuation.

  16. Ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix protein is important for viral budding.

    Directory of Open Access Journals (Sweden)

    Yao E Wang

    2010-11-01

    Full Text Available Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4 pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS and the leucine-rich nuclear export signal (NES found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50 of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.

  17. GOLGA2/GM130, cis-Golgi Matrix Protein, is a Novel Target of Anticancer Gene Therapy

    OpenAIRE

    Chang, Seung-Hee; Hong, Seong-Ho; Jiang, Hu-Lin; Minai-Tehrani, Arash; Yu, Kyeong-Nam; Lee, Jae-Ho; Kim, Ji-Eun; Shin, Ji-Young; Kang, Bitna; Park, Sungjin; Han, Kiwon; Chae, Chanhee; Cho, Myung-Haing

    2012-01-01

    Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an im...

  18. [Sustained release of recombinant human bone morphogenetic protein-2 combined with stromal vascular fraction cells in promoting posterolateral spinal fusion in rat model].

    Science.gov (United States)

    Yuan, Wei; Zheng, Jun; Qian, Jinyu; Zhou, Xiaoxiao; Wang, Minghui; Wang, Xiuhui

    2017-07-01

    To observe the effect of stromal vascular fraction cells (SVFs) from rat fat tissue combined with sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) in promoting the lumbar fusion in rat model. SVFs were harvested from subcutaneous fat of bilateral inguinal region of 4-month-old rat through the collagenase I digestion. The sustained release carrier was prepared via covalent bond of the rhBMP-2 and β-tricalcium phosphate (β-TCP) by the biominetic apatite coating process. The sustained release effect was measured by BCA method. Thirty-two rats were selected to establish the posterolateral lumbar fusion model and were divided into 4 groups, 8 rats each group. The decalcified bone matrix (DBX) scaffold+PBS, DBX scaffold+rhBMP-2/β-TCP sustained release carrier, DBX scaffold+SVFs, and DBX scaffold+rhBMP-2/β-TCP sustained release carrier+SVFs were implanted in groups A, B, C, and D respectively. X-ray films, manual spine palpation, and high-resolution micro-CT were used to evaluate spinal fusion at 8 weeks after operation; bone mineral density (BMD) and bone volume fraction were analyzed; the new bone formation was evaluated by HE staining and Masson's trichrome staining, osteocalcin (OCN) was detected by immunohistochemical staining. The cumulative release amount of rhBMP-2 was about 40% at 2 weeks, indicating sustained release effect of rhBMP-2; while the control group was almost released within 2 weeks. At 8 weeks, the combination of manual spine palpation, X-ray, and micro-CT evaluation showed that group D had the strongest bone formation (100%, 8/8), followed by group B (75%, 6/8), group C (37.5%, 3/8), and group A (12.5%, 1/8). Micro-CT analysis showed BMD and bone volume fraction were significantly higher in group D than groups A, B, and C ( P cells with bone matrix deposition, and an active osteogenic process similar to the mineralization of long bones in group D. The bone formation of group B was weaker than that of group D, and

  19. Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity

    International Nuclear Information System (INIS)

    Zhirnov, O.P.; Manykin, A.A.; Rossman, J.S.; Klenk, H.D.

    2016-01-01

    Influenza virus has two major structural modules, an external lipid envelope and an internal ribonucleocapsid containing the genomic RNA in the form of the ribonucleoprotein (RNP) complex, both of which are interlinked by the matrix protein M1. Here we studied M1-RNP cohesion within virus exposed to acidic pH in vitro. The effect of acidification was dependent on the cleavage of the surface glycoprotein HA. Acidic pH caused a loss of intravirion RNP-M1 cohesion and activated RNP polymerase activity in virus with cleaved HA (HA1/2) but not in the uncleaved (HA0) virus. The in vitro acidified HA1/2 virus rapidly lost infectivity whereas the HA0 one retained infectivity, following activation by trypsin, suggesting that premature activation and release of the RNP is detrimental to viral infectivity. Rimantadine, an inhibitor of the M2 ion channel, was found to protect the HA1/2 virus interior against acidic disintegration, confirming that M2-dependent proton translocation is essential for the intravirion RNP release and suggesting that the M2 ion channel is only active in virions with cleaved HA. Acidic treatment of both HA0 and HA1/2 influenza viruses induces formation of spikeless bleb-like protrusion of ~25 nm in diameter on the surface of the virion, though only the HA1/2 virus was permeable to protons and permitted RNP release. It is likely that this bleb corresponds to the M2-enriched and M1-depleted focus arising from pinching off of the virus during the completion of budding. Cooperatively, the data suggest that the influenza virus has an asymmetric structure where the M1-mediated organization of the RNP inside the virion is a prerequisite for infectious entry into target cell. - Highlights: • The influenza A virus has a novel asymmetric internal structure. • The structure is largely maintained by M1-RNP cohesion within the virion. • This asymmetry plays an important role during viral entry, facilitating virus uncoating and the initiation of a productive

  20. Molecular decay of enamel matrix protein genes in turtles and other edentulous amniotes

    Directory of Open Access Journals (Sweden)

    Meredith Robert W

    2013-01-01

    Full Text Available Abstract Background Secondary edentulism (toothlessness has evolved on multiple occasions in amniotes including several mammalian lineages (pangolins, anteaters, baleen whales, birds, and turtles. All edentulous amniote clades have evolved from ancestors with enamel-capped teeth. Previous studies have documented the molecular decay of tooth-specific genes in edentulous mammals, all of which lost their teeth in the Cenozoic, and birds, which lost their teeth in the Cretaceous. By contrast with mammals and birds, tooth loss in turtles occurred in the Jurassic (201.6-145.5 Ma, providing an extended time window for tooth gene degradation in this clade. The release of the painted turtle and Chinese softshell turtle genomes provides an opportunity to recover the decayed remains of tooth-specific genes in Testudines. Results We queried available genomes of Testudines (Chrysemys picta [painted turtle], Pelodiscus sinensis [Chinese softshell turtle], Aves (Anas platyrhynchos [duck], Gallus gallus [chicken], Meleagris gallopavo [turkey], Melopsittacus undulatus [budgerigar], Taeniopygia guttata [zebra finch], and enamelless mammals (Orycteropus afer [aardvark], Choloepus hoffmanni [Hoffmann’s two-toed sloth], Dasypus novemcinctus [nine-banded armadillo] for remnants of three enamel matrix protein (EMP genes with putative enamel-specific functions. Remnants of the AMBN and ENAM genes were recovered in Chrysemys and retain their original synteny. Remnants of AMEL were recovered in both testudines, although there are no shared frameshifts. We also show that there are inactivated copies of AMBN, AMEL and ENAM in representatives of divergent avian lineages including Galloanserae, Passeriformes, and Psittaciformes, and that there are shared frameshift mutations in all three genes that predate the basal split in Neognathae. Among enamelless mammals, all three EMP genes exhibit inactivating mutations in Orycteropus and Choloepus. Conclusions Our results

  1. Intravirion cohesion of matrix protein M1 with ribonucleocapsid is a prerequisite of influenza virus infectivity

    Energy Technology Data Exchange (ETDEWEB)

    Zhirnov, O.P., E-mail: zhirnov@inbox.ru [D.I. Ivanovsky Institute of Virology, Moscow 123098 (Russian Federation); Manykin, A.A. [D.I. Ivanovsky Institute of Virology, Moscow 123098 (Russian Federation); Rossman, J.S. [School of Biosciences, University of Kent, Canterbury CT27NJ (United Kingdom); Klenk, H.D. [Institute of Virology, Philipps University, Marburg 35037 (Germany)

    2016-05-15

    Influenza virus has two major structural modules, an external lipid envelope and an internal ribonucleocapsid containing the genomic RNA in the form of the ribonucleoprotein (RNP) complex, both of which are interlinked by the matrix protein M1. Here we studied M1-RNP cohesion within virus exposed to acidic pH in vitro. The effect of acidification was dependent on the cleavage of the surface glycoprotein HA. Acidic pH caused a loss of intravirion RNP-M1 cohesion and activated RNP polymerase activity in virus with cleaved HA (HA1/2) but not in the uncleaved (HA0) virus. The in vitro acidified HA1/2 virus rapidly lost infectivity whereas the HA0 one retained infectivity, following activation by trypsin, suggesting that premature activation and release of the RNP is detrimental to viral infectivity. Rimantadine, an inhibitor of the M2 ion channel, was found to protect the HA1/2 virus interior against acidic disintegration, confirming that M2-dependent proton translocation is essential for the intravirion RNP release and suggesting that the M2 ion channel is only active in virions with cleaved HA. Acidic treatment of both HA0 and HA1/2 influenza viruses induces formation of spikeless bleb-like protrusion of ~25 nm in diameter on the surface of the virion, though only the HA1/2 virus was permeable to protons and permitted RNP release. It is likely that this bleb corresponds to the M2-enriched and M1-depleted focus arising from pinching off of the virus during the completion of budding. Cooperatively, the data suggest that the influenza virus has an asymmetric structure where the M1-mediated organization of the RNP inside the virion is a prerequisite for infectious entry into target cell. - Highlights: • The influenza A virus has a novel asymmetric internal structure. • The structure is largely maintained by M1-RNP cohesion within the virion. • This asymmetry plays an important role during viral entry, facilitating virus uncoating and the initiation of a productive

  2. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.

    Science.gov (United States)

    Kessler, P D; Podsakoff, G M; Chen, X; McQuiston, S A; Colosi, P C; Matelis, L A; Kurtzman, G J; Byrne, B J

    1996-11-26

    Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the beta-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.

  3. Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

    Science.gov (United States)

    Drakouli, Sotiria; Lyberopoulou, Aggeliki; Papathanassiou, Maria; Mylonis, Ilias; Georgatsou, Eleni

    2017-08-01

    Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function. © 2017 Federation of European Biochemical Societies.

  4. Electrochemistry and biosensing reactivity of heme proteins adsorbed on the structure-tailored mesoporous Nb2O5 matrix

    International Nuclear Information System (INIS)

    Xu Xin; Tian Bozhi; Zhang Song; Kong Jilie; Zhao Dongyuan; Liu Baohong

    2004-01-01

    The highly ordered mesoporous niobium oxides fabricated by self-adjusted synthesis have been used as immobilization matrices of heme proteins including Cytochrome c (Cyt C) and horseradish peroxidase (HRP) for their large surface areas, narrow pore size distributions and good biocompatibility. The assembling process was investigated by cyclic voltammetry, amperometry and potential step chronoamperometry in details. Niobium oxide matrices with different structural features were templated with the surfactants and the selectivity of these hosts to specific protein characteristics was determined. It was observed that proteins could be readily assembled onto the mesoporous films with detectable retention of bioactivity. The Nb 2 O 5 matrix with a tailored pore size and counterpoised surface charge to that of hemes allowed for a maximum adsorption capacity of biomolecules. The adsorbed redox molecules exhibited direct electrochemical behavior and gave a pair of well-defined quasi-reversible cyclic voltammetric peaks, indicating that the mesoporous niobium oxide matrix could effectively promote the direct electron transfer between the protein redox site adsorbed and the electrode surface. The midpoint redox potentials of adsorbed Cyt-c and HRP were 14 and -122 mV versus SCE, respectively. Furthermore, the immobilized HRP onto Nb 2 O 5 derived electrode presented good bioactivity and thus was fabricated as an amperometric biosensor for the response of hydrogen peroxide in the range from 0.1 μM to 0.1 mM

  5. Structure and dynamics of Ebola virus matrix protein VP40 by a coarse-grained Monte Carlo simulation

    Science.gov (United States)

    Pandey, Ras; Farmer, Barry

    Ebola virus matrix protein VP40 (consisting of 326 residues) plays a critical role in viral assembly and its functions such as regulation of viral transcription, packaging, and budding of mature virions into the plasma membrane of infected cells. How does the protein VP40 go through structural evolution during the viral life cycle remains an open question? Using a coarse-grained Monte Carlo simulation we investigate the structural evolution of VP40 as a function of temperature with the input of a knowledge-based residue-residue interaction. A number local and global physical quantities (e.g. mobility profile, contact map, radius of gyration, structure factor) are analyzed with our large-scale simulations. Our preliminary data show that the structure of the protein evolves through different state with well-defined morphologies which can be identified and quantified via a detailed analysis of structure factor.

  6. On plate graphite supported sample processing for simultaneous lipid and protein identification by matrix assisted laser desorption ionization mass spectrometry.

    Science.gov (United States)

    Calvano, Cosima Damiana; van der Werf, Inez Dorothé; Sabbatini, Luigia; Palmisano, Francesco

    2015-05-01

    The simultaneous identification of lipids and proteins by matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) after direct on-plate processing of micro-samples supported on colloidal graphite is demonstrated. Taking advantages of large surface area and thermal conductivity, graphite provided an ideal substrate for on-plate proteolysis and lipid extraction. Indeed proteins could be efficiently digested on-plate within 15 min, providing sequence coverages comparable to those obtained by conventional in-solution overnight digestion. Interestingly, detection of hydrophilic phosphorylated peptides could be easily achieved without any further enrichment step. Furthermore, lipids could be simultaneously extracted/identified without any additional treatment/processing step as demonstrated for model complex samples such as milk and egg. The present approach is simple, efficient, of large applicability and offers great promise for protein and lipid identification in very small samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Matrix-assisted laser desorption/ionisation mass spectrometry imaging and its development for plant protein imaging

    Directory of Open Access Journals (Sweden)

    Millar A Harvey

    2011-07-01

    Full Text Available Abstract Matrix-Assisted Laser Desorption/Ionisation (MALDI mass spectrometry imaging (MSI uses the power of high mass resolution time of flight (ToF mass spectrometry coupled to the raster of lasers shots across the cut surface of tissues to provide new insights into the spatial distribution of biomolecules within biological tissues. The history of this technique in animals and plants is considered and the potential for analysis of proteins by this technique in plants is discussed. Protein biomarker identification from MALDI-MSI is a challenge and a number of different approaches to address this bottleneck are discussed. The technical considerations needed for MALDI-MSI are reviewed and these are presented alongside examples from our own work and a protocol for MALDI-MSI of proteins in plant samples.

  8. A Simple Sonication Improves Protein Signal in Matrix-Assisted Laser Desorption Ionization Imaging

    Science.gov (United States)

    Lin, Li-En; Su, Pin-Rui; Wu, Hsin-Yi; Hsu, Cheng-Chih

    2018-02-01

    Proper matrix application is crucial in obtaining high quality matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). Solvent-free sublimation was essentially introduced as an approach of homogeneous coating that gives small crystal size of the organic matrix. However, sublimation has lower extraction efficiency of analytes. Here, we present that a simple sonication step after the hydration in standard sublimation protocol significantly enhances the sensitivity of MALDI MSI. This modified procedure uses a common laboratory ultrasonicator to immobilize the analytes from tissue sections without noticeable delocalization. Improved imaging quality with additional peaks above 10 kDa in the spectra was thus obtained upon sonication treatment. [Figure not available: see fulltext.

  9. Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

    International Nuclear Information System (INIS)

    Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep; Sinha, Surajit; Dash, Prasanta K.; Siddappa, Nagadenahalli B.; Ranga, Udaykumar; Mitra, Debashis; Chattopadhyay, Samit

    2010-01-01

    Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFα leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

  10. Formulation and evaluation of sustained release matrix tablets of pioglitazone hydrochloride using processed Aloe vera mucilage as release modifier

    Directory of Open Access Journals (Sweden)

    Manoj Choudhary

    2015-01-01

    Full Text Available Background: Natural gums and mucilage which hydrates and swells on contact with aqueous media are used as additives in the formulation of hydrophilic drug delivery system. Aim: The purpose of this study was to develop a new monolithic matrix system for complete delivery of Pioglitazone hydrochloride (HCl, in a zero-order manner over an extended time period using processed Aloe vera gel mucilage (PAG as a release modifier. Materials and Methods: The matrices were prepared by dry blending of selected ratios of polymer and ingredients using direct compression technique. Physicochemical properties of dried powdered mucilage of A. vera were studied. Various formulations of pioglitazone HCl and A. vera mucilage were prepared using different drug: Polymer ratios viz., 1:1, 1:2, 1:3, 1:4, 1:5 for PAG by direct compression technique. Results: The formulated matrix tablets were found to have better uniformity of weight and drug content with low statistical deviation. The swelling behavior and in vitro release rate characteristics were also studied. Conclusion: The study proved that the dried A. vera mucilage can be used as a matrix forming material for controlled release of Pioglitazone HCl matrix tablets.

  11. Molecular energy dissipation in nanoscale networks of Dentin Matrix Protein 1 is strongly dependent on ion valence

    Science.gov (United States)

    Adams, J; Fantner, G E; Fisher, L W; Hansma, P K

    2008-01-01

    The fracture resistance of biomineralized tissues such as bone, dentin, and abalone is greatly enhanced through the nanoscale interactions of stiff inorganic mineral components with soft organic adhesive components. A proper understanding of the interactions that occur within the organic component, and between the organic and inorganic components, is therefore critical for a complete understanding of the mechanics of these tissues. In this paper, we use Atomic Force Microscope (AFM) force spectroscopy and dynamic force spectroscopy to explore the effect of ionic interactions within a nanoscale system consisting of networks of Dentin Matrix Protein 1 (DMP1) (a component of both bone and dentin organic matrix), a mica surface, and an AFM tip. We find that DMP1 is capable of dissipating large amounts of energy through an ion-mediated mechanism, and that the effectiveness increases with increasing ion valence. PMID:18843380

  12. Mueller-matrix mapping of biological tissues in differential diagnosis of optical anisotropy mechanisms of protein networks

    Energy Technology Data Exchange (ETDEWEB)

    Ushenko, V A; Sidor, M I [Yuriy Fedkovych Chernivtsi National University, Chernivtsi (Ukraine); Marchuk, Yu F; Pashkovskaya, N V; Andreichuk, D R [Bukovinian State Medical University, Chernivtsi (Ukraine)

    2015-03-31

    We report a model of Mueller-matrix description of optical anisotropy of protein networks in biological tissues with allowance for the linear birefringence and dichroism. The model is used to construct the reconstruction algorithms of coordinate distributions of phase shifts and the linear dichroism coefficient. In the statistical analysis of such distributions, we have found the objective criteria of differentiation between benign and malignant tissues of the female reproductive system. From the standpoint of evidence-based medicine, we have determined the operating characteristics (sensitivity, specificity and accuracy) of the Mueller-matrix reconstruction method of optical anisotropy parameters and demonstrated its effectiveness in the differentiation of benign and malignant tumours. (laser applications and other topics in quantum electronics)

  13. Molecular energy dissipation in nanoscale networks of dentin matrix protein 1 is strongly dependent on ion valence

    International Nuclear Information System (INIS)

    Adams, J; Fantner, G E; Hansma, P K; Fisher, L W

    2008-01-01

    The fracture resistance of biomineralized tissues such as bone, dentin, and abalone is greatly enhanced through the nanoscale interactions of stiff inorganic mineral components with soft organic adhesive components. A proper understanding of the interactions that occur within the organic component, and between the organic and inorganic components, is therefore critical for a complete understanding of the mechanics of these tissues. In this paper, we use atomic force microscope (AFM) force spectroscopy and dynamic force spectroscopy to explore the effect of ionic interactions within a nanoscale system consisting of networks of dentin matrix protein 1 (DMP1) (a component of both bone and dentin organic matrix), a mica surface and an AFM tip. We find that DMP1 is capable of dissipating large amounts of energy through an ion-mediated mechanism, and that the effectiveness increases with increasing ion valence

  14. Molecular energy dissipation in nanoscale networks of dentin matrix protein 1 is strongly dependent on ion valence

    Energy Technology Data Exchange (ETDEWEB)

    Adams, J; Fantner, G E; Hansma, P K [Department of Physics, Broida Hall, University of California, Santa Barbara, CA 93106 (United States); Fisher, L W [Craniofacial and Skeletal Diseases Branch, NIDCR, NIH, DHHS, Bethesda, MD 20892 (United States)], E-mail: adams@physics.ucsb.edu, E-mail: fantner@physics.ucsb.edu, E-mail: lfisher@dir.nidcr.nih.gov, E-mail: prasant@physics.ucsb.edu

    2008-09-24

    The fracture resistance of biomineralized tissues such as bone, dentin, and abalone is greatly enhanced through the nanoscale interactions of stiff inorganic mineral components with soft organic adhesive components. A proper understanding of the interactions that occur within the organic component, and between the organic and inorganic components, is therefore critical for a complete understanding of the mechanics of these tissues. In this paper, we use atomic force microscope (AFM) force spectroscopy and dynamic force spectroscopy to explore the effect of ionic interactions within a nanoscale system consisting of networks of dentin matrix protein 1 (DMP1) (a component of both bone and dentin organic matrix), a mica surface and an AFM tip. We find that DMP1 is capable of dissipating large amounts of energy through an ion-mediated mechanism, and that the effectiveness increases with increasing ion valence.

  15. ASH1L Suppresses Matrix Metalloproteinase through Mitogen-activated Protein Kinase Signaling Pathway in Pulpitis.

    Science.gov (United States)

    Bei, Yin; Tianqian, Hui; Fanyuan, Yu; Haiyun, Luo; Xueyang, Liao; Jing, Yang; Chenglin, Wang; Ling, Ye

    2017-02-01

    Pulpitis is an inflammation of dental pulp produced by a response to external stimuli. The response entails substantial cellular and molecular activities. Both genetic and epigenetic regulators contribute to the occurrence of pulpitis. However, the epigenetic mechanisms are still poorly understood. In this research, we studied the role of the absent, small, or homeotic-like (ASH1L) gene in the process of pulpitis. Human dental pulp cells (HDPCs) were stimulated with proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Gene expression profiling was performed to assess the occurrence of epigenetic regulators. Pulp tissue from rat experimental pulpitis was subjected to immunofluorescence to detect the occurrence of ASH1L and trimethylation of lysine 4 histone 3 (H3K4me3). The presence of ASH1L in HDPCs that had been generated by TNF-α stimulation was analyzed by Western blot procedures and cellular immunofluorescence. Once detected, ASH1L was silenced through the use of specific small interfering RNA. The effects of ASH1L on the occurrence and operation of matrix metalloproteinases (MMPs) were then tested by analysis of quantitative polymerase chain reactions, Western blotting, and zymography. Chromatin immunoprecipitation was performed to detect whether ASH1L and H3K4me3 were present in the promoter regions of MMPs. We then used Western blot procedures to examine the nuclear factor kappa B and the mitogen-activated protein kinase (MAPK) responses to the silencing of ASH1L. We also examined the specific pathway involved in ASH1L regulation of the MMPs. After stimulating HDPCs with TNF-α, ASH1L emerged as 1 of the most strongly induced epigenetic mediators. We found that TNF-α treatment induced the expression of ASH1L through the nuclear factor kappa B and MAPK signal pathways. ASH1L was found in both the nucleus and the cytoplasm. TNF-α treatment was particularly active in inducing the accumulation of ASH1L in cellular cytoplasm. As is also consistent

  16. Preparation of sustained release matrix pellets by melt agglomeration in the fluidized bed: influence of formulation variables and modelling of agglomerate growth.

    Science.gov (United States)

    Pauli-Bruns, Anette; Knop, Klaus; Lippold, Bernhard C

    2010-03-01

    The one-step preparation of sustained release matrix pellets, using a melting procedure in a fluidized bed apparatus, was tested in a 2(3) full factorial design of experiments, using microcrystalline wax as lipophilic binder, theophylline as model drug and talc as additional matrix forming agent. The three influence parameters were (A) size of binder particles, (B) fraction of theophylline in solid particles and (C) fraction of microcrystalline wax in formulation. The response variables were agglomerate size and size distribution, dissolution time, agglomerate crush resistance, sphericity, yield and porosity. Nearly spherical pellets comprising a smooth, closed surface could be obtained with the used method, exhibiting the hollow core typical for the immersion and layering mechanism. The reproducibility was very good concerning all responses. The size of agglomerates is proportional to the size of the binder particles, which serve as cores for pellet formation in the molten state in the fluidized bed. Additionally, the agglomerate size is influenced by the volume of the solid particles in relation to the binder particles, with more solid particles leading to larger agglomerates and vice versa. Dissolution times vary in a very wide range, resulting from the interplay between amount of drug in relation to the meltable matrix substance microcrystalline wax and the non-meltable matrix substance talc. The change of binder particle size does not lead to a structural change of the matrix; both dissolution times and porosity are not significantly altered. Agglomerate crush resistance is low due to the hollow core of the pellets. However, it is significantly increased if the volume fraction of microcrystalline wax in the matrix is high, which means that the matrix is mechanically better stabilized. A theoretical model has been established to quantitatively explain agglomerate growth and very good accordance of the full particle size distributions between predicted and

  17. Identification of a Novel Dentin Matrix Protein-1 (DMP-1) Mutation and Dental Anomalies in a Kindred with Autosomal Recessive Hypophosphatemia

    OpenAIRE

    Turan, Serap; Aydin, Cumhur; Bereket, Abdullah; Akcay, Teoman; Güran, Tülay; Yaralioglu, Betul Akmen; Bastepe, Murat; Jüppner, Harald

    2009-01-01

    An autosomal recessive form of hypophosphatemia (ARHP) was recently shown to be caused by homozygous mutations in DMP1, the gene encoding dentin matrix protein-1 (DMP-1), a non-collagenous bone matrix protein with an important role in the development and mineralization of bone and teeth. Here, we report a previously not reported consanguineous ARHP kindred in which the three affected individuals carry a novel homozygous DMP-1 mutation. The index case presented at the age of 3 years with bowin...

  18. Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin-like proteins.

    Science.gov (United States)

    Pérez-Munive, Clara; Blumenthal, Sonal S D; de la Espina, Susana Moreno Díaz

    2012-01-01

    Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins.

  19. Canine Distemper Virus Matrix Protein Influences Particle Infectivity, Particle Composition, and Envelope Distribution in Polarized Epithelial Cells and Modulates Virulence ▿

    OpenAIRE

    Dietzel, Erik; Anderson, Danielle E.; Castan, Alexandre; von Messling, Veronika; Maisner, Andrea

    2011-01-01

    In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis o...

  20. Comparison of efficacies of different bone substitutes adhered to osteoblasts with and without extracellular matrix proteins

    Directory of Open Access Journals (Sweden)

    Li-Ling Tseng

    2013-12-01

    Conclusion: The results indicated that ECM proteins increased cell attachment to bone substitutes in vitro. The preferential affinity of different bone substitutes to certain ECM proteins was evident. Cerasorb and BoneCeramic had better MG63 human osteosarcoma cell adhesion ability than Bio-Oss and MBCP.

  1. The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

    Science.gov (United States)

    Duan, Zhiqiang; Chen, Jian; Xu, Haixu; Zhu, Jie; Li, Qunhui; He, Liang; Liu, Huimou; Hu, Shunlin; Liu, Xiufan

    2014-03-01

    The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Really old-palaeoimmunology: immunohistochemical analysis of extracellular matrix proteins in historic and pre-historic material.

    Science.gov (United States)

    Wick, G; Kalischnig, G; Maurer, H; Mayerl, C; Müller, P U

    2001-09-01

    In this review, we summarize data concerning the respective preservation and deterioration of antigenic determinants of various collagenous and non-collagenous extracellular matrix (ECM) proteins in palaeontologic material of different ages. ECM proteins are the major quantitative constituents of mammalian organisms and were, therefore, selected as important representative proteins for these analyses. The specimens, studied by immunofluorescence and immunohistochemical techniques, included the skin of 500-1500 year-old human mummies from Peru, skin and striated muscle from the 5300-year-old glacier mummy ("Iceman") from Tyrol, Austria, and a 50-million-year-old bat with preserved soft body parts from the fossil excavation site of Messel, Germany. In frozen sections of the former two sources, epitopes recognized by specific antibodies for triple-helical antigenic determinants of different types of collagen resistant against conventional proteases were preserved, while non-helical domains, as well as the non-collagenous ECM proteins, could no longer be demonstrated. The fossil bat, although showing evidence of fibrous, collagen-like structures in conventional histology, revealed no collagenous or non-collagenous ECM proteins by any technique. It later turned out that this was due to the replacement of the original soft parts in these fossils by lawns of bacteria. These studies introduced immunological techniques into palaeontology and opened new approaches for studying physiologically- and pathologically-altered structures in tissues of animals and humans of considerable historical age.

  3. Protein crystallization with microseed matrix screening: application to human germline antibody Fabs

    International Nuclear Information System (INIS)

    Obmolova, Galina; Malia, Thomas J.; Teplyakov, Alexey; Sweet, Raymond W.; Gilliland, Gary L.

    2014-01-01

    The power of microseed matrix screening is demonstrated in the crystallization of a panel of antibody Fab fragments. The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization

  4. The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding.

    Science.gov (United States)

    Prchal, Jan; Srb, Pavel; Hunter, Eric; Ruml, Tomáš; Hrabal, Richard

    2012-10-26

    We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein. Copyright © 2012. Published by Elsevier Ltd.

  5. Cartilage oligomeric matrix protein deficiency promotes early onset and the chronic development of collagen-induced arthritis

    DEFF Research Database (Denmark)

    Geng, Hui; Carlsen, Stefan; Nandakumar, Kutty

    2008-01-01

    ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a homopentameric protein in cartilage. The development of arthritis, like collagen-induced arthritis (CIA), involves cartilage as a target tissue. We have investigated the development of CIA in COMP-deficient mice. METHODS: COMP......-deficient mice in the 129/Sv background were backcrossed for 10 generations against B10.Q mice, which are susceptible to chronic CIA. COMP-deficient and wild-type mice were tested for onset, incidence, and severity of arthritis in both the collagen and collagen antibody-induced arthritis models. Serum anti......-collagen II and anti-COMP antibodies as well as serum COMP levels in arthritic and wild-type mice were measured by enzyme-linked immunosorbent assay. RESULTS: COMP-deficient mice showed a significant early onset and increase in the severity of CIA in the chronic phase, whereas collagen II-antibody titers were...

  6. Effect of apoptosis and response of extracellular matrix proteins after chemotherapy application on human breast cancer cell spheroids.

    Science.gov (United States)

    Oktem, G; Vatansever, S; Ayla, S; Uysal, A; Aktas, S; Karabulut, B; Bilir, A

    2006-02-01

    Multicellular tumor spheroid (MTS) represents a three-dimensional structural form of tumors in laboratory conditions, and it has the characteristics of avascular micrometastases or intervascular spaces of big tumors. Recent studies indicate that extracellular matrix (ECM) proteins play a critical role in tumor metastasis, therefore normal and cancer cells require an ECM for survival, proliferation and differentiation. Doxorubicin and Docetaxel are widely used in the therapy of breast cancer, as well as in in vivo and in vitro studies. In this study, we examined the effect of apoptosis and proliferation of cells on the human breast cancer cell line, MCF-7, by using p53, bcl-2 and Ki67 gene expression, and the tendency to metastasis with extracellular matrix proteins, laminin and type IV collagen after chemotherapy in the spheroid model. The apoptotic cell death in situ was detected by TUNEL method. TUNEL-positive cells and positive immunoreactivities of laminin, type IV collagen, p53 and, bcl-2 were detected in the control group. There was no laminin and type IV collagen immunoreactivities in spheroids of drug groups. While TUNEL-positive cells and p53 immunoreactivity were detected in Docetaxel, Doxorubicin and Docetaxel/Doxorubicin groups, p53 immunoreactivity was not observed in the Docetaxel group. There was no bcl-2 immunoreactivity in either drug group. In addition, we did not detect Ki67 immunoreactivity in both control and drug treatment groups. However, the absence of Ki67 protein in MCF-7 breast multicellular tumor spheroids is possibly related to the cells in G0 or S phase. These chemotherapeutic agents may affect the presence of ECM proteins in this in vitro model of micrometastasis of spheroids. These findings suggest that the possible mechanism of cell death in Doxorubicin and Docetaxel/Doxorubicin treatment groups is related to apoptosis through the p53 pathway. However, we considered the possibility that there is another control mechanism for the

  7. The hyaluronan and proteoglycan link proteins: Organizers of the brain extracellular matrix and key molecules for neuronal function and plasticity.

    Science.gov (United States)

    Oohashi, Toshitaka; Edamatsu, Midori; Bekku, Yoko; Carulli, Daniela

    2015-12-01

    The hyaluronan and proteoglycanbinding link protein (Hapln) is a key molecule in the formation and control of hyaluronan-based condensed perineuronal matrix in the adult brain. This review summarizes the recent advances in understanding the role of Haplns in the formation and control of two distinct types of perineuronal matrices, one for "classical" PNN and the other for the specialized extracellular matrix (ECM) at the node of Ranvier in the central nervous system (CNS). We introduce the structural components of each ECM organization including the basic concept of supramolecular structure named "HLT model". We furthermore summarize the developmental and physiological role of perineuronal ECMs from the studies of Haplns and related molecules. Finally, we also discuss the potential mechanism modulating PNNs in the adult CNS. This layer of organized matrices may exert a direct effect via core protein or sugar moiety from the structure or by acting as a binding site for biologically active molecules, which are important for neuronal plasticity and saltatory conduction. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Microfibril-associated Protein 4 Binds to Surfactant Protein A (SP-A) and Colocalizes with SP-A in the Extracellular Matrix of the Lung

    DEFF Research Database (Denmark)

    Schlosser, Anders; Thomsen, Theresa H.; Shipley, J. Michael

    2006-01-01

    for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril-associated protein 4 (MFAP4) and SP-A. MFAP4 is a collagen-binding molecule containing a C-terminal fibrinogen-like domain and a N-terminal located integrin-binding motif. We produced recombinant MFAP4......-A composed of the neck region and carbohydrate recognition domain of SP-A indicating that the interaction between MFAP4 and SP-A is mediated via the collagen domain of SP-A. Monoclonal antibodies directed against MFAP4 and SP-A were used for immunohistochemical analysis, which demonstrates that the two...... molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP-A via the collagen region in vitro, and that MFAP4 and SP-A colocates in different lung compartments...

  9. Differences in extracellular matrix proteins between Friesian horses with aortic rupture, unaffected Friesians and Warmblood horses.

    Science.gov (United States)

    Ploeg, M; Gröne, A; van de Lest, C H A; Saey, V; Duchateau, L; Wolsein, P; Chiers, K; Ducatelle, R; van Weeren, P R; de Bruijn, M; Delesalle, C

    2017-09-01

    Unlike in Warmblood horses, aortic rupture is quite common in Friesian horses, in which a hereditary trait is suspected. The aortic connective tissue in affected Friesians shows histological changes such as medial necrosis, elastic fibre fragmentation, mucoid material accumulation and fibrosis with aberrant collagen morphology. However, ultrastructural examination of the collagen fibres of the mid-thoracic aorta has been inconclusive in further elucidating the pathogenesis of the disease. To assess several extracellular matrix (ECM) components biochemically in order to explore a possible underlying breed-related systemic ECM defect in Friesians with aortic rupture. Cadaver study. Tissues from affected Friesians (n = 18), unaffected Friesians (n = 10) and Warmblood horses (n = 30) were compared. Samples were taken from the thoracic aorta at the level of the rupture site, from two locations caudal to the rupture and from the deep digital flexor tendon. Total collagen content, post-translational modifications of collagen formation including lysine hydroxylation, and hydroxylysylpyridinoline (HP), lysylpyridinoline (LP) and pyrrole cross-links were analysed. Additionally, elastin cross-links, glycosaminoglycan content and matrix metalloproteinase (MMP) activity were assessed. Significantly increased MMP activity and increased LP and HP cross-linking, lysine hydroxylation and elastin cross-linking were found at the site of rupture in affected Friesians. These changes may reflect processes involved in healing and aneurysm formation. Unaffected Friesians had less lysine hydroxylation and pyrrole cross-linking within the tendons compared with Warmblood horses. No differences in the matrix of the aorta were found between normal Warmbloods and Friesian horses. Small sample size. The differences in collagen parameters in tendon tissue may reflect differences in connective tissue metabolism between Friesians and Warmblood horses. © 2017 EVJ Ltd.

  10. Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

    Science.gov (United States)

    Masuda, Hiro-taka; Ishihara, Seiichiro; Harada, Ichiro; Mizutani, Takeomi; Ishikawa, Masayori; Kawabata, Kazushige; Haga, Hisashi

    2014-01-01

    We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

  11. Integrin-mediated adhesion of human mesenchymal stem cells to extracellular matrix proteins adsorbed to polymer surfaces

    International Nuclear Information System (INIS)

    Dånmark, S; Mustafa, K; Finne-Wistrand, A; Albertsson, A-C; Patarroyo, M

    2012-01-01

    In vitro, degradable aliphatic polyesters are widely used as cell carriers for bone tissue engineering, despite their lack of biological cues. Their biological active surface is rather determined by an adsorbed layer of proteins from the surrounding media. Initial cell fate, including adhesion and proliferation, which are key properties for efficient cell carriers, is determined by the adsorbed layer of proteins. Herein we have investigated the ability of human bone marrow derived stem cells (hBMSC) to adhere to extracellular matrix (ECM) proteins, including fibronectin and vitronectin which are present in plasma and serum. hBMSC expressed integrins for collagens, laminins, fibronectin and vitronectin. Accordingly, hBMSC strongly adhered to these purified ECM proteins by using the corresponding integrins. Although purified fibronectin and vitronectin adsorbed to aliphatic polyesters to a lower extent than to cell culture polystyrene, these low levels were sufficient to mediate adhesion of hBMSC. It was found that plasma- and serum-coated polystyrene adsorbed significant levels of both fibronectin and vitronectin, and fibronectin was identified as the major adhesive component of plasma for hBMSC; however, aliphatic polyesters adsorbed minimal levels of fibronectin under similar conditions resulting in impaired cell adhesion. Altogether, the results suggest that the efficiency of aliphatic polyesters cell carriers could be improved by increasing their ability to adsorb fibronectin. (paper)

  12. Circulating matrix gamma-carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome

    NARCIS (Netherlands)

    Cranenburg, E. C. M.; van Spaendonck-Zwarts, K. Y.; Bonafe, L.; Crettol, L. Mittaz; Rodiger, L. A.; Dikkers, F. G.; van Essen, A. J.; Superti-Furga, A.; Alexandrakis, E.; Vermeer, C.; Schurgers, L. J.; Laverman, G. D.

    Background and objectives: Matrix gamma-carboxyglutamate protein (MGP), a vitamin K-dependent protein, is recognized as a potent local inhibitor of vascular calcification. Studying patients with Keutel syndrome (KS), a rare autosomal recessive disorder resulting from MGP mutations, provides an

  13. Circulating matrix gamma-carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome

    NARCIS (Netherlands)

    Cranenburg, E. C. M.; van Spaendonck-Zwarts, K. Y.; Bonafe, L.; Mittaz Crettol, L.; Rödiger, L. A.; Dikkers, F. G.; van Essen, A. J.; Superti-Furga, A.; Alexandrakis, E.; Vermeer, C.; Schurgers, L. J.; Laverman, G. D.

    2011-01-01

    Background and objectives: Matrix gamma-carboxyglutamate protein (MGP), a vitamin K-dependent protein, is recognized as a potent local inhibitor of vascular calcification. Studying patients with Keutel syndrome (KS), a rare autosomal recessive disorder resulting from MGP mutations, provides an

  14. Matrix rigidity regulates cancer cell growth by modulating cellular metabolism and protein synthesis.

    Directory of Open Access Journals (Sweden)

    Robert W Tilghman

    Full Text Available Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy.This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa, cells on soft substrates (150-300 Pa exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins and glycolysis (e.g., phosphofructokinase-1, whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway.The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical

  15. Correlation Between Placental Matrix Metalloproteinase 9 and Tumor Necrosis Factor-α Protein Expression Throughout Gestation in Normal Human Pregnancy.

    Science.gov (United States)

    Basu, Jayasri; Agamasu, Enyonam; Bendek, Bolek; Salafia, Carolyn M; Mishra, Aruna; Lopez, Julia Vasquez; Kroes, Jessica; Dragich, Sharon Claire; Thakur, Ashley; Mikhail, Magdy

    2018-04-01

    Matrix metalloproteinases (MMPs), specifically MMP-9 plays a role in human placentation. The enzyme confers an invasive ability to cytotrophoblasts and degrades the endometrial matrix as the cells infiltrate the decidua to keep up with placental growth. Since tumor necrosis factor-α (TNF-α) can induce the synthesis of MMP-9, we investigated the patterns of changes in and correlation between placental villous MMP-9 and TNF-α expressions throughout normal human gestation. Placentas were obtained from 179 normal pregnant women who underwent elective abortion or term delivery. Chorionic villi isolated from placental samples were grouped as first, second, and third trimester (7 0/7 -13 0/7 , 13 1/7 -23 6/7 , and 37 0/7 -42 4/7 weeks, respectively). Chorionic villous TNF-α and MMP-9 proteins were assayed using enzyme immunoassay kits. There were significant differences in MMP-9 and TNF-α protein expressions among the trimester groups ( P = .001). The MMP-9 protein increased progressively with an increase in gestational age (GA), but TNF-α peaked in the second trimester. Within each trimester group, we searched for the effects of variation of GA in days on the 2 variables. A significant positive correlation between MMP-9 and GA was noted in the first trimester ( r = 0.364, P = .005). No other comparisons were significant. When GA was controlled for, partial correlation revealed a significant positive correlation between TNF-α and MMP-9 only in the second trimester ( r = 0.300, P = .018). We hypothesize that the TNF-α peak and the positive correlation between TNF-α and MMP-9 in the second trimester of normal human gestation could contribute toward a successful pregnancy outcome.

  16. Phototherapy up-regulates dentin matrix proteins expression and synthesis by stem cells from human-exfoliated deciduous teeth.

    Science.gov (United States)

    Turrioni, Ana Paula S; Basso, Fernanda G; Montoro, Liege A; Almeida, Leopoldina de Fátima D de; Costa, Carlos A de Souza; Hebling, Josimeri

    2014-10-01

    The aim of this study was to evaluate the effects of infrared LED (850nm) irradiation on dentin matrix proteins expression and synthesis by cultured stem cells from human exfoliated deciduous teeth (SHED). Near-exfoliation primary teeth were extracted (n=3), and SHED cultures were characterized by immunofluorescence using STRO-1, CD44, CD146, Nanog and OCT3/4 antibodies, before experimental protocol. The SHEDs were seeded (3×10(4) cells/cm(2)) with DMEM containing 10% FBS. After 24-h incubation, the culture medium was replaced by osteogenic differentiation medium, and the cells were irradiated with LED light at energy densities (EDs) of 0 (control), 2, or 4J/cm(2) (n=8). The irradiated SHEDs were then evaluated for alkaline phosphatase (ALP) activity, total protein (TP) production, and collagen synthesis (SIRCOL™ Assay), as well as ALP, collagen type I (Col I), dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein (DMP-1) gene expression (qPCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α=0.05). Increased ALP activity and collagen synthesis, as well as gene expression of DSPP and ALP, were observed for both EDs compared with non-irradiated cells. The ED of 4J/cm(2) also increased gene expression of COL I and DMP-1. In conclusion, infrared LED irradiation was capable of biostimulating SHEDs by increasing the expression and synthesis of proteins related with mineralized tissue formation, with overall better results for the energy dose of 4J/cm(2). Phototherapy is an additional approach for the clinical application of LED in Restorative Dentistry. Infrared LED irradiation of the cavity's floor could biostimulate subjacent pulp cells, improving local tissue healing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Extracellular Matrix (ECM) Multilayer Membrane as a Sustained Releasing Growth Factor Delivery System for rhTGF-β3 in Articular Cartilage Repair

    Science.gov (United States)

    Park, Sang-Hyug; Kim, Moon Suk; Kim, Young Jick; Choi, Byung Hyune; Lee, Chun Tek; Park, So Ra; Min, Byoung-Hyun

    2016-01-01

    Recombinant human transforming growth factor beta-3 (rhTGF-β3) is a key regulator of chondrogenesis in stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-β3 using a multilayered extracellular matrix (ECM) membrane. We hypothesize that the sustained release of rhTGF-β3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS) are investigated using rhTGF-β3 as a candidate drug. The bioactivity of the released rhTGF-ß3 was evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs) using western blot and circular dichroism (CD) analyses in vitro. The cartilage reparability was evaluated through implanting EMLDS with endogenous and exogenous MSC in both in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-ß3 was clearly observed over a prolonged period of time in vitro and the released rhTGF-β3 maintained its structural stability and biological activity. Successful cartilage repair was also demonstrated when rabbit MSCs were treated with rhTGF-β3-loaded EMLDS ((+) rhTGF-β3 EMLDS) in an in vivo model and when rabbit chondrocytes and MSCs were treated in ex vivo models. Therefore, the multilayer ECM membrane could be a useful drug delivery system for cartilage repair. PMID:27258120

  18. Extracellular Matrix (ECM Multilayer Membrane as a Sustained Releasing Growth Factor Delivery System for rhTGF-β3 in Articular Cartilage Repair.

    Directory of Open Access Journals (Sweden)

    Soon Sim Yang

    Full Text Available Recombinant human transforming growth factor beta-3 (rhTGF-β3 is a key regulator of chondrogenesis in stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-β3 using a multilayered extracellular matrix (ECM membrane. We hypothesize that the sustained release of rhTGF-β3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS are investigated using rhTGF-β3 as a candidate drug. The bioactivity of the released rhTGF-ß3 was evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs using western blot and circular dichroism (CD analyses in vitro. The cartilage reparability was evaluated through implanting EMLDS with endogenous and exogenous MSC in both in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-ß3 was clearly observed over a prolonged period of time in vitro and the released rhTGF-β3 maintained its structural stability and biological activity. Successful cartilage repair was also demonstrated when rabbit MSCs were treated with rhTGF-β3-loaded EMLDS ((+ rhTGF-β3 EMLDS in an in vivo model and when rabbit chondrocytes and MSCs were treated in ex vivo models. Therefore, the multilayer ECM membrane could be a useful drug delivery system for cartilage repair.

  19. Identification of the proteins responsible for SAR DNA binding in nuclear matrix of ''Cucurbita pepo''

    International Nuclear Information System (INIS)

    Rzepecki, R.; Markiewicz, E.; Szopa, J.

    1995-01-01

    The nuclear matrices from White bush (''Cucurbita pepo var. patisonina'') cell nuclei have been isolated using three methods: I, standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; II, the same with pre-treatment of cell nuclei with 0.5 mM CuSO 4 (stabilisation step); and III, method with extraction by lithium diiodosalicylate (LIS), and compared the polypeptide pattern. The isolated matrices specifically bind SAR DNA derived from human β-interferon gene in the exogenous SAR binding assay and in the gel mobility shift assay. Using IgG against the 32 kDa endonuclease we have found in the DNA-protein blot assay that this protein is one of the proteins binding SAR DNA. We have identified three proteins with molecular mass of 65 kDa, 60 kDa and 32 kDa which are responsible for SAR DNA binding in the gel mobility shift assay experiments. (author). 21 refs, 3 figs

  20. Flax fabric reinforced arylated soy protein composites: A brittle-matrix behaviour

    CSIR Research Space (South Africa)

    Kumar, R

    2012-05-01

    Full Text Available Biocomposites were successfully prepared by the reinforcement of soy protein isolate (SPI) with different weight fractions of woven flax fabric. The flax-fabric-reinforced SPI-based composites were then arylated with 2,2-diphenyl-2-hydroxyethanoic...

  1. Identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells.

    Science.gov (United States)

    Ho, Steven C L; Yang, Yuansheng

    2014-08-01

    Promoters are essential on plasmid vectors to initiate transcription of the transgenes when generating therapeutic recombinant proteins expressing mammalian cell lines. High and sustained levels of gene expression are desired during therapeutic protein production while gene expression is useful for cell engineering. As many finely controlled promoters exhibit cell and product specificity, new promoters need to be identified, optimized and carefully evaluated before use. Suitable promoters can be identified using techniques ranging from simple molecular biology methods to modern high-throughput omics screenings. Promoter engineering is often required after identification to either obtain high and sustained expression or to provide a wider range of gene expression. This review discusses some of the available methods to identify and engineer promoters for therapeutic recombinant protein expression in mammalian cells.

  2. Evaluation of Ocimum basilicum L. seed mucilage as rate controlling matrix for sustained release of propranolol HCl

    Directory of Open Access Journals (Sweden)

    Majid Saeedi

    2015-01-01

    Full Text Available Polysaccharide mucilage derived from the seeds of Ocimum basilicum L. (family Lamiaceae was investigated for use in matrix formulations containing propranolol hydrochloride. Basil mucilage was extracted and several tablets were formulated. The effect of mucilage on drug release rate was evaluated in comparison with tablets containing two kinds of hydroxypropyl methylcellulose (HPMC K4M and HPMC K100M as standard polymer. The release data were fitted to several models for kinetic evaluation. The results showed that hardness decreased and friability of tablets increased as the concentration of mucilage increased. The rate of release of propranolol HCl from O. basilicm mucilage matrices was mainly controlled by the drug: mucilage ratio. Drug release was slower from the HPMC K4M and HPMCK100M containing tablets compared to the mucilage containing matrices than the drug release from matrices containing O. basilicum seed mucilage in similar ratios.  Formulations containing O. basilicm mucilage were found to exhibit suitable release pattern. The results of kinetic analysis showed that in tablets containing O. basilicm mucilage the highest correlation coefficient was achieved with the zero order model. The swelling and erosion studies revealed that, as the proportion of mucilage in tablets was increased, there was a corresponding increase in percent swelling and a decrease in percent erosion of tablets.

  3. Development of Carrageenan Hydrogel as a Sustained Release Matrix Containing Tocotrienol-Rich Palm-Based Vitamin E

    International Nuclear Information System (INIS)

    Yee, C.M.; Zafarizal Aldrin Azizul Hasan; Norashikin Ahmad; Hazimah, A.H.

    2016-01-01

    Topically applied hydrogel system as a general therapeutic mask for transdermal delivery of hydrophobic actives is not efficient due to the differences in polarity between the actives and the polymer network. This work presents a study on developing hydrogels based on carrageenan as a matrix for the delivery of a hydrophobic type of active, i.e. tocotrienol-rich palm-based vitamin E (TRPE) into the skin. The strength and flexibility of the hydrogel were increased by the inclusion of guar gum, potassium citrate and glycerine. The thermogravimetric analysis (TGA) results indicated a higher quantity of water in the hydrogel with glycerine while differential scanning calorimetry (DSC) showed three types of water molecules existed in the hydrogel. The hydrogel was non-irritating according to OECD Test Guideline No. 439 for in vitro skin irritation test. The hydrogel with TRPE fluids was able to permeate the polysulfone membrane and bioavailability of TRPE improved with the inclusion of PEG-40 hydrogenated castor oil mixture. Therefore, a carrageenan-based hydrogel with locust bean, guar gum, glycerine, potassium citrate and TRPE was successfully developed with good strength and flexibility and without any potential irritancy. The good bioavailability of TRPE-loaded in the hydrogel can be used for skin care application. (author)

  4. Evaluation of Plantago major L. seed mucilage as a rate controlling matrix for sustained release of propranolol hydrochloride.

    Science.gov (United States)

    Saeedi, Majid; Morteza-Semnani, Katayoun; Sagheb-Doust, Mehdi

    2013-03-01

    Polysaccharide mucilage derived from the seeds of Plantago major L. (family Plantaginaceae) was investigated for use in matrix formulations containing propranolol hydrochloride. HPMC K4M and tragacanth were used as standards for comparison. The hardness, tensile strength, and friability of tablets increased as the concentration of mucilage increased, indicating good compactibility of mucilage powders. The rate of release of propranolol hydrochloride from P. major mucilage matrices was mainly controlled by the drug/mucilage ratio. Formulations containing P. major mucilage were found to exhibit a release rate comparable to HPMC containing matrices at a lower drug/polymer ratio (drug/HPMC 2:1). These results demonstrated that P. major mucilage is a better release retardant compared to tragacanth at an equivalent content. The results of kinetic analysis showed that in F3 (containing 1:2 drug/mucilage) the highest correlation coefficient was achieved with the zero order model. The swelling and erosion studies revealed that as the proportion of mucilage in tablets was increased, there was a corresponding increase in percent swelling and a decrease in percent erosion of tablets. The DSC and FT-IR studies showed that no formation of complex between the drug and mucilage or changes in crystallinity of the drug had occurred.

  5. Detection of lung cancer using plasma protein profiling by matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Shevchenko, Valeriy E; Arnotskaya, Natalia E; Zaridze, David G

    2010-01-01

    There are no satisfactory plasma biomarkers which are available for the early detection and monitoring of lung cancer, one of the most frequent cancers worldwide. The aim of this study is to explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) to plasma proteomic patterns to distinguish lung cancer patients from healthy individuals. The EDTA plasma samples have been pre-fractionated using magnetic bead kits functionalized with weak cation exchange coatings. We compiled MS protein profiles for 90 patients with squamous cell carcinomas (SCC) and compared them with profiles from 187 healthy controls. The MALDI-ToF spectra were analyzed statistically using ClinProTools bioinformatics software. Depending on the sample used, up to 441 peaks/spectrum could be detected in a mass range of 1000-20,000 Da; 33 of these proteins had statistically differential expression levels between SCC and control plasma (P 90%) in external validation test. These results suggest that plasma MALDI-ToF MS protein profiling can distinguish patients with SCC and also from healthy individuals with relatively high sensitivity and specificity and that MALDI- ToF MS is a potential tool for the screening of lung cancer.

  6. Energy Matrix of Structurally Important Side-Chain/Side-Chain Interactions in Proteins

    Czech Academy of Sciences Publication Activity Database

    Berka, K.; Laskowski, R. A.; Hobza, P.; Vondrášek, Jiří

    2010-01-01

    Roč. 6, č. 7 (2010), s. 2191-2203 ISSN 1549-9618 R&D Projects: GA ČR GAP208/10/0725; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : protein structure * DFT method * force fields * interaction energy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 5.138, year: 2010

  7. Sustained Release of Protein Therapeutics from Subcutaneous Thermosensitive Biocompatible and Biodegradable Pentablock Copolymers (PTSgels

    Directory of Open Access Journals (Sweden)

    Elizabeth Schaefer

    2016-01-01

    Full Text Available Objective. To evaluate thermosensitive, biodegradable pentablock copolymers (PTSgel for sustained release and integrity of a therapeutic protein when injected subcutaneously. Materials and Methods. Five PTSgels with PEG-PCL-PLA-PCL-PEG block arrangements were synthesized. In vitro release of IgG from PTSgels and concentrations was evaluated at 37°C. Released IgG integrity was characterized by SDS-PAGE. In vitro disintegration for 10GH PTSgel in PBS was monitored at 37°C over 72 days using gravimetric loss and GPC analysis. Near-infrared IgG in PTSgel was injected subcutaneously and examined by in vivo imaging and histopathology for up to 42 days. Results. IgG release was modulated from approximately 7 days to more than 63 days in both in vitro and in vivo testing by varying polymer composition, concentration of PTSgel aqueous solution, and concentration of IgG. Released IgG in vitro maintained structural integrity by SDS-PAGE. Subcutaneous PTSgels were highly biocompatible and in vitro IgG release occurred in parallel with the disappearance of subcutaneous gel in vivo. Conclusions. Modulation of release of biologics to fit the therapeutic need can be achieved by varying the biocompatible and biodegradable PTSgel composition. Release of IgG parallels disappearance of the polymeric gel; hence, little or no PTSgel remains after drug release is complete.

  8. Atypical protein kinase C activity is required for extracellular matrix degradation and invasion by Src-transformed cells.

    Science.gov (United States)

    Rodriguez, Elena M; Dunham, Elizabeth E; Martin, G Steven

    2009-10-01

    Atypical protein kinase C (aPKC) isoforms have been shown to mediate Src-dependent signaling in response to growth factor stimulation. To determine if aPKC activity contributes to the transformed phenotype of cells expressing oncogenic Src, we have examined the activity and function of aPKCs in 3T3 cells expressing viral Src (v-Src). aPKC activity and tyrosine phosphorylation were found to be elevated in some but not all clones of mouse fibroblasts expressing v-Src. aPKC activity was inhibited either by addition of a membrane-permeable pseudosubstrate, by expression of a dominant-negative aPKC, or by RNAi-mediated knockdown of specific aPKC isoforms. aPKC activity contributes to morphological transformation and stress fiber disruption, and is required for migration of Src-transformed cells and for their ability to polarize at the edge of a monolayer. The lambda isoform of aPKC is specifically required for invasion through extracellular matrix in Boyden chamber assays and for degradation of the extracellular matrix in in situ zymography assays. Tyrosine phosphorylation of aPKClambda is required for its ability to promote cell invasion. The defect in invasion upon aPKC inhibition appears to result from a defect in the assembly and/or function of podosomes, invasive adhesions on the ventral surface of the cell that are sites of protease secretion. aPKC was also found to localize to podosomes of v-Src transformed cells, suggesting a direct role for aPKC in podosome assembly and/or function. We conclude that basal or elevated aPKC activity is required for the ability of Src-transformed cells to degrade and invade the extracellular matrix. Copyright 2009 Wiley-Liss, Inc.

  9. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    International Nuclear Information System (INIS)

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-01-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, 32 P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV

  10. Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometry

    DEFF Research Database (Denmark)

    Persson, S; Sönksen, C P; Frigaard, N U

    2000-01-01

    We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1...... microL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were...... proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll...

  11. Plasma concentrations of extracellular matrix protein fibulin-1 are related to cardiovascular risk markers in chronic kidney disease and diabetes

    DEFF Research Database (Denmark)

    Scholze, Alexandra; Bladbjerg, Else-Marie; Sidelmann, Johannes J

    2013-01-01

    ABSTRACT: BACKGROUND: Fibulin-1 is one of a few extracellular matrix proteins present in blood in high concentrations. We aimed to define the relationship between plasma fibulin-1 levels and risk markers of cardiovascular disease. METHODS: Plasma fibulin-1 was determined in subjects with chronic...... to determine central hemodynamic and arterial stiffness indices. RESULTS: We observed a positive correlation of fibulin-1 levels with age (r = 0.38; p = 0.033), glycated hemoglobin (r = 0.80; p = 0.003), creatinine (r = 0.35; p = 0.045), and fibrinogen (r = 0.39; p = 0.027). Glomerular filtration rate...... and fibulin-1 were inversely correlated (r = -0.57; p = 0.022). There was a positive correlation between fibulin-1 and central pulse pressure (r = 0.44; p = 0.011) and central augmentation pressure (r = 0.55; p = 0.001). In a multivariable regression model, diabetes, creatinine, fibrinogen and central...

  12. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    Energy Technology Data Exchange (ETDEWEB)

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-02-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

  13. Evaluation of Urinary Nuclear Matrix Protein-22 as Tumor Marker Versus Tissue Polypeptide Specific Antigen in Bilharzial and Bladder Cancer

    International Nuclear Information System (INIS)

    Ahmed, W.A.; El-Kabany, H.

    2004-01-01

    Urinary nuclear matrix protein-22 (NMP-22) and tissue polypeptide specific antigen (TPS) were determined as potential marker for early detection of bladder tumors in patients with high risk (Bilharzial-patients), monitoring and follow up bladder cancer patients. The objective was to determine sensitivity and specificity of markers for bilharzial and cancer lesions. The levels of two parameters were determined pre and post operation. A total of 110 individuals, 20 healthy, 20 bilharzial patients and 70 bladder cancer patients with confirmed diagnosis were investigated. Urine samples were assayed for NMP-22 and TPS test kits. Some bladder cancer patients were selected to follow up. NMP-22 showed highly significant increase (P,0.001) more than TPS (P<0.01) in bladder cancer patients when compared with bilharzial and control group. Overall sensitivity is 7.8% for TPS and 98.5% for NMP-22

  14. Extracellular Matrix Proteins Expression Profiling in Chemoresistant Variants of the A2780 Ovarian Cancer Cell Line

    Directory of Open Access Journals (Sweden)

    Radosław Januchowski

    2014-01-01

    Full Text Available Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly—over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis.

  15. AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Xia Luo

    2015-01-01

    Full Text Available Background/Aims: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. Methods: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f. Results: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1312 (encoding constitutively active AMPKα1 whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1. In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. Conclusion: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

  16. Identifying stabilizing key residues in proteins using interresidue interaction energy matrix

    Czech Academy of Sciences Publication Activity Database

    Biedermannová, Lada; Hobza, Pavel; Vondrášek, Jiří

    2008-01-01

    Roč. 72, č. 1 (2008), s. 402-413 ISSN 0887-3585 R&D Projects: GA ČR GA203/05/0009; GA ČR GA203/06/1727; GA ČR(CZ) GD203/05/H001; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : protein stabilisation * an-initio calculation * interaction energy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.419, year: 2008

  17. General transfer matrix formalism to calculate DNA-protein-drug binding in gene regulation: application to OR operator of phage lambda.

    Science.gov (United States)

    Teif, Vladimir B

    2007-01-01

    The transfer matrix methodology is proposed as a systematic tool for the statistical-mechanical description of DNA-protein-drug binding involved in gene regulation. We show that a genetic system of several cis-regulatory modules is calculable using this method, considering explicitly the site-overlapping, competitive, cooperative binding of regulatory proteins, their multilayer assembly and DNA looping. In the methodological section, the matrix models are solved for the basic types of short- and long-range interactions between DNA-bound proteins, drugs and nucleosomes. We apply the matrix method to gene regulation at the O(R) operator of phage lambda. The transfer matrix formalism allowed the description of the lambda-switch at a single-nucleotide resolution, taking into account the effects of a range of inter-protein distances. Our calculations confirm previously established roles of the contact CI-Cro-RNAP interactions. Concerning long-range interactions, we show that while the DNA loop between the O(R) and O(L) operators is important at the lysogenic CI concentrations, the interference between the adjacent promoters P(R) and P(RM) becomes more important at small CI concentrations. A large change in the expression pattern may arise in this regime due to anticooperative interactions between DNA-bound RNA polymerases. The applicability of the matrix method to more complex systems is discussed.

  18. Evaluating the efficacy of a structure-derived amino acid substitution matrix in detecting protein homologs by BLAST and PSI-BLAST

    Directory of Open Access Journals (Sweden)

    Nalin CW Goonesekere

    2009-06-01

    Full Text Available Nalin CW GoonesekereDepartment of Chemistry and Biochemistry, University of Northern iowa, Cedar Falls, IA, USAAbstract: The large numbers of protein sequences generated by whole genome sequencing projects require rapid and accurate methods of annotation. The detection of homology through computational sequence analysis is a powerful tool in determining the complex evolutionary and functional relationships that exist between proteins. Homology search algorithms employ amino acid substitution matrices to detect similarity between proteins sequences. The substitution matrices in common use today are constructed using sequences aligned without reference to protein structure. Here we present amino acid substitution matrices constructed from the alignment of a large number of protein domain structures from the structural classification of proteins (SCOP database. We show that when incorporated into the homology search algorithms BLAST and PSI-blaST, the structure-based substitution matrices enhance the efficacy of detecting remote homologs. Keywords: computational biology, protein homology, amino acid substitution matrix, protein structure

  19. Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards

    DEFF Research Database (Denmark)

    Mirgorodskaya, O A; Kozmin, Y P; Titov, M I

    2000-01-01

    A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards for...... inhibitor, were quantified by MALDI-time-of-flight (TOF) mass spectrometry.......A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards...

  20. In-vitro GIT Tolerance of Microencapsulated Bifidobacterium bifidum ATCC 35914 Using Polysaccharide-Protein Matrix.

    Science.gov (United States)

    Iqbal, Rabia; Zahoor, Tahir; Huma, Nuzhat; Jamil, Amer; Ünlü, Gülhan

    2018-03-12

    Longevity of probiotic is the main concern for getting maximum benefits when added in food product. Bifidobacterium, a probiotic, tends to lose its viability during gastrointestinal track (GIT) transit and storage of food. Their viability can be enhanced through microencapsulation technology. In this study, Bifidobacterium bifidum (B. bifidum) ATCC 35914 was encapsulated by using two experimental plans. In the first plan, chitosan (CH) at 0.6, 0.8, and 1.0% and sodium alginate (SA) at 4, 5, and 6% were used. Based on encapsulation efficiency, 6% sodium alginate and 0.8% chitosan were selected for single coating of the bacteria, and the resulting micro beads were double coated with different concentrations (5, 7.5, and 10%) of whey protein concentrate (WPC) in the second plan. Encapsulation efficiency and GIT tolerance were determined by incubating the micro beads in simulated gastrointestinal juices (SIJ) at variable pH and exposure times, and their release (liberation of bacterial cells) profile was also observed in SIJ. The microencapsulated bacterial cells showed significantly (P < 0.01) higher viability as compared to the unencapsulated (free) cells during GIT assay. The double-coated micro beads SA 6%-WPC 5% and CH 0.8%-WPC 5% were proven to have the higher survival at pH 3.0 after 90 min of incubation time and at pH 7.0 after 3-h exposure in comparison to free cells in simulated conditions of the stomach and intestine, respectively. Moreover, double coating with whey protein concentrate played a significant role in the targeted (10 6-9  CFU/mL) delivery under simulated intestinal conditions.

  1. Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model.

    Science.gov (United States)

    Dawson, R A; Goberdhan, N J; Freedlander, E; MacNeil, S

    1996-03-01

    The aim of this study was to investigate whether prior culture of cells on ECM proteins might positively influence the performance of keratinocytes when cells are transferred to a dermal in vitro wound bed model. Keratinocytes were cultured using a method for producing cultured epithelial autografts for severely burned patients (essentially using Green's medium, a mitogen-rich medium containing fetal calf serum, cholera toxin, EGF, insulin, transferrin and triiodothyronine). Cells were cultured either on irradiated 3T3 fibroblasts (as in the standard Rheinwald and Green technique) or, alternatively, on collagen I, collagen IV, matrigel, RGD, vitronectin or fibronectin. Under these conditions matrigel, collagen I and IV enhanced initial attachment, RGD, vitronectin, fibronectin and irradiated 3T3 fibroblasts did not. Proliferation of cells was positively influenced by matrigel, collagen I and IV and irradiated 3T3 fibroblasts; of these, however, only matrigel and 3T3 fibroblasts had sustained significant effects on keratinocyte proliferation over 4 days. Cells on fibronectin showed significantly reduced proliferation. An acellular non-viable dermis was then used to mimic the homograft allodermis onto which cultured epithelial autograft sheets are grafted clinically and cells cultured on the various ECM proteins for 96 h were transferred to this in vitro wound model. None of the substrates enhanced keratinocyte performance on this model. It was concluded that under these conditions some ECM proteins can significantly affect keratinocyte attachment and, to a lesser extent, proliferation but that the culture of keratinocytes on these ECM proteins does not appear to confer any lasting benefit to the attachment of these keratinocytes to an in vitro wound-bed model.

  2. Decreased expression of extracellular matrix proteins and trophic factors in the amygdala complex of depressed mice after chronic immobilization stress

    Directory of Open Access Journals (Sweden)

    Jung Soonwoong

    2012-06-01

    Full Text Available Abstract Background The amygdala plays an essential role in controlling emotional behaviors and has numerous connections to other brain regions. The functional role of the amygdala has been highlighted by various studies of stress-induced behavioral changes. Here we investigated gene expression changes in the amygdala in the chronic immobilization stress (CIS-induced depression model. Results Eight genes were decreased in the amygdala of CIS mice, including genes for neurotrophic factors and extracellular matrix proteins. Among these, osteoglycin, fibromodulin, insulin-like growth factor 2 (Igf2, and insulin-like growth factor binding protein 2 (Igfbp2 were further analyzed for histological expression changes. The expression of osteoglycin and fibromodulin simultaneously decreased in the medial, basolateral, and central amygdala regions. However, Igf2 and Igfbp2 decreased specifically in the central nucleus of the amygdala. Interestingly, this decrease was found only in the amygdala of mice showing higher immobility, but not in mice displaying lower immobility, although the CIS regimen was the same for both groups. Conclusions These results suggest that the responsiveness of the amygdala may play a role in the sensitivity of CIS-induced behavioral changes in mice.

  3. GOLGA2/GM130, cis-Golgi matrix protein, is a novel target of anticancer gene therapy.

    Science.gov (United States)

    Chang, Seung-Hee; Hong, Seong-Ho; Jiang, Hu-Lin; Minai-Tehrani, Arash; Yu, Kyeong-Nam; Lee, Jae-Ho; Kim, Ji-Eun; Shin, Ji-Young; Kang, Bitna; Park, Sungjin; Han, Kiwon; Chae, Chanhee; Cho, Myung-Haing

    2012-11-01

    Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an important role in glycosylation and transport of protein in the secretory pathway. In this study, the effects of short hairpin RNA (shRNA) constructs targeting GOLGA2/GM130 (shGOLGA2) on autophagy and lung cancer growth were evaluated in vitro and in vivo. Downregulation of GOLGA2/GM130 led to induction of autophagy and inhibition of glycosylation in A549 cells and in the lungs of K-ras(LA1) mice. Furthermore, downregulation of GOLGA2/GM130 decreased angiogenesis and cancer cell invasion in vitro and suppressed tumorigenesis in lung cancer mice model. The tumor specificity of sequence targeting GOLGA2/GM130 was also demonstrated. Taken together, these results suggest that induction of autophagy by shGOLGA2 may induce cell death rather than cell survival. Therefore, downregulation of GOLGA2/GM130 may be a potential therapeutic option for lung cancer.

  4. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    International Nuclear Information System (INIS)

    Woloschak, G.E.

    1994-01-01

    Experiments were designed to examine the effects Of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide revealed that cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure. (2) Cycloheximide did not affect accumulation of MRNA for actin genes; and that cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin MRNA accumulation following exposure to ionizing radiation. in addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  5. Expression of cytoskeletal and matrix genes following exposure to ionizing radiation: Dose-rate effects and protein synthesis requirements

    International Nuclear Information System (INIS)

    Woloschak, G.E.; Felcher, P.; Chang-Liu, Chin-Mei

    1992-01-01

    Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements (γ- and β-actin and α-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either α-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide, however, revealed several interesting and novel findings: (1) Cycloheximide repressed accumulation of α-tubulin following exposure to high dose-rate neutrons or γ rays; this did not occur following similar low dose-rate exposure (2) Cycloheximide did not affect accumulation of mRNA for actin genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to γ rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of α-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation. In addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons

  6. Förster-type energy transfer as a probe for changes in local fluctuations of the protein matrix.

    Science.gov (United States)

    Somogyi, B; Matkó, J; Papp, S; Hevessy, J; Welch, G R; Damjanovich, S

    1984-07-17

    Much evidence, on both theoretical and experimental sides, indicates the importance of local fluctuations (in energy levels, conformational substates, etc.) of the macromolecular matrix in the biological activity of proteins. We describe here a novel application of the Förster-type energy-transfer process capable of monitoring changes both in local fluctuations and in conformational states of macromolecules. A new energy-transfer parameter, f, is defined as an average transfer efficiency, [E], normalized by the actual average quantum efficiency of the donor fluorescence, [phi D]. A simple oscillator model (for a one donor-one acceptor system) is presented to show the sensitivity of this parameter to changes in amplitudes of local fluctuations. The different modes of averaging (static, dynamic, and intermediate cases) occurring for a given value of the average transfer rate, [kt], and the experimental requirements as well as limitations of the method are also discussed. The experimental tests were performed on the ribonuclease T1-pyridoxamine 5'-phosphate conjugate (a one donor-one acceptor system) by studying the change of the f parameter with temperature, an environmental parameter expectedly perturbing local fluctuations of proteins. The parameter f increased with increasing temperature as expected on the basis of the oscillator model, suggesting that it really reflects changes of fluctuation amplitudes (significant changes in the orientation factor, k2, as well as in the spectral properties of the fluorophores can be excluded by anisotropy measurements and spectral investigations). Possibilities of the general applicability of the method are also discussed.

  7. Exocrine pancreas glutamate secretion help to sustain enterocyte nutritional needs under protein restriction.

    Science.gov (United States)

    Araya, S; Kuster, E; Gluch, D; Mariotta, L; Lutz, C; Reding, T V; Graf, R; Verrey, F; Camargo, S M R

    2018-04-01

    Glutamine (Gln) is the most concentrated amino acid in blood and considered conditionally essential. Its requirement is increased during physiological stress, such as malnutrition or illness, despite its production by muscle and other organs. In the malnourished state, Gln has been suggested to have a trophic effect on the exocrine pancreas and small intestine. However, the Gln transport capacity, the functional relationship of these two organs, and the potential role of the Gln-glutamate (Glu) cycle are unknown. We observed that pancreatic acinar cells express lower levels of Glu than Gln transporters. Consistent with this expression pattern, the rate of Glu influx into acinar cells was approximately sixfold lower than that of Gln. During protein restriction, acinar cell glutaminase expression was increased and Gln accumulation was maintained. Moreover, Glu secretion by acinar cells into pancreatic juice and thus into the lumen of the small intestine was maintained. In the intestinal lumen, Glu absorption was preserved and Glu dehydrogenase expression was augmented, potentially providing the substrates for increasing energy production via the TCA cycle. Our findings suggest that one mechanism by which Gln exerts a positive effect on exocrine pancreas and small intestine involves the Gln metabolism in acinar cells and the secretion of Glu into the small intestine lumen. The exocrine pancreas acinar cells not only avidly accumulate Gln but metabolize Gln to generate energy and to synthesize Glu for secretion in the pancreatic juice. Secreted Glu is suggested to play an important role during malnourishment in sustaining small intestinal homeostasis. NEW & NOTEWORTHY Glutamine (Gln) has been suggested to have a trophic effect on exocrine pancreas and small intestine in malnourished states, but the mechanism is unknown. In this study, we suggest that this trophic effect derives from an interorgan relationship between exocrine pancreas and small intestine for Gln

  8. The Roles of Water in the Protein Matrix: A Largely Untapped Resource for Drug Discovery.

    Science.gov (United States)

    Spyrakis, Francesca; Ahmed, Mostafa H; Bayden, Alexander S; Cozzini, Pietro; Mozzarelli, Andrea; Kellogg, Glen E

    2017-08-24

    The value of thoroughly understanding the thermodynamics specific to a drug discovery/design study is well known. Over the past decade, the crucial roles of water molecules in protein structure, function, and dynamics have also become increasingly appreciated. This Perspective explores water in the biological environment by adopting its point of view in such phenomena. The prevailing thermodynamic models of the past, where water was seen largely in terms of an entropic gain after its displacement by a ligand, are now known to be much too simplistic. We adopt a set of terminology that describes water molecules as being "hot" and "cold", which we have defined as being easy and difficult to displace, respectively. The basis of these designations, which involve both enthalpic and entropic water contributions, are explored in several classes of biomolecules and structural motifs. The hallmarks for characterizing water molecules are examined, and computational tools for evaluating water-centric thermodynamics are reviewed. This Perspective's summary features guidelines for exploiting water molecules in drug discovery.

  9. The Force Exerted by the Membrane Potential During Protein Import into the Mitochondrial Matrix

    Science.gov (United States)

    Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

    2002-01-01

    The electrostatic force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated and found to vary from 1.4 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 12 to 6.5 Angstroms, its measured range. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a nonaqueous pore gives a force of 3.1 pN per unit charge, which represents an upper limit. When applied to mitochondrial import experiments on the protein harness, these results imply that a force of 11 plus or minus 4 pN is sufficient to catalyze the unfolding of harness during import. Comparison of these results with unfolding forces measured using atomic force microscopy suggests that the two are not inconsistent.

  10. Epidermal growth factor-containing fibulin-like extracellular matrix protein 1 expression and regulation in uterine leiomyoma.

    Science.gov (United States)

    Marsh, Erica E; Chibber, Shani; Wu, Ju; Siegersma, Kendra; Kim, Julie; Bulun, Serdar

    2016-04-01

    To determine the presence, differential expression, and regulation of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) in uterine leiomyomas. Laboratory in vivo and in vitro study with the use of human leiomyoma and myometrial tissue and primary cells. Academic medical center. Leiomyoma and myometrial tissue samples and cultured cells. 5-Aza-2'-deoxycytidine (5-aza-dC) treatment. Fold-change difference between EFEMP1 and fibulin-3 expression in leiomyoma tissue and cells compared with matched myometrial samples, and fold-change difference in EFEMP1 expression with 5-Aza-dC treatment. In vivo, EFEMP1 expression was 3.19-fold higher in myometrial tissue than in leiomyoma tissue. EFEMP1 expression in vitro was 5.03-fold higher in myometrial cells than in leiomyoma cells. Western blot and immunohistochemistry staining of tissue and cells confirmed similar findings in protein expression. Treatment of leiomyoma cells with 5-Aza-dC resulted in increased expression of EFEMP1 in vitro. The EFEMP1 gene and its protein product, fibulin-3, are both significantly down-regulated in leiomyoma compared with myometrium when studied both in vivo and in vitro. The increase in EFEMP1 expression in leiomyoma cells with 5-Aza-dC treatment suggest that differential methylation is responsible, in part, for the differences seen in gene expression. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  11. AMP-activated protein kinase activation mediates CCL3-induced cell migration and matrix metalloproteinase-2 expression in human chondrosarcoma

    Science.gov (United States)

    2013-01-01

    Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. PMID:24047437

  12. Vitamin k intake and plasma desphospho-uncarboxylated matrix Gla-protein levels in kidney transplant recipients.

    Directory of Open Access Journals (Sweden)

    Paul Y Boxma

    Full Text Available Vitamin K is essential for activation of γ-carboxyglutamate (Gla-proteins including the vascular calcification inhibitor matrix Gla-protein (MGP. Insufficient vitamin K intake leads to production of uncarboxylated, mostly inactive proteins and contributes to an increased cardiovascular risk. In kidney transplant recipients, cardiovascular risk is high but vitamin K intake and status have not been defined. We investigated dietary vitamin K intake, vascular vitamin K status and its determinants in kidney transplant recipients. We estimated vitamin K intake in a cohort of kidney transplant recipients (n = 60 with stable renal function (creatinine clearance 61 [42-77] (median [interquartile range] ml/min, who were 75 [35-188] months after transplantation, using three-day food records and food frequency questionnaires. Vascular vitamin K status was assessed by measuring plasma desphospho-uncarboxylated MGP (dp-ucMGP. Total vitamin K intake was below the recommended level in 50% of patients. Lower vitamin K intake was associated with less consumption of green vegetables (33 vs 40 g/d, p = 0.06 and increased dp-ucMGP levels (621 vs 852 pmol/L, p500 pmol/L in 80% of patients. Multivariate regression identified creatinine clearance, coumarin use, body mass index, high sensitivity-CRP and sodium excretion as independent determinants of dp-ucMGP levels. In a considerable part of the kidney transplant population, vitamin K intake is too low for maximal carboxylation of vascular MGP. The high dp-ucMGP levels may result in an increased risk for arterial calcification. Whether increasing vitamin K intake may have health benefits for kidney transplant recipients should be addressed by future studies.

  13. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

    Science.gov (United States)

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C; Warfield, Kelly L; Aman, M Javad; Holtsberg, Frederick W

    2016-02-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Vitamin k intake and plasma desphospho-uncarboxylated matrix Gla-protein levels in kidney transplant recipients.

    Science.gov (United States)

    Boxma, Paul Y; van den Berg, Else; Geleijnse, Johanna M; Laverman, Gozewijn D; Schurgers, Leon J; Vermeer, Cees; Kema, Ido P; Muskiet, Frits A; Navis, Gerjan; Bakker, Stephan J L; de Borst, Martin H

    2012-01-01

    Vitamin K is essential for activation of γ-carboxyglutamate (Gla)-proteins including the vascular calcification inhibitor matrix Gla-protein (MGP). Insufficient vitamin K intake leads to production of uncarboxylated, mostly inactive proteins and contributes to an increased cardiovascular risk. In kidney transplant recipients, cardiovascular risk is high but vitamin K intake and status have not been defined. We investigated dietary vitamin K intake, vascular vitamin K status and its determinants in kidney transplant recipients. We estimated vitamin K intake in a cohort of kidney transplant recipients (n = 60) with stable renal function (creatinine clearance 61 [42-77] (median [interquartile range]) ml/min), who were 75 [35-188] months after transplantation, using three-day food records and food frequency questionnaires. Vascular vitamin K status was assessed by measuring plasma desphospho-uncarboxylated MGP (dp-ucMGP). Total vitamin K intake was below the recommended level in 50% of patients. Lower vitamin K intake was associated with less consumption of green vegetables (33 vs 40 g/d, p = 0.06) and increased dp-ucMGP levels (621 vs 852 pmol/L, p500 pmol/L) in 80% of patients. Multivariate regression identified creatinine clearance, coumarin use, body mass index, high sensitivity-CRP and sodium excretion as independent determinants of dp-ucMGP levels. In a considerable part of the kidney transplant population, vitamin K intake is too low for maximal carboxylation of vascular MGP. The high dp-ucMGP levels may result in an increased risk for arterial calcification. Whether increasing vitamin K intake may have health benefits for kidney transplant recipients should be addressed by future studies.

  15. The key role of proinflammatory cytokines, matrix proteins, RANKL/OPG and Wnt/β-catenin in bone healing of hip arthroplasty patients.

    Science.gov (United States)

    Cassuto, Jean; Folestad, Agnetha; Göthlin, Jan; Malchau, Henrik; Kärrholm, Johan

    2018-02-01

    1NP increased immediately after surgery and returned to basal level at 6W followed by a new peak at 10Y returning to basal at 13Y. IL-8 and IL-1β peaked at 5Y post-THA and returned to basal level at 10Y. RANKL/OPG and Wnt/β-catenin remained at preoperative levels until 5Y post-THA when a sustained increase in OPG level, paralleled by a sustained decrease in sclerostin, started and lasted until 18Y. Despite a strong increase by RANKL at 13Y, the OPG/RANKL-ratio remained high between 5Y and 18Y. Dkk-1 and sFRP-1 remained at basal level until 5Y followed by a peak at 7Y and a return to basal level at 15Y. Similarly, RANKL increased after 5Y, peaked at 13Y and returned to basal levels at 18Y, thus coinciding with Wnt-1. In contrast, Wnt3a, Dkk-3, Dkk-4, sFRP-3 and Wif-1 did not differ from preoperative levels or healthy subjects during the course of the follow-up. The primary peak of proinflammatory cytokines involved in the initiation of bone healing after trauma is in line with previous results. The primary phase of increased matrix proteins, P1NP and BALP paralleled by RANKL, OPG and Wnt/β-catenin remaining at preoperative level until 5Y, support a strong formation of mineralized matrix and to a lesser degree bone during this phase. The secondary proinflammatory peak at 5Y is likely a trigger of coupled bone remodeling and neosynthesis as it is followed by increased levels of the bone anabolic turnover marker, BALP, and mediators of the RANKL/OPG and Wnt/β-catenin pathways. A continuous increase by OPG level and the bone turnover marker, BALP, lasting from 5Y until 18Y and paralleled by a similar decrease in sclerostin level support their being key regulators of bone anabolism, whereas the transient and opposed activities of RANKL, Wnt-1, Dkk-1 and sFRP-1 serve as fine tuning tools during the coupled remodeling phase. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

    Science.gov (United States)

    Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

    2011-12-01

    The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

  17. 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) promotes invasion and activation of matrix metalloproteinases

    International Nuclear Information System (INIS)

    Xie, Zhihui; Yuan, Hongyan; Yin, Yuzhi; Zeng, Xiao; Bai, Renkui; Glazer, Robert I

    2006-01-01

    Metastasis is a major cause of morbidity and mortality in breast cancer with tumor cell invasion playing a crucial role in the metastatic process. PDK1 is a key molecule that couples PI3K to cell proliferation and survival signals in response to growth factor receptor activation, and is oncogenic when expressed in mouse mammary epithelial cells. We now present evidence showing that PDK1-expressing cells exhibit enhanced anchorage-dependent and -independent cell growth and are highly invasive when grown on Matrigel. These properties correlate with induction of MMP-2 activity, increased MT1-MMP expression and a unique gene expression profile. Invasion assays in Matrigel, MMP-2 zymogram analysis, gene microarray analysis and mammary isografts were used to characterize the invasive and proliferative function of cells expressing PDK1. Tissue microarray analysis of human breast cancers was used to measure PDK1 expression in invasive tumors by IHC. Enhanced invasion on Matrigel in PDK1-expressing cells was accompanied by increased MMP-2 activity resulting from stabilization against proteasomal degradation. Increased MMP-2 activity was accompanied by elevated levels of MT1-MMP, which is involved in generating active MMP-2. Gene microarray analysis identified increased expression of the ECM-associated genes decorin and type I procollagen, whose gene products are substrates of MT1-MMP. Mammary fat pad isografts of PDK1-expressing cells produced invasive adenocarcinomas. Tissue microarray analysis of human invasive breast cancer indicated that PDK1pSer241 was strongly expressed in 90% of samples. These results indicate that PDK1 serves as an important effector of mammary epithelial cell growth and invasion in the transformed phenotype. PDK1 mediates its effect in part by MT1-MMP induction, which in turn activates MMP-2 and modulates the ECM proteins decorin and collagen. The presence of increased PDK1 expression in the majority of invasive breast cancers suggests its

  18. Resonance assignments of the myristoylated Y28F/Y67F mutant of the Mason-Pfizer monkey virus matrix protein

    Czech Academy of Sciences Publication Activity Database

    Doležal, Michal; Hrabal, R.; Ruml, T.; Rumlová, Michaela

    2015-01-01

    Roč. 9, č. 2 (2015), s. 229-233 ISSN 1874-2718 Institutional support: RVO:61388963 Keywords : isotopic labeling * matrix protein * M-PMV * myristoylation * resonance assignment * reverse labeling Subject RIV: CE - Biochemistry Impact factor: 0.687, year: 2015

  19. Assignment of 1H, 13C, and 15N resonances of WT matrix protein and its R55F mutant from Mason-Pfizer monkey virus

    Czech Academy of Sciences Publication Activity Database

    Vlach, J.; Lipov, J.; Veverka, V.; Rumlová, Michaela; Ruml, T.; Hrabal, R.

    2005-01-01

    Roč. 31, - (2005), s. 381-382 ISSN 0925-2738 R&D Projects: GA ČR GA203/03/0490 Institutional research plan: CEZ:AV0Z4055905 Keywords : Mason-Pfizer monkey virus * NMR resonance assignment * matrix protein Subject RIV: CE - Biochemistry Impact factor: 2.180, year: 2005

  20. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules

    NARCIS (Netherlands)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including

  1. Cartilage oligomeric matrix protein (COMP) in rheumatoid arthritis and its correlation with sonographic knee cartilage thickness and disease activity.

    Science.gov (United States)

    Sakthiswary, Rajalingham; Rajalingam, Shamala; Hussein, Heselynn; Sridharan, Radhika; Asrul, Abdul Wahab

    2017-12-01

    The aim of the study is to investigate the correlation of serum cartilage oligomeric matrix protein (COMP) levels with articular cartilage damage based on sonographic knee cartilage thickness (KCT) and disease activity in rheumatoid arthritis (RA). A total of 61 RA patients and 27 healthy controls were recruited in this study. Serum samples were obtained from all subjects to determine the serum COMP levels. All subjects had bilateral ultrasound scan of their knees. The KCT was based on the mean of measurements at three sites: the medial condyle, lateral condyle and intercondylar notch. Besides, the RA patients were assessed for their disease activity based on 28-joint-based Disease Activity Score (DAS 28). Serum COMP concentrations were significantly elevated in the RA patients compared to the controls (p = 0.001). The serum COMP levels had an inverse relationship with bilateral KCT in RA subjects and the healthy controls. COMP correlated significantly with disease activity based on DAS 28 (r = 0.299, p = 0.010), disease duration (r = 0.439, p = correlation between serum COMP and DAS 28 scores was comparable to the traditional markers of inflammation: erythrocyte sedimentation rate (ESR) (r = 0.372, p = 0.003) and C-reactive protein (CRP) (r = 0.305, p = 0.017). The serum COMP is a promising biomarker in RA which reflects disease activity and damage to the articular cartilage.

  2. Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

    Science.gov (United States)

    Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

    2014-01-01

    Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

  3. Biophysical characterization of the olfactomedin domain of myocilin, an extracellular matrix protein implicated in inherited forms of glaucoma.

    Directory of Open Access Journals (Sweden)

    Susan D Orwig

    Full Text Available Myocilin is an eye protein found in the trabecular extracellular matrix (TEM, within the anatomic region that controls fluid flow. Variants of myocilin, localized to its olfactomedin (OLF domain, have been linked to inherited forms of glaucoma, a disease associated with elevated intraocular pressure. OLF domains have also been implicated in psychiatric diseases and cancers by their involvement in signaling, neuronal growth, and development. However, molecular characterization of OLFs has been hampered by challenges in recombinant expression, a hurdle we have recently overcome for the myocilin OLF domain (myoc-OLF. Here, we report the first detailed solution biophysical characterization of myoc-OLF to gain insight into its structure and function. Myoc-OLF is stable in the presence of glycosaminoglycans, as well as in a wide pH range in buffers with functional groups reminiscent of such glycosaminoglycans. Circular dichroism (CD reveals significant β-sheet and β-turn secondary structure. Unexpectedly, the CD signature is reminiscent of α-chymotrypsin as well as another ocular protein family, the βγ-crystallins. At neutral pH, intrinsic tryptophan fluorescence and CD melts indicate a highly cooperative transition with a melting temperature of ∼55 °C. Limited proteolysis combined with mass spectrometry reveals that the compact core structural domain of OLF consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. The data presented here inform new testable hypotheses for interactions with specific TEM components, and will assist in design of therapeutic agents for myocilin glaucoma.

  4. DNA-PK dependent targeting of DNA-ends to a protein complex assembled on matrix attachment region DNA sequences

    International Nuclear Information System (INIS)

    Mauldin, S.K.; Getts, R.C.; Perez, M.L.; DiRienzo, S.; Stamato, T.D.

    2003-01-01

    Full text: We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end-binding was observed. Calculation of relative binding activities indicates that DNA-end binding activities to MAR sequences was 7 to 21 fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV, scaffold attachment factor A, topoisomerase II, and poly(ADP-ribose) polymerase preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends. After electroporation of a 32P-labeled DNA probe into human cells and cell fractionation, 87% of the total intercellular radioactivity remained in nuclei after a 0.5M NaCl extraction suggesting the probe was strongly bound in the nucleus. The above observations raise the possibility that DNA-PK targets DNA-ends to a repair and/or DNA damage signaling complex which is assembled on MAR sites in the nucleus

  5. Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium.

    Science.gov (United States)

    Shishkov, Alexander; Bogacheva, Elena; Fedorova, Natalia; Ksenofontov, Alexander; Badun, Gennadii; Radyukhin, Victor; Lukashina, Elena; Serebryakova, Marina; Dolgov, Alexey; Chulichkov, Alexey; Dobrov, Evgeny; Baratova, Lyudmila

    2011-12-01

    The structure of the C-terminal domain of the influenza virus A matrix M1 protein, for which X-ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally-activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C-terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C-terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C-terminal domain is an almost flat layer with a three-α-helical structure. To explain the high level of tritium label incorporation into the C-terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI-TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C-terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell. © 2011 The Authors Journal compilation © 2011 FEBS.

  6. Increased serum cartilage oligomeric matrix protein levels and decreased patellar bone mineral density in patients with chondromalacia patellae.

    Science.gov (United States)

    Murphy, E; FitzGerald, O; Saxne, T; Bresnihan, B

    2002-11-01

    Chondromalacia patellae is a potentially disabling disorder characterised by features of patellar cartilage degradation. To evaluate markers of cartilage and bone turnover in patients with chondromalacia patellae. 18 patients with chondromalacia patellae were studied. Serum cartilage oligomeric matrix protein (s-COMP) and bone sialoprotein (s-BSP) levels were measured by enzyme linked immunosorbent assay (ELISA) and compared with those of age and sex matched healthy control subjects. Periarticular bone mineral density (BMD) of both knee joints was assessed by dual energy x ray absorptiometry (DXA). s-COMP levels were significantly raised in all patients with chondromalacia patellae compared with healthy control subjects (p=0.0001). s-BSP levels did not differ significantly between the groups (p=0.41). BMD of the patella was significantly reduced in patients with chondromalacia patellae compared with the control subjects (p=0.016). In patients with bilateral chondromalacia patellae, BMD of the patella was lower in the more symptomatic knee joint (p=0.005). Changes in periarticular BMD were localised to the patella and were not present in femoral regions. Neither s-COMP (p=0.18) nor s-BSP (p=0.40) levels correlated with patellar BMD. Increased s-COMP levels, reflecting cartilage degradation, and reduced BMD localised to the patella may represent clinically useful markers in the diagnosis and monitoring of patients with chondromalacia patellae. Measures of cartilage degradation did not correlate with loss of patellar bone density, suggesting dissociated pathophysiological mechanisms.

  7. Study on the correlation between extracellular matrix protein-1 and the growth, metastasis and angiogenesis of laryngeal carcinoma.

    Science.gov (United States)

    Meng, Xin-Yu; Liu, Juan; Lv, Feng; Liu, Ming-Qiu; Wan, Jing-Ming

    2015-01-01

    To investigate the correlation between extracellular matrix protein-1 (ECM1) and the growth, metastasis and angiogenesis of laryngeal carcinoma. Forty-five samples with laryngeal benign and malignant tumors confirmed by pathology in Laiwu City People's Hospital from March 2006 to March 2011 were collected, in which there were 29 cases with laryngeal carcinoma and 16 with benign tumors. The expression of ECM1 and factor VIII-related antigens in patients with laryngeal carcinoma and those with benign tumors was respectively detected using immunohistochemical method, and the correlation between ECM1 staining grade and microvessel density (MVD) was analyzed. In laryngeal carcinoma tissue, ECM1 was mainly expressed in cytoplasm, less in cytomembrane or intercellular substance. With abundant expression in the tissue of laryngeal benign tumors (benign mesenchymoma and hemangioma), ECM1 was primarily expressed in the connective tissue, which was different from the expression in laryngeal carcinoma tissue. The proportion of positive ECM1 staining (++) in patients with laryngeal carcinoma was dramatically higher than those with benign tumors (pcorrelation analysis revealed that ECM1 staining grade in laryngeal carcinoma tissue had a significantly-positive correlation with MVD (r=0.866, p=0.000). ECM1 expression in laryngeal carcinoma is closely associated with tumor cell growth, metastasis and angiogenesis, which can be considered as an effective predictor in the occurrence and postoperative recurrence of laryngeal carcinoma.

  8. Outcome of nonunion fractures in dogs treated with fixation, compression resistant matrix, and recombinant human bone morphogenetic protein-2.

    Science.gov (United States)

    Massie, Anna M; Kapatkin, Amy S; Fuller, Mark C; Verstraete, Frank J M; Arzi, Boaz

    2017-03-20

    To report the use of compression resistant matrix (CRM) infused with recombinant human bone morphogenetic protein (rhBMP-2) prospectively in the healing of nonunion long-bone fractures in dogs. A longitudinal cohort of dogs that were presented with nonunion fractures were classified and treated with CRM soaked with rhBMP-2 and fracture fixation. They were followed with serial radiographs and evaluated for healing times and complications according to the time frame and definitions previously established for orthopaedic clinical cases. Eleven nonunion fractures in nine dogs were included. Median healing time was 10 weeks (range: 7-20 weeks). Major perioperative complications due to bandage morbidity were encountered in two of 11 limbs and resolved. All other complications were minor. They occurred perioperatively in eight of 11 limbs. Minor follow-up complications included short-term in one of two limbs, mid-term in one of three, and long-term in four of five limbs. Nine limbs returned to full function and two limbs returned to acceptable function at the last follow-up. Nonunion fractures given a poor prognosis via standard-of-care treatment were successfully repaired using CRM with rhBMP-2 accompanying fixation. These dogs, previously at high risk of failure, returned to full or acceptable function.

  9. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  10. Extracellular matrix protein fibulin-1 plasma levels are associated with increased cardiovascular risk in chronic kidney disease

    DEFF Research Database (Denmark)

    Scholze, Alexandra

    INTRODUCTION AND AIMS: Fibulin-1 is one of the few extracellular matrix proteins present in blood in high concentrations. We aimed to define the relationship between plasma fibulin-1 levels and risk markers of cardiovascular disease in patients with chronic kidney disease. METHODS: Plasma fibulin-1...... hemodynamic and arterial stiffness indices. RESULTS: We observed a positive correlation of fibulin-1 levels with age (r=0.38; p=0.033), glycated hemoglobin (r=0.80; p=0.003), creatinine (r=0.35; p=0.045), and fibrinogen (r=0.39; p=0.027). Glomerular filtration rate and fibulin-1 were inversely correlated (r......=-0.57; p=0.022). There was a positive correlation between fibulin-1 and central pulse pressure (r=0.44; p=0.011) and central augmentation pressure (r=0.55; p=0.001). In a multivariable regression model, diabetes, creatinine, fibrinogen and central augmentation pressure were independent predictors...

  11. Structure of the extracellular domain of matrix protein 2 of influenza A virus in complex with a protective monoclonal antibody.

    Science.gov (United States)

    Cho, Ki Joon; Schepens, Bert; Seok, Jong Hyeon; Kim, Sella; Roose, Kenny; Lee, Ji-Hye; Gallardo, Rodrigo; Van Hamme, Evelien; Schymkowitz, Joost; Rousseau, Frederic; Fiers, Walter; Saelens, Xavier; Kim, Kyung Hyun

    2015-04-01

    The extracellular domain of influenza A virus matrix protein 2 (M2e) is conserved and is being evaluated as a quasiuniversal influenza A vaccine candidate. We describe the crystal structure at 1.6 Å resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surfaces of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the center by the highly conserved tryptophan residue in M2e. This is the first description of the three-dimensional structure of M2e. M2e of influenza A is under investigation as a universal influenza A vaccine, but its three-dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the center of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reported in vivo escape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Weaponizing human EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) for 21st century cancer therapeutics.

    Science.gov (United States)

    Zhou, Yi-Hong; Hu, Yuanjie; Yu, Liping; Ke, Chao; Vo, Christopher; Hsu, Hao; Li, Zhenzhi; Di Donato, Anne T; Chaturbedi, Abhishek; Hwang, Ji Won; Siegel, Eric R; Linskey, Mark E

    2016-01-01

    De-regulated EFEMP1 gene expression in solid tumors has been widely reported with conflicting roles. We dissected EFEMP1 to identify domains responsible for its cell context-dependent dual functions, with the goal being to construct an EFEMP1-derived tumor-suppressor protein (ETSP) that lacked tumor-promoting function. Exon/intron boundaries of EFEMP1 were used as boundaries of functional modules in constructing EFEMP1 variants, with removal of various module(s), and/or mutating an amino acid residue to convert a weak integrin binding-site into a strong one. A series of in vitro assays on cancerous features, and subcutaneous and intracranial xenograft-formation assays, were carried out for effects from overexpression of wild-type and variant forms of EFEMP1 in two glioma subpopulations characterized as tumor mass-forming cells (TMCs) or stem-like tumor initiating cells (STICs), where EFEMP1 showed cellcontext- dependent dual functions. One of the EFEMP1 variants was identified as the sought-after ETSP, which had a stronger tumor-suppression function in TMCs by targeting EGFR and angiogenesis, and a new tumor-suppression function in STICs by targeting NOTCH signaling and MMP2-mediated cell invasion. Therefore, ETSP may form the basis for further important research to develop a novel cancer therapy to treat many types of cancer by its tumor suppressor effect in the extracellular matrix compartment.

  13. Cartilage link protein 1 (Crtl1), an extracellular matrix component playing an important role in heart development.

    Science.gov (United States)

    Wirrig, Elaine E; Snarr, Brian S; Chintalapudi, Mastan R; O'neal, Jessica L; Phelps, Aimee L; Barth, Jeremy L; Fresco, Victor M; Kern, Christine B; Mjaatvedt, Corey H; Toole, Bryan P; Hoffman, Stanley; Trusk, Thomas C; Argraves, W Scott; Wessels, Andy

    2007-10-15

    To expand our insight into cardiac development, a comparative DNA microarray analysis was performed using tissues from the atrioventricular junction (AVJ) and ventricular chambers of mouse hearts at embryonic day (ED) 10.5-11.0. This comparison revealed differential expression of approximately 200 genes, including cartilage link protein 1 (Crtl1). Crtl1 stabilizes the interaction between hyaluronan (HA) and versican, two extracellular matrix components essential for cardiac development. Immunohistochemical studies showed that, initially, Crtl1, versican, and HA are co-expressed in the endocardial lining of the heart, and in the endocardially derived mesenchyme of the AVJ and outflow tract (OFT). At later stages, this co-expression becomes restricted to discrete populations of endocardially derived mesenchyme. Histological analysis of the Crtl1-deficient mouse revealed a spectrum of cardiac malformations, including AV septal and myocardial defects, while expression studies showed a significant reduction in versican levels. Subsequent analysis of the hdf mouse, which carries an insertional mutation in the versican gene (CSPG2), demonstrated that haploinsufficient versican mice display septal defects resembling those seen in Crtl1(-/-) embryos, suggesting that reduced versican expression may contribute to a subset of the cardiac abnormalities observed in the Crtl1(-/-) mouse. Combined, these findings establish an important role for Crtl1 in heart development.

  14. A novel subnucleocapsid nanoplatform for mucosal vaccination against influenza virus that targets the ectodomain of matrix protein 2.

    Science.gov (United States)

    Hervé, Pierre-Louis; Raliou, Mariam; Bourdieu, Christiane; Dubuquoy, Catherine; Petit-Camurdan, Agnès; Bertho, Nicolas; Eléouët, Jean-François; Chevalier, Christophe; Riffault, Sabine

    2014-01-01

    In this study, subnucleocapsid nanorings formed by the recombinant nucleoprotein (N) of the respiratory syncytial virus were evaluated as a platform to anchor heterologous antigens. The ectodomain of the influenza virus A matrix protein 2 (M2e) is highly conserved and elicits protective antibodies when it is linked to an immunogenic carrier, making it a promising target to develop universal influenza vaccines. In this context, one or three M2e copies were genetically linked to the C terminus of N to produce N-M2e and N-3M2e chimeric recombinant nanorings. Mice were immunized intranasally with N-M2e or N-3M2e or with M2e or 3M2e control peptides. N-3M2e-vaccinated mice showed the strongest mucosal and systemic antibody responses. These mice presented a reduced viral load and minor weight loss, and all survived upon challenge with influenza virus A/PR8/34 (H1N1) (PR8). We compared the intranasal route to the subcutaneous route of N-3M2e immunization. Only the intranasal route induced a strong local IgA response and led to the protection of mice upon challenge. Finally, we demonstrated that the induction of anti-M2e antibodies by N-3M2e is not impaired by preexisting anti-N immunity. Overall, these results show that the N nanoring is a potent carrier for mucosal delivery of vaccinal antigens.

  15. Matrix protein of vesicular stomatitis virus: a potent inhibitor of vascular endothelial growth factor and malignant ascites formation.

    Science.gov (United States)

    Zhou, Y; Wen, F; Zhang, P; Tang, R; Li, Q

    2013-03-01

    Malignant ascites is common in various types of cancers and is difficult to manage. Vascular endothelial growth factor (VEGF) has a pivotal role in malignant ascites. The matrix protein of vesicular stomatitis virus (VSVMP) has been shown to inhibit host gene expression and induce the apoptosis of cancer cells. The present study was designed to determine whether VSVMP suppresses the formation of ascites in ascites-producing peritoneal carcinomatosis. BALB/c female mice, 6-8 weeks old, bearing peritoneal tumors of H22 or MethA cells received an intraperitoneal administration of 50 μg VSVMP/250 μg liposome complexes, 50 μg empty plasmid/250 μg liposome complexes or 0.9% NaCl solution, respectively, every 2 days for 3 weeks. Administration of VSVMP resulted in a significant inhibition in ascites formation, improvement in health condition and prolonged survival of the treated mice. Decreased peritoneum osmolarity and reduced tumor vascularity coincided with dramatic reductions in the VEGF level in ascites fluid and plasma. Examination of floating tumor cells collected from the peritoneal wash revealed an apparently increased number of apoptotic cells and profound downregulation of VEGF mRNA in the VSVMP-treated mice. Our data indicate for the first time that in BALB/c mice bearing H22 or MethA cell peritoneal tumors, VSVMP may inhibit VEGF production and suppress angiogenesis, consequently abolishing ascites formation.

  16. Staurosporine and extracellular matrix proteins mediate the conversion of small cell lung carcinoma cells into a neuron-like phenotype.

    Directory of Open Access Journals (Sweden)

    Tamara Murmann

    Full Text Available Small cell lung carcinomas (SCLCs represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions.

  17. Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification.

    Science.gov (United States)

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Rodriguez-Navarro, Alejandro B; Nys, Yves; Gautron, Joël

    2015-09-01

    Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

  18. Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification

    Directory of Open Access Journals (Sweden)

    Pauline Marie

    2015-09-01

    Full Text Available Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1 widespread deposition of amorphous calcium carbonate (ACC, (2 ACC transformation into crystalline calcite aggregates, (3 formation of larger calcite crystal units and (4 rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

  19. Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

    DEFF Research Database (Denmark)

    Callesen, Anne K; Christensen, René Depont; Madsen, Jonna S

    2008-01-01

    for serum protein profiling we investigated a range of sample preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom......Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum......-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different sample preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard...

  20. The non-structural protein 5 and matrix protein are antigenic targets of T cell immunity to genotype 1 porcine reproductive and respiratory syndrome viruses

    Directory of Open Access Journals (Sweden)

    Helen eMokhtar

    2016-02-01

    Full Text Available The porcine reproductive and respiratory syndrome virus (PRRSV is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focussed on envelope glycoproteins to target virus-neutralising antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress towards market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralising antibodies, it has been proposed that T cell mediated immunity plays a key role. We therefore hypothesised that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely-related (subtype 1 or divergent (subtype 3 PRRSV-1 strain. Dominant T cell IFN-γ responses were directed against the non-structural protein 5 (NSP5, and to a lesser extent, the matrix (M protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by co-expression of TNF-α and mobilisation of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved amongst strains of both PRRSV genotypes. Thus M and NSP5 represent attractive vaccine candidate T cell antigens which should be evaluated further in the context of PRRSV vaccine development.

  1. Thermo- and pH-responsive polymer brushes-grafted gigaporous polystyrene microspheres as a high-speed protein chromatography matrix.

    Science.gov (United States)

    Qu, Jian-Bo; Xu, Yu-Liang; Liu, Jun-Yi; Zeng, Jing-Bin; Chen, Yan-Li; Zhou, Wei-Qing; Liu, Jian-Guo

    2016-04-08

    Dual thermo- and pH-responsive chromatography has been proposed using poly(N-isopropylacrylamide-co-butyl methacrylate-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAM-co-BMA-co-DMAPAAM)) brushes grafted gigaporous polystyrene microspheres (GPM) as matrix. Atom transfer radical polymerization (ATRP) initiator was first coupled onto GPM through Friedel-Crafts acylation with 2-bromoisobutyryl bromide. The dual-responsive polymer brushes were then grafted onto GPM via surface-initiated ATRP. The surface composition, gigaporous structure, protein adsorption and dual-responsive chromatographic properties of the matrix (GPM-P(NIPAM-co-BMA-co-DMAPAAM) were characterized in detail. Results showed that GPM were successfully grafted with thermoresponsive cationic polymer brushes and that the gigaporous structure was well maintained. A column packed with GPM-P(NIPAM-co-BMA-co-DMAPAAM presented low backpressure, good permeability and appreciable thermo-responsibility. By changing pH of the mobile phase and temperature of the column in turn, the column can separate three model proteins at the mobile phase velocity up to 2528cmh(-1). A separation mechanism of this matrix was also proposed. All results indicate that the dual thermo- and pH-responsive chromatography matrix has great potentials in 'green' high-speed protein chromatography. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Identification of oxidised proteins in the matrix of rice leaf mitochondria by immunoprecipitation and two-dimensional liquid chromatography-tandem mass spectrometry

    DEFF Research Database (Denmark)

    Kristensen, B.K.; Askerlund, P.; Bykova, N.V.

    2004-01-01

    .2 mM CuSO4 for 10 min at room temperature). The oxidised proteins in both samples were tagged with dinitrophenylhydrazine (DNP), which forms a covalent bond with carbonyl groups. The DNP-tagged proteins were immunoprecipitated using anti-DNP antibodies and digested with trypsin. The mixture...... of peptides was analysed by nano-HPLC coupled online to an ESI-Quad-TOF mass spectrometer. The peptides were separated by stepwise ion exchange chromatography followed by reverse phase chromatography (2D-LC), and analysed by MS/MS. Proteins were identified by un-interpreted fragment ion database searches...... blots showed that neither the isolation of mitochondria, nor their subfractionation introduced carbonyl groups. We therefore conclude that a number of proteins are oxidised in the matrix of rice leaf mitochondria in vivo and further identify a group of proteins that are particularly susceptible to mild...

  3. Influence of secondary structure on in-source decay of protein in matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Takayama, Mitsuo; Osaka, Issey; Sakakura, Motoshi

    2012-01-01

    The susceptibility of the N-Cα bond of the peptide backbone to specific cleavage by in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was studied from the standpoint of the secondary structure of three proteins. A naphthalene derivative, 5-amino-1-naphtol (5,1-ANL), was used as the matrix. The resulting c'-ions, which originate from the cleavage at N-Cα bonds in flexible secondary structures such as turn and bend, and are free from intra-molecular hydrogen-bonded α-helix structure, gave relatively intense peaks. Furthermore, ISD spectra of the proteins showed that the N-Cα bonds of specific amino acid residues, namely Gly-Xxx, Xxx-Asp, and Xxx-Asn, were more susceptible to MALDI-ISD than other amino acid residues. This is in agreement with the observation that Gly, Asp and Asn residues usually located in turns, rather than α-helix. The results obtained indicate that protein molecules embedded into the matrix crystal in the MALDI experiments maintain their secondary structures as determined by X-ray crystallography, and that MALDI-ISD has the capability for providing information concerning the secondary structure of protein.

  4. A Novel Subnucleocapsid Nanoplatform for Mucosal Vaccination against Influenza Virus That Targets the Ectodomain of Matrix Protein 2

    Science.gov (United States)

    Hervé, Pierre-Louis; Raliou, Mariam; Bourdieu, Christiane; Dubuquoy, Catherine; Petit-Camurdan, Agnès; Bertho, Nicolas; Eléouët, Jean-François

    2014-01-01

    In this study, subnucleocapsid nanorings formed by the recombinant nucleoprotein (N) of the respiratory syncytial virus were evaluated as a platform to anchor heterologous antigens. The ectodomain of the influenza virus A matrix protein 2 (M2e) is highly conserved and elicits protective antibodies when it is linked to an immunogenic carrier, making it a promising target to develop universal influenza vaccines. In this context, one or three M2e copies were genetically linked to the C terminus of N to produce N-M2e and N-3M2e chimeric recombinant nanorings. Mice were immunized intranasally with N-M2e or N-3M2e or with M2e or 3M2e control peptides. N-3M2e-vaccinated mice showed the strongest mucosal and systemic antibody responses. These mice presented a reduced viral load and minor weight loss, and all survived upon challenge with influenza virus A/PR8/34 (H1N1) (PR8). We compared the intranasal route to the subcutaneous route of N-3M2e immunization. Only the intranasal route induced a strong local IgA response and led to the protection of mice upon challenge. Finally, we demonstrated that the induction of anti-M2e antibodies by N-3M2e is not impaired by preexisting anti-N immunity. Overall, these results show that the N nanoring is a potent carrier for mucosal delivery of vaccinal antigens. PMID:24155388

  5. Effects of extracellular matrix proteins on macrophage differentiation, growth, and function: comparison of liquid and agar culture systems

    Science.gov (United States)

    Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Both spaceflight and skeletal unloading suppress the haematopoietic differentiation of macrophages (Sonnenfeld et al., Aviat. Space Environ. Med., 61:648-653, 1990; Armstrong et al., J. Appl. Physiol., 75:2734-2739, 1993). The mechanism behind this reduction in haematopoiesis has yet to be elucidated. However, changes in bone marrow extracellular matrix (ECM) may be involved. To further understand the role of ECM products in macrophage differentiation, we have performed experiments evaluating the effects of fibronectin, laminin, collagen type I, and collagen type IV on macrophage development and function. Bone marrow-derived macrophages cultured on four different ECM substrates in liquid culture medium showed less growth than those cultured on plastic. Significant morphological differences were seen on each of the substrates used. Phenotypically and functionally, as measured by class II major histocompatibility molecule (MHCII) expression, MAC-2 expression, and the secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), these macrophages were similar. In contrast, bone marrow-derived macrophages cultured in suspension, using agar, showed no difference in growth when exposed to ECM proteins. However, IL-6 and TNF-alpha secretion was affected by fibronectin, laminin, collagen type I, and collagen type IV in a concentration-dependent manner. We conclude that the ECM products fibronectin, laminin, collagen type I, and collagen type IV have profound effects on macrophage development and function. Additionally, we suggest that an ECM-supplemented agar culture system provides an environment more analogous to in vivo bone marrow than does a traditional liquid culture system.

  6. Enamel matrix protein derivative plus synthetic bone substitute for the treatment of mandibular Class II furcation defects: a case series.

    Science.gov (United States)

    Queiroz, Lucas Araujo; Santamaria, Mauro; Casati, Marcio; Silverio, Karina; Nociti-Junior, Francisco; Sallum, Enilson

    2015-03-01

    The aim of this study is to report on the treatment of mandibular Class II furcation defects with enamel matrix protein derivative (EMD) combined with a βTCP/HA (β-tricalcium phosphate/hydroxyapatite) alloplastic material. Thirteen patients were selected. All patients were nonsmokers, systemically healthy, and diagnosed with chronic periodontitis; had not taken medications known to interfere with periodontal tissue health and healing; presented one Class II mandibular furcation defect with horizontal probing equal to or greater than 4 mm at buccal site. The clinical parameters evaluated were probing depth (PD), relative gingival margin position (RGMP), relative vertical clinical attachment level (RVCAL), and relative horizontal clinical attachment level (RHCAL). A paired Student t test was used to detect differences between the baseline and 6-month measurements, with the level of significance of .05. After 6 months, the treatment produced a statistically significant reduction in PD and a significant gain in RVCAL and RHCAL, but no observable change in RGMP. RVCAL ranged from 13.77 (± 1.31) at baseline to 12.15 (± 1.29) after 6 months, with a mean change of -1.62 ± 1.00 mm (P < .05). RHCAL ranged from 5.54 (± 0.75) to 2.92 (± 0.92), with a mean change of -2.62 ± 0.63 mm (P < .05). After 6 months, 76.92% of the patients improved their diagnosis to Class I furcation defects while 23.08% remained as Class II. The present study has shown that positive clinical results may be expected from the combined treatment of Class II furcation defects with EMD and βTCP/HA, especially considering the gain of horizontal attachment level. Despite this result, controlled clinical studies are needed to confirm our outcomes.

  7. Spatial and temporal analysis of extracellular matrix proteins in the developing murine heart: a blueprint for regeneration.

    Science.gov (United States)

    Hanson, Kevin P; Jung, Jangwook P; Tran, Quyen A; Hsu, Shao-Pu P; Iida, Rioko; Ajeti, Visar; Campagnola, Paul J; Eliceiri, Kevin W; Squirrell, Jayne M; Lyons, Gary E; Ogle, Brenda M

    2013-05-01

    The extracellular matrix (ECM) of the embryonic heart guides assembly and maturation of cardiac cell types and, thus, may serve as a useful template, or blueprint, for fabrication of scaffolds for cardiac tissue engineering. Surprisingly, characterization of the ECM with cardiac development is scattered and fails to comprehensively reflect the spatiotemporal dynamics making it difficult to apply to tissue engineering efforts. The objective of this work was to define a blueprint of the spatiotemporal organization, localization, and relative amount of the four essential ECM proteins, collagen types I and IV (COLI, COLIV), elastin (ELN), and fibronectin (FN) in the left ventricle of the murine heart at embryonic stages E12.5, E14.5, and E16.5 and 2 days postnatal (P2). Second harmonic generation (SHG) imaging identified fibrillar collagens at E14.5, with an increasing density over time. Subsequently, immunohistochemistry (IHC) was used to compare the spatial distribution, organization, and relative amounts of each ECM protein. COLIV was found throughout the developing heart, progressing in amount and organization from E12.5 to P2. The amount of COLI was greatest at E12.5 particularly within the epicardium. For all stages, FN was present in the epicardium, with highest levels at E12.5 and present in the myocardium and the endocardium at relatively constant levels at all time points. ELN remained relatively constant in appearance and amount throughout the developmental stages except for a transient increase at E16.5. Expression of ECM mRNA was determined using quantitative polymerase chain reaction and allowed for comparison of amounts of ECM molecules at each time point. Generally, COLI and COLIII mRNA expression levels were comparatively high, while COLIV, laminin, and FN were expressed at intermediate levels throughout the time period studied. Interestingly, levels of ELN mRNA were relatively low at early time points (E12.5), but increased significantly by P2. Thus

  8. Pore size is a critical parameter for obtaining sustained protein release from electrochemically synthesized mesoporous silicon microparticles

    Directory of Open Access Journals (Sweden)

    Ester L. Pastor

    2015-10-01

    Full Text Available Mesoporous silicon has become a material of high interest for drug delivery due to its outstanding internal surface area and inherent biodegradability. We have previously reported the preparation of mesoporous silicon microparticles (MS-MPs synthesized by an advantageous electrochemical method, and showed that due to their inner structure they can adsorb proteins in amounts exceeding the mass of the carrier itself. Protein release from these MS-MPs showed low burst effect and fast delivery kinetics with complete release in a few hours. In this work, we explored if tailoring the size of the inner pores of the particles would retard the protein release process. To address this hypothesis, three new MS-MPs prototypes were prepared by electrochemical synthesis, and the resulting carriers were characterized for morphology, particle size, and pore structure. All MS-MP prototypes had 90 µm mean particle size, but depending on the current density applied for synthesis, pore size changed between 5 and 13 nm. The model protein α-chymotrypsinogen was loaded into MS-MPs by adsorption and solvent evaporation. In the subsequent release experiments, no burst release of the protein was detected for any prototype. However, prototypes with larger pores (>10 nm reached 100% release in 24–48 h, whereas prototypes with small mesopores (<6 nm still retained most of their cargo after 96 h. MS-MPs with ∼6 nm pores were loaded with the osteogenic factor BMP7, and sustained release of this protein for up to two weeks was achieved. In conclusion, our results confirm that tailoring pore size can modify protein release from MS-MPs, and that prototypes with potential therapeutic utility for regional delivery of osteogenic factors can be prepared by convenient techniques.

  9. Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix.

    Science.gov (United States)

    Neri, L M; Bortul, R; Zweyer, M; Tabellini, G; Borgatti, P; Marchisio, M; Bareggi, R; Capitani, S; Martelli, A M

    1999-06-01

    The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the

  10. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein

    Science.gov (United States)

    Kessler, Paul D.; Podsakoff, Gregory M.; Chen, Xiaojuan; McQuiston, Susan A.; Colosi, Peter C.; Matelis, Laura A.; Kurtzman, Gary J.; Byrne, Barry J.

    1996-01-01

    Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the β-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies. PMID:8943064

  11. A biotin-drug extraction and acid dissociation (BEAD) procedure to eliminate matrix and drug interference in a protein complex anti-drug antibody (ADA) isotype specific assay.

    Science.gov (United States)

    Niu, Hongmei; Klem, Thomas; Yang, Jinsong; Qiu, Yongchang; Pan, Luying

    2017-07-01

    Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone–related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor

    Science.gov (United States)

    Kong, Li; Zhao, Yun-Peng; Tian, Qing-Yun; Feng, Jian-Quan; Kobayashi, Tatsuya; Merregaert, Joseph; Liu, Chuan-Ju

    2016-01-01

    Chondrogenesis and endochondral ossification are precisely controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. Here, we report that extracellular matrix protein 1 (ECM1) is a previously unrecognized regulator of chondrogenesis. ECM1 is induced in the course of chondrogenesis and its expression in chondrocytes strictly depends on parathyroid hormone–related peptide (PTHrP) signaling pathway. Overexpression of ECM1 suppresses, whereas suppression of ECM1 enhances, chondrocyte differentiation and hypertrophy in vitro and ex vivo. In addition, target transgene of ECM1 in chondrocytes or osteoblasts in mice leads to striking defects in cartilage development and endochondral bone formation. Of importance, ECM1 seems to be critical for PTHrP action in chondrogenesis, as blockage of ECM1 nearly abolishes PTHrP regulation of chondrocyte hypertrophy, and overexpression of ECM1 rescues disorganized growth plates of PTHrP-null mice. Furthermore, ECM1 and progranulin chondrogenic growth factor constitute an interaction network and act in concert in the regulation of chondrogenesis.—Kong, L., Zhao, Y.-P., Tian, Q.-Y., Feng, J.-Q., Kobayashi, T., Merregaert, J., Liu, C.-J. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone–related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor. PMID:27075243

  13. Extracellular matrix proteins matrix metallopeptidase 9 (MMP9) and soluble intercellular adhesion molecule 1 (sICAM-1) and correlations with clinical staging in euthymic bipolar disorder.

    Science.gov (United States)

    Reininghaus, Eva Z; Lackner, Nina; Birner, Armin; Bengesser, Susanne; Fellendorf, Frederike T; Platzer, Martina; Rieger, Alexandra; Queissner, Robert; Kainzbauer, Nora; Reininghaus, Bernd; McIntyre, Roger S; Mangge, Harald; Zelzer, Sieglinde; Fuchs, Dietmar; Dejonge, Silvia; Müller, Norbert

    2016-03-01

    Matrix metallopeptidase 9 (MMP9) and soluble intercellular adhesion molecule 1 (sICAM-1) are both involved in the restructuring of connective tissues. Evidence also implicates MMP9 and sICAM in cardiovascular and neoplastic diseases, where blood levels may be a marker of disease severity or prognosis. In individuals with bipolar disorder (BD), higher risk for cardiovascular illness has been extensively reported. The aim of this investigation was to measure and compare peripheral levels of serum MMP9 and sICAM in adults with euthymic BD and healthy controls (HC). Furthermore, we focussed on correlations with illness severity and metabolic parameters. MMP9 levels among the BD sample (n = 112) were significantly higher than among the HC (n = 80) (MMP9: F = 9.885, p = 0.002, η(2)  = 0.058) after controlling for confounding factors. Patients with BD in a later, progressive stage of disease showed significantly higher MMP9 as well as sICAM-1 levels compared to patients with BD in an earlier stage of disease (MMP9: F = 5.8, p = 0.018, η(2)  = 0.054; sICAM-1: F = 5.6, p = 0.020, η(2)  = 0.052). Correlation analyses of cognitive measures revealed a negative association between performance on the d2 Test of Attention and MMP9 (r = -0.287, p = 0.018) in the BD sample. Despite the sample being euthymic (i.e., according to conventional criteria) at the time of analysis, we found significant correlations between MMP9 as well as sICAM-1 and subthreshold depressive/hypomanic symptoms. A collection of disparate findings herein point to a role of MMP9 and cICAM-1 in the patho-progressive process of BD: the increased levels of serum MMP9 and sICAM-1, the correlation between higher levels of these parameters, progressive stage, and cognitive dysfunction in BD, and the positive correlation with subthreshold symptoms. As sICAM-1 and MMP9 are reliable biomarkers of inflammatory and early atherosclerotic disease, these markers may provide indications of the

  14. Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

    Science.gov (United States)

    Wijesinghe, Kaveesha J; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E; Gerstman, Bernard S; Chapagain, Prem P; Li, Sheng; Stahelin, Robert V

    2017-04-14

    Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Hyaluronan microgel as a potential carrier for protein sustained delivery by tailoring the crosslink network

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Chunhong [Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Zhao, Jianhao, E-mail: jhzhao@jnu.edu.cn [Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China); Tu, Mei; Zeng, Rong; Rong, Jianhua [Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632 (China); Engineering Research Center of Artificial Organs and Materials, Ministry of Education, Guangzhou 510632 (China)

    2014-03-01

    Hyaluronan (HA) microgels with different crosslink network, i.e. HGPs-1, HGPs-1.5, HGPs-3, HGPs-6 and HGPs-15, were synthesized using divinyl sulfone (DVS) as the crosslinker in an inverse microemulsion system for controlling the sustained delivery of bovine serum albumin (BSA). With increasing the crosslinker content, the average particle size slightly increased from 1.9 ± 0.3 μm to 3.6 ± 0.5 μm by dynamic laser scattering analysis. However, the crosslinker content had no significant effect on the morphology of HA microgels by scanning and transmission electron microscopes. Fourier transform infrared spectroscopy and elemental analysis proved more sulfur participated in the crosslink reaction when raising the crosslinker amount. The water swelling test confirmed the increasing crosslink density with the crosslinker content by calculating the average molecular weight between two crosslink points to be 8.25 ± 2.51 × 10{sup 5}, 1.26 ± 0.43 × 10{sup 5}, 0.96 ± 0.09 × 10{sup 5}, 0.64 ± 0.03 × 10{sup 5}, and 0.11 ± 0.01 × 10{sup 5} respectively. The degradation of HA microgels by hyaluronidase slowed down by enhancing the crosslink density, only about 5% of HGPs-15 was degraded as opposed to over 90% for HGPs-1. BSA loading had no obvious influence on the surface morphology of HA microgels but seemed to induce their aggregation. The increase of crosslink density decreased the BSA loading capacity but facilitated its long-term sustained delivery. When the molar ratio of DVS to repeating unit of HA reached 3 or higher, similar delivery profiles were obtained. Among all these HA microgels, HGPs-3 was the optimal carrier for BSA sustained delivery in this system because it possessed both high BSA loading capacity and long-term delivery profile simultaneously. - Highlights: • HA microgels with different crosslink densities were prepared. • The crosslinker content had little effect on the morphology and size of HA microgels. • The crosslink density

  16. Study of the relationship between mononuclear inflammatory infiltrate and Ki-67 and basement membrane and extracellular matrix protein expression in radicular cysts.

    Science.gov (United States)

    Mourão, R V C; Júnior, E C Pinheiro; Barros Silva, P G; Turatti, E; Mota, M R L; Alves, A P N N

    2016-05-01

    To evaluate the relationship between mononuclear inflammatory infiltrate and the expression of a proliferative immunomarker (Ki-67) as well as to evaluate basement membrane and extracellular matrix proteins (laminin and collagen type IV) in radicular cysts and dentigerous cysts (DC). Immunohistochemical analyses were performed in heavily inflamed radicular cysts (HIRC), slightly inflamed radicular cysts (SIRC) and DC (n = 20) using Ki-67 (Dako(®) , 1 : 50), anticollagen type IV (DBS(®) , 1 : 40) and antilaminin (DBS(®) , 1 : 20). The data were analysed using anova/Tukey's test (Ki-67) and Kruskal-Wallis/Dunn's test (collagen type IV and laminin) (P collagen type IV in the basement membrane of the SIRC group was significantly more continuous (P = 0.0475) than in the HIRC group. DC had significantly less collagen type IV in extracellular matrix immunoexpression than HIRC and SIRC (P = 0.0246). Laminin was absent in the basement membrane in the SIRC and DC groups, and the extracellular matrix of the HIRC was weak and punctate. The presence of inflammatory factors in the radicular cyst wall modified the expression of proliferation factors in the epithelial lining and the expression of collagen type IV and laminin in the basement membrane, but did not modify extracellular matrix behaviour in radicular cysts. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  17. Comparative expression of the four enamel matrix protein genes, amelogenin, ameloblastin, enamelin and amelotin during amelogenesis in the lizard Anolis carolinensis.

    Science.gov (United States)

    Gasse, Barbara; Sire, Jean-Yves

    2015-01-01

    In a recent study, we have demonstrated that amelotin (AMTN) gene structure and its expression during amelogenesis have changed during tetrapod evolution. Indeed, this gene is expressed throughout enamel matrix deposition and maturation in non-mammalian tetrapods, while in mammals its expression is restricted to the transition and maturation stages of amelogenesis. Previous studies of amelogenin (AMEL) gene expression in a lizard and a salamander have shown similar expression pattern to that in mammals, but to our knowledge there are no data regarding ameloblastin (AMBN) and enamelin (ENAM) expression in non-mammalian tetrapods. The present study aims to look at, and compare, the structure and expression of four enamel matrix protein genes, AMEL, AMBN, ENAM and AMTN during amelogenesis in the lizard Anolis carolinensis. We provide the full-length cDNA sequence of A. carolinensis AMEL and AMBN, and show for the first time the expression of ENAM and AMBN in a non-mammalian species. During amelogenesis in A. carolinensis, AMEL, AMBN and ENAM expression in ameloblasts is similar to that described in mammals. It is noteworthy that AMEL and AMBN expression is also found in odontoblasts. Our findings indicate that AMTN is the only enamel matrix protein gene that is differentially expressed in ameloblasts between mammals and sauropsids. Changes in AMTN structure and expression could be the key to explain the structural differences between mammalian and reptilian enamel, i.e. prismatic versus non-prismatic.

  18. Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein Response.

    Science.gov (United States)

    Lahlali, Thomas; Plissonnier, Marie-Laure; Romero-López, Cristina; Michelet, Maud; Ducarouge, Benjamin; Berzal-Herranz, Alfredo; Zoulim, Fabien; Mehlen, Patrick; Parent, Romain

    2016-05-01

    Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown. Isolation of translational complexes, determination of RNA secondary structures by selective 2'-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro-grown hepatocytic cells, wild-type, and netrin-1 transgenic mice. HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA-like endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated region-driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C-mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice. UPR-resistant, internal ribosome entry site-driven netrin-1 translation leads to

  19. Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

    Directory of Open Access Journals (Sweden)

    Zarei Saeed

    2011-08-01

    Full Text Available Abstract Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also

  20. Protein secondary structure and stability determined by combining exoproteolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Villanueva, Josep; Villegas, Virtudes; Querol, Enrique; Avilés, Francesc X; Serrano, Luis

    2002-09-01

    In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability. Copyright 2002 John Wiley & Sons, Ltd.

  1. Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.

    Science.gov (United States)

    Duan, Zhiqiang; Xu, Haixu; Ji, Xinqin; Zhao, Jiafu; Xu, Houqiang; Hu, Yan; Deng, Shanshan; Hu, Shunlin; Liu, Xiufan

    2018-12-31

    The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336-433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

  2. Effect of hyperbaric oxygenation on carbohydrate metabolism protein synthesis in the myocardium during sustained hypodynamia

    Science.gov (United States)

    Makarov, G. A.

    1980-01-01

    Glycolysis and the intensity of protein synthesis were studied in 140 white male rats in subcellular fractions of the myocardium during 45 day hypodynamia and hyperbaric oxygenation. Hypodynamia increased: (1) the amount of lactic acids; (2) the amount of pyruvic acid; (3) the lactate/pyruvate coefficient; and (4) the activities of aldolase and lactate dehydrogenase. Hyperbaric oxygenation was found to have a favorable metabolic effect on the animals with hypodynamia.

  3. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Ines eLassowskat

    2014-10-01

    Full Text Available Mitogen-activated protein kinases (MAPKs target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phosphoproteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the PEN pathway required for penetration resistance to filamentous pathogens. Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org.

  4. The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor β1 in Hepatic Stellate Cells.

    Science.gov (United States)

    Arriola Benitez, Paula Constanza; Rey Serantes, Diego; Herrmann, Claudia Karina; Pesce Viglietti, Ayelén Ivana; Vanzulli, Silvia; Giambartolomei, Guillermo Hernán; Comerci, Diego José; Delpino, María Victoria

    2016-02-01

    The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

    Science.gov (United States)

    Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

    2010-09-15

    The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  6. Heat Shock Protein 90 Inhibitor Decreases Collagen Synthesis of Keloid Fibroblasts and Attenuates the Extracellular Matrix on the Keloid Spheroid Model.

    Science.gov (United States)

    Lee, Won Jai; Lee, Ju Hee; Ahn, Hyo Min; Song, Seung Yong; Kim, Yong Oock; Lew, Dae Hyun; Yun, Chae-Ok

    2015-09-01

    The 90-kDa heat-shock protein (heat-shock protein 90) is an abundant cytosolic chaperone, and inhibition of heat-shock protein 90 by 17-allylamino-17-demethoxygeldanamycin (17-AAG) compromises transforming growth factor (TGF)-β-mediated transcriptional responses by enhancing TGF-β receptor I and II degradation, thus preventing Smad2/3 activation. In this study, the authors evaluated whether heat-shock protein 90 regulates TGF-β signaling in the pathogenesis and treatment of keloids. Keloid fibroblasts were treated with 17-AAG (10 μM), and mRNA levels of collagen types I and III were determined by real-time reverse- transcriptase polymerase chain reaction. Also, secreted TGF-β1 was assessed by enzyme-linked immunosorbent assay. The effect of 17-AAG on protein levels of Smad2/3 complex was determined by Western blot analysis. In addition, in 17-AAG-treated keloid spheroids, the collagen deposition and expression of major extracellular matrix proteins were investigated by means of Masson trichrome staining and immunohistochemistry. The authors found that heat-shock protein 90 is overexpressed in human keloid tissue compared with adjacent normal tissue, and 17-AAG decreased mRNA levels of type I collagen, secreted TGF-ß1, and Smad2/3 complex protein expression in keloid fibroblasts. Masson trichrome staining revealed that collagen deposition was decreased in 17-AAG-treated keloid spheroids, and immunohistochemical analysis showed that expression of collagen types I and III, elastin, and fibronectin was markedly decreased in 17-AAG-treated keloid spheroids. These results suggest that the antifibrotic action of heat-shock protein 90 inhibitors such as 17-AAG may have therapeutic effects on keloids.

  7. Matriz de indicadores de sustentabilidade para a gestão de resíduos sólidos urbanos Matrix of sustainability indicatorsfor the urban solid waste management

    Directory of Open Access Journals (Sweden)

    Leila Santos Santiago

    2012-06-01

    Full Text Available O artigo apresenta uma matriz de indicadores de sustentabilidade para a gestão de resíduos sólidos urbanos e a forma de sua construção. A construção foi realizada baseando-se em informações adquiridas em referências nacionais e internacionais. Os indicadores foram organizados em uma matriz preliminar, a qual passou por um processo de validação externa, com especialistas, por meio do método Delphi. Para a realização deste método foram necessárias duas rodadas. A matriz final possui 6 dimensões de sustentabilidade: política, tecnológica, econômica/financeira, ambiental/ecológica, conhecimento e inclusão social, contemplando 42 indicadores e 126 descritores. A matriz elaborada poderá ser utilizada como instrumento de avaliação e planejamento da gestão dos resíduos sólidos em municípios.This paper presents a matrix of Sustainability Indicators for the Urban Solid Waste Management and the way of its construction. The construction was performed based on information acquired in national and international references. The indicators were organized into a primary matrix, which has gone an external validation process, with experts through the Delphi method. To perform this method two rounds were necessary. The final matrix has 6 dimensions of sustainability: politics, technology, economic/financial, environmental/ecological, knowledge and social inclusion, comprising 42 indicators, 126 descriptors. The matrix produced can be used as a tool for evaluation and planning of the solid waste management in municipalities.

  8. Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

    DEFF Research Database (Denmark)

    Sasaki, T; Brakebusch, C; Engel, J

    1998-01-01

    Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resis...... in the extracellular matrix of several mouse tissues....... in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1...

  9. Preparation and in-vitro in-vivo evaluation of sustained release matrix diclofenac sodium tablets using PVP-K90 and natural gums.

    Science.gov (United States)

    Iqbal, Zafar; Khan, Raza; Nasir, Fazli; Khan, Jamshaid Ali; Rashid, Abdur; Khan, Abbas; Khan, Abad

    2011-10-01

    Conventional dosage form is nowadays mostly replaced by sustained release formulation in order to increase drug efficacy and patient compliance. The sustained release properties of the PVP K90 alone and in combination with guar gum, xanthan gum and gum tragacanth were evaluated using diclofenac sodium (100 mg/tablet) as a model drug. Tablets were processed using wet granulation method and evaluated for sustained drug release properties. The drug release from the formulations was studied in relationship with Commercially available Diclofenac Sodium SR, used as a reference tablets and results were expressed as similarity (f1) and differential factor (f2). The tablets prepared using PVP K90 160 mg/tablet sustained the release of diclofenac sodium for 12 hours. Formulations where the PVP K90 was partially replaced with different gums also sustained the release of drug for 12 hours. The release of the drug from these formulations mainly followed Higuchi model and super case-II and Non-Fickian diffusion. The in-vivo drug release was studied in healthy human volunteers using non-blinded cross over, two period design using Diclofenac Sodium SR Tablets as a reference drug. The relative bioavailability of the formulation containing PVP K90 and gum tragacanth was 0.91. The studies showed that the use of the PVP K90 in combination with gum tragacanth both in-vitro and in-vivo sustained the release of the drug.

  10. Knowledge and tools to enhance resilience of beef grazing systems for sustainable animal protein production.

    Science.gov (United States)

    Steiner, Jean L; Engle, David M; Xiao, Xiangming; Saleh, Ali; Tomlinson, Peter; Rice, Charles W; Cole, N Andy; Coleman, Samuel W; Osei, Edward; Basara, Jeffrey; Middendorf, Gerad; Gowda, Prasanna; Todd, Richard; Moffet, Corey; Anandhi, Aavudai; Starks, Patrick J; Ocshner, Tyson; Reuter, Ryan; Devlin, Daniel

    2014-11-01

    Ruminant livestock provides meat and dairy products that sustain health and livelihood for much of the world's population. Grazing lands that support ruminant livestock provide numerous ecosystem services, including provision of food, water, and genetic resources; climate and water regulation; support of soil formation; nutrient cycling; and cultural services. In the U.S. southern Great Plains, beef production on pastures, rangelands, and hay is a major economic activity. The region's climate is characterized by extremes of heat and cold and extremes of drought and flooding. Grazing lands occupy a large portion of the region's land, significantly affecting carbon, nitrogen, and water budgets. To understand vulnerabilities and enhance resilience of beef production, a multi-institutional Coordinated Agricultural Project (CAP), the "grazing CAP," was established. Integrative research and extension spanning biophysical, socioeconomic, and agricultural disciplines address management effects on productivity and environmental footprints of production systems. Knowledge and tools being developed will allow farmers and ranchers to evaluate risks and increase resilience to dynamic conditions. The knowledge and tools developed will also have relevance to grazing lands in semiarid and subhumid regions of the world. © 2014 New York Academy of Sciences.

  11. Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Nilsson, C L; Puchades, M; Westman, A; Blennow, K; Davidsson, P

    1999-01-01

    Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, beta2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition, >25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.

  12. Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

    DEFF Research Database (Denmark)

    Callesen, Anne K; Mohammed, Shabaz; Bunkenborg, Jakob

    2005-01-01

    for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI...

  13. Meat juice: An alternative matrix for assessing animal health by measuring acute phase proteins. Correlations of pig-MAP and haptoglobin concentrations in pig meat juice and plasma.

    Science.gov (United States)

    Piñeiro, M; Gymnich, S; Knura, S; Piñeiro, C; Petersen, B

    2009-10-01

    Quantification of acute phase proteins (APPs) in blood can be used for monitoring animal health and welfare on farms, and could be also of interest for the detection of diseased animals during the meat inspection process. However serum or plasma is not always available for end-point analysis at slaughter. Meat juice might provide an adequate, alternative matrix that can be easily obtained for post-mortem analysis at abattoirs. The concentrations of pig Major Acute phase Protein (pig-MAP) and haptoglobin, two of the main APPs in pigs, were determined in approximately 300 paired samples of plasma and meat juice from the diaphragm (pars costalis), obtained after freezing and thawing the muscle. APPs concentrations in meat juice were closely correlated to those in plasma (r=0.695 for haptoglobin, r=0.858 for pig-MAP, panimal health in pig production, with implications for food safety and meat quality.

  14. Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer.

    Directory of Open Access Journals (Sweden)

    Victoria Catalán

    Full Text Available Excess adipose tissue represents a major risk factor for the development of colon cancer with inflammation and extracellular matrix (ECM remodeling being proposed as plausible mechanisms. The aim of this study was to investigate whether obesity can influence circulating levels of inflammation-related extracellular matrix proteins in patients with colon cancer (CC, promoting a microenvironment favorable for tumor growth.Serum samples obtained from 79 subjects [26 lean (LN and 53 obese (OB] were used in the study. Enrolled subjects were further subclassified according to the established diagnostic protocol for CC (44 without CC and 35 with CC. Anthropometric measurements as well as circulating metabolites and hormones were determined. Circulating concentrations of the ECM proteins osteopontin (OPN, chitinase-3-like protein 1 (YKL-40, tenascin C (TNC and lipocalin-2 (LCN-2 were determined by ELISA.Significant differences in circulating OPN, YKL-40 and TNC concentrations between the experimental groups were observed, being significantly increased due to obesity (P<0.01 and colon cancer (P<0.05. LCN-2 levels were affected by obesity (P<0.05, but no differences were detected regarding the presence or not of CC. A positive association (P<0.05 with different inflammatory markers was also detected.To our knowledge, we herein show for the first time that obese patients with CC exhibit increased circulating levels of OPN, YKL-40 and TNC providing further evidence for the influence of obesity on CC development via ECM proteins, representing promising diagnostic biomarkers or target molecules for therapeutics.

  15. Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN) on monocytes/macrophages.

    Science.gov (United States)

    Ge, Heng; Yuan, Wei; Liu, Jidong; He, Qing; Ding, Song; Pu, Jun; He, Ben

    2015-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages. The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells) in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway. 1) It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN) that increased after being exposed to inflammatory signals (PMA and H2O2). 2) Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG) the simple type. 3) Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression). Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

  16. Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN on monocytes/macrophages.

    Directory of Open Access Journals (Sweden)

    Heng Ge

    Full Text Available Extracellular matrix metalloproteinase inducer (EMMPRIN is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages.The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway.1 It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN that increased after being exposed to inflammatory signals (PMA and H2O2. 2 Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG the simple type. 3 Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression.Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

  17. Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

    Science.gov (United States)

    Tsfasman, Tatyana; Kost, Vladimir; Markushin, Stanislav; Lotte, Vera; Koptiaeva, Irina; Bogacheva, Elena; Baratova, Ludmila; Radyukhin, Victor

    2015-12-02

    The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating.

    Science.gov (United States)

    Sorkio, Anni; Hongisto, Heidi; Kaarniranta, Kai; Uusitalo, Hannu; Juuti-Uusitalo, Kati; Skottman, Heli

    2014-02-01

    Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

  19. Improvement of beef cattle genetics provided increasing sustainability of beef cattle production and protein consumption in Thailand

    Energy Technology Data Exchange (ETDEWEB)

    Boonyanuwat, K. [Beef Cattle Research and Development Group, Division of Animal Husbandry, Department of Livestock Development, Bangkok (Thailand)], E-mail: kalayabo@yahoo.com; Sirisom, P [Tak Livestock Breeding and Research Center, Meung (Thailand); Putharatanung, A [Nongkwang Livestock Research and Breeding Center, Photharam (Thailand)

    2009-07-01

    .00%. They were the smallest size. Farmers raised them by main of grass-fed and some added with concentrate. Meat quality from this group was the lowest. The meat texture of this group is the most firm. It was suitable for Thai food cooking. Meat from this group was used to make meatball and Thai food. The second group was around 38.50%. They were fattening 3-6 month. Meat from this group was medium quality. Farmers fed them with grass, agricultural by product, and concentrate. Meat from this group was used for general Thai food and steak cooking. The third and forth group were around 20.00%. In this group, it was added with other beef cattle: Kampangsaen and Ponyangkhum beef cattle. They were fed higher concentrate, up to 90% of concentrate in the last month of fattening. They were fattening 6-12 month. Farmers fed them following feeding plan of cooperative, so they had the greatest ADG and % carcass. Quality of meat from this group was the best, most tender. Meat from this group was used for steak and shabushabu. Meat from this group had quality the same as import meat. Some of them were better than import meat. It was tenderer. The fifth group was less than 0.5%. They were on experiment to establish new breed by DLD. This group was optimized for Thai farmers, small size and low mature age. Meat from this group had the most tenderness and marbling. Meat from this group had more quality than import meat. It was used for steak and shabushabu. Farmers could earn money from beef cattle raising occupation. Beef cattle genetic improvement provided sustainable productivity, profitability, quality of products, and sustained economic growth in rural Thailand. Beef meat production would be the mainly red meat production and provided protein sources for consumer in Thailand. (author)

  20. Distribution of Cathepsin K in Late Stage of Tooth Germ Development and Its Function in Degrading Enamel Matrix Proteins in Mouse.

    Directory of Open Access Journals (Sweden)

    Tao Jiang

    Full Text Available Cathepsin K (CTSK is a member of cysteine proteinase family, and is predominantly expressed in osteoclastsfor degradationof bone matrix proteins. Given the similarity in physical properties of bone and dental mineralized tissues, including enamel, dentin and cementum, CTSK is likely to take part in mineralization process during odontogenesis. On the other hand, patients with pycnodysostosis caused by mutations of the CTSK gene displayedmultipledental abnormalities, such as hypoplasia of the enamel, obliterated pulp chambers, hypercementosis and periodontal disease. Thereforeitis necessary to study the metabolic role of CTSK in tooth matrix proteins. In this study, BALB/c mice at embryonic day 18 (E18, post-natal day 1 (P1, P5, P10 and P20 were used (5 mice at each time pointfor systematic analyses of CTSK expression in the late stage of tooth germ development. We found that CTSK was abundantly expressed in the ameloblasts during secretory and maturation stages (P5 and P10 by immunohistochemistry stainings.During dentinogenesis, the staining was also intense in the mineralization stage (P5 and P10,but not detectable in the early stage of dentin formation (P1 and after tooth eruption (P20.Furthermore, through zymography and digestion test in vitro, CTSK was proved to be capable of hydrolyzing Emdogain and also cleaving Amelogenininto multiple products. Our resultsshed lights on revealing new functions of CTSK and pathogenesis of pycnodysostosis in oral tissues.

  1. Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway

    International Nuclear Information System (INIS)

    Matsumoto, Ken-ichi; Minamitani, Takeharu; Orba, Yasuko; Sato, Mami; Sawa, Hirofumi; Ariga, Hiroyoshi

    2004-01-01

    The results of our previous study showed that tumor invasion and metastasis are promoted in extracellular matrix (ECM) tenascin-X-deficient (TNX-/-) mice via increased expression of matrix metalloproteinases (MMPs). However, little is known about the relationship between TNX deficiency and activation of MMP genes. In this study, we investigated the molecular mechanism by which TNX deficiency activates the MMP-2 gene. We examined the intracellular signaling pathways that regulate gene expression of the proteinase in isolated fibroblasts. Results of gelatin zymography showed that MMP-2 was induced to a greater extent in TNX-/- fibroblasts embedded in type I collagen than in wild-type fibroblasts. RT-PCR analysis revealed that the increased level of MMP-2 expression was caused at the transcription level. Conversely, stable overexpression of TNX in a fibroblast cell line reduced MMP-2 expression and suppressed MMP-2 promoter activity. In addition, treatment of TNX-/- fibroblasts with SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and genistein, a tyrosine kinase inhibitor, suppressed the increased level of proMMP-2 and increased MMP-2 promoter activity in TNX-/- fibroblasts. Furthermore, increased activation of JNK and tyrosine phosphorylation of certain proteins were observed in TNX-/- fibroblasts. These findings suggest that induction of MMP-2 by TNX deficiency is mediated, at least in part, through the JNK and protein tyrosine kinase phosphorylation pathway

  2. Assembly of Ebola virus matrix protein VP40 is regulated by latch-like properties of N and C terminal tails.

    Directory of Open Access Journals (Sweden)

    Leslie P Silva

    Full Text Available The matrix protein VP40 coordinates numerous functions in the viral life cycle of the Ebola virus. These range from the regulation of viral transcription to morphogenesis, packaging and budding of mature virions. Similar to the matrix proteins of other nonsegmented, negative-strand RNA viruses, VP40 proceeds through intermediate states of assembly (e.g. octamers but it remains unclear how these intermediates are coordinated with the various stages of the life cycle. In this study, we investigate the molecular basis of synchronization as governed by VP40. Hydrogen/deuterium exchange mass spectrometry was used to follow induced structural and conformational changes in VP40. Together with computational modeling, we demonstrate that both extreme N and C terminal tail regions stabilize the monomeric state through a direct association. The tails appear to function as a latch, released upon a specific molecular trigger such as RNA ligation. We propose that triggered release of the tails permits the coordination of late-stage events in the viral life cycle, at the inner membrane of the host cell. Specifically, N-tail release exposes the L-domain motifs PTAP/PPEY to the transport and budding complexes, whereas triggered C-tail release could improve association with the site of budding.

  3. Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: Members of the nuclear DAPC associate with the nuclear matrix

    International Nuclear Information System (INIS)

    Fuentes-Mera, Lizeth; Rodriguez-Munoz, Rafael; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Gonzalez, Everardo; Mornet, Dominique; Cisneros, Bulmaro

    2006-01-01

    Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, β-sarcoglycan, β-dystroglycan, α- and β-syntrophin, α1- and β-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, β-dystroglycan, nNOS, β-sarcoglycan, α/β syntrophin, α1-dystrobrevin and β-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, β-dystroglycan and β-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture

  4. Valsartan attenuates pulmonary hypertension via suppression of mitogen activated protein kinase signaling and matrix metalloproteinase expression in rodents.

    Science.gov (United States)

    Lu, Yuyan; Guo, Haipeng; Sun, Yuxi; Pan, Xin; Dong, Jia; Gao, Di; Chen, Wei; Xu, Yawei; Xu, Dachun

    2017-08-01

    It has previously been demonstrated that the renin-angiotensin system is involved in the pathogenesis and development of pulmonary hypertension (PH). However, the efficacy of angiotensin II type I (AT1) receptor blockers in the treatment of PH is variable. The present study examined the effects of the AT1 receptor blocker valsartan on monocrotaline (MCT)‑induced PH in rats and chronic hypoxia‑induced PH in mice. The results demonstrated that valsartan markedly attenuated development of PH in rats and mice, as indicated by reduced right ventricular systolic pressure, diminished lung vascular remodeling and decreased right ventricular hypertrophy, compared with vehicle treated animals. Immunohistochemical analyses of proliferating cell nuclear antigen expression revealed that valsartan suppressed smooth muscle cell proliferation. Western blot analysis demonstrated that valsartan limited activation of p38, c‑Jun N‑terminal kinase 1/2 and extracellular signal‑regulated kinase 1/2 signaling pathways and significantly reduced MCT‑induced upregulation of pulmonary matrix metalloproteinases‑2 and ‑9, and transforming growth factor‑β1 expression. The results suggested that valsartan attenuates development of PH in rodents by reducing expression of extracellular matrix remodeling factors and limiting smooth muscle cell proliferation to decrease pathological vascular remodeling. Therefore, valsartan may be a valuable future therapeutic approach for the treatment of PH.

  5. Cold and Heat Stress Diversely Alter Both Cauliflower Respiration and Distinct Mitochondrial Proteins Including OXPHOS Components and Matrix Enzymes

    Science.gov (United States)

    Rurek, Michał; Czołpińska, Magdalena; Pawłowski, Tomasz Andrzej; Krzesiński, Włodzimierz; Spiżewski, Tomasz

    2018-01-01

    Complex proteomic and physiological approaches for studying cold and heat stress responses in plant mitochondria are still limited. Variations in the mitochondrial proteome of cauliflower (Brassica oleracea var. botrytis) curds after cold and heat and after stress recovery were assayed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) in relation to mRNA abundance and respiratory parameters. Quantitative analysis of the mitochondrial proteome revealed numerous stress-affected protein spots. In cold, major downregulations in the level of photorespiratory enzymes, porine isoforms, oxidative phosphorylation (OXPHOS) and some low-abundant proteins were observed. In contrast, carbohydrate metabolism enzymes, heat-shock proteins, translation, protein import, and OXPHOS components were involved in heat response and recovery. Several transcriptomic and metabolic regulation mechanisms are also suggested. Cauliflower plants appeared less susceptible to heat; closed stomata in heat stress resulted in moderate photosynthetic, but only minor respiratory impairments, however, photosystem II performance was unaffected. Decreased photorespiration corresponded with proteomic alterations in cold. Our results show that cold and heat stress not only operate in diverse modes (exemplified by cold-specific accumulation of some heat shock proteins), but exert some associations at molecular and physiological levels. This implies a more complex model of action of investigated stresses on plant mitochondria. PMID:29547512

  6. A pH- and temperature-responsive bioresorbable injectable hydrogel based on polypeptide block copolymers for the sustained delivery of proteins in vivo.

    Science.gov (United States)

    Turabee, Md Hasan; Thambi, Thavasyappan; Duong, Huu Thuy Trang; Jeong, Ji Hoon; Lee, Doo Sung

    2018-02-27

    Sustained delivery of protein therapeutics is limited owing to the fragile nature of proteins. Despite its great potential, delivery of proteins without any loss of bioactivity remains a challenge in the use of protein therapeutics in the clinic. To surmount this shortcoming, we report a pH- and temperature-responsive in situ-forming injectable hydrogel based on comb-type polypeptide block copolymers for the controlled delivery of proteins. Polypeptide block copolymers, composed of hydrophilic polyethylene glycol (PEG), temperature-responsive poly(γ-benzyl-l-glutamate) (PBLG), and pH-responsive oligo(sulfamethazine) (OSM), exhibit pH- and temperature-induced sol-to-gel transition behavior in aqueous solutions. Polypeptide block copolymers were synthesized by combining N-carboxyanhydride-based ring-opening polymerization and post-functionalization of the chain-end using N-hydroxy succinimide ester activated OSM. The physical properties of polypeptide-based hydrogels were tuned by varying the composition of temperature- and pH-responsive PBLG and OSM in block copolymers. Polypeptide block copolymers were non-toxic to human embryonic kidney cells at high concentrations (2000 μg mL -1 ). Subcutaneous administration of polypeptide block copolymer sols formed viscoelastic gel instantly at the back of Sprague-Dawley (SD) rats. The in vivo gels exhibited sustained degradation and were found to be bioresorbable in 6 weeks without any noticeable inflammation at the injection site. Anionic characteristics of hydrogels allow efficient loading of a cationic model protein, lysozyme, through electrostatic interaction. Lysozyme-loaded polypeptide block copolymer sols readily formed a viscoelastic gel in vivo and sustained lysozyme release for at least a week. Overall, the results demonstrate an elegant approach to control the release of certain charged proteins and open a myriad of therapeutic possibilities in protein therapeutics.

  7. Methamphetamine transiently increases the blood-brain barrier permeability in the hippocampus: role of tight junction proteins and matrix metalloproteinase-9.

    Science.gov (United States)

    Martins, Tânia; Baptista, Sofia; Gonçalves, Joana; Leal, Ermelindo; Milhazes, Nuno; Borges, Fernanda; Ribeiro, Carlos F; Quintela, Oscar; Lendoiro, Elena; López-Rivadulla, Manuel; Ambrósio, António F; Silva, Ana P

    2011-09-09

    Methamphetamine (METH) is a powerful stimulant drug of abuse that has steadily gained popularity worldwide. It is known that METH is highly neurotoxic and causes irreversible damage of brain cells leading to neurological and psychiatric abnormalities. Recent studies suggested that METH-induced neurotoxicity might also result from its ability to compromise blood-brain barrier (BBB) function. Due to the crucial role of BBB in the maintenance of brain homeostasis and protection against toxic molecules and pathogenic organisms, its dysfunction could have severe consequences. In this study, we investigated the effect of an acute high dose of METH (30mg/kg) on BBB permeability after different time points and in different brain regions. For that, young adult mice were sacrificed 1h, 24h or 72h post-METH administration. METH increased BBB permeability, but this effect was detected only at 24h after administration, being therefore a transitory effect. Interestingly, we also found that the hippocampus was the most susceptible brain region to METH, comparing to frontal cortex and striatum. Moreover, in an attempt to identify the key players in METH-induced BBB dysfunction we further investigated potential alterations in tight junction (TJ) proteins and matrix metalloproteinase-9 (MMP-9). METH was able to decrease the protein levels of zonula occludens (ZO)-1, claudin-5 and occludin in the hippocampus 24h post-injection, and increased the activity and immunoreactivity of MMP-9. The pre-treatment with BB-94 (30mg/kg), a matrix metalloproteinase inhibitor, prevented the METH-induced increase in MMP-9 immunoreactivity in the hippocampus. Overall, the present data demonstrate that METH transiently increases the BBB permeability in the hippocampus, which can be explained by alterations on TJ proteins and MMP-9. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Improvement of gluten-free bread properties by the incorporation of bovine plasma proteins and different saccharides into the matrix.

    Science.gov (United States)

    Rodriguez Furlán, Laura T; Pérez Padilla, Antonio; Campderrós, Mercedes E

    2015-03-01

    The aim of this work was to improve the quality of gluten-free bread, incorporating plasma bovine proteins concentrated by ultrafiltration and freeze-dried with saccharides (inulin and sucrose). The influence of these compounds on textural properties and final bread quality was assessed. The textural studies revealed that with the addition of proteins and inulin, homogeneous and smaller air cells were achieved improving the textural properties while the bread hardness was comparable with breads with gluten. The volume of gluten-free breads increased with increasing proteins and inulin concentrations, reaching a maximum at a protein concentration of 3.5% (w/w). The addition of the enhancers improved moisture retention of the loaves after cooking and an increase of lightness of crumb with respect to the control was observed. The sensory analysis found no statistically significant difference in sensory attributes evaluated with respect to the control, so these ingredients do not negatively affect the organoleptic properties of bread. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Molecular aspects of the interaction between Mason-Pfizer monkey virus matrix protein and artificial phospholipid membrane

    Czech Academy of Sciences Publication Activity Database

    Junková, P.; Prchal, J.; Spiwok, V.; Pleskot, Roman; Kadlec, Jan; Krásný, L.; Hynek, R.; Hrabal, R.; Ruml, T.

    2016-01-01

    Roč. 84, č. 11 (2016), s. 1717-1727 ISSN 0887-3585 Institutional support: RVO:61388963 Keywords : covalent labelling * mass spectrometry * multiscale molecular dynamics * protein-membrane interaction * phosphatidylinositol-(4,5)-bisphosphate * liposomes Subject RIV: CE - Biochemistry Impact factor: 2.289, year: 2016

  10. Preparation and characterization of cross-linked excipient of coprocessed xanthan gum-acacia gum as matrix for sustained release tablets

    Science.gov (United States)

    Surini, Silvia; Wati, Dina Risma; Syahdi, Rezi Riadhi

    2018-02-01

    Sustained release tablet is solid dosage form which is designed to release drugs slowly in the body. This research was intended to prepare and characterize the cross-linked excipients of co-processed xanthan gum-acacia gum (CL-Co-XGGA) as matrices for sustained release tablets with gliclazide as a model drug. CL-Co-XGGA excipients were cross-linked materials of co-processed excipients of xanthan gum-acacia gum (Co-XGGA) using sodium trimetaphosphate. Co-processed excipients of xanthan gum-acacia gum were prepared in the ratio of each excipient 1:2, 1:1 and 2:1. Co-XGGA and CL-Co-XGGA excipients were characterized physically, chemically and functionally. Then, the sustained release (SR) tablets were formulated by wet granulation method using CL-Co-XGGA excipients as matrices. Also, the dissolution study of the gliclazide SR tablets was carried out in phosphate buffer medium pH 7,4 containing sodium lauryl sulphate 0.2% for 12 hours. The results showed that the degree of substitution (DS) of CL-Co-XGGA 1:2, 1:1, 2:1 excipients were respectively 0.067, 0.082 and 0.08. Besides that, the excipients gel strengths were 14.03, 17.27 and 20,70 gF, respectively. The cross-linked excipients had improved flow properties and swelling capability compared to the Co-XGGA excipients. The results of the gliclazide SR tablets evaluations showed that all tablets were passed all tablet requirements. Moreover, the gliclazide release from SR tablets F1 - F6 revealed the sustained release profile, which was following zero order kinetics (F1, F2, F3, F6) and Higuchi kinetics (F4 and F5). It could be concluded that the obtained CL-Co-XGGA excipients might be used as matrices for sustained release tablets and could retard drug release up to 8 until 32 hours.

  11. Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

    Science.gov (United States)

    Oliveira, Rosane; de Morais, Zenaide Maria; Gonçales, Amane Paldes; Romero, Eliete Caló; Vasconcellos, Silvio Arruda; Nascimento, Ana L T O

    2011-01-01

    Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

  12. Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

    Directory of Open Access Journals (Sweden)

    Rosane Oliveira

    Full Text Available Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30 and LIC12238. We have employed Escherichia coli BL21 (SI strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively. In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa. Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

  13. Fusobacterium nucleatum binding to complement regulatory protein CD46 modulates the expression and secretion of cytokines and matrix metalloproteinases by oral epithelial cells.

    Science.gov (United States)

    Mahtout, Hayette; Chandad, Fatiha; Rojo, Jose M; Grenier, Daniel

    2011-02-01

    Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum-bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.

  14. Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear Factor-κB Pathways.

    Science.gov (United States)

    Chiang, Hsiu-Mei; Chan, Shih-Yun; Chu, Yin; Wen, Kuo-Ching

    2015-05-13

    Ultraviolet (UV) irradiation is one of the most important extrinsic factors contributing to skin photodamage. After UV irradiation, a series of signal transductions in the skin will be activated, leading to inflammatory response and photoaged skin. In this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for its photoprotective effects. The results revealed that 5-25 μM fisetin inhibits cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, MMP-3, MMP-9 expression induced by ultraviolet B (UVB) irradiation in human skin fibroblasts. In addition, fisetin suppressed UVB-induced collagen degradation. With regard to its effect on upper-stream signal transduction, we found that fisetin reduced the expression of ultraviolet (UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB) degradation and increased the amount of p65, which is a major subunit of nuclear factor-κB (NF-κB), in cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP response element-binding protein (CREB) Ser-133 phosphorylation level in the phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway. Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), and nitric oxide (NO) generation. The mentioned effects and mechanisms suggest that fisetin can be used in the development of photoprotective agents.

  15. Fabrication of a novel hemin-based monolithic column and its application in separation of protein from complex bio-matrix.

    Science.gov (United States)

    Jiang, Xiaoya; Zhang, Doudou; Li, Xueying; Wang, Xixi; Bai, Ligai; Liu, Haiyan; Yan, Hongyuan

    2017-05-10

    A novel polymer-based monolithic column was prepared via redox initiation system within the confines of a stainless steel column with 4.6mm i.d. In the processes, hemin and lauryl methacrylate were used as co-monomers; ethylene dimethacrylate as crosslinking agent; n-butyl alcohol, ethanediol, and N, N-dimethylformamide as tri-porogens; benzoyl peroxide and N, N-dimethyl aniline as redox initiation system. The resulting polymer-based monolithic columns were characterized by scanning electron microscopy, nitrogen adsorption-desorption instrument, and mercury intrusion porosimeter, respectively. The results illustrated that the improved monolith had relative uniform porous structure, good permeability, and low back pressure. Aromatic compounds were used to test the chromatographic behavior of the monolith, resulting in highest column efficiency of 19 880 plates per meter with reversed-phase mechanism. Furthermore, the homemade monolith was used as the stationary phase of high performance liquid chromatography to separate proteins from complex bio-matrix, including human plasma, egg white, and snailase. The results showed that the monolithic column occupied good separation ability with these complex bio-samples. Excellent specific character of the homemade hemin-based monolith was that it could simultaneously remove high-abundance proteins (including human serum albumin, immunoglobulin G, and human fibrinogen) from human plasma and separate other proteins to different fractions. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Extracellular Protein Interactions Mediated by the Neural Cell Adhesion Molecule, NCAM: Heterophilic Interactions Between NCAM and Cell Adhesion Molecules, Extracellular Matrix Proteins, and Viruses

    DEFF Research Database (Denmark)

    Nielsen, Janne; Kulahin, Nikolaj; Walmod, Peter

    2008-01-01

    interactions, thereby modulating a range of biological processes. This review summarizes interactions between NCAM and other CAMs and ECM proteins. Additionally, the role of NCAM as a receptor for rabies virus, and its implications in rabies infections is briefly described. Interactions between NCAM and its...

  17. YKL-40, a mammalian member of the chitinase family, is a matrix protein of specific granules in human neutrophils

    DEFF Research Database (Denmark)

    Volck, B; Price, P A; Johansen, J S

    1998-01-01

    YKL-40, also called human cartilage glycoprotein-39 (HC gp-39), is a member of family 18 glycosyl hydrolases. YKL-40 is secreted by chondrocytes, synovial cells, and macrophages, and recently it has been reported that YKL-40 has a role as an autoantigen in rheumatoid arthritis (RA). The function...... of patients with RA, and the cells are assumed to play a role in joint destruction in that disorder. Therefore, we examined whether neutrophils are a source of YKL-40. YKL-40 was found to colocalize and comobilize with lactoferrin (the most abundant protein of specific granules) but not with gelatinase...... YKL-40 at the myelocyte-metamyelocyte stage, the stage of maturation at which other specific granule proteins are formed. Assuming that YKL-40 has a role as an autoantigen in RA by inducing T cell-mediated autoimmune response, YKL-40 released from neutrophils in the inflamed joint could be essential...

  18. Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: loss of chondrocyte stability.

    Science.gov (United States)

    Krawczak, David A; Westendorf, Jennifer J; Carlson, Cathy S; Lewis, Jack L

    2009-06-01

    The aim of this study was to determine the effects of bone morphogenetic protein-2 (BMP-2) on articular chondrocyte tissues grown as monolayers in vitro for up to 8 weeks. Articular chondrocytes were isolated from New Zealand White rabbits and plated in monolayer cultures. The cultures were supplemented with 100 ng/mL of BMP-2 for up to 8 weeks and the extracellular matrix (ECM) composition, material properties, and messenger RNA (mRNA) expression were analyzed. mRNA expression of cartilage-specific genes, type II collagen, and aggrecan showed that BMP-2 enhanced chondrocyte stability for up to 3 weeks. After 3 weeks in culture, there was substantially more type I collagen expression and more osteopontin and runt-related transcription factor 2 expression in 5- and 8-week cultures treated with BMP-2 than in controls. Additionally, matrix metalloproteinase-13 and ADAMTS-5 (A disintegrin-like and metalloproteinase with thrombospondin 5) were upregulated in 5- and 8-week cultures treated with BMP-2, coinciding with a loss of ECM density, collagen, and proteoglycan. Eight-week tissue stimulated with BMP-2 was more fragile and tore more easily when removed from the culture dish as compared to controls, suggesting temporal limitations to the effectiveness of BMP-2 in monolayer systems and perhaps other models to enhance the generation of a cartilage-like tissue for tissue engineering purposes.

  19. [Expression of erythroblastic leukemia viral oncogene homolog 3 (ErbB-3) binding protein-1, matrix metalloproteinases, eplthelial cadherin in adenoid cystic carcinoma and correlation analysis].

    Science.gov (United States)

    Sun, Jian; Yu, You-cheng; Luo, Yi-xi; Tian, Zhen

    2012-12-01

    To investigate the expression of ErbB-3 binding protein-1 (EBP-1), matrix metalloproteinase 9 (MMP-9) and E-cadherin (E-cad) in adenoid cystic carcinoma and their correlation. Immunohistochemistry(PV6000 method) was used to detect EBP-1, MMP-9 and E-cad expression in 66 cases of adenoid cystic carcinoma tissues and matched para-cancerous normal tissues. In this study all cases were successfully followed up. The positive expression rate of EBP-1 in adenoid cystic carcinoma tissues was 85%. EBP-1 expression was significantly correlated to pathological pattern and clinical stage (P correlation between EBP-1 and E-cad expression, and positive correlation between EBP-1 and MMP-9. EBP-1 and its correlation with MMP-9 and E-cad may be used as useful indicators for clinical assessment of tumor biological behavior and prognosis in patients with adenoid cystic carcinoma.

  20. The Gene Expression of Human Endothelial Cells Is Modulated by Subendothelial Extracellular Matrix Proteins: Short-Term Response to Laminar Shear Stress

    Czech Academy of Sciences Publication Activity Database

    Chlupáč, Jaroslav; Filová, Elena; Havlíková, Jana; Matějka, R.; Riedel, Tomáš; Houska, Milan; Brynda, Eduard; Pamula, E.; Rémy, M.; Bareille, R.; Fernandez, P.; Daculsi, R.; Bourget, Ch.; Bačáková, Lucie; Bordenave, L.

    2014-01-01

    Roč. 20, 15-16 (2014), s. 2253-2264 ISSN 2152-4947 R&D Projects: GA ČR(CZ) GAP108/10/1106; GA ČR(CZ) GAP108/11/1857; GA ČR(CZ) GA305/08/0108; GA ČR GAP205/12/1702; GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109; GA MZd(CZ) NT11270 Grant - others:GA MŠk(CZ) Barrande 2005-06-036-1 Institutional support: RVO:67985823 ; RVO:61389013 Keywords : atherosclerosis * endothelial cells * extracellular matrix proteins * tissue engineering Subject RIV: EI - Biotechnology ; Bionics; CD - Macromolecular Chemistry (UMCH-V) Impact factor: 4.448, year: 2014

  1. A collapsin response mediator protein 2 isoform controls myosin II-mediated cell migration and matrix assembly by trapping ROCK II

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Morgan-Fisher, Marie; Wait, Robin

    2012-01-01

    Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among...... nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two......-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions...

  2. Amelogenin, an extracellular matrix protein, in the treatment of venous leg ulcers and other hard-to-heal wounds: experimental and clinical evidence

    DEFF Research Database (Denmark)

    Romanelli, M.; Dini, V.; Vowden, P.

    2008-01-01

    Amelogenins are extracellular matrix proteins that, under physiological conditions, self-assemble into globular aggregates up to micron-sizes. Studies with periodontal fibroblasts indicate that attachment to these structures increases the endogenous secretion of multiple growth factors and cell...... that the application of amelogenin (Xelma, Molnlycke Health Care, Gothenburg, Sweden) as an adjunct treatment to compression results in significant reduction in ulcer size, improvement in the state of ulcers, reduced pain, and a larger proportion of ulcers with low levels of exudate, compared with treatment...... therapy demonstrated in the RCT are being repeated in "real life" situations and that amelogenin therapy may also have a role to play in the treatment of other wound types such as diabetic foot ulcers Udgivelsesdato: 2008...

  3. Autosomal dominant precocious osteoarthropathy due to a mutation of the cartilage oligomeric matrix protein (COMP) gene: further expansion of the phenotypic variations of COMP defects

    Energy Technology Data Exchange (ETDEWEB)

    Kawaji, Hiroyuki [Department of Orthopaedic Surgery, Sanyudo Hospital, 6-1-219 Chuou, Yonezawa, Yamagata 992-0045 (Japan); Nishimura, Gen [Department of Radiology, Nasu Chuou Hospital, Tochigi (Japan); Watanabe, Sobei; Sasaki, Akira; Sano, Tokuhisa [Department of Orthopaedic Surgery, Tohoku Kohsei-Nenkin Hospital, Miyagi (Japan); Mabuchi, Akihiko; Ikeda, Toshiyuki; Ikegawa, Shiro [Laboratory for Bone and Joint Diseases, SNP Research Center, Tokyo (Japan); Ohashi, Hirofumi [Division of Medical Genetics, Saitama Children' s Medical Center, Saitama (Japan)

    2002-12-01

    We report on a Japanese family of four generations with an autosomal dominant precocious osteoarthropathy. The cardinal clinical manifestations of affected individuals were painful weight-bearing large joints, which started in late childhood or adolescence. The radiological hallmarks included coxa plana, mild epiphyseal dysplasia of the knee, and round talar domes with tibiotalar slant in childhood, which evolved into degenerative joint diseases in adulthood. The disease phenotype was cosegregated with a mutation of the cartilage oligomeric matrix protein (COMP) gene in the family members, who underwent molecular evaluation. COMP mutations have been reported in a mild form of multiple epiphyseal dysplasia (MED), Ribbing type, as well as allied disorders with more severe manifestations, such as MED Fairbank type and pseudoachondroplasia. Unlike previously reported cases with the Ribbing type, the present patients did not have short stature or brachydactyly. This report expands further the phenotypic variations of COMP defects. (orig.)

  4. Modulation of extracellular matrix proteins and hepatate stellate cell activation following gadolinium chloride induced Kuffer cell blockade in an experimental model of liver fibrosis/cirrhosis

    Directory of Open Access Journals (Sweden)

    Nilgün Tekkesin

    2013-06-01

    Full Text Available Hepatic fibrosis is now regarded as a common response to chronic liver injury; regardless of its nature (viral infections, alcohol abuse and metal overload. It is also characterized by excessive deposition of extracellular matrix (ECM components. The ECM is a dynamic complex of macromolecules that includes collagens, glycoproteins, and proteoglycans, such as laminin and fibronectin; it has been shown that it does not only support the tissue structure, but also plays a major role in cell adhesion, proliferation, and differentiation. Remodelling of the ECM may be the signal that facilitates lobular reorganization during liver regeneration after a liver injury. Much work has been done concerning the ECM synthesis and protein contents.

  5. Increased levels of the calcification marker matrix Gla Protein and the inflammatory markers YKL-40 and CRP in patients with type 2 diabetes and ischemic heart disease

    DEFF Research Database (Denmark)

    Thomsen, Stine B; Rathcke, Camilla N; Zerahn, Bo

    2010-01-01

    . In the present study levels of markers of calcification (MGP) and inflammation (YKL-40, hsCRP) were evaluated in patients with T2 D and/or ischemic heart disease (IHD). MATERIALS AND METHODS: The study population consisted of 1) patients with T2D (n = 45); 2) patients with IHD (n = 37); patients with both T2D......OBJECTIVE AND DESIGN: Low grade inflammation is of pathogenic importance in atherosclerosis and in the development of cardiovascular disease (CVD) and type 2 diabetes (T2D). Matrix GLA protein (MGP), an inhibitor of medial calcification of arteries, is increased in patients with atherosclerosis...... and IHD (n = 20) and 4) healthy controls (n = 20). Biochemical parameters were measured in venous blood samples. RESULTS: Levels of MGP, YKL-40 and hsCRP were increased in patients with IHD and/or T2D (p T2D and IHD had higher MGP levels (p

  6. Matrix theory

    CERN Document Server

    Franklin, Joel N

    2003-01-01

    Mathematically rigorous introduction covers vector and matrix norms, the condition-number of a matrix, positive and irreducible matrices, much more. Only elementary algebra and calculus required. Includes problem-solving exercises. 1968 edition.

  7. Sustainable protein from biogas. The perspective of susteine; Duurzaam eiwit uit biogas. Verkenning van het perspectief van susteine

    Energy Technology Data Exchange (ETDEWEB)

    Oosterhuis, N. [Easthouse Business Solutions, Rolde (Netherlands); Hooijer, H. [We-Do Interim Management and Consultancy, Culemborg (Netherlands)

    2007-12-15

    Production of single-cell protein (Susteine) on the basis of biogas (methane) from manure digestion might be an interesting option to reduce the amounts of manure from cattle, pigs and poultry and also might be an option for a sustainable production of high-value protein. An earlier made investigation leaded to the conclusion that many questions had to be answered to make a better picture of this opportunity for the Dutch agricultural society. In this study we investigated the feasibility of such a process and product. It is concluded that the product will be accepted in the market as long as the HACCP-requirements which are applicable in the feed production are fulfilled. The product as such, assuming that the quality is comparable to the product as was produced by Norferm, could get an attractive position especially in the application for piglets. A quantity of 10.000 t/y could easily be sold in this market segment. Of course the product first has to be registered, which needs additional studies. Technically we foresee no major hurdles in the production of Susteine on basis of methane from biogas. The biogas has to be purified to such a quality that SCP-production is feasible, however, technologies are available. It needs a further investigation to establish the quality requirements for such a process. The scale of operation of the biogas installation creates a financial-economic problem. In order to produce 10.000 t protein / year, a quantity of 30-35 million m{sup 3} of biogas is needed, for which 2 million ton of manure has to be digested. In case a centralized production of gas and protein is foreseen, the number of transport movements will be unacceptable. Besides the quantity of manure to be transported also approximately the same amount of digested material has to be transported. A unique opportunity in The Netherlands is the presence of a pipe network for natural gas. Such a network could serve as a transport system of locally produced, purified, biogas

  8. Static Mechanical Loading Influences the Expression of Extracellular Matrix and Cell Adhesion Proteins in Vaginal Cells Derived From Premenopausal Women With Severe Pelvic Organ Prolapse.

    Science.gov (United States)

    Kufaishi, Hala; Alarab, May; Drutz, Harold; Lye, Stephen; Shynlova, Oksana

    2016-08-01

    Primary human vaginal cells derived from women with severe pelvic organ prolapse (POP-HVCs) demonstrate altered cellular characteristics as compared to cells derived from asymptomatic women (control-HVCs). Using computer-controllable Flexcell stretch unit, we examined whether POP-HVCs react differently to mechanical loading as compared to control-HVCs by the expression of extracellular matrix (ECM) components, cell-ECM adhesion proteins, and ECM degrading and maturating enzymes. Vaginal tissue biopsies from premenopausal patients with Pelvic Organ Prolapse Quantification System stage ≥3 (n = 8) and asymptomatic controls (n = 7) were collected during vaginal hysterectomy or repair. Human vaginal cells were isolated by enzymatic digestion, seeded on collagen (COLI)-coated plates, and stretched (24 hours, 25% elongation). Total RNA was extracted, and 84 genes were screened using Human ECM and Adhesion Molecules polymerase chain reaction array; selected genes were verified by quantitative reverse transcription-polymerase chain reaction. Stretch-conditioned media (SCM) were collected and analyzed by protein array, immunoblotting, and zymography. In mechanically stretched control-HVCs, transcript levels of integrins (ITGA1, ITGA4, ITGAV, and ITGB1) and matrix metalloproteinases (MMPs) 2, 8, and 13 were downregulated (P SCM from POP-HVCs compared to control-HVCs. Primary human vaginal cells derived from women with severe pelvic organ prolapse and control-HVCs react differentially to in vitro mechanical stretch. Risk factors that induce stretch may alter ECM composition and cell-ECM interaction in pelvic floor tissue leading to the abatement of pelvic organ support and subsequent POP development. © The Author(s) 2016.

  9. Enhanced hyaline cartilage matrix synthesis in collagen sponge scaffolds by using siRNA to stabilize chondrocytes phenotype cultured with bone morphogenetic protein-2 under hypoxia.

    Science.gov (United States)

    Legendre, Florence; Ollitrault, David; Hervieu, Magalie; Baugé, Catherine; Maneix, Laure; Goux, Didier; Chajra, Hanane; Mallein-Gerin, Frédéric; Boumediene, Karim; Galera, Philippe; Demoor, Magali

    2013-07-01

    Cartilage healing by tissue engineering is an alternative strategy to reconstitute functional tissue after trauma or age-related degeneration. However, chondrocytes, the major player in cartilage homeostasis, do not self-regenerate efficiently and lose their phenotype during osteoarthritis. This process is called dedifferentiation and also occurs during the first expansion step of autologous chondrocyte implantation (ACI). To ensure successful ACI therapy, chondrocytes must be differentiated and capable of synthesizing hyaline cartilage matrix molecules. We therefore developed a safe procedure for redifferentiating human chondrocytes by combining appropriate physicochemical factors: hypoxic conditions, collagen scaffolds, chondrogenic factors (bone morphogenetic protein-2 [BMP-2], and insulin-like growth factor I [IGF-I]) and RNA interference targeting the COL1A1 gene. Redifferentiation of dedifferentiated chondrocytes was evaluated using gene/protein analyses to identify the chondrocyte phenotypic profile. In our conditions, under BMP-2 treatment, redifferentiated and metabolically active chondrocytes synthesized a hyaline-like cartilage matrix characterized by type IIB collagen and aggrecan molecules without any sign of hypertrophy or osteogenesis. In contrast, IGF-I increased both specific and noncharacteristic markers (collagens I and X) of chondrocytes. The specific increase in COL2A1 gene expression observed in the BMP-2 treatment was shown to involve the specific enhancer region of COL2A1 that binds the trans-activators Sox9/L-Sox5/Sox6 and Sp1, which are associated with a decrease in the trans-inhibitors of COL2A1, c-Krox, and p65 subunit of NF-kappaB. Our procedure in which BMP-2 treatment under hypoxia is associated with a COL1A1 siRNA, significantly increased the differentiation index of chondrocytes, and should offer the opportunity to develop new ACI-based therapies in humans.

  10. Interferon-γ-inducible protein-10 in chronic hepatitis C: Correlations with insulin resistance, histological features & sustained virological response.

    Science.gov (United States)

    Crisan, Dana; Grigorescu, Mircea Dan; Radu, Corina; Suciu, Alina; Grigorescu, Mircea

    2017-04-01

    One of the multiple factors contributing to virological response in chronic hepatitis C (CHC) is interferon-gamma-inducible protein-10 (IP-10). Its level reflects the status of interferon-stimulated genes, which in turn is associated with virological response to antiviral therapy. The aim of this study was to evaluate the role of serum IP-10 levels on sustained virological response (SVR) and the association of this parameter with insulin resistance (IR) and liver histology. Two hundred and three consecutive biopsy proven CHC patients were included in the study. Serum levels of IP-10 were determined using ELISA method. IR was evaluated by homeostasis model assessment-IR (HOMA-IR). Histological features were assessed invasively by liver biopsy and noninvasively using FibroTest, ActiTest and SteatoTest. Predictive factors for SVR and their interrelations were assessed. A cut-off value for IP-10 of 392 pg/ml was obtained to discriminate between responders and non-responders. SVR was obtained in 107 patients (52.70%). Area under the receiver operating characteristic curve for SVR was 0.875 with a sensitivity of 91.6 per cent, specificity 74.7 per cent, positive predictive value 80.3 per cent and negative predictive value 88.7 per cent. Higher values of IP-10 were associated with increasing stages of fibrosis (P<0.01) and higher grades of inflammation (P=0.02, P=0.07) assessed morphologically and noninvasively through FibroTest and ActiTest. Significant steatosis and IR were also associated with increased levels of IP-10 (P=0.01 and P=0.02). In multivariate analysis, IP-10 levels and fibrosis stages were independently associated with SVR. Our findings showed that the assessment of serum IP-10 level could be a predictive factor for SVR and it was associated with fibrosis, necroinflammatory activity, significant steatosis and IR in patients with chronic HCV infection.

  11. Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.

    Science.gov (United States)

    Pavarotti, Martín; Capmany, Anahí; Vitale, Nicolas; Colombo, María Isabel; Damiani, María Teresa

    2012-02-01

    Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCβII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCβII but not PKCβI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCβII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  12. Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

    Science.gov (United States)

    Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

    2017-11-01

    The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules.

    Science.gov (United States)

    Oliveira, Tatiane R; Longhi, Mariana T; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2010-03-01

    The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis. Copyright 2009 Elsevier Masson SAS. All rights reserved.

  14. Unravelling the nuclear matrix proteome

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Knol, Jaco C; Jimenez, Connie R

    2009-01-01

    The nuclear matrix (NM) model posits the presence of a protein/RNA scaffold that spans the mammalian nucleus. The NM proteins are involved in basic nuclear function and are a promising source of protein biomarkers for cancer. Importantly, the NM proteome is operationally defined as the proteins...

  15. Common Prairie feeds with different soluble and insoluble fractions used for CPM diet formulation in dairy cattle: Impact of carbohydrate-protein matrix structure on protein and other primary nutrient digestion

    Science.gov (United States)

    Peng, Quanhui; Wang, Zhisheng; Zhang, Xuewei; Yu, Peiqiang

    2014-03-01

    An experiment was conducted to investigate the relationship of carbohydrates molecular spectral characteristics to rumen degradability of primary nutrients in Prairie feeds in dairy cattle. In total, 12 different types of feeds were selected, each type of feed was from three different source with total 37 samples. Six types of them were energy-sourced feeds and the others were protein-sourced feeds. The carbohydrates molecular spectral intensity of various functional groups were collected using Fourier transform infrared attenuated total reflectance (ATR-FT/IR) spectroscopy. In the in situ study, the results showed that the rumen digestibility and digestible fractions of primary nutrients (DM, OM, NCP, and CP) were significantly different (P digestibility and digestible fractions of DM, OM and NCP. Spectral intensities of H_1150, H_1015, A_1, and A_3 were weakly negatively associated with in situ rumen degradation of CP. Spectral intensities of A_1240 and H_1240, mainly associated with cellulosic compounds, were correlated with rumen CP degradation. The multiple regression analysis demonstrated that the spectral intensities of A_3 and H_1415 played the most important role and could be used as a potential tool to predict rumen protein degradation of feeds in dairy cattle. In conclusion, this study showed that the carbohydrates as a whole have an effect on protein rumen degradation, rather than cellulose alone, indicating carbohydrate-protein matrix structure impact protein utilization in dairy cattle. The non-invasive molecular spectral technique (ATR-FT/IR) could be used as a rapid potential tool to predict rumen protein degradation of feedstuffs by using molecular spectral bands intensities in carbohydrate fingerprint region.

  16. Common Prairie feeds with different soluble and insoluble fractions used for CPM diet formulation in dairy cattle: impact of carbohydrate-protein matrix structure on protein and other primary nutrient digestion.

    Science.gov (United States)

    Peng, Quanhui; Wang, Zhisheng; Zhang, Xuewei; Yu, Peiqiang

    2014-01-01

    An experiment was conducted to investigate the relationship of carbohydrates molecular spectral characteristics to rumen degradability of primary nutrients in Prairie feeds in dairy cattle. In total, 12 different types of feeds were selected, each type of feed was from three different source with total 37 samples. Six types of them were energy-sourced feeds and the others were protein-sourced feeds. The carbohydrates molecular spectral intensity of various functional groups were collected using Fourier transform infrared attenuated total reflectance (ATR-FT/IR) spectroscopy. In the in situ study, the results showed that the rumen digestibility and digestible fractions of primary nutrients (DM, OM, NCP, and CP) were significantly different (P<0.05) among the feeds. The spectral bands features were significantly different (P<0.05) among the feeds. Spectral intensities of A_Cell, H_1415 and H_1370 were weakly positively correlated with in situ rumen digestibility and digestible fractions of DM, OM and NCP. Spectral intensities of H_1150, H_1015, A_1, and A_3 were weakly negatively associated with in situ rumen degradation of CP. Spectral intensities of A_1240 and H_1240, mainly associated with cellulosic compounds, were correlated with rumen CP degradation. The multiple regression analysis demonstrated that the spectral intensities of A_3 and H_1415 played the most important role and could be used as a potential tool to predict rumen protein degradation of feeds in dairy cattle. In conclusion, this study showed that the carbohydrates as a whole have an effect on protein rumen degradation, rather than cellulose alone, indicating carbohydrate-protein matrix structure impact protein utilization in dairy cattle. The non-invasive molecular spectral technique (ATR-FT/IR) could be used as a rapid potential tool to predict rumen protein degradation of feedstuffs by using molecular spectral bands intensities in carbohydrate fingerprint region. Copyright © 2013 Elsevier B

  17. Direct detection of carbapenemase-associated proteins of Acinetobacter baumannii using nanodiamonds coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Chang, Kai-Chih; Chung, Chin-Yi; Yeh, Chen-Hsing; Hsu, Kuo-Hsiu; Chin, Ya-Ching; Huang, Sin-Siang; Liu, Bo-Rong; Chen, Hsi-An; Hu, Anren; Soo, Po-Chi; Peng, Wen-Ping

    2018-04-01

    The appearance and spread of carbapenem-resistant Acinetobacter baumannii (CRAB) pose a challenge for optimization of antibiotic therapies and outbreak preventions. The carbapenemase production can be detected through culture-based methods (e.g. Modified Hodge Test-MHT) and DNA based methods (e.g. Polymerase Chain Reaction-PCR). The culture-based methods are time-consuming, whereas those of PCR assays need only a few hours but due to its specificity, can only detect known genetic targets encoding carbapenem-resistance genes. Therefore, new approaches to detect carbapenemase-producing A. baumannii are of great importance. Here, we have developed a rapid and novel method using detonation nanodiamonds (DNDs) as a platform for concentration and extraction of A. baumannii carbapenemase-associated proteins prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS) analysis. To concentrate and extract the A. baumannii carbapenemase-associated proteins, we tested several protein precipitation conditions and found a 0.5% trifluoroacetic acid (TFA) solution within the bacterial suspension could result in strong ion signals with DNDs. A total of 66 A. baumannii clinical-isolates including 51 carbapenem-resistant strains and 15 carbapenem-susceptible strains were tested. Our result showed that among the 51 carbapenem-resistant strains 49 strains had a signal at m/z ~40,279 (±87); among the 15 carbapenem-susceptible strains, 4 strains showed a signal at m/z ~40,279. With on-diamond digestion, we confirmed that the captured protein at m/z ~40,279 was related to ADC family extended-spectrum class C beta-lactamase, from A. baumannii. Using this ADC family protein as a biomarker (m/z ~ 40,279) for carbapenem susceptibility testing of A. baumannii, the sensitivity and the specificity could reach 96% and 73% as compared to traditional imipenem susceptibility testing (MIC results). However, the sensitivity and specificity of this method

  18. Evaluation of a simple protein extraction method for species identification of clinically relevant staphylococci by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    Science.gov (United States)

    Matsuda, Naoto; Matsuda, Mari; Notake, Shigeyuki; Yokokawa, Hirohide; Kawamura, Yoshiaki; Hiramatsu, Keiichi; Kikuchi, Ken

    2012-12-01

    In clinical microbiology, bacterial identification is labor-intensive and time-consuming. A solution for this problem is the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In this study, we evaluated a modified protein extraction method of identification performed on target plates (on-plate extraction method) with MALDI-TOF (Bruker Microflex LT with Biotyper version 3.0) and compared it to 2 previously described methods: the direct colony method and a standard protein extraction method (standard extraction method). We evaluated the species of 273 clinical strains and 14 reference strains of staphylococci. All isolates were characterized using the superoxide dismutase A sequence as a reference. For the species identification, the on-plate, standard extraction, and direct colony methods identified 257 isolates (89.5%), 232 isolates (80.8%), and 173 isolates (60.2%), respectively, with statistically significant differences among the three methods (P extraction method is at least as good as standard extraction in identification rate and has the advantage of a shorter processing time.

  19. The secreted protein ANGPTL2 promotes metastasis of osteosarcoma cells through integrin α5β1, p38 MAPK, and matrix metalloproteinases.

    Science.gov (United States)

    Odagiri, Haruki; Kadomatsu, Tsuyoshi; Endo, Motoyoshi; Masuda, Tetsuro; Morioka, Masaki Suimye; Fukuhara, Shigetomo; Miyamoto, Takeshi; Kobayashi, Eisuke; Miyata, Keishi; Aoi, Jun; Horiguchi, Haruki; Nishimura, Naotaka; Terada, Kazutoyo; Yakushiji, Toshitake; Manabe, Ichiro; Mochizuki, Naoki; Mizuta, Hiroshi; Oike, Yuichi

    2014-01-21

    The tumor microenvironment can enhance the invasive capacity of tumor cells. We showed that expression of angiopoietin-like protein 2 (ANGPTL2) in osteosarcoma (OS) cell lines increased and the methylation of its promoter decreased with time when grown as xenografts in mice compared with culture. Compared with cells grown in normal culture conditions, the expression of genes encoding DNA demethylation-related enzymes increased in tumor cells implanted into mice or grown in hypoxic, serum-starved culture conditions. ANGPTL2 expression in OS cell lines correlated with increased tumor metastasis and decreased animal survival by promoting tumor cell intravasation mediated by the integrin α5β1, p38 mitogen-activated protein kinase, and matrix metalloproteinases. The tolloid-like 1 (TLL1) protease cleaved ANGPTL2 into fragments in vitro that did not enhance tumor progression when overexpressed in xenografts. Expression of TLL1 was weak in OS patient tumors, suggesting that ANGPTL2 may not be efficiently cleaved upon secretion from OS cells. These findings demonstrate that preventing ANGPTL2 signaling stimulated by the tumor microenvironment could inhibit tumor cell migration and metastasis.

  20. The Influence of Arginine on the Response of Enamel Matrix Derivative (EMD Proteins to Thermal Stress: Towards Improving the Stability of EMD-Based Products.

    Directory of Open Access Journals (Sweden)

    Alessandra Apicella

    Full Text Available In a current procedure for periodontal tissue regeneration, enamel matrix derivative (EMD, which is the active component, is mixed with a propylene glycol alginate (PGA gel carrier and applied directly to the periodontal defect. Exposure of EMD to physiological conditions then causes it to precipitate. However, environmental changes during manufacture and storage may result in modifications to the conformation of the EMD proteins, and eventually premature phase separation of the gel and a loss in therapeutic effectiveness. The present work relates to efforts to improve the stability of EMD-based formulations such as Emdogain™ through the incorporation of arginine, a well-known protein stabilizer, but one that to our knowledge has not so far been considered for this purpose. Representative EMD-buffer solutions with and without arginine were analyzed by 3D-dynamic light scattering, UV-Vis spectroscopy, transmission electron microscopy and Fourier transform infrared spectroscopy at different acidic pH and temperatures, T, in order to simulate the effect of pH variations and thermal stress during manufacture and storage. The results provided evidence that arginine may indeed stabilize EMD against irreversible aggregation with respect to variations in pH and T under these conditions. Moreover, stopped-flow transmittance measurements indicated arginine addition not to suppress precipitation of EMD from either the buffers or the PGA gel carrier when the pH was raised to 7, a fundamental requirement for dental applications.

  1. Buddleja officinalis suppresses high glucose-induced vascular smooth muscle cell proliferation: role of mitogen-activated protein kinases, nuclear factor-kappaB and matrix metalloproteinases.

    Science.gov (United States)

    Lee, Yun Jung; Kim, Jin Sook; Kang, Dae Gill; Lee, Ho Sub

    2010-02-01

    Diabetes mellitus is a well-established risk factor for vascular diseases caused by atherosclerosis. In the development of diabetic atherogenesis, vascular smooth muscle cell proliferation is recognized as a key event. Thus, we aimed to investigate whether an ethanol extract of Buddleja officinalis (EBO) suppresses high glucose-induced proliferation in primary cultured human aortic smooth muscle cells (HASMC). [(3)H]-thymidine incorporation revealed that incubation of HASMC with a high concentration of glucose (25 mmol/L) increased cell proliferation. The expression levels of cell cycle protein were also increased by treatment with high glucose concentration. Pretreatment of HASMC with EBO significantly attenuated the increase of high glucose-induced cell proliferation as well as p38 mitogen-activated protein kinases (MAPK) and JNK phosphorylation. EBO suppressed high glucose-induced matrix metalloproteinase (MMP)-9 activity in a dose-dependent manner. In addition, EBO suppressed nuclear factor-kappaB (NF-kappaB) nuclear translocation and transcriptional activity in high glucose conditions. Taken together, the present data suggest that EBO could suppress high glucose-induced atherosclerotic processes through inhibition of p38, JNK, NF-kappaB and MMP signal pathways in HASMC.

  2. Detecting early kidney damage in horses with colic by measuring matrix metalloproteinase -9 and -2, other enzymes, urinary glucose and total proteins

    Directory of Open Access Journals (Sweden)

    Salonen Hanna

    2007-01-01

    Full Text Available Abstract Background The aim of the study was to investigate urine matrix metalloproteinase (MMP-2 and -9 activity, alkaline phosphatase/creatinine (U-AP/Cr and gamma-glutamyl-transpeptidase/creatinine (U-GGT/Cr ratios, glucose concentration, and urine protein/creatinine (U-Prot/Cr ratio and to compare data with plasma MMP-2 and -9 activity, cystatin-C and creatinine concentrations in colic horses and healthy controls. Horses with surgical colic (n = 5 were compared to healthy stallions (n = 7 that came for castration. Blood and urine samples were collected. MMP gelatinolytic activity was measured by zymography. Results We found out that horses with colic had significantly higher urinary MMP-9 complex and proMMP-9 activities than horses in the control group. Colic horses also had higher plasma MMP-2 activity than the control horses. Serum creatinine, although within reference range, was significantly higher in the colic horses than in the control group. There was no significant increase in urinary alkaline phosphatase, gamma-glutamyltranspeptidase or total proteins in the colic horses compared to the control group. A human cystatin-C test (Dako Cytomation latex immunoassay® based on turbidimetry did not cross react with equine cystatin-C. Conclusion The results indicate that plasma MMP-2 may play a role in the pathogenesis of equine colic and urinary MMP-9 in equine kidney damage.

  3. A Collapsin Response Mediator Protein 2 Isoform Controls Myosin II-Mediated Cell Migration and Matrix Assembly by Trapping ROCK II

    Science.gov (United States)

    Morgan-Fisher, Marie; Wait, Robin; Couchman, John R.; Wewer, Ulla M.

    2012-01-01

    Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells. PMID:22431514

  4. Improvement of reduced serum cartilage oligomeric matrix protein levels in systemic juvenile idiopathic arthritis patients treated with the anti-interleukin-6 receptor monoclonal antibody tocilizumab.

    Science.gov (United States)

    Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

    2009-01-01

    In this study, we determined serum cartilage oligomeric matrix protein (COMP) levels in systemic juvenile idiopathic arthritis (sJIA) patients during both the active and the remission phases to investigate how the growth cartilage turnover changed under tocilizumab treatment. Specimens were collected from 201 healthy children under 16 years of age with no growth impairment, and paired sera were collected from 11 sJIA patients treated with tocilizumab. Disease activity was assessed from white blood cell count, erythrocyte sedimentation rate, C-reactive protein, and ferritin, and the COMP concentration was determined by sandwich enzyme-linked immunosorbent assay. Serum COMP concentrations were found independent of age, and the mean value in healthy children was 17.74+/-5.6 U/L. The mean serum COMP in sJIA patients during the active phase was 10.75+/-3.9 U/L, lower than that of healthy children. The mean serum COMP in the remission phase (14.89+/-3.9 U/L) was significantly higher than that in the active period (P<0.05). These results suggested that in sJIA patients, a reduced serum COMP concentration is a useful marker of active disease and growth impairment, and that the growth cartilage turnover suppressed during the active phase is improved in the remission phase under tocilizumab treatment.

  5. Increased expression of Matrix Metalloproteinase 9 in liver from NZB/W F1 mice received antibody against human parvovirus B19 VP1 unique region protein

    Directory of Open Access Journals (Sweden)

    Hsu Gwo-Jong

    2009-01-01

    Full Text Available Abstract Background Human parvovirus B19 infection has been postulated to the anti-phospholipid syndrome (APS in autoimmunity. However, the influence of anti-B19-VP1u antibody in autoimmune diseases is still obscure. Methods To elucidate the effect of anti-B19-VP1u antibodies in systemic lupus erythematosus (SLE, passive transfer of rabbit anti-B19-VP1u IgG was injected intravenously into NZB/W F1 mice. Results Significant reduction of platelet count and prolonged thrombocytopenia time were detected in anti-B19-VP1u IgG group as compared to other groups, whereas significant increases of anti-B19-VP1u, anti-phospholipid (APhL, and anti-double strand DNA (dsDNA antibody binding activity were detected in anti-B19-VP1u group. Additionally, significant increases of matrix metalloproteinase-9 (MMP9 activity and protein expression were detected in B19-VP1u IgG group. Notably, phosphatidylinositol 3-phosphate kinase (PI3K and phosphorylated extracellular signal-regulated kinase (ERK proteins were involved in the induction of MMP9. Conclusion These experimental results firstly demonstrated the aggravated effects of anti-B19-VP1u antibody in disease activity of SLE.

  6. Interactions of GRF(1-29)NH2 with plasma proteins and their effects on the release of the peptide from a PLAGA matrix.

    Science.gov (United States)

    Mariette, B; Coudane, J; Vert, M

    2005-09-02

    The administration of the GRF(1-29)NH2 Growth Hormone Releasing Hormone analog is known as relevant of the concept of drug delivery system using a bioresorbable matrix. However, the release of this peptide from poly(dl-lactic acid-co-glycolic acid) matrices is affected by its insolubility at neutral in salted media and in plasma as well. In order to investigate the origin and the nature of the insolubility in these media in more details, the precipitates collected when the peptide was set in contact with saline, isotonic pH=7.4 phosphate buffer and plasma were analyzed by various techniques, namely weighting, gel chromatography, 1D- and 2D-immunoelectrophoresis, and dialysis to discern the soluble from the insoluble or aggregated fractions. It is shown that precipitation in protein-free salted media is due to a salting out phenomenon complemented by the neutralization of the solubilizing electrostatic charges in the isotonic buffer. In contrast, the precipitation in plasma is due to inter polyelectrolyte-type complexation that involved polyanionic proteins having a rather low isoelectric point like albumin, transferin, haptoglobulin and IgG immunoglobulins. When a rather large quantity of GRF(1-29)NH2 was entrapped in bioresorbable pellets working at a percolating regime after subcutaneous implantation in rats, the peptide was slowly released despite the complexation with plasma proteins. However only a very small part of the peptide was found in blood, this small part being still large enough to cause a detectable increase of the circulating growth hormone concentration. Attempts made to increase the solubility of the peptide in plasma were successful when the peptide was combined with arginine, an amino acid known to promote the poor hormonal activity of injected GRF(1-29)NH2 solutions under clinical conditions.

  7. Presence of Insulin-Like Growth Factor Binding Proteins Correlates With Tumor-Promoting Effects of Matrix Metalloproteinase 9 in Breast Cancer

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    Jae-Hyun Park

    2015-05-01

    Full Text Available The stroma of breast cancer can promote the disease’s progression, but whether its composition and functions are shared among different subtypes is poorly explored. We compared stromal components of a luminal [mouse mammary tumor virus (MMTV–Neu] and a triple-negative/basal-like [C3(1–Simian virus 40 large T antigen (Tag] genetically engineered breast cancer mouse model. The types of cytokines and their expression levels were very different in the two models, as was the extent of innate immune cell infiltration; however, both models showed infiltration of innate immune cells that expressed matrix metalloproteinase 9 (MMP9, an extracellular protease linked to the progression of many types of cancer. By intercrossing with Mmp9 null mice, we found that the absence of MMP9 delayed tumor onset in the C3(1-Tag model but had no effect on tumor onset in the MMTV-Neu model. We discovered that protein levels of insulin-like growth factor binding protein-1 (IGFBP-1, an MMP9 substrate, were increased in C3(1-Tag;Mmp9−/− compared to C3(1-Tag;Mmp9+/+ tumors. In contrast, IGFBP-1 protein expression was low in MMTV-Neu tumors regardless of Mmp9 status. IGFBP-1 binds and antagonizes IGFs, preventing them from activating their receptors to promote cell proliferation and survival. Tumors from C3(1-Tag;Mmp9−/− mice had reduced IGF-1 receptor phosphorylation, consistent with slower tumor onset. Finally, gene expression analysis of human breast tumors showed that high expression of IGFBP mRNA was strongly correlated with good prognosis but not when MMP9 mRNA was also highly expressed. In conclusion, MMP9 has different effects on breast cancer progression depending on whether IGFBPs are expressed.

  8. Expression and clinical significance of extracellular matrix protein 1 and vascular endothelial growth factor-C in lymphatic metastasis of human breast cancer

    International Nuclear Information System (INIS)

    Wu, Qiu-Wan; She, Hong-Qiang; Liang, Jing; Huang, Yu-Fan; Yang, Qing-Mo; Yang, Qiao-Lu; Zhang, Zhi-Ming

    2012-01-01

    Extracellular matrix protein 1 (ECM1) and vascular endothelial growth factor-C (VEGF-C) are secretory glycoproteins that are associated with lymphangiogenesis; these proteins could, therefore, play important roles in the lymphatic dissemination of tumors. However, very little is known about their potential roles in lymphangiogenesis. The aim of this study was to investigate whether correlations exist between ECM1 and VEGF-C in human breast cancer, lymphangiogenesis, and the clinicopathological characteristics of the disease. ECM1 and VEGF-C mRNA and protein expression levels in 41 patients were investigated using real-time reverse transcriptase polymerase chain reaction (RT-PCR), or immunohistochemical (IHC) staining of breast cancer tissue, matched noncancerous breast epithelial tissues, and suspicious metastatic axillary lymph nodes. D2-40 labelled lymph vessels and lymphatic microvessel density (LMVD) were counted. Correlations between ECM1 or VEGF-C protein expression levels, LMVD, and clinicopathological parameters were statistically tested. The rate of ECM1 positive staining in breast cancer tissues was higher (31/41, 75.6%) than that in the corresponding epithelial tissues (4/41, 9.8%, P < 0.001) and lymph nodes (13/41, 31.7%, P < 0.001). Similarly, the VEGF-C expression rate in cancer specimens was higher (33/41, 80.5%) than in epithelial tissues (19/41, 46.3%, P < 0.01) or lymph nodes (15/41, 36.6%, P < 0.01). Higher ECM1 and VEGF-C mRNA expression levels were also detected in the tumor tissues, compared to the non-cancerous tissue types or lymph nodes (P < 0.05). ECM1 protein expression was positively correlated with the estrogen receptor status (P < 0.05) and LMVD (P < 0.05). LMVD in the ECM1- and VEGF-C-positive tumor specimens was higher than that in the tissue types with negative staining (P < 0.05). Both ECM1 and VEGF-C were overexpressed in breast cancer tissue samples. ECM1 expression was positively correlated with estrogen responsiveness and the

  9. Tracing metabolic routes of dietary carbohydrate and protein in rainbow trout (Oncorhynchus mykiss) using stable isotopes ([¹³C]starch and [¹⁵N]protein): effects of gelatinisation of starches and sustained swimming.

    Science.gov (United States)

    Felip, Olga; Ibarz, Antoni; Fernández-Borràs, Jaume; Beltrán, Marta; Martín-Pérez, Miguel; Planas, Josep V; Blasco, Josefina

    2012-03-01

    Here we examined the use of stable isotopes, [¹³C]starch and [¹⁵N]protein, as dietary tracers to study carbohydrate assimilation and distribution and protein utilisation, respectively, by rainbow trout (Oncorhynchus mykiss). The capacity of glucose uptake and use by tissues was studied, first, by varying the digestibility of carbohydrate-rich diets (30 % carbohydrate), using raw starch and gelatinised starch (GS) and, second, by observing the effects of two regimens of activity (voluntary swimming, control; sustained swimming at 1·3 body lengths/s, exercise) on the GS diet. Isotopic ratio enrichment (¹³C and ¹⁵N) of the various tissue components (protein, lipid and glycogen) was measured in the liver, muscles, viscera and the rest of the fish at 11 and 24 h after a forced meal. A level of 30 % of digestible carbohydrates in the food exceeded the capacity of rainbow trout to use this nutrient, causing long-lasting hyperglycaemia that raises glucose uptake by tissues, and the synthesis of glycogen and lipid in liver. Total 13C recovered 24 h post-feeding in the GS group was lower than at 11 h, indicating a proportional increase in glucose oxidation, although the deposition of lipids in white muscle (WM) increased. Prolonged hyperglycaemia was prevented by exercise, since sustained swimming enhances the use of dietary carbohydrates, mainly through conversion to lipids in liver and oxidation in muscles, especially in red muscle (RM). Higher recoveries of total 15N for exercised fish at 24 h, mainly into the protein fraction of both RM and WM, provide evidence that sustained swimming improves protein deposition, resulting in an enhancement of the protein-sparing effect.

  10. Amelogenin, an extracellular matrix protein, in the treatment of venous leg ulcers and other hard-to-heal wounds: Experimental and clinical evidence  ||FREE PAPER||

    Directory of Open Access Journals (Sweden)

    Marco Romanelli

    2008-06-01

    Full Text Available Marco Romanelli1, Valentina Dini1, Peter Vowden2, Magnus S Ågren31Department of Dermatology, University of Pisa, Pisa, Italy; 2Vascular Unit, Bradford Royal Infirmary, Bradford, United Kingdom; 3Department of Surgery K, Bispebjerg Hospitals, Copenhagen University Hospital, Copenhagen, DenmarkAbstract: Amelogenins are extracellular matrix proteins that, under physiological conditions, self-assemble into globular aggregates up to micron-sizes. Studies with periodontal fibroblasts indicate that attachment to these structures increases the endogenous secretion of multiple growth factors and cell proliferation. Pre-clinical and clinical studies indicate that cutaneous wounds benefit from treatment with amelogenins. A randomized controlled trial (RCT involving patients with hard-to-heal venous leg ulcers (VLUs (ie, ulcers with a surface area ≥10 cm2 and duration of ≥6 months showed that the application of amelogenin (Xelma®, Molnlycke Health Care, Gothenburg, Sweden as an adjunct treatment to compression results in significant reduction in ulcer size, improvement in the state of ulcers, reduced pain, and a larger proportion of ulcers with low levels of exudate, compared with treatment with compression alone. Amelogenin therapy was also shown to be safe to use in that there were no significant differences in adverse events noted between patients treated with amelogenin plus compression and those treated with compression alone. Case study evaluations indicate that the benefits of amelogenin therapy demonstrated in the RCT are being repeated in “real life” situations and that amelogenin therapy may also have a role to play in the treatment of other wound types such as diabetic foot ulcers.Keywords: extracellular matrix, amelogenin, venous leg ulcers, diabetic foot ulcers, pyoderma gangrenosum

  11. Activation of phagocytic cells by Staphylococcus epidermidis biofilms: effects of extracellular matrix proteins and the bacterial stress protein GroEL on netosis and MRP-14 release.

    Science.gov (United States)

    Dapunt, Ulrike; Gaida, Matthias M; Meyle, Eva; Prior, Birgit; Hänsch, Gertrud M

    2016-07-01

    The recognition and phagocytosis of free-swimming (planktonic) bacteria by polymorphonuclear neutrophils have been investigated in depth. However, less is known about the neutrophil response towards bacterial biofilms. Our previous work demonstrated that neutrophils recognize activating entities within the extracellular polymeric substance (EPS) of biofilms (the bacterial heat shock protein GroEL) and that this process does not require opsonization. Aim of this study was to evaluate the release of DNA by neutrophils in response to biofilms, as well as the release of the inflammatory cytokine MRP-14. Neutrophils were stimulated with Staphylococcus epidermidis biofilms, planktonic bacteria, extracted EPS and GroEL. Release of DNA and of MRP-14 was evaluated. Furthermore, tissue samples from patients suffering from biofilm infections were collected and evaluated by histology. MRP-14 concentration in blood samples was measured. We were able to show that biofilms, the EPS and GroEL induce DNA release. MRP-14 was only released after stimulation with EPS, not GroEL. Histology of tissue samples revealed MRP-14 positive cells in association with neutrophil infiltration and MRP-14 concentration was elevated in blood samples of patients suffering from biofilm infections. Our data demonstrate that neutrophil-activating entities are present in the EPS and that GroEL induces DNA release by neutrophils. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export.

    Science.gov (United States)

    Brunotte, Linda; Flies, Joe; Bolte, Hardin; Reuther, Peter; Vreede, Frank; Schwemmle, Martin

    2014-07-18

    In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Interleukin-1beta-induced release of matrix proteins into culture media causes inhibition of mineralization of nodules formed by periodontal ligament cells in vitro.

    Science.gov (United States)

    Chien, H H; Lin, W L; Cho, M I

    1999-05-01

    The mechanism by which interleukin-1beta (IL-1) inhibits the formation of mineralized tissue nodules by periodontal ligament (PDL) cells in vitro was investigated through the processes of morphological analysis, immunoprecipitation, and Northern blot analysis. PDL cells were obtained from a 2-day-old coagulum in tooth socket and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bone serum (FBS) and antibiotics. Confluent cells were grown for up to 3 weeks in the presence of ascorbic acid (AA), beta-glycerophosphate (GP), and dexamethasone (Dex), or IL-1. PDL cells cultured in the presence of GP and AA did not differentiate, but those treated with Dex, GP, and AA (Dex group) underwent differentiation, showing four stages (confluent, multilayer, nodule, and mineralization) of disparate morphological characteristics. In contrast, the cells treated with IL-1, Dex, GP, and AA (IL-1 group) did form multilayers but failed to form mineralized nodules. Electron microscopy demonstrated that the Dex-induced mineralized nodules contain multilayers of fibroblastic cells, numerous collagen fibrils, and dense globular as well as fused electron dense patches that are associated with numerous apatite crystals. The nodule-like structures in the IL-1 group were also comprised of multilayered fibroblastic cells, but they contained only a small number of collagen fibrils, and no dense globular or fused patches. Von Kossa staining confirmed the presence of numerous mineralized nodules in the Dex group and their scarceness in the IL-1 group. Northern blot analysis of IL-1-treated cells, however, revealed the presence of mRNAs for type I collagen (Col I), secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC), whose expression patterns and levels were comparable to those of the Dex group. Immunoprecipitation analysis of OPN and BSP in the cell/matrix layers and the culture

  14. Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

    Science.gov (United States)

    Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

    2016-11-01

    To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  15. Matrix-Gla Protein rs4236 [A/G] gene polymorphism and serum and GCF levels of MGP in patients with subgingival dental calculus.

    Science.gov (United States)

    Doğan, Gülnihal Emrem; Demir, Turgut; Aksoy, Hülya; Sağlam, Ebru; Laloğlu, Esra; Yildirim, Abdulkadir

    2016-10-01

    Matrix-Gla Protein (MGP) is one of the major Gla-containing protein associated with calcification process. It also has a high affinity for Ca 2+ and hydroxyapatite. In this study we aimed to evaluate the MGP rs4236 [A/G] gene polymorphism in association with subgingival dental calculus. Also a possible relationship between MGP gene polymorphism and serum and GCF levels of MGP were examined. MGP rs4236 [A/G] gene polymorphism was investigated in 110 patients with or without subgingival dental calculus, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. Additionally, serum and GCF levels of MGP of the patients were compared according to subgingival dental calculus. Comparison of patients with and without subgingival dental calculus showed no statistically significant difference in MGP rs4236 [A/G] gene polymorphism (p=0.368). MGP concentrations in GCF of patients with subgingival dental calculus were statistically higher than those without subgingival dental calculus (p=0.032). However,