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Sample records for extracellular proteins limit

  1. Extracellular secretion of recombinant proteins

    Science.gov (United States)

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  2. Imaging Extracellular Protein Concentration with Nanoplasmonic Sensors

    OpenAIRE

    Byers, Jeff M.; Christodoulides, Joseph A.; Delehanty, James B.; Raghu, Deepa; Raphael, Marc P.

    2015-01-01

    Extracellular protein concentrations and gradients queue a wide range of cellular responses, such as cell motility and division. Spatio-temporal quantification of these concentrations as produced by cells has proven challenging. As a result, artificial gradients must be introduced to the cell culture to correlate signal and response. Here we demonstrate a label-free nanoplasmonic imaging technique that can directly map protein concentrations as secreted by single cells in real time and which ...

  3. In vitro Determination of Extracellular Proteins from Xylella fastidiosa

    Science.gov (United States)

    Mendes, Juliano S.; Santiago, André S.; Toledo, Marcelo A. S.; Horta, Maria A. C.; de Souza, Alessandra A.; Tasic, Ljubica; de Souza, Anete P.

    2016-01-01

    The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3–30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components. PMID:28082960

  4. 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 ...

  5. Immunomodulatory activity of extracellular heat shock proteins and their autoantibodies.

    Science.gov (United States)

    Yokota, Shin-ichi; Fujii, Nobuhiro

    2010-05-01

    HSP are groups of stress-inducible proteins which contribute to quality control by assisting the correct folding of both nascent and denatured proteins, and promoting the degradation of unrecoverable denatured proteins. HSP also help to maintain cellular homeostasis and protect from cell death through a mechanism called thermotolerance. Cells subjected to mild stress induce HSP which then protect them against subsequent stress. However, in cells subjected to severe stress, HSP promote apoptosis. Besides these intracellular events, HSP also exist in extracellular fluids, and have been shown to contribute to immunomodulation. In innate immunity extracellular HSP, like various microbial substances, induce various proinflammatory cytokines. In acquired immunity they interact with antigenic polypeptides and assist in antigen presentation. The extracellular HSP are so-called adjuvant. Release of HSP from cells is triggered by stress and trauma, and is thus regarded as an immunological "danger signal". In addition, anti-HSP autoantibodies are frequently found in patients with autoimmune diseases and inflammatory disorders, and these autoantibodies can modulate the "danger signal" triggered by extracellular HSP.

  6. Extracellular Proteins: Novel Key Components of Metal Resistance in Cyanobacteria?

    Science.gov (United States)

    Giner-Lamia, Joaquín; Pereira, Sara B; Bovea-Marco, Miquel; Futschik, Matthias E; Tamagnini, Paula; Oliveira, Paulo

    2016-01-01

    Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities, and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias toward the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria.

  7. Proteins of the brain extracellular fluid: evidence for release of S-100 protein.

    Science.gov (United States)

    Shashoua, V E; Hesse, G W; Moore, B W

    1984-06-01

    Extracellular protein fractions were obtained (1) by mild, isotonic irrigation of freshly perfused brain tissue; (2) by collection of proteins released into superfusing medium by physiologically viable slices of rat hippocampus; and (3) by sampling the CSF of anesthetized rats. Analysis of the S-100 protein content of these fractions gave values of 2.8, 4.2, and 1.8 micrograms S-100/mg protein, respectively. These values were three- to sixfold higher than the S-100 content of the soluble cytoplasmic protein fractions from the same tissue. This several-fold higher S-100 content of the extracellular protein fractions relative to the intracellular cytoplasmic protein fractions indicates that S-100 is selectively released into the extracellular spaces of the brain. We suggest that the biological function of this CNS protein may involve intercellular transfer.

  8. Extracellular proteins: Novel key components of metal resistance in cyanobacteria?

    Directory of Open Access Journals (Sweden)

    Joaquin eGiner-Lamia

    2016-06-01

    Full Text Available Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias towards the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria.

  9. Secretion and extracellular space travel of Wnt proteins.

    Science.gov (United States)

    Gross, Julia Christina; Boutros, Michael

    2013-08-01

    Wnt signaling pathways control many processes during development, stem cell maintenance and homeostasis, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. Wnts are hydrophobic proteins, however, quite paradoxically, they can travel over distances to induce cell-type specific responses. While there has been an initial focus on elucidating the intracellular signaling cascade, discoveries in the past few years have shed light on a highly complex, and regulated secretory process that guides Wnt proteins through the exocytic pathway. Wnt proteins are at least in portion packaged onto extracellular carriers such as exosomes. Similar to dysregulation of components in the Wnt receiving cell, failure to regulate Wnt secretion has been linked to cancer. Here, we review recent discoveries on factors and processes implicated in Wnt secretion. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Prediction of extracellular matrix proteins based on distinctive sequence and domain characteristics.

    Science.gov (United States)

    Jung, Juhyun; Ryu, Taewoo; Hwang, Yongdeuk; Lee, Eunjung; Lee, Doheon

    2010-01-01

    Extracellular matrix (ECM) proteins are secreted to the exterior of the cell, and function as mediators between resident cells and the external environment. These proteins not only support cellular structure but also participate in diverse processes, including growth, hormonal response, homeostasis, and disease progression. Despite their importance, current knowledge of the number and functions of ECM proteins is limited. Here, we propose a computational method to predict ECM proteins. Specific features, such as ECM domain score and repetitive residues, were utilized for prediction. Based on previously employed and newly generated features, discriminatory characteristics for ECM protein categorization were determined, which significantly improved the performance of Random Forest and support vector machine (SVM) classification. We additionally predicted novel ECM proteins from non-annotated human proteins, validated with gene ontology and earlier literature. Our novel prediction method is available at biosoft.kaist.ac.kr/ecm.

  11. The potential for targeting extracellular LOX proteins in human malignancy

    Directory of Open Access Journals (Sweden)

    Mayorca-Guiliani A

    2013-11-01

    Full Text Available Alejandro Mayorca-Guiliani, Janine T Erler Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark Abstract: The extracellular matrix (ECM is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX enzymes cross-links collagens and elastin and, therefore, is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient deaths. However, LOX biosynthesis results in by-product with antiproliferative properties in certain cancers, and LOX enzymes may have different effects depending on the molecular network in which they are active. Therefore, the design of therapies targeting the LOX family needs to be guided by the molecular makeup of the individual disease and will probably require other agents to act on both the LOX enzymes and their associated network. Keywords: cancer, extracellular matrix, lysyl oxidase, metastasis

  12. Extracellular peptidase hunting for improvement of protein production in plant cells and roots

    Directory of Open Access Journals (Sweden)

    Jérôme eLallemand

    2015-02-01

    Full Text Available Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion, in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA and human serum immunoglobulins G (hIgGs. Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

  13. The role of brain extracellular proteins in neuroplasticity and learning.

    Science.gov (United States)

    Shashoua, V E

    1985-06-01

    Double labeling studies of the pattern of protein synthesis in goldfish and mouse brain identified a class of glycoproteins (the ependymins) whose turnover rate was enhanced after training. A variety of control experiments indicated that these macromolecules have an important role in the molecular and cell biology of learning. Antisera to the ependymins when injected into the brains of trained goldfish cause amnesia of a newly acquired behavior. Isolation and localization studies by immunocytochemical methods indicate that the ependymins are released into the brain extracellular fluid by a class of neurosecretory cells. In mammalian brain ependymin-containing cells are highly concentrated in the limibic system. The ependymins are constituted from two disulfide-linked acidic polypeptide chains (M.W.37K and 31K). They contain at least 5% covalently bound carbohydrate per chain with mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid as the predominant components. The highly soluble ependymins can rapidly polymerize to form an insoluble fibrous matrix if calcium is removed from solution by the addition of a Ca2+-chelating agent or dialysis. The self-aggregation property of the ependymins can be triggered by the depletion of Ca2+ from the extracellular space. Studies of the kinetics of the aggregation phenomenon by measurements of turbidity changes indicate that the process can be terminated but not reversed by restoring Ca2+ to its normal CSF level. Immunohistochemical studies of the brains of trained goldfish show the presence of punctate statining sites in the perimeter of certain cells located in specific brain regions. This suggests that ependymin aggregation might occur in vivo during learning. A molecular hypothesis relating the aggregation properties of the ependymins to neuroplasticity and learning is proposed.

  14. Extracellular protein analysis of activated sludge and their functions in wastewater treatment plant by shotgun proteomics.

    Science.gov (United States)

    Zhang, Peng; Shen, Yu; Guo, Jin-Song; Li, Chun; Wang, Han; Chen, You-Peng; Yan, Peng; Yang, Ji-Xiang; Fang, Fang

    2015-07-10

    In this work, proteins in extracellular polymeric substances extracted from anaerobic, anoxic and aerobic sludges of wastewater treatment plant (WWTP) were analyzed to probe their origins and functions. Extracellular proteins in WWTP sludges were identified using shotgun proteomics, and 130, 108 and 114 proteins in anaerobic, anoxic and aerobic samples were classified, respectively. Most proteins originated from cell and cell part, and their most major molecular functions were catalytic activity and binding activity. The results exhibited that the main roles of extracellular proteins in activated sludges were multivalence cations and organic molecules binding, as well as in catalysis and degradation. The catalytic activity proteins were more widespread in anaerobic sludge compared with those in anoxic and aerobic sludges. The structure difference between anaerobic and aerobic sludges could be associated with their catalytic activities proteins. The results also put forward a relation between the macro characteristics of activated sludges and micro functions of extracellular proteins in biological wastewater treatment process.

  15. Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates.

    Science.gov (United States)

    Sotillo, Javier; Pearson, Mark; Potriquet, Jeremy; Becker, Luke; Pickering, Darren; Mulvenna, Jason; Loukas, Alex

    2016-01-01

    Herein we show for the first time that Schistosoma mansoni adult worms secrete exosome-like extracellular vesicles ranging from 50 to 130nm in size. Extracellular vesicles were collected from the excretory/secretory products of cultured adult flukes and purified by Optiprep density gradient, resulting in highly pure extracellular vesicle preparations as confirmed by transmission electron microscopy and Nanosight tracking analysis. Extracellular vesicle proteomic analysis showed numerous known vaccine candidates, potential virulence factors and molecules implicated in feeding. These findings provide new avenues for the exploration of host-schistosome interactions and offer a potential mechanism by which some vaccine antigens exert their protective efficacy. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Occurrence State and Molecular Structure Analysis of Extracellular Proteins with Implications on the Dewaterability of Waste-Activated Sludge.

    Science.gov (United States)

    Wu, Boran; Ni, Bing-Jie; Horvat, Kristine; Song, Liyan; Chai, Xiaoli; Dai, Xiaohu; Mahajan, Devinder

    2017-08-15

    The occurrence state and molecular structure of extracellular proteins were analyzed to reveal the influencing factors on the water-holding capacities of protein-like substances in waste-activated sludge (WAS). The gelation process of extracellular proteins verified that advanced oxidation processes (AOPs) for WAS dewaterability improvement eliminated the water affinity of extracellular proteins and prevented these macromolecules from forming stable colloidal aggregates. Isobaric tags for relative and absolute quantitation proteomics identified that most of the extracellular proteins were originally derived from the intracellular part and the proteins originally located in the extracellular part were mainly membrane-associated. The main mechanism of extracellular protein transformation during AOPs could be represented by the damage of the membrane or related external encapsulating structure and the release of intracellular substances. For the selected representative extracellular proteins, the strong correlation (R 2 > 0.97, p proteins on the interstitial water removal from WAS.

  17. The Staphylococcus aureus extracellular matrix protein (Emp) has a fibrous structure and binds to different extracellular matrices.

    Science.gov (United States)

    Geraci, Jennifer; Neubauer, Svetlana; Pöllath, Christine; Hansen, Uwe; Rizzo, Fabio; Krafft, Christoph; Westermann, Martin; Hussain, Muzaffar; Peters, Georg; Pletz, Mathias W; Löffler, Bettina; Makarewicz, Oliwia; Tuchscherr, Lorena

    2017-10-20

    The extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of β-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.

  18. Fibroblastic synoviocytes secrete plasma proteins via α2 -macroglobulins serving as intracellular and extracellular chaperones.

    Science.gov (United States)

    Zhao, Ke-Wei; Murray, Elsa J Brochmann; Murray, Samuel S

    2015-11-01

    Changes in plasma protein levels in synovial fluid (SF) have been implicated in osteoarthritis and rheumatoid arthritis. It was previously thought that the presence of plasma proteins in SF reflected ultrafiltration or extravasation from the vasculature, possibly due to retraction of inflamed endothelial cells. Recent proteomic analyses have confirmed the abundant presence of plasma proteins in SF from control and arthritic patients. Systematic depletion of high-abundance plasma proteins from SF and conditioned media from synoviocytes cultured in serum, and protein analysis under denaturing/reducing conditions have limited our understanding of sources and the native structures of "plasma protein" complexes in SF. Using Western blotting, qPCR, and mass spectrometry, we found that Hig-82 lapine fibroblastic synovicytes cultured under serum-free conditions expressed and secreted plasma proteins, including the cytokine-binding protein secreted phosphoprotein 24 kDa (Spp24) and many of the proteases and protease inhibitors found in SF. Treating synoviocytes with TGF-β1 or BMP-2 for 24 h upregulated the expression of plasma proteins, including Spp24, α2 -HS-glycoprotein, α1 -antitrypsin, IGF-1, and C-reactive protein. Furthermore, many of the plasma proteins of mass <151 kDa were secreted as disulfide-bound complexes with members of the α2 -macroglobulin (A2M) family, which serve as intracellular and extracellular chaperones, not protease inhibitors. Using brefeldin A to block vesicular traffic and protease inhibitors to inhibit endogenous activation of naïve A2M, we demonstrated that the complexes were formed in the endoplasmic reticulum lumen and that Ca(2+) cysteine protease-dependent processes are involved. © Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

  19. Altered protein levels in the isolated extracellular matrix of failing human hearts with dilated cardiomyopathy.

    Science.gov (United States)

    DeAguero, Joshua L; McKown, Elizabeth N; Zhang, Liwen; Keirsey, Jeremy; Fischer, Edgar G; Samedi, Von G; Canan, Benjamin D; Kilic, Ahmet; Janssen, Paul M L; Delfín, Dawn A

    Dilated cardiomyopathy (DCM) is associated with extensive pathological cardiac remodeling and involves numerous changes in the protein expression profile of the extracellular matrix of the heart. We obtained seven human, end-stage, failing hearts with DCM (DCM-failing) and nine human, nonfailing donor hearts and compared their extracellular matrix protein profiles. We first showed that the DCM-failing hearts had indeed undergone extensive remodeling of the left ventricle myocardium relative to nonfailing hearts. We then isolated the extracellular matrix from a subset of these hearts and performed a proteomic analysis on the isolated matrices. We found that the levels of 26 structural proteins were altered in the DCM-failing isolated cardiac extracellular matrix compared to nonfailing isolated cardiac extracellular matrix. Overall, most of the extracellular matrix proteins showed reduced levels in the DCM-failing hearts, while all of the contractile proteins showed increased levels. There was a mixture of increased and decreased levels of cytoskeletal and nuclear transport proteins. Using immunoprobing, we verified that collagen IV (α2 and α6 isoforms), zyxin, and myomesin protein levels were reduced in the DCM-failing hearts. We expect that these data will add to the understanding of the pathology associated with heart failure with DCM. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins.

    Science.gov (United States)

    Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia; Navarro-Garcia, Fernando

    2017-03-28

    The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia, require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens.IMPORTANCE The type 3 secretion system is essential for injection of virulence factors, which are delivered directly into the cytosol of the host cells for usurping and subverting host processes. Recent studies have shown that these effectors proteins indeed travel inside an "injectisome" conduit through a single step of translocation by connecting the bacterium and host cell cytoplasms. However, all findings are not compatible with this model. For example, both YopH, a protein detected on the surface of Yersinia, and EspC, an autotransporter protein secreted by enteropathogenic E. coli, require a functional T3

  1. 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

  2. The potential for targeting extracellular LOX proteins in human malignancy

    DEFF Research Database (Denmark)

    Mayorca Guiliani, Alejandro Enrique; Erler, Janine T

    2013-01-01

    The extracellular matrix (ECM) is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX) enzymes cross-links collagens and elastin and, therefore...

  3. Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles.

    Science.gov (United States)

    Durcin, Maëva; Fleury, Audrey; Taillebois, Emiliane; Hilairet, Grégory; Krupova, Zuzana; Henry, Céline; Truchet, Sandrine; Trötzmüller, Martin; Köfeler, Harald; Mabilleau, Guillaume; Hue, Olivier; Andriantsitohaina, Ramaroson; Martin, Patrice; Le Lay, Soazig

    2017-01-01

    Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple

  4. Antibody Response to Human Extracellular HER2 Subdomain Proteins in Mice.

    Science.gov (United States)

    Sadri-Ardalani, Fateme; Ahmadi, Moslem; Hemmati, Azam; Emami, Shaghayegh; Farid, Samira; Amiri, Mohammad Mehdi; Jeddi-Tehrani, Mahmood; Shabani, Mahdi; Shokri, Fazel

    2017-06-01

    In addition to passive immunotherapy using anti-HER2 monoclonal antibodies, active immunotherapy via HER2 targeting is an interesting approach to inducing specific anti-tumor immune responses. We have recently reported the immunogenicity of HER2 subdomains following DNA immunization and HER2 protein boosting. In the present study, we evaluated the immunogenicity of different HER2 extracellular subdomains for the induction of anti-HER2 antibody response in BALB/c mice. To investigate and characterize antibody responses to human recombinant proteins of HER2 extracellular subdomains in immunized mice. Four subdomains of HER2 extracellular domain were expressed in E.coli; subsequently, purified recombinant proteins were intraperitoneally injected in BALB/c mice with Freund's adjuvant. The anti-HER2 antibody response was detected by ELISA, immunoblotting and flow cytometry. All the four HER2 subdomains along with the full extracellular domain (fECD) were able to induce specific anti-HER2 antibodies. Although anti-HER2 subdomains antibodies could not react with eukaryotic recombinant fECD protein by ELISA, they were able to recognize this protein by immunoblotting under both reduced and non-reduced conditions. Furthermore, only the sera of mice immunized with fECD protein could recognize native HER2 on HER2 overexpressing tumor cells (>99%) by flow cytometry. Moreover, fECD immunized mice sera inhibited the proliferation of tumor cells by XTT assay. The prokaryotic recombinant proteins of HER2 extracellular subdomains are immunogenic, yet the induced specific antibodies do not react with the native HER2 protein due to the paucity of post-translation modifications and /or distortion of the native conformation of isolated HER2 extracellular subdomains which might be potentially effective for induction of cell mediated immune response against HER2.

  5. Matrix gla protein: An extracellular matrix protein regulates myostatin expression in the muscle developmental program.

    Science.gov (United States)

    Ahmad, Sarafraz; Jan, Arif Tasleem; Baig, Mohammad Hassan; Lee, Eun Ju; Choi, Inho

    2017-03-01

    Skeletal muscle development involves interactions between intracellular and extracellular factors that act in concert to regulate the myogenic process. Matrix gla protein (MGP), a well-known inhibitor of calcification in soft tissues, has been reported to be highly up-regulated during myogenesis. Our interest in the regulation of muscle satellite cells (MSCs) by extracellular matrix (ECM) led us to investigate the effects of MGP during the progression of myogenesis. Participation of MGP in the myogenic process was investigated in vitro using C2C12 cells, and knockdown of its gene was performed to determine its effects on the expression of myogenic regulatory factors (MRFs) and other ECM genes. In addition, interactions between MGP, Fibromodulin (FMOD), and Myostatin (MSTN) were investigated by conducting co-immunoprecipitation and in silico studies. Matrix gla protein knockdown (MGPkd) shows pronounced effects during myogenesis as evidenced by the down regulation of myogenic marker (MYOG and MYOD), and ECM (COL1α1 and FMOD) genes. Down-regulation of MSTN expression in MGPkd cells suggests its role in coordinating the regulation of MSTN expression. Having strong affinity for ACVRIIB receptor, in silico data confirms MGP interference in the interaction of MSTN with ACVRIIB. These findings show MGP inhibits MSTN functionally by disrupting its binding to receptor. The present study provides insights of an ECM protein that participates in the regulation of the myogenic program by inhibiting the activity of the myogenic negative regulator MSTN, which suggests that MGP might be used for designing novel inhibitors that can promote muscle regeneration or treat muscle atrophy. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics.

    Science.gov (United States)

    Rocco, C J; Davey, M E; Bakaletz, L O; Goodman, S D

    2017-04-01

    Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co-aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Flocculation behaviour of hematite-kaolinite suspensions in presence of extracellular bacterial proteins and polysaccharides.

    Science.gov (United States)

    Poorni, S; Natarajan, K A

    2014-02-01

    Cells of Bacillus subtilis exhibited higher affinity towards hematite than to kaolinite. Bacterial cells were grown and adapted in the presence of hematite and kaolinite. Higher amounts of mineral-specific proteinaceous compounds were secreted in the presence of kaolinite while hematite-grown cells produced higher amounts of exopolysaccharides. Extracellular proteins (EP) exhibited higher adsorption density on kaolinite which was rendered more hydrophobic. Hematite surfaces were rendered more hydrophilic due to increased adsorption of extracellular polysaccharides (ECP). Significant surface chemical changes were produced due to interaction between minerals and extracellular proteins and polysaccharides. Iron oxides such as hematite could be effectively removed from kaolinite clays using selective bioflocculation of hematite after interaction with EP and ECP extracted from mineral-grown cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Extracellular Phosphate Induces the Expression of Dentin Matrix Protein 1 Through the FGF Receptor in Osteoblasts.

    Science.gov (United States)

    Nishino, Jin; Yamazaki, Miwa; Kawai, Masanobu; Tachikawa, Kanako; Yamamoto, Keiko; Miyagawa, Kazuaki; Kogo, Mikihiko; Ozono, Keiichi; Michigami, Toshimi

    2017-05-01

    Dentin matrix protein 1 (Dmp1) is an extracellular matrix protein involved in phosphate metabolism and biomineralization, and its expression markedly increases during the maturation of osteoblasts into osteocytes. We previously reported that an increased level of inorganic phosphate (Pi) in media up-regulated the expression of Dmp1 in primary osteocytes isolated from mouse bones. In the present study, we found that elevated extracellular Pi strongly induced the expression of Dmp1 in osteoblasts and explored its underlying mechanism of action. In an osteoblastic cell line MC3T3-E1, increases in extracellular Pi induced the phosphorylation of ERK1/2 and up-regulated the expression of Dmp1, fibroblast growth factor 2 (Fgf2), and Fgf receptor 1 (Fgfr1). A co-treatment with the MEK inhibitor U0126 abolished the increase in the expression of Dmp1 and Fgfr1 by elevated Pi, suggesting the involvement of the MEK/ERK pathway in this up-regulation. Elevated extracellular Pi also resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), which was diminished by knockdown of Slc20a1 encoding Pit1 sodium-phosphate co-transporter. The co-treatment with an inhibitor against FGFR (SU5402) abolished the up-regulation of Dmp1 induced by elevated extracellular Pi. In primary osteoblasts, a treatment with 4 mM Pi transiently increased the expression of early growth response 1 (Egr1) before the up-regulation of Dmp1. These results indicate that FGFR mediates the direct effects of extracellular Pi on the expression of Dmp1 in osteoblasts and enhance the close relationship between the signaling evoked by elevated extracellular Pi and FGF/FGFR signaling. J. Cell. Biochem. 118: 1151-1163, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. BIBF1120 inhibits fibroblasts proliferation and production of the extracellular matrix protein fibulin-1

    NARCIS (Netherlands)

    Burgess, Janette; Munk, Lizzie; Jaffar, Jade; Black, Judith; Oliver, Brian

    2015-01-01

    Introduction: In patients with idiopathic pulmonary fibrosis (IPF) transforming growth factorbeta 1 (TGFβ1) induces excessive extracellular matrix (ECM) protein deposition leading to fibrosis. Our recent studies have shown that the glycoprotein fibulin-1 is increased in serum and lung tissue from

  10. 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.

  11. Extracellular fluid proteins of goldfish brain: studies of concentration and labeling patterns.

    Science.gov (United States)

    Shashoua, V E

    1981-10-01

    An extraction procedure for the isolation of proteins from the extracellular fluid (ECF) of goldfish brain was developed and applied in an investigation of the time course and pattern of labeling of ECF proteins. The results indicate that two out of the many protein bands present, which migrated at 32,000 and 26,000 daltons on SDS-polyacrylamide electrophoretic gels, could incorporate as much as 50% of the label of the ECF fraction, even though their concentration was only 14%. Measurements of the protein content of the ECF and its volume (24% of the brain) by the inulin method were used to calculate the protein concentration in the extracellular space of goldfish brain. This gave a value of 1.6-2%, i.e., about 50% of the value obtained for the protein concentration of the cytoplasmic fraction devoid of particulate matter. Such a result suggests that the goldfish brain intracellular and extracellular fluids, separated by the neural membranes, contain relatively comparable levels of proteins.

  12. 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

  13. Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli.

    Science.gov (United States)

    Zhou, Yuling; Lu, Zhenghui; Wang, Xiang; Selvaraj, Jonathan Nimal; Zhang, Guimin

    2018-02-01

    As a common expression host, Escherichia coli has received more and more attention due to the recently developed secretory expression system, which offers advantages like reduced downstream bioprocesses and improved product quality. These advantages, coupled with high-density fermentation technology, make it a preferred system for large-scale production of many proteins utilized in industry and agriculture at a reduced process cost. To improve the secretion efficiency of target proteins, various strategies, including signal peptide optimization, periplasmic leakage, and chaperones co-expression have been developed. In addition, the optimization of the fermentation conditions such as temperature, inducer, and medium were also taken into account for the extracellular production in the high-density fermentation to reduce the cost of production. Here, these strategies ranging from genetic engineering to fermentation optimization were summarized for the future guidance of extracellular production of recombinant proteins using E. coli.

  14. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

    Science.gov (United States)

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

  15. Guanine derivatives modulate extracellular matrix proteins organization and improve neuron-astrocyte co-culture.

    Science.gov (United States)

    Decker, Helena; Francisco, Sheila S; Mendes-de-Aguiar, Cláudia B N; Romão, Luciana F; Boeck, Carina R; Trentin, Andréa G; Moura-Neto, Vivaldo; Tasca, Carla I

    2007-07-01

    Guanine derivatives (GD) have been shown to exert relevant extracellular effects as intercellular messengers, neuromodulators in the central nervous system, and trophic effects on astrocytes and neurons. Astrocytes have been pointed out as the major source of trophic factors in the nervous system, however, several trophic effects of astrocytic-released soluble factors are mediated through modulation of extracellular matrix (ECM) proteins. In this study, we investigated the effects of guanosine-5'-monophosphate (GMP) and guanosine (GUO) on the expression and organization of ECM proteins in cerebellar astrocytes. Moreover, to evaluate the effects of astrocytes pre-treated with GMP or GUO on cerebellar neurons we used a neuron-astrocyte coculture model. GMP or GUO alters laminin and fibronectin organization from a punctate to a fibrillar pattern, however, the expression levels of the ECM proteins were not altered. Guanine derivatives-induced alteration of ECM proteins organization is mediated by activation of mitogen activated protein kinases (MAPK), CA(2+)-calmodulin-dependent protein kinase II (CaMK-II), protein kinase C (PKC), and protein kinase A (PKA) pathways. Furthermore, astrocytes treated with GMP or GUO promoted an increased number of cerebellar neurons in coculture, without altering the neuritogenesis pattern. No proliferation of neurons or astrocytes was observed due to GMP or GUO treatment. Our results show that guanine derivatives promote a reorganization of the ECM proteins produced by astrocytes, which might be responsible for a better interaction with neurons in cocultures.

  16. Photorhabdus adhesion modification protein (Pam binds extracellular polysaccharide and alters bacterial attachment

    Directory of Open Access Journals (Sweden)

    Joyce Susan A

    2010-05-01

    Full Text Available Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C and human (37°C temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect

  17. Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment

    LENUS (Irish Health Repository)

    Jones, Robert T

    2010-05-12

    Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C) and human (37°C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of

  18. Nitric oxide diffusion to red blood cells limits extracellular, but not intraphagosomal, peroxynitrite formation by macrophages.

    Science.gov (United States)

    Prolo, Carolina; Álvarez, María Noel; Ríos, Natalia; Peluffo, Gonzalo; Radi, Rafael; Romero, Natalia

    2015-10-01

    Macrophage-derived nitric oxide ((•)NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by (•)NO itself or (•)NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O(2)(•-)). In vivo, the facile extracellular diffusion of (•)NO as well as different competing consumption routes limit its bioavailability for the reaction with O(2)(•-) and, hence, peroxynitrite formation. In this work, we evaluated the extent by which (•)NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O(2)(•-) and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of (•)NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of (•)NO and O(2)(•-)-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of (•)NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O(2)(•-) reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of (•)NO and O(2)(•-)-forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, (

  19. Paparan zat besi pada ekspresi protein spesifik extracellular polymeric substance biofilm Aggregatibacter actinomycetemcomitans

    Directory of Open Access Journals (Sweden)

    Marchella Hendrayanti W

    2014-06-01

    Full Text Available Background: The study of biofilms bacteria could be an alternative of preventive treatment in reducing prevalence of aggressive periodontitis in the community, because biofilm protects the bacteria from environmental conditions, including the attack of immune system and antimicrobial. Aggregatibacter actinomycetemcomitans is a major cause of bacterial aggressive periodontitis. Purpose: This study aims to examine the iron exposure to specific protein expression of extracellular polymeric substance (EPS of Aggregatibacter actinomycetemcomitans biofilm. Methods: Protein containing EPS biofilm was isolated from cultures of A.actinomycetemcomitans. The protein was processed through several procedures: electrophoresis , electroelution , immunization of rabbits , serum isolation , and purification of antibodies. After the Western blotting procedure the antibody was used. Protein containing EPS biofilms exposed to iron, then once again isolated from cultures of A. actinomycetemcomitans. The electrophoresis and Western blotting were done on the isolated protein. Results: The result showed that the the expression of specific proteins in EPS biofilm decreased in response to iron exposure. Conclusions: Iron exposure could influenced the specific protein expression in EPS biofilm of Aggregatibacter actinomycetemcomitans.Latar belakang: Penelitian terhadap bakteri biofilm dapat menjadi alternatif perawatan preventif dalam menurunkan prevalensi periodontitis agresif di masyarakat, karena biofilm melindungi bakteri terhadap kondisi lingkungan, termasuk serangan sistem imun dan antimikroba. Aggregatibacter actinomycetemcomitans merupakan bakteri penyebab utama periodontitis agresif. Tujuan: Studi ini bertujuan meneliti paparan zat besi terhadap ekspresi protein spesifik extracellular polymeric substance (EPS Aggregatibacter actinomycetemcomitans. Metode: Protein yang mengandung EPS biofilm diisolasi dari kultur A. actinomycetemcomitans. Protein yang diisolasi

  20. Extracellular vesicle-derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression.

    Science.gov (United States)

    Kim, Jung-Hwan; Jeun, Eun-Ji; Hong, Chun-Pyo; Kim, Seong-Hoon; Jang, Min Seong; Lee, Eun-Jung; Moon, Sook Jin; Yun, Chang Ho; Im, Sin-Hyeog; Jeong, Seok-Geun; Park, Beom-Young; Kim, Kyong-Tai; Seoh, Ju-Young; Kim, Yoon-Keun; Oh, Sung-Jong; Ham, Jun-Sang; Yang, Bo-Gie; Jang, Myoung Ho

    2016-02-01

    The incidence of food allergies has increased dramatically during the last decade. Recently, probiotics have been studied for the prevention and treatment of allergic disease. We examined whether Bifidobacterium longum KACC 91563 and Enterococcus faecalis KACC 91532 have the capacity to suppress food allergies. B longum KACC 91563 and E faecalis KACC 91532 were administered to BALB/c wild-type mice, in which food allergy was induced by using ovalbumin and alum. Food allergy symptoms and various immune responses were assessed. B longum KACC 91563, but not E faecalis KACC 91532, alleviated food allergy symptoms. Extracellular vesicles of B longum KACC 91563 bound specifically to mast cells and induced apoptosis without affecting T-cell immune responses. Furthermore, injection of family 5 extracellular solute-binding protein, a main component of extracellular vesicles, into mice markedly reduced the occurrence of diarrhea in a mouse food allergy model. B longum KACC 91563 induces apoptosis of mast cells specifically and alleviates food allergy symptoms. Accordingly, B longum KACC 91563 and family 5 extracellular solute-binding protein exhibit potential as therapeutic approaches for food allergies. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  1. Altered expression of extracellular matrix proteins and integrins in oral lichen planus (OLP).

    Science.gov (United States)

    Becker, J; Schuppan, D

    1995-04-01

    The immunohistochemical distribution of collagens type I, III, IV, V, VI, of undulin and tenascin, and of integrins alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and beta 4, was studied in 14 biopsies of oral lichen planus (OLP), 5 biopsies of orthokeratinized gingiva and 4 biopsies of oral fibrous hyperplasia. The localization of extracellular matrix proteins showed altered expression in OLP when compared to normal oral mucosa, with two principal patterns corresponding to the reticular or atrophic type. Whereas in the reticular type a focal loss of immunoreactivity for collagen types I, III, V, VI and undulin was noted in areas with a cellular infiltrate, in the atrophic variant almost complete loss of immunoreactivity of the subepithelial extracellular matrix was found. There was no clear correlation between the distribution of extracellular matrix proteins and their integrin receptors. The present findings suggest that the autoimmune reaction in OLP might not be primarily targeted to oral keratinocytes but to an unknown antigen in the connective tissue stroma. The changes in the subepithelial extracellular matrix associated with the inflammatory reaction might, especially in the atrophic variant, impair the cross-talk between epithelium and mesenchyme and favour both the loss of barrier function and the development of erosions in the clinical course of the disease.

  2. Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase System

    Science.gov (United States)

    Kim, Jongmin; Shin, Hyunwoo; Kim, Jiyoon; Kim, Junho; Park, Jaesung

    2015-01-01

    We present a simple and rapid method to isolate extracellular vesicles (EVs) by using a polyethylene glycol/dextran aqueous two-phase system (ATPS). This system isolated more than ~75% of melanoma-derived EVs from a mixture of EVs and serum proteins. To increase the purity of EVs, a batch procedure was combined as additional steps to remove protein contaminants, and removed more than ~95% of the protein contaminants. We also performed RT-PCR and western blotting to verify the diagnostic applicability of the isolated EVs, and detected mRNA derived from melanoma cells and CD81 in isolated EVs. PMID:26090684

  3. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Bokoch, Michael P; Zou, Yaozhong; Rasmussen, Søren Gøgsig Faarup

    2010-01-01

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation...... receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate...

  4. Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase System.

    Directory of Open Access Journals (Sweden)

    Jongmin Kim

    Full Text Available We present a simple and rapid method to isolate extracellular vesicles (EVs by using a polyethylene glycol/dextran aqueous two-phase system (ATPS. This system isolated more than ~75% of melanoma-derived EVs from a mixture of EVs and serum proteins. To increase the purity of EVs, a batch procedure was combined as additional steps to remove protein contaminants, and removed more than ~95% of the protein contaminants. We also performed RT-PCR and western blotting to verify the diagnostic applicability of the isolated EVs, and detected mRNA derived from melanoma cells and CD81 in isolated EVs.

  5. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.

    Directory of Open Access Journals (Sweden)

    Constantin F Urban

    2009-10-01

    Full Text Available Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs. NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

  6. Respiratory syncytial virus fusion protein promotes TLR-4-dependent neutrophil extracellular trap formation by human neutrophils.

    Directory of Open Access Journals (Sweden)

    Giselle A Funchal

    Full Text Available Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.

  7. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.

    Directory of Open Access Journals (Sweden)

    Daehwan Chung

    Full Text Available Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its

  8. CELL-DENSITY MODULATES GROWTH, EXTRACELLULAR-MATRIX, AND PROTEIN-SYNTHESIS OF CULTURED RAT MESANGIAL CELLS

    NARCIS (Netherlands)

    WOLTHUIS, A; BOES, A

    1993-01-01

    Mesangial cell (MC) hyperplasia and accumulation of extracellular matrix are hallmarks of chronic glomerular disease. The present in vitro study examined the effects of cell density on growth, extracellular matrix formation, and protein synthesis of cultured rat MCs. A negative linear relationship

  9. Prions on the run: How extracellular vesicles serve as delivery vehicles for self-templating protein aggregates.

    Science.gov (United States)

    Liu, Shu; Hossinger, André; Göbbels, Sarah; Vorberg, Ina M

    2017-03-04

    Extracellular vesicles (EVs) are actively secreted, membrane-bound communication vehicles that exchange biomolecules between cells. EVs also serve as dissemination vehicles for pathogens, including prions, proteinaceous infectious agents that cause transmissible spongiform encephalopathies (TSEs) in mammals. Increasing evidence accumulates that diverse protein aggregates associated with common neurodegenerative diseases are packaged into EVs as well. Vesicle-mediated intercellular transmission of protein aggregates can induce aggregation of homotypic proteins in acceptor cells and might thereby contribute to disease progression. Our knowledge of how protein aggregates are sorted into EVs and how these vesicles adhere to and fuse with target cells is limited. Here we review how TSE prions exploit EVs for intercellular transmission and compare this to the transmission behavior of self-templating cytosolic protein aggregates derived from the yeast prion domain Sup 35 NM. Artificial NM prions are non-toxic to mammalian cell cultures and do not cause loss-of-function phenotypes. Importantly, NM particles are also secreted in association with exosomes that horizontally transmit the prion phenotype to naive bystander cells, a process that can be monitored with high accuracy by automated high throughput confocal microscopy. The high abundance of mammalian proteins with amino acid stretches compositionally similar to yeast prion domains makes the NM cell model an attractive model to study self-templating and dissemination properties of proteins with prion-like domains in the mammalian context.

  10. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

    Directory of Open Access Journals (Sweden)

    Kosalková Katarina

    2012-01-01

    Full Text Available Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold in cells grown with casein or casein phosphopeptides (CPPs. CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs, whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase, which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  11. The changes of proteins and polysaccharides in extracellular polymeric substance for Spirogyra fluviatilis under different salinity

    Science.gov (United States)

    Lee, Yichao; Chang, Shuiping

    2017-05-01

    Spirogyra is a genus of widely distributed, large green fresh water algae. This study discovered that changes in salinity can induce Spirogyra fluviatilis to produce amounts of extracellular polymeric substance (EPS) when controlling other environmental conditions. If culturing S. fluviatilis with salinity greater than a 3.0‰ medium for 4 hours, the secretion EPS will be changed. And the level of polysaccharides and proteins, the primary components of EPS, is slightly increased in accordance with the increase in the salinity. But the proteins to polysaccharides ratio changes are not significantly

  12. Surface localized and extracellular Glyceraldehyde-3-phosphate dehydrogenase of Bacillus anthracis is a plasminogen binding protein.

    Science.gov (United States)

    Matta, Sumit Kumar; Agarwal, Shivangi; Bhatnagar, Rakesh

    2010-11-01

    The role of anchorless proteins on the surface of most pathogenic microorganisms has long been studied in context to their interactions with multiple host proteins, facilitating the dissemination of pathogen within the host tissues. In order to gain more insights into anthrax pathogenesis, we hereby report the presence of a prominent moonlighting enzyme, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface and in the extracellular medium of Bacillus anthracis. Out of the three heterologously expressed recombinant isoforms, rGapA (334 amino acids in native form; GapA) showed a significant NAD+ mediated GAPDH activity, whereas rGapB (342 amino acids in native form; GapB) showed a slight activity with NADP+. The rGapN (479 amino acids in native form; GapN) was enzymatically inactive with either NAD+ or NADP+. GapA was ascertained to be present in the extracellular medium and on the surface of B. anthracis. On the other hand, GapN was absent from both the surface and extracellular medium, whereas GapB was scarcely present on the surface of B. anthracis. Human plasminogen predominantly interacted with the rGapA isoform at physiological concentrations and the interaction was found to be lysine dependent. Immunization with rGapA resulted in a significant protection upon challenge with Bacillus anthracis in the murine model. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. A Protein Involved in the Assembly of an Extracellular Calcium Storage Matrix*

    Science.gov (United States)

    Glazer, Lilah; Shechter, Assaf; Tom, Moshe; Yudkovski, Yana; Weil, Simy; Aflalo, Eliahu David; Pamuru, Ramachandra Reddy; Khalaila, Isam; Bentov, Shmuel; Berman, Amir; Sagi, Amir

    2010-01-01

    Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBankTM data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate. PMID:20150428

  14. Single proteins that serve linked functions in intracellular and extracellular microenvironments

    Energy Technology Data Exchange (ETDEWEB)

    Radisky, Derek C.; Stallings-Mann, Melody; Hirai, Yohei; Bissell, Mina J.

    2009-06-03

    Maintenance of organ homeostasis and control of appropriate response to environmental alterations requires intimate coordination of cellular function and tissue organization. An important component of this coordination may be provided by proteins that can serve distinct, but linked, functions on both sides of the plasma membrane. Here we present a novel hypothesis in which non-classical secretion can provide a mechanism through which single proteins can integrate complex tissue functions. Single genes can exert a complex, dynamic influence through a number of different processes that act to multiply the function of the gene product(s). Alternative splicing can create many different transcripts that encode proteins of diverse, even antagonistic, function from a single gene. Posttranslational modifications can alter the stability, activity, localization, and even basic function of proteins. A protein can exist in different subcellular localizations. More recently, it has become clear that single proteins can function both inside and outside the cell. These proteins often lack defined secretory signal sequences, and transit the plasma membrane by mechanisms separate from the classical ER/Golgi secretory process. When examples of such proteins are examined individually, the multifunctionality and lack of a signal sequence are puzzling - why should a protein with a well known function in one context function in such a distinct fashion in another? We propose that one reason for a single protein to perform intracellular and extracellular roles is to coordinate organization and maintenance of a global tissue function. Here, we describe in detail three specific examples of proteins that act in this fashion, outlining their specific functions in the extracellular space and in the intracellular space, and we discuss how these functions may be linked. We present epimorphin/syntaxin-2, which may coordinate morphogenesis of secretory organs (as epimorphin) with control of

  15. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  16. Proteins of the extracellular fluid of mouse brain: extraction and partial characterization.

    Science.gov (United States)

    Hofstein, R; Hesse, G; Shashoua, V E

    1983-05-01

    An extraction procedure for the isolation of proteins from the brain extracellular fluid (ECF) was developed and applied to studies of the ECF components of mouse brain. Perfused intact brains were incubated in an isotonic medium for periods of up to 2 h at 0 degrees C to allow the release of ECF into the medium without disruption of the integrity of the tissue. The validity of the extraction procedure was established by (a) the fact that the total yield of ECF proteins was constant per unit weight of brain tissue, (b) the absence of tyrosine hydroxylase, an enzyme marker of the cytoplasmic fraction, from the extracts, and (c) the distinctive features of the one- and two-dimensional gel electrophoretic patterns of ECF proteins as compared with those of the cytoplasmic and membrane fractions. The results indicate that the extracellular fluid of mouse brain contains a mixture of proteins with a wide distribution of molecular weights (10,000-100,000 daltons) at a concentration level of about 0.3%.

  17. 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

  18. Extracellular volume quantification in isolated hypertension - changes at the detectable limits?

    Science.gov (United States)

    Treibel, Thomas A; Zemrak, Filip; Sado, Daniel M; Banypersad, Sanjay M; White, Steven K; Maestrini, Viviana; Barison, Andrea; Patel, Vimal; Herrey, Anna S; Davies, Ceri; Caulfield, Mark J; Petersen, Steffen E; Moon, James C

    2015-08-12

    Diffuse myocardial fibrosis (DMF) is important in cardiovascular disease, however until recently could only be assessed by invasive biopsy. We hypothesised that DMF measured by T1 mapping is elevated in isolated systemic hypertension. In a study of well-controlled hypertensive patients from a specialist tertiary centre, 46 hypertensive patients (median age 56, range 21 to 78, 52 % male) and 50 healthy volunteers (median age 45, range 28 to 69, 52 % male) underwent clinical CMR at 1.5 T with T1 mapping (ShMOLLI) using the equilibrium contrast technique for extracellular volume (ECV) quantification. Patients underwent 24-hours Automated Blood Pressure Monitoring (ABPM), echocardiographic assessment of diastolic function, aortic stiffness assessment and measurement of NT-pro-BNP and collagen biomarkers. Late gadolinium enhancement (LGE) revealed significant unexpected underlying pathology in 6 out of 46 patients (13 %; myocardial infarction n = 3; hypertrophic cardiomyopathy (HCM) n = 3); these were subsequently excluded. Limited, non-ischaemic LGE patterns were seen in 11 out of the remaining 40 (28 %) patients. Hypertensives on therapy (mean 2.2 agents) had a mean ABPM of 152/88 mmHg, but only 35 % (14/40) had left ventricular hypertrophy (LVH; LV mass male > 90 g/m(2); female > 78 g/m(2)). Native myocardial T1 was similar in hypertensives and controls (955 ± 30 ms versus 965 ± 38 ms, p = 0.16). The difference in ECV did not reach significance (0.26 ± 0.02 versus 0.27 ± 0.03, p = 0.06). In the subset with LVH, the ECV was significantly higher (0.28 ± 0.03 versus 0.26 ± 0.02, p infarction, HCM) not detected by echocardiography previously or even during this study. T1 mapping revealed increased diffuse myocardial fibrosis, but the increases were small and only occurred with LVH.

  19. A Novel Molecular Diagnostic of Glioblastomas: Detection of an Extracellular Fragment of Protein Tyrosine Phosphatase μ

    Directory of Open Access Journals (Sweden)

    Susan M. Burden-Gulley

    2010-04-01

    Full Text Available We recently found that normal human brain and low-grade astrocytomas express the receptor protein tyrosine phosphatase mu (PTPμ and that the more invasive astrocytomas, glioblastoma multiforme (GBM, downregulate full-length PTPμ expression. Loss of PTPμ expression in GBMs is due to proteolytic cleavage that generates an intracellular and potentially a cleaved and released extracellular fragment of PTPμ. Here, we identify that a cleaved extracellular fragment containing the domains required for PTPμ-mediated adhesion remains associated with GBM tumor tissue. We hypothesized that detection of this fragment would make an excellent diagnostic tool for the localization of tumor tissue within the brain. To this end, we generated a series of fluorescently tagged peptide probes that bind the PTPμ fragment. The peptide probes specifically recognize GBM cells in tissue sections of surgically resected human tumors. To test whether the peptide probes are able to detect GBM tumors in vivo, the PTPμ peptide probes were tested in both mouse flank and intracranial xenograft human glioblastoma tumor model systems. The glial tumors were molecularly labeled with the PTPμ peptide probes within minutes of tail vein injection using the Maestro FLEX In Vivo Imaging System. The label was stable for at least 3 hours. Together, these results indicate that peptide recognition of the PTPμ extracellular fragment provides a novel molecular diagnostic tool for detection of human glioblastomas. Such a tool has clear translational applications and may lead to improved surgical resections and prognosis for patients with this devastating disease.

  20. [myc-p62 fusion protein suppresses the phosphorylation of extracellular signal-regulated kinase].

    Science.gov (United States)

    Zhang, Yunjing; Feng, Yingying; Zhang, Li; Xu, Xiaojie; Wang, Tao; Fan, Zhongyi; Ye, Qinong; Zhang, Rong

    2015-05-01

    To construct a eukaryotic expression vector of human autophagy maker protein P62 labeled with myc tag, and detect its biological function. Human P62 gene was amplified from human breast DNA library by PCR and cloned into pXJ-40-myc vector. HEK293T cells were transfected with the recombinant plasmid myc-p62. Western blotting was conducted to detect the fusion protein expression and the effect on the phosphorylation of extracellular signal-regulated kinase (ERK). Double enzyme identification and sequencing result showed that P62 eukaryotic expression vector labeled with myc tag was successfully constructed and the inserted fragment was correct. Western blotting indicated that the fusion protein was successfully expressed and was able to inhibit ERK phosphorylation. The eukaryotic expression vector of myc-p62 was successfully constructed and proved to have an inhibitory effect on ERK phosphorylation.

  1. 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.

  2. 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.

  3. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Jinghuan Li

    2015-05-01

    Full Text Available Extracellular vesicles (EVs play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors, some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

  4. 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.

  5. 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.......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...

  6. Extracellular vesicles: a platform for the structure determination of membrane proteins by Cryo-EM.

    Science.gov (United States)

    Zeev-Ben-Mordehai, Tzviya; Vasishtan, Daven; Siebert, C Alistair; Whittle, Cathy; Grünewald, Kay

    2014-11-04

    Membrane protein-enriched extracellular vesicles (MPEEVs) provide a platform for studying intact membrane proteins natively anchored with the correct topology in genuine biological membranes. This approach circumvents the need to conduct tedious detergent screens for solubilization, purification, and reconstitution required in classical membrane protein studies. We have applied this method to three integral type I membrane proteins, namely the Caenorhabditis elegans cell-cell fusion proteins AFF-1 and EFF-1 and the glycoprotein B (gB) from Herpes simplex virus type 1 (HSV1). Electron cryotomography followed by subvolume averaging allowed the 3D reconstruction of EFF-1 and HSV1 gB in the membrane as well as an analysis of the spatial distribution and interprotein interactions on the membrane. MPEEVs have many applications beyond structural/functional investigations, such as facilitating the raising of antibodies, for protein-protein interaction assays or for diagnostics use, as biomarkers, and possibly therapeutics. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Pneumococcal Predictive Proteins Selected by Microbial Genomic Approach Are Serotype Cross-Reactive and Bind to Host Extracellular Matrix Proteins.

    Science.gov (United States)

    Argondizzo, Ana Paula Corrêa; Rocha-de-Souza, Cláudio Marcos; de Almeida Santiago, Marta; Galler, Ricardo; Reis, Joice Neves; Medeiros, Marco Alberto

    2017-08-01

    Streptococcus pneumoniae is a colonizer of the human nasopharynx, which accounts for most of the community-acquired pneumonia cases and can cause non-invasive and invasive diseases. Current available vaccines are serotype-specific and the use of recombinant proteins associated with virulence is an alternative to compose vaccines and to overcome these problems. In a previous work, we describe the identification of proteins in S. pneumoniae by reverse vaccinology and the genetic diversity of these proteins in clinical isolates. It was possible to purify a half of 20 selected proteins in soluble form. The expression of these proteins on the pneumococcal cells surface was confirmed by flow cytometry. We demonstrated that some of these proteins were able to bind to extracellular matrix proteins and were recognized by sera from patients with pneumococcal meningitis infection caused by several pneumococcal serotypes. In this context, our results suggest that these proteins may play a role in pneumococcal pathogenesis and might be considered as potential vaccine candidates.

  8. PYK2 mediates BzATP-induced extracellular matrix proteins synthesis.

    Science.gov (United States)

    Torigoe, Go; Nagao, Mayu; Tanabe, Natsuko; Manaka, Soichiro; Kariya, Taro; Kawato, Takayuki; Sekino, Jumpei; Kato, Shunichiro; Maeno, Masao; Suzuki, Naoto; Shimizu, Noriyoshi

    2017-12-16

    Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2'(3)-Ο-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts. BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression. These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion

    NARCIS (Netherlands)

    Ellen, Albert F.; Albers, Sonja-Verena; Driessen, Arnold J. M.

    Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three

  10. The ZP domain is a conserved module for polymerization of extracellular proteins.

    Science.gov (United States)

    Jovine, Luca; Qi, Huayu; Williams, Zev; Litscher, Eveline; Wassarman, Paul M

    2002-06-01

    Many eukaryotic extracellular proteins share a sequence of unknown function, called the zona pellucida (ZP) domain. Among these proteins are the mammalian sperm receptors ZP2 and ZP3, non-mammalian egg coat proteins, Tamm-Horsfall protein (THP), glycoprotein-2 (GP-2), alpha- and beta-tectorins, transforming growth factor (TGF)-beta receptor III and endoglin, DMBT-1 (deleted in malignant brain tumour-1), NompA (no-mechanoreceptor-potential-A), Dumpy and cuticlin-1 (refs 1,2). Here, we report that the ZP domain of ZP2, ZP3 and THP is responsible for polymerization of these proteins into filaments of similar supramolecular structure. Most ZP domain proteins are synthesized as precursors with carboxy-terminal transmembrane domains or glycosyl phosphatidylinositol (GPI) anchors. Our results demonstrate that the C-terminal transmembrane domain and short cytoplasmic tail of ZP2 and ZP3 are not required for secretion, but are essential for assembly. Finally, we suggest a molecular basis for dominant human hearing disorders caused by point mutations within the ZP domain of alpha-tectorin.

  11. Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease.

    Science.gov (United States)

    Pocsfalvi, Gabriella; Raj, Delfin A A; Fiume, Immacolata; Vilasi, Annalisa; Trepiccione, Francesco; Capasso, Giovambattista

    2015-06-01

    Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Extracellular fluid proteins of goldfish brain: evidence for the presence of proteases and esterases.

    Science.gov (United States)

    Shashoua, V E; Holmquist, B

    1986-09-01

    Preparations of enriched fractions of extracellular fluid (ECF) proteins from goldfish brain were found to contain protease(s) and esterase(s). The N-substituted furanacryloyl (FA) peptides FA-Phe-Gly-Gly and FA-Phe-OMe were used as model substrates for determining protease and esterase activity, respectively, in a spectrophotometric assay. Studies of the profile of substrate specificity and identification of the types of compounds that were effective as inhibitors showed that these ECF enzymes have some distinctive properties. GSH, but not GSSG, and EDTA inhibited the protease(s) without influencing the esterase(s), whereas L-1-tosylamide-2-phenylethylchloromethyl ketone blocked both protease and esterase activities of ECF. Most of the protease and esterase properties of ECF could be bound to concanavalin A-Sepharose affinity chromatographic columns in association with ependymin--a brain extracellular protein. These observations indicate that ECF may contain a metalloprotease(s) and raise the possibility that the ependymins might be a substrate for these ECF enzymes.

  13. Crystal structure of the extracellular domain of human myelin protein zero

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Brunzelle, Joseph S.; Kovari, Iulia A.; Sohi, Jasloveleen; Kamholz, John; Kovari, Ladislau C. (WSU-MED); (NWU)

    2012-03-27

    Charcot-Marie-Tooth disease (CMT), a hereditary motor and sensory neuropathy, is the most common genetic neuropathy with an incidence of 1 in 2600. Several forms of CMT have been identified arising from different genomic abnormalities such as CMT1 including CMT1A, CMT1B, and CMTX. CMT1 with associated peripheral nervous system (PNS) demyelination, the most frequent diagnosis, demonstrates slowed nerve conduction velocities and segmental demyelination upon nerve biopsy. One of its subtypes, CMT1A, presents a 1.5-Mb duplication in the p11-p12 region of the human chromosome 17 which encodes peripheral myelin protein 22 (PMP22). CMT1B, a less common form, arises from the mutations in the myelin protein zero (MPZ) gene on chromosome 1, region q22-q23, which encodes the major structural component of the peripheral myelin. A rare type of CMT1 has been found recently and is caused by point mutations in early growth response gene 2 (EGR2), encoding a zinc finger transcription factor in Schwann cells. In addition, CMTX, an X-linked form of CMT, arises from a mutation in the connexin-32 gene. Myelin protein zero, associated with CMT1B, is a transmembrane protein of 219 amino acid residues. Human MPZ consists of three domains: 125 residues constitute the glycosylated immunoglobulin-like extracellular domain; 27 residues span the membrane; and 67 residues comprise the highly basic intracellular domain. MPZ makes up approximately 50% of the protein content of myelin, and is expressed predominantly in Schwann cells, the myelinating cell of the PNS. Myelin protein zero, a homophilic adhesion molecule, is a member of the immunoglobulin super-family and is essential for normal myelin structure and function. In addition, MPZ knockout mice displayed abnormal myelin that severely affects the myelination pathway, and overexpression of MPZ causes congenital hypomyelination of peripheral nerves. Myelin protein zero mutations account for {approx}5% of patients with CMT. To date, over 125

  14. Extracellular Matrix Proteins Mediate HIV-1 gp120 Interactions with α4β7.

    Science.gov (United States)

    Plotnik, David; Guo, Wenjin; Cleveland, Brad; von Haller, Priska; Eng, Jimmy K; Guttman, Miklos; Lee, Kelly K; Arthos, James; Hu, Shiu-Lok

    2017-11-01

    Gut-homing α4β7high CD4+ T lymphocytes have been shown to be preferentially targeted by human immunodeficiency virus type 1 (HIV-1) and are implicated in HIV-1 pathogenesis. Previous studies demonstrated that HIV-1 envelope protein gp120 binds and signals through α4β7 and that this likely contributes to the infection of α4β7high T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α4β7 binding. However, lack of α4β7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α4β7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α4β7 interactions. We show that Chinese hamster ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that copurified with gp120. CHO cell fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to α4β7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to α4β7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion-exchange chromatography abrogated V2-independent gp120-α4β7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-α4β7 interactions similarly to CHO cell fibronectin. These findings provide an explanation for the apparently contradictory observations regarding the gp120-α4β7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology.IMPORTANCE Immune tissues within the gut are severely damaged by HIV-1, and this plays an important role in the development of AIDS. Integrin α4β7 plays a major role in the trafficking of lymphocytes, including CD4+ T cells, into gut lymphoid tissues. Previous reports

  15. The extracellular proteome of Bifidobacterium animalis subsp. lactis BB‐12 reveals proteins with putative roles in probiotic effects

    DEFF Research Database (Denmark)

    Gilad, Ofir; Svensson, Birte; Viborg, Alexander Holm

    2011-01-01

    Bifidobacterium animalis subsp. lactis BB‐12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2‐DE coupled with MALDI‐TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either...

  16. An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

    Directory of Open Access Journals (Sweden)

    Jessica McCready

    2014-04-01

    Full Text Available Extracellular Hsp90 (eHsp90 activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP and Lysyl oxidase 2-like protein (LOXL2 that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion.

  17. An Impermeant Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell Migration that Involves Lysyl Oxidase 2-like Protein

    Energy Technology Data Exchange (ETDEWEB)

    McCready, Jessica [Department of Natural Sciences, Assumption College, Worcester, MA 01609 (United States); Wong, Daniel S. [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States); Burlison, Joseph A.; Ying, Weiwen [Synta Pharmaceuticals, Lexington, MA 02421 (United States); Jay, Daniel G., E-mail: daniel.jay@tufts.edu [Department of Developmental Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Cell and Molecular Physiology Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111 (United States)

    2014-04-30

    Extracellular Hsp90 (eHsp90) activates a number of client proteins outside of cancer cells required for migration and invasion. Therefore, eHsp90 may serve as a novel target for anti-metastatic drugs as its inhibition using impermeant Hsp90 inhibitors would not affect the numerous vital intracellular Hsp90 functions in normal cells. While some eHsp90 clients are known, it is important to establish other proteins that act outside the cell to validate eHsp90 as a drug target to limit cancer spread. Using mass spectrometry we identified two precursor proteins Galectin 3 binding protein (G3BP) and Lysyl oxidase 2-like protein (LOXL2) that associate with eHsp90 in MDA-MB231 breast cancer cell conditioned media and confirmed that LOXL2 binds to eHsp90 in immunoprecipitates. We introduce a novel impermeant Hsp90 inhibitor STA-12-7191 derived from ganetespib and show that it is markedly less toxic to cells and can inhibit cancer cell migration in a dose dependent manner. We used STA-12-7191 to test if LOXL2 and G3BP are potential eHsp90 clients. We showed that while LOXL2 can increase wound healing and compensate for STA-12-7191-mediated inhibition of wound closure, addition of G3BP had no affect on this assay. These findings support of role for LOXL2 in eHsp90 stimulated cancer cell migration and provide preliminary evidence for the use of STA-12-7191 to inhibit eHsp90 to limit cancer invasion.

  18. RAGE-mediated extracellular matrix proteins accumulation exacerbates HySu-induced pulmonary hypertension.

    Science.gov (United States)

    Jia, Daile; He, Yuhu; Zhu, Qian; Liu, Huan; Zuo, Caojian; Chen, Guilin; Yu, Ying; Lu, Ankang

    2017-05-01

    Extracellular matrix (ECM) proteins accumulation contributes to the progression of pulmonary arterial hypertension (PAH), a rare and fatal cardiovascular condition defined by high pulmonary arterial pressure, whether primary, idiopathic, or secondary to other causes. The receptor for advanced glycation end products (RAGE) is constitutively expressed in the lungs and plays an important role in ECM deposition. Nonetheless, the mechanisms by which RAGE mediates ECM deposition/formation in pulmonary arteries and its roles in PAH progression remain unclear. Expression of RAGE and its activating ligands, S100/calgranulins and high mobility group box 1 (HMGB1), were increased in both human and mouse pulmonary arterial smooth muscle cells (PASMCs) under hypoxic conditions and were also strikingly upregulated in pulmonary arteries in hypoxia plus SU5416 (HySu)-induced PAH in mice. RAGE deletion alleviated pulmonary arterial pressure and restrained extracellular matrix accumulation in pulmonary arteries in HySu-induced PAH murine model. Moreover, blocking RAGE activity with a neutralizing antibody in human PASMCs, or RAGE deficiency in mouse PASMCs exposed to hypoxia, suppressed the expression of fibrotic proteins by reducing TGF-β1 expression. RAGE reconstitution in deficient mouse PASMCs restored hypoxia-stimulated TGF-β1 production via ERK1/2 and p38 MAPK pathway activation and subsequently increased ECM protein expression. Interestingly, HMGB1 acting on RAGE, not toll-like receptor 4 (TLR4), induced ECM deposition in PASMCs. Finally, in both idiopathic PAH patients and HySu-induced PAH mice, soluble RAGE (sRAGE) levels in serum were significantly elevated compared to those in controls. Activation of RAGE facilitates the development of hypoxia-induced pulmonary hypertension by increase of ECM deposition in pulmonary arteries. Our results indicate that sRAGE may be a potential biomarker for PAH diagnosis and disease severity, and that RAGE may be a promising target for

  19. An ensemble method with hybrid features to identify extracellular matrix proteins.

    Science.gov (United States)

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2015-01-01

    The extracellular matrix (ECM) is a dynamic composite of secreted proteins that play important roles in numerous biological processes such as tissue morphogenesis, differentiation and homeostasis. Furthermore, various diseases are caused by the dysfunction of ECM proteins. Therefore, identifying these important ECM proteins may assist in understanding related biological processes and drug development. In view of the serious imbalance in the training dataset, a Random Forest-based ensemble method with hybrid features is developed in this paper to identify ECM proteins. Hybrid features are employed by incorporating sequence composition, physicochemical properties, evolutionary and structural information. The Information Gain Ratio and Incremental Feature Selection (IGR-IFS) methods are adopted to select the optimal features. Finally, the resulting predictor termed IECMP (Identify ECM Proteins) achieves an balanced accuracy of 86.4% using the 10-fold cross-validation on the training dataset, which is much higher than results obtained by other methods (ECMPRED: 71.0%, ECMPP: 77.8%). Moreover, when tested on a common independent dataset, our method also achieves significantly improved performance over ECMPP and ECMPRED. These results indicate that IECMP is an effective method for ECM protein prediction, which has a more balanced prediction capability for positive and negative samples. It is anticipated that the proposed method will provide significant information to fully decipher the molecular mechanisms of ECM-related biological processes and discover candidate drug targets. For public access, we develop a user-friendly web server for ECM protein identification that is freely accessible at http://iecmp.weka.cc.

  20. Cutting edge: extracellular high mobility group box-1 protein is a proangiogenic cytokine.

    Science.gov (United States)

    Mitola, Stefania; Belleri, Mirella; Urbinati, Chiara; Coltrini, Daniela; Sparatore, Bianca; Pedrazzi, Marco; Melloni, Edon; Presta, Marco

    2006-01-01

    The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.

  1. Extracellular matrix proteins as temporary coating for thin-film neural implants

    Science.gov (United States)

    Ceyssens, Frederik; Deprez, Marjolijn; Turner, Neill; Kil, Dries; van Kuyck, Kris; Welkenhuysen, Marleen; Nuttin, Bart; Badylak, Stephen; Puers, Robert

    2017-02-01

    Objective. This study investigates the suitability of a thin sheet of extracellular matrix (ECM) proteins as a resorbable coating for temporarily reinforcing fragile or ultra-low stiffness thin-film neural implants to be placed on the brain, i.e. microelectrocorticographic (µECOG) implants. Approach. Thin-film polyimide-based electrode arrays were fabricated using lithographic methods. ECM was harvested from porcine tissue by a decellularization method and coated around the arrays. Mechanical tests and an in vivo experiment on rats were conducted, followed by a histological tissue study combined with a statistical equivalence test (confidence interval approach, 0.05 significance level) to compare the test group with an uncoated control group. Main results. After 3 months, no significant damage was found based on GFAP and NeuN staining of the relevant brain areas. Significance. The study shows that ECM sheets are a suitable temporary coating for thin µECOG neural implants.

  2. Interactions of human embryonic stem cell-derived cardiovascular progenitor cells with immobilized extracellular matrix proteins.

    Science.gov (United States)

    Lu, Jizhen; Kaestle, Katrin; Huang, Jijun; Liu, Qiao; Zhang, Peng; Gao, Ling; Gardiner, James; Thissen, Helmut; Yang, Huang-Tian

    2017-04-01

    Human embryonic stem cell-derived cardiovascular progenitor cells (hESC-CVPCs) hold great promise for cell-based therapies of heart diseases. However, little is known about their niche microenvironment and in particular the required extracellular matrix (ECM) components. Here we screened combinations of surface-immobilized ECM proteins to identify substrates that support the attachment and survival of hESC-CVPCs. Covalent immobilization of ECM proteins laminin (Lm), fibronectin (Fn), collagen I (CI), collagen III (CIII), and collagen IV (CIV) in multiple combinations and concentrations was achieved by reductive amination on transparent acetaldehyde plasma polymer (AAPP) interlayer coatings. We identified that CI, CIII, CIV, and Fn and their combinations were important for hESC-CVPC attachment and survival, while Lm was dispensable. Moreover, for coatings displaying single ECM proteins, CI and CIII performed better than CIV and Fn, while coatings displaying the combined ECM proteins CIII + CIV and Fn + CIII + CIV at 100 µg/mL were comparable to Matrigel in regard to supporting hESC-CVPC attachment and viability. Our results identify ECM proteins required for hESC-CVPCs and demonstrate that coatings displaying multiple immobilized ECM proteins offer a suitable microenvironment for the attachment and survival of hESC-CVPCs. This knowledge contributes to the development of approaches for maintaining hESC-CVPCs and therefore to advances in cardiovascular regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1094-1104, 2017. © 2017 Wiley Periodicals, Inc.

  3. Disruption of microbial biofilms by an extracellular protein isolated from epibiotic tropical marine strain of Bacillus licheniformis

    Digital Repository Service at National Institute of Oceanography (India)

    Dusane, D.H.; Damare, S.R.; Nancharaiah, Y.V.; Ramaiah, N.; Venugopalan, V.P.; Kumar, A.R.; Zinjarde, S.S.

    Biofilms by an Extracellular Protein Isolated from Epibiotic Tropical Marine Strain of Bacillus licheniformis Devendra H. Dusane1¤, Samir R. Damare2, Yarlagadda V. Nancharaiah3, N. Ramaiah2, Vayalam P. Venugopalan3, Ameeta Ravi Kumar1, Smita S. Zinjarde1* 1..., Damare SR, Nancharaiah YV, Ramaiah N, Venugopalan VP, et al. (2013) Disruption of Microbial Biofilms by an Extracellular Protein Isolated from Epibiotic Tropical Marine Strain of Bacillus licheniformis. PLoS ONE 8(5): e64501. doi:10.1371/journal...

  4. Comparative profiling of extractable proteins in extracellular matrices of porcine cholecyst and jejunum intended for preparation of tissue engineering scaffolds.

    Science.gov (United States)

    Muhamed, Jaseer; Rajan, Akhila; Surendran, Arun; Jaleel, Abdul; Anilkumar, Thapasimuthu V

    2017-04-01

    Scaffolds prepared from cholecyst and jejunum have differential immunological potential, despite similar biocompatibility, when used as subcutaneous grafts. The reason for differential immunogenicity is probably due to differences in the nature of protein composition and biomolecules in the extracellular matrices (ECMs) of source organs that are used for preparation of the scaffolds. Against this background, the present study aims to identify the extractable proteins of ECMs derived from porcine cholecyst and jejunum. The proteins were extracted and identified through a conventional database search following sodium dodecyl sulfate-polyacrylamide gel-electrophoresis separation and mass spectroscopy. The resultant protein profile was analyzed and at least 154 proteins in cholecyst-derived extracellular matrix (CDE) and 186 proteins in jejunum-derived extracellular matrix (JDE) were identified. Both the matrices contained several extracelluar proteins including fibronectin, nidogen, decorin, and lumican that are known to participate in wound healing responses. However, the CDE had fewer cellular proteins than JDE, especially the latter contained class-I and class-II histocompatibility antigens which are incriminated as potent immunogens responsible for graft rejection. The results of the study suggested that the ECMs used for the scaffold preparation need not be "acellular" and differences in the protein composition of the ECMs might have caused the differential wound healing responses. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 489-496, 2017. © 2015 Wiley Periodicals, Inc.

  5. Expression of Extracellular Matrix Proteins in Basal Membranes During Fetal Nephron Development in Mice

    Directory of Open Access Journals (Sweden)

    Beyhan GÜRCÜ

    2016-04-01

    Full Text Available In this study, we investigated the distribution of laminin, collagen type IV, nidogen and fibronectine during metanephric development in fetal mouse kidney by immunohistochemistry. Stain density of basement membranes of tubules, glomerules and mesangial matrix were compared in pre-capillary, immature glomerular and mature glomerular stages of fetal kidney. All the matrix proteins were strongly stained in precapillary stage. In immature glomerular stage, a strong staining was observed for fibronectin. Staining intensity was slightly decreased for the other proteins in this stage. In mature glomerular stage, diminished staining for all proteins was observed similar to the previous stage, except fibronectin. The strongest immunoreactions were found for fibronectin and nidogen in all investigated stages. In general, there was a similar staining intensity for all glycoproteins during maturation except for laminin. It was thought that the distribution of extracellular matrix molecules plays an important role for the kidney development. Interactions amoung these molecules probably crucial on cell behavior like migration, proliferation and differentiation in normal development of the nephron.

  6. Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C.

    Science.gov (United States)

    Su, Lingqia; Jiang, Qi; Yu, Lingang; Wu, Jing

    2017-02-08

    Our laboratory has reported a strategy for improving the extracellular production of recombinant proteins through co-expression with Thermobifida fusca cutinase, which increases membrane permeability via its phospholipid hydrolysis activity. However, the foam generated by the lysophospholipid product makes the fermentation process difficult to control in a fermentor. Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce sn1,2-diacylglycerides and organic phosphate, which do not induce foam formation. Therefore, co-expression with Bacillus cereus PLC was investigated as a method to improve the extracellular production of recombinant proteins. When B. cereus PLC was expressed in Escherichia coli without its signal peptide, 95.3% of the total PLC activity was detected in the culture supernatant. PLC expression enhanced membrane permeability without obvious cell lysis. Then, six test enzymes, three secretory and three cytosolic, were co-expressed with B. cereus PLC. The enhancement of extracellular production correlated strongly with the molecular mass of the test enzyme. Extracellular production of Streptomyces sp. FA1 xylanase (43 kDa), which had the lowest molecular mass among the secretory enzymes, was 4.0-fold that of its individual expression control. Extracellular production of glutamate decarboxylase (51 kDa), which had the lowest molecular mass among the cytosolic enzymes, reached 26.7 U/mL; 88.3% of the total activity produced. This strategy was effectively scaled up using a 3-L fermentor. No obvious foam was generated during this fermentation process. This is the first study to detail the enhanced extracellular production of recombinant proteins through co-expression with PLC. This new strategy, which is especially appropriate for lower molecular mass proteins, allows large-scale protein production in an easily controlled fermentation process.

  7. Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects

    Energy Technology Data Exchange (ETDEWEB)

    Desai, Jayashree [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Shannon, Mark E. [Applied Biosystems; Johnson, Mahlon D. [University of Tennessee Graduate School of Medicine; Ruff, David W. [Applied Biosystems; Hughes, Lori A [ORNL; Kerley, Marilyn K [ORNL; Carpenter, D A [ORNL; Johnson, Dabney K [ORNL; Rinchik, Eugene M. [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Culiat, Cymbeline T [ORNL

    2006-01-01

    The mammalian Nell1 gene encodes a protein kinase C-b1 (PKC-b1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell16R, a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell16R has a T!A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell16R mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell16R mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

  8. Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

    Science.gov (United States)

    Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

    2017-10-01

    Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant

  9. Proteomic analysis of ovarian cancer cells reveals dynamic processes of protein secretion and shedding of extra-cellular domains.

    Directory of Open Access Journals (Sweden)

    Vitor M Faça

    Full Text Available BACKGROUND: Elucidation of the repertoire of secreted and cell surface proteins of tumor cells is relevant to molecular diagnostics, tumor imaging and targeted therapies. We have characterized the cell surface proteome and the proteins released into the extra-cellular milieu of three ovarian cancer cell lines, CaOV3, OVCAR3 and ES2 and of ovarian tumor cells enriched from ascites fluid. METHODOLOGY AND FINDINGS: To differentiate proteins released into the media from protein constituents of media utilized for culture, cells were grown in the presence of [(13C]-labeled lysine. A biotinylation-based approach was used to capture cell surface associated proteins. Our general experimental strategy consisted of fractionation of proteins from individual compartments followed by proteolytic digestion and LC-MS/MS analysis. In total, some 6,400 proteins were identified with high confidence across all specimens and fractions. CONCLUSIONS AND SIGNIFICANCE: Protein profiles of the cell lines had substantial similarity to the profiles of human ovarian cancer cells from ascites fluid and included protein markers known to be associated with ovarian cancer. Proteomic analysis indicated extensive shedding from extra-cellular domains of proteins expressed on the cell surface, and remarkably high secretion rates for some proteins (nanograms per million cells per hour. Cell surface and secreted proteins identified by in-depth proteomic profiling of ovarian cancer cells may provide new targets for diagnosis and therapy.

  10. Staphylococcus aureus biofilm removal by targeting biofilm-associated extracellular proteins

    Directory of Open Access Journals (Sweden)

    Sudhir K Shukla

    2017-01-01

    Methods: Biofilm assay was done in 96-well microtitre plate to evaluate the effect of proteinase K on biofilms of bovine mastitis S. Aureus isolates. Extracellular polymeric substances were extracted and evaluated for their composition (protein, polysaccharides and extracellular DNA, before and after the proteinase K treatment. Results: Biofilm assay showed that 2 μg/ml proteinase K significantly inhibited biofilm development in bap-positive S. aureus V329 as well as other S. aureus isolates (SA7, SA10, SA33, SA352, but not in bap-mutant M556 and SA392 (a weak biofilm-producing strain. Proteinase K treatment on S. aureus planktonic cells showed that there was no inhibition of planktonic growth up to 32 μg/ml of proteinase K. Proteinase K treatment on 24 h old preformed biofilms showed an enhanced dispersion of bap-positive V329 and SA7, SA10, SA33 and SA352 biofilms; however, proteinase K did not affect the bap-mutant S. aureus M556 and SA392 biofilms. Biofilm compositions study before and after proteinase K treatment indicated that Bap might also be involved in eDNA retention in the biofilm matrix that aids in biofilm stability. When proteinase K was used in combination with antibiotics, a synergistic effect in antibiotic efficacy was observed against all biofilm-forming S. aureus isolates. Interpretation & conclusions: Proteinase K inhibited biofilms growth in S. aureus bovine mastitis isolates but did not affect their planktonic growth. An enhanced dispersion of preformed S. aureus biofilms was observed on proteinase K treatment. Proteinase K treatment with antibiotics showed a synergistic effect against S. aureus biofilms. The study suggests that dispersing S. aureus by protease can be of use while devising strategies againstS. aureus biofilms.

  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...

  12. Epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting S100A4 protein

    DEFF Research Database (Denmark)

    Klingelhöfer, Jörg; Møller, Henrik D.; Sumer, Eren U

    2009-01-01

    The function of S100A4, a member of the calcium-binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro-metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms...

  13. Shewanella Oneidensis MR-1 Msh Pilin Proteins are Involved in Extracellular Electron Transfer in Microbial Fuel Cells

    Science.gov (United States)

    2011-01-01

    purchased from ATCC (700550) and the two Msh deletion mutants were gifts from Dr. Daad Saffarini (University of Wisconsin-Milwaukee). In-frame chromosomal...than the actual extracellular structural pilin proteins (nanofilaments). Acknowledgments We would like to thank Daad Saffarini, Rachida Bouhenni and

  14. The retention of extracellular matrix proteins and angiogenic and mitogenic cytokines in a decellularized porcine dermis.

    Science.gov (United States)

    Hoganson, David M; O'Doherty, Elisabeth M; Owens, Gwen E; Harilal, Dina O; Goldman, Scott M; Bowley, Chris M; Neville, Craig M; Kronengold, Russell T; Vacanti, Joseph P

    2010-09-01

    Decellularized dermis materials demonstrate considerable utility in surgical procedures including hernia repair and breast reconstruction. A new decellularized porcine dermis material has been developed that retains many native extracellular matrix (ECM) proteins and cytokines. This material has substantial mechanical strength with maximum tensile strength of 141.7 +/- 85.4 (N/cm) and suture pull through strength of 47.0 +/- 14.0 (N). After processing, many ECM proteins remained in the material including collagen III, collagen IV, collagen VII, laminin and fibronectin. Glycosaminoglycans, including hyaluronic acid, were also preserved. Among several cytokines whose levels were quantified, more vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-beta) were retained within this material than in comparable decellularized dermis materials. The retention of bioactivity was demonstrated in a cell culture assay. Because this decellularized porcine dermis material both retains significant strength and has substantial biological activity, it may promote rapid integration and repair in clinical applications. Copyright 2010. Published by Elsevier Ltd.

  15. Extracellular Vesicles from Parasitic Helminths Contain Specific Excretory/Secretory Proteins and Are Internalized in Intestinal Host Cells

    Science.gov (United States)

    Marcilla, Antonio; Trelis, María; Cortés, Alba; Sotillo, Javier; Cantalapiedra, Fernando; Minguez, María Teresa; Valero, María Luz; Sánchez del Pino, Manuel Mateo; Muñoz-Antoli, Carla; Toledo, Rafael; Bernal, Dolores

    2012-01-01

    The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30–100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents. PMID:23029346

  16. Extracellular matrix protein in calcified endoskeleton: a potential additive for crystal growth and design

    Science.gov (United States)

    Azizur Rahman, M.; Fujimura, Hiroyuki; Shinjo, Ryuichi; Oomori, Tamotsu

    2011-06-01

    In this study, we demonstrate a key function of extracellular matrix proteins (ECMPs) on seed crystals, which are isolated from calcified endoskeletons of soft coral and contain only CaCO 3 without any living cells. This is the first report that an ECMP protein extracted from a marine organism could potentially influence in modifying the surface of a substrate for designing materials via crystallization. We previously studied with the ECMPs from a different type of soft coral ( Sinularia polydactyla) without introducing any seed crystals in the process , which showed different results. Thus, crystallization on the seed in the presence of ECMPs of present species is an important first step toward linking function to individual proteins from soft coral. For understanding this interesting phenomenon, in vitro crystallization was initiated in a supersaturated solution on seed particles of calcite (1 0 4) with and without ECMPs. No change in the crystal growth shape occurred without ECMPs present during the crystallization process. However, with ECMPs, the morphology and phase of the crystals in the crystallization process changed dramatically. Upon completion of crystallization with ECMPs, an attractive crystal morphology was found. Scanning electron microscopy (SEM) was utilized to observe the crystal morphologies on the seeds surface. The mineral phases of crystals nucleated by ECMPs on the seeds surface were examined by Raman spectroscopy. Although 50 mM Mg 2+ is influential in making aragonite in the crystallization process, the ECMPs significantly made calcite crystals even when 50 mM Mg 2+ was present in the process. Crystallization with the ECMP additive seems to be a technically attractive strategy to generate assembled micro crystals that could be used in crystals growth and design in the Pharmaceutical and biotechnology industries.

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

    Science.gov (United States)

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

    2008-03-01

    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.

  18. Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin

    DEFF Research Database (Denmark)

    Barnea, G; Grumet, M; Milev, P

    1994-01-01

    The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically...... labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous...

  19. Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    DEFF Research Database (Denmark)

    Rossol, Manuela; Pierer, Matthias; Raulien, Nora

    2012-01-01

    Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1ß during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular...... calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca(2+) pathway. The resulting increase in the intracellular calcium concentration...

  20. Limited hydrolysis of soybean protein concentrate and isolate with ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-20

    Jul 20, 2009 ... Soy protein concentrate (SPC) and soy protein isolate (SPI) were limited hydrolyzed with trypsin or neutrase under controlled hydrolysis conditions. Eight soybean protein hydrolysates, 4 SPC hydroly- sates and 4 SPI hydrolysates were prepared with degree of hydrolysis (DH) of 1 or 2%. SDS-PAGE was.

  1. 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.

  2. Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering*

    Science.gov (United States)

    Hill, Ryan C.; Calle, Elizabeth A.; Dzieciatkowska, Monika; Niklason, Laura E.; Hansen, Kirk C.

    2015-01-01

    The use of extracellular matrix (ECM)1 scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs. PMID:25660013

  3. Quantification of extracellular matrix proteins from a rat lung scaffold to provide a molecular readout for tissue engineering.

    Science.gov (United States)

    Hill, Ryan C; Calle, Elizabeth A; Dzieciatkowska, Monika; Niklason, Laura E; Hansen, Kirk C

    2015-04-01

    The use of extracellular matrix (ECM) scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin

    Science.gov (United States)

    Tseng, Boo Shan; Zhang, Wei; Harrison, Joe J.; Quach, Tam P.; Song, Jisun Lee; Penterman, Jon; Singh, Pradeep K.; Chopp, David L.; Packman, Aaron I.; Parsek, Matthew R.

    2013-01-01

    SUMMARY Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the biofilm matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance, as previous studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid P. aeruginosa biofilms. To our surprise, we showed that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions, but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration. PMID:23751003

  5. Interaction of silver nanoparticles (SNPs) with bacterial extracellular proteins (ECPs) and its adsorption isotherms and kinetics.

    Science.gov (United States)

    Khan, S Sudheer; Srivatsan, P; Vaishnavi, N; Mukherjee, Amitava; Chandrasekaran, N

    2011-08-15

    Indiscriminate and increased use of silver nanoparticles (SNPs) in consumer products leads to the release of it into the environment. The fate and transport of SNPs in environment remains unknown. We have studied the interaction of SNPs with extracellular protein (ECP) produced by two environmental bacterial species and the adsorption behavior in aqueous solutions. The effect of pH and salt concentrations on the adsorption was also investigated. The adsorption process was found to be dependent on surface charge (zeta potential). The capping of SNPs by ECP was confirmed by Fourier transform infrared spectroscopy and X-ray diffraction. The adsorption of ECP on SNPs was analyzed by Langmuir and Freundlich models, suggesting that the equilibrium adsorption data fitted well with Freundlich model. The equilibrium adsorption data were modeled using the pseudo-first-order and pseudo-second-order kinetic equations. The results indicated that pseudo-second-order kinetic equation would better describe the adsorption kinetics. The capping was stable at environmental pH and salt concentration. The destabilization of nanoparticles was observed at alkaline pH. The study suggests that the stabilization of nanoparticles in the environment might lead to the accumulation and transport of nanomaterials in the environment, and ultimately destabilizes the functioning of the ecosystem. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    Energy Technology Data Exchange (ETDEWEB)

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Neto Paiva, Claudia; Torres Bozza, Marcelo [Departamento de Imunologia, Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil); Rosado Fantappie, Marcelo, E-mail: fantappie@bioqmed.ufrj.br [Instituto de Bioquimica Medica, Programa de Biotecnologia e Biologia Molecular, Universidade Federal do Rio de Janeiro, CCS, Ilha do Fundao, Rio de Janeiro 21941-590 (Brazil)

    2009-12-25

    Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1{Delta}C) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1{Delta}C were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

  7. Cannabinoid inhibits HIV-1 Tat-stimulated adhesion of human monocyte-like cells to extracellular matrix proteins

    Science.gov (United States)

    Raborn, Erinn S.; Jamerson, Melissa; Marciano-Cabral, Francine; Cabral, Guy A.

    2014-01-01

    Aims The aim of this study was to assess the effect of select cannabinoids on human immunodeficiency virus type 1 (HIV-1) transactivating (Tat) protein-enhanced monocyte-like cell adhesion to proteins of the extracellular matrix (ECM). Main Methods Collagen IV, laminin, or an ECM gel were used to construct extracellular matrix layers. Human U937 monocyte-like cells were exposed to Tat in the presence of Δ9-tetrahydrocannabinol (THC), CP55,940, and other select cannabinoids. Cell attachment to ECM proteins was assessed using an adhesion assay. Key findings THC and CP55,940 inhibited Tat-enhanced attachment of U937 cells to ECM proteins in a mode that was linked to the cannabinoid receptor type 2 (CB2R). The cannabinoid treatment of Tat-activated U937 cells was associated with altered β1-integrin expression and distribution of polymerized actin, suggesting a modality by which these cannabinoids inhibited adhesion to the ECM. Significance The blood-brain barrier (BBB) is a complex structure that is composed of cellular elements and an extracellular matrix (ECM). HIV-1 Tat promotes transmigration of monocytes across this barrier, a process that includes interaction with ECM proteins. The results indicate that cannabinoids that activate the CB2R inhibit the ECM adhesion process. Thus, this receptor has potential to serve as a therapeutic agent for ablating neuroinflammation associated with HIV-elicited influx of monocytes across the BBB. PMID:24742657

  8. Fungal degradation of wood: initial proteomic analysis of extracellular proteins of Phanerochaete chrysosporium grown on oak substrate.

    Science.gov (United States)

    Abbas, Ahmed; Koc, Hasan; Liu, Feng; Tien, Ming

    2005-01-01

    Two-dimensional (2-D) gel electrophoresis was used to separate the extracellular proteins produced by the white-rot fungus Phanerochaete chrysosporium. Solid-substrate cultures grown on red oak wood chips yielded extracellular protein preparations which were not suitable for 2-D gel analysis. However, pre-washing the wood chips with water helped decrease the amount of brown material which caused smearing on the acidic side of the isoelectric focusing gel. The 2-D gels from these wood-grown cultures revealed more than 45 protein spots. These spots were subjected to in-gel digestion with trypsin followed by either peptide fingerprint analysis by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or by liquid chromatography (LC)/MS/MS sequencing. Data from both methods were analyzed by Protein Prospector and the local P. chrysosporium annotated database. MALDI-TOF/MS only identified two proteins out of 25 analyzed. This was most likely due to problems associated with glycosylation. Protein sequencing by LC/MS/MS of the same 25 proteins resulted in identification of 16 proteins. Most of the proteins identified act on either cellulose or hemicellulose or their hydrolysis products. Thus far no lignin peroxidase, Mn peroxidase or laccases have been detected.

  9. Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

    Science.gov (United States)

    Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

    2017-02-01

    Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. Copyright © 2017 the American Physiological Society.

  10. Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

    Science.gov (United States)

    Luo, Ting; Krüger, Thomas; Knüpfer, Uwe; Kasper, Lydia; Wielsch, Natalie; Hube, Bernhard; Kortgen, Andreas; Bauer, Michael; Giamarellos-Bourboulis, Evangelos J; Dimopoulos, George; Brakhage, Axel A; Kniemeyer, Olaf

    2016-08-05

    During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera.

  11. Extracellular administration of BCL2 protein reduces apoptosis and improves survival in a murine model of sepsis.

    Directory of Open Access Journals (Sweden)

    Akiko Iwata

    2011-02-01

    Full Text Available Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival.We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2 family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP. We show that intraperitoneal injection of picomole range doses of recombinant human (rh BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo.Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins.

  12. 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

  13. 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.

  14. 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.

  15. Histochemical study of fibrillar proteins of the extracellular matrix in benign and malignant mammary neoplasms in dogs

    OpenAIRE

    Martins,Ana Maria Cristina Rabello Pinto da Fonseca; Tamaso,Elia; Guerra,José Luiz

    2002-01-01

    The aim of the present study was to study some of the proteins that form the extracellular matrix of 54 benign and malignant mammary neoplasms in dogs, using histochemical methods: Picrosirius and polarization microscopy for collagenous fibers, Gordon -Sweats's method for reticular fibers and Weigert's fucsine-resorcine method for elastic fibers. A large variability in quantity, distribution and characteristics of the matrix components was observed in the different types of neoplasms. Collage...

  16. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    DEFF Research Database (Denmark)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig

    2002-01-01

    the extracellular signal-regulated kinase (ERK) cascade to regulate GRK2 cellular levels. ERK activation by receptor stimulation elevated endogenous GRK2 while antagonist treatment decreased cellular GRK2. Activating ERK by overexpressing constitutive active MEK-1 or Ras elevated GRK2 protein levels while blocking...... ERK using PD98059 or dominant negative Ras abolished this effect. These data suggest ERK is a critical regulator of GRK2 levels....

  17. Inhibition of histone deacetylase activity in human endometrial stromal cells promotes extracellular matrix remodelling and limits embryo invasion.

    Directory of Open Access Journals (Sweden)

    Carlos Estella

    Full Text Available Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs to degrade extracellular matrix (ECM proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs. The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion.

  18. Inhibition of Histone Deacetylase Activity in Human Endometrial Stromal Cells Promotes Extracellular Matrix Remodelling and Limits Embryo Invasion

    Science.gov (United States)

    Atkinson, Stuart P.; Quiñonero, Alicia; Martínez, Sebastián; Pellicer, Antonio; Simón, Carlos

    2012-01-01

    Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs) to degrade extracellular matrix (ECM) proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs). The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs) that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs) related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA) increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion. PMID:22291969

  19. HES1 promotes extracellular matrix protein expression and inhibits proliferation and migration in human trabecular meshwork cells under oxidative stress.

    Science.gov (United States)

    Xu, Linqi; Zhang, Yan; Guo, Ruru; Shen, Wencui; Qi, Yan; Wang, Qingsong; Guo, Zhenglong; Qi, Chen; Yin, Haifang; Wang, Jiantao

    2017-03-28

    Glaucoma is the leading cause of irreversible blindness. The most prevalent form of glaucoma is primary open-angle glaucoma (POAG). Oxidative stress is one of the major pathogenic factors of the POAG, and can elicit molecular and functional changes in trabecular meshwork cells, causing increased aqueous humor outflow resistance and elevated intraocular pressure. However, the regulatory mechanisms underlying oxidative stress-induced cell phenotypic changes remain elusive. Herein, we exposed primary human trabecular meshwork cells to the oxidative stress induced by 300 μM H2O2 for 2 h, and found significantly up-regulated expression of extracellular matrix proteins and a transcription factor, hairy and enhancer of split-1 (HES1). The cell functions, including migration and proliferation, were impaired by the oxidative stress. Furthermore, HES1 shRNA abrogated the extracellular matrix protein up-regulation and rescued the functional defects caused by the oxidative stress; conversely, HES1 overexpression resulted in the molecular and functional changes similar to those induced by H2O2. These results suggest that HES1 promotes extracellular matrix protein expression and inhibits proliferative and migratory functions in the trabecular meshwork cells under oxidative stress, thereby providing a novel pathogenic mechanism underlying and a potential therapeutic target to the POAG.

  20. A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix

    Science.gov (United States)

    Shechter, Assaf; Glazer, Lilah; Cheled, Shira; Mor, Eyal; Weil, Simy; Berman, Amir; Bentov, Shmuel; Aflalo, Eliahu D.; Khalaila, Isam; Sagi, Amir

    2008-01-01

    Despite the proclamation of Lowenstam and Weiner that crustaceans are the “champions of mineral mobilization and deposition of the animal kingdom,” relatively few proteins from the two main calcification sites in these animals, i.e., the exoskeleton and the transient calcium storage organs, have been identified, sequenced, and their roles elucidated. Here, a 65-kDa protein (GAP 65) from the gastrolith of the crayfish, Cherax quadricarinatus, is fully characterized and its function in the mineralization of amorphous calcium carbonate (ACC) of the extracellular matrix is demonstrated. GAP 65 is a negatively charged glycoprotein that possesses three predicted domains: a chitin-binding domain 2, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase domain. Expression of GAP 65 was localized to columnar epithelial cells of the gastrolith disk during premolt. In vivo administration of GAP 65 dsRNA resulted in a significant reduction of GAP 65 transcript levels in the gastrolith disk. Such gene silencing also caused dramatic structural and morphological deformities in the chitinous-ACC extracellular matrix structure. ACC deposited in these gastroliths appeared to be sparsely packed with large elongated cavities compared with the normal gastrolith, where ACC is densely compacted. ACC spherules deposited in these gastroliths are significantly larger than normal. GAP 65, moreover, inhibited calcium carbonate crystallization in vitro and stabilized synthetic ACC. Thus, GAP 65 is the first protein shown to have dual function, involved both in extracellular matrix formation and in mineral deposition during biomineralization. PMID:18480260

  1. Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover

    Science.gov (United States)

    Mooney, D. J.; Hansen, L. K.; Langer, R.; Vacanti, J. P.; Ingber, D. E.

    1994-01-01

    Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and

  2. Clinical significance of immunohistochemically detected extracellular matrix proteins and their spatial distribution in primary cancer.

    Science.gov (United States)

    Čunderlíková, B

    2016-09-01

    Our understanding of cancer has evolved mainly from results of studies utilizing experimental models. Simplification inherent to in vitro cell culture models enabled potential ways of cell behaviour in response to various external stimuli to be described, but it has led also to disappointments in clinical trials, presumably due to the lack of crucial tissue components, including extracellular matrix (ECM). ECM and its role in healthy and diseased tissues are being explored extensively and significance of ECM for cell behaviour has been evidenced experimentally. Part of the information gathered in such research that is relevant for natural conditions of a human body can be identified by carefully designed analyses of human tissue samples. This review summarizes published information on clinical significance of ECM in cancer and examines whether effects of ECM on cell behaviour evidenced in vitro, could be supported by clinically based data acquired from analysis of tissue samples. Based on current approaches of clinical immunohistochemical analyses, impact of ECM components on tumour cell behaviour is vague. Except of traditionally considered limitations, other reasons may include lack of stratification of analyzed cases based on clinicopathologic parameters, inclusion of patients treated postoperatively by different treatments or neglecting complexity of interactions among tumour constituents. Nevertheless, reliable immunohistochemical studies represent a source of crucial information for design of tumour models comprising ECM corresponding to real clinical situation. Knowledge gathered from such immunohistochemical studies combined with achievements in tissue engineering hold promise for reversal of the unfavourable trends in the current translational oncologic research. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. The Multifunctional Role of the Pallilysin-Associated Treponema pallidum Protein, Tp0750, in Promoting Fibrinolysis and Extracellular Matrix Component Degradation

    Science.gov (United States)

    Houston, Simon; Russell, Shannon; Hof, Rebecca; Roberts, Alanna K.; Cullen, Paul; Irvine, Kyle; Smith, Derek S.; Borchers, Christoph H.; Tonkin, Michelle L.; Boulanger, Martin J.; Cameron, Caroline E.

    2014-01-01

    Summary The mechanisms that facilitate dissemination of the highly invasive spirochete, Treponema pallidum, are incompletely understood. Previous studies showed the treponemal metalloprotease pallilysin (Tp0751) possesses fibrin clot degradation capability, suggesting a role in treponemal dissemination. In the current study we report characterization of the functionally-linked protein Tp0750. Structural modelling predicts Tp0750 contains a von Willebrand factor type A (vWFA) domain, a protein-protein interaction domain commonly observed in extracellular matrix (ECM)-binding proteins. We report Tp0750 is a serine protease that degrades the major clot components fibrinogen and fibronectin. We also demonstrate Tp0750 cleaves a matrix metalloprotease (MMP) peptide substrate that is targeted by several MMPs, enzymes central to ECM remodelling. Through proteomic analyses we show Tp0750 binds the endothelial fibrinolytic receptor, annexin A2, in a specific and dose-dependent manner. These results suggest Tp0750 constitutes a multifunctional protein that is able to (1) degrade infection-limiting clots by both inhibiting clot formation through degradation of host coagulation cascade proteins and promoting clot dissolution by complexing with host proteins involved in the fibrinolytic cascade and (2) facilitate ECM degradation via MMP-like proteolysis of host components. We propose that through these activities Tp0750 functions in concert with pallilysin to enable T. pallidum dissemination. PMID:24303899

  4. 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...... was determined in patients with chronic kidney disease (n=32; median age, 63 years; inter-quartile range, 51 to 73 years). Serological biomarkers related to cardiovascular disease (fibrinogen, interleukin 6, C-reactive protein) were measured. Arterial applanation tonometry was used to determine central...... in chronic kidney disease....

  5. High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.

    Science.gov (United States)

    Zhang, Kang; Su, Lingqia; Duan, Xuguo; Liu, Lina; Wu, Jing

    2017-02-20

    We recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, PamyQ from Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536. Using the enzyme β-cyclodextrin glycosyltransferase (β-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the PamyQ' promoter produced the greatest extracellular β-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter PHpaII-PamyQ' produced the highest extracellular β-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter PHpaII-PamyQ' also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of β-CGTase using the dual-promoter PHpaII-PamyQ' system was investigated in a 3-L fermenter. Extracellular expression of β-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications. The dual-promoter PHpaII-PamyQ' system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.

  6. Papillote and Piopio: Drosophila ZP-domain proteins required for cell adhesion to the apical extracellular matrix and microtubule organization.

    Science.gov (United States)

    Bökel, Christian; Prokop, Andreas; Brown, Nicholas H

    2005-02-01

    Adhesion between epithelial cells and extracellular substrates is normally mediated through basal adhesion complexes. However, some cells also possess comparable junctions on their apical surface. Here, we describe two new Drosophila proteins, Piopio and Papillote, that are required for the link between the apical epithelial surface and the overlying apical extracellular matrix (aECM). The two proteins share a zona pellucida (ZP) domain with mammalian aECM components, including the tectorins found in the vertebrate inner ear. Tagged versions of both proteins localized to the apical epithelial surface. Mutations in piopio, papillote and dumpy (another gene encoding a ZP-domain protein) cause defects in the innermost layer of the aECM and its detachment from the epidermis. Loss of Piopio, but not Papillote or Dumpy, causes the absence of specialized microtubule bundles from pupal wings, suggesting that Piopio plays a role in microtubule organization. Thus, ZP domain-containing proteins may have shared functions within the aECM, while also exhibiting specific interactions with the cytoskeleton.

  7. Proteins of novel lactic acid bacteria from Apis mellifera mellifera: an insight into the production of known extra-cellular proteins during microbial stress.

    Science.gov (United States)

    Butler, Èile; Alsterfjord, Magnus; Olofsson, Tobias C; Karlsson, Christofer; Malmström, Johan; Vásquez, Alejandra

    2013-10-22

    Lactic acid bacteria (LAB) has been considered a beneficial bacterial group, found as part of the microbiota of diverse hosts, including humans and various animals. However, the mechanisms of how hosts and LAB interact are still poorly understood. Previous work demonstrates that 13 species of Lactobacillus and Bifidobacterium from the honey crop in bees function symbiotically with the honeybee. They protect each other, their hosts, and the surrounding environment against severe bee pathogens, bacteria, and yeasts. Therefore, we hypothesized that these LAB under stress, i.e. in their natural niche in the honey crop, are likely to produce bioactive substances with antimicrobial activity. The genomic analysis of the LAB demonstrated varying genome sizes ranging from 1.5 to 2.2 mega-base pairs (Mbps) which points out a clear difference within the protein gene content, as well as specialized functions in the honeybee microbiota and their adaptation to their host. We demonstrate a clear variation between the secreted proteins of the symbiotic LAB when subjected to microbial stressors. We have identified that 10 of the 13 LAB produced extra-cellular proteins of known or unknown function in which some are arranged in interesting putative operons that may be involved in antimicrobial action, host interaction, or biofilm formation. The most common known extra-cellular proteins secreted were enzymes, DNA chaperones, S-layer proteins, bacteriocins, and lysozymes. A new bacteriocin may have been identified in one of the LAB symbionts while many proteins with unknown functions were produced which must be investigated further. The 13 LAB symbionts likely play different roles in their natural environment defending their niche and their host and participating in the honeybee's food production. These roles are partly played through producing extracellular proteins on exposure to microbial stressors widely found in natural occurring flowers. Many of these secreted proteins may have a

  8. Microsurgical anatomy of membranous layers of the pituitary gland and the expression of extracellular matrix collagenous proteins.

    Science.gov (United States)

    Ceylan, Savas; Anik, Ihsan; Koc, Kenan; Kokturk, Sibel; Ceylan, Sureyya; Cine, Naci; Savli, Hakan; Sirin, Gozde; Sam, Bulent; Gazioglu, Nurperi

    2011-12-01

    There are several reports about the microanatomical and histological features of sellar and parasellar membranous structures and clinical studies about MMP proteinase as a predictive factor. However, studies on collagen contents of sellar and parasellar membranous structures are limited. We demonstrated the membranous structures surrounding the pituitary gland and defined extracellular matrix (ECM) collagenous proteins, collagen I-IV expression patterns of sellar and parasellar connective tissues. The study was carried out on ten fresh postmortem human bodies at the Forensic Medicine Institution. Cavernous sinuses were resected with sellar structures and were stored at -80°C liquid nitrogen tanks. Medial wall of the cavernous sinus, pituitary capsule and pituitary tissue samples were obtained for RT-PCR. Opposite side specimens were used for histological and immune staining studies. Collagens I-IV were studied by immunohistochemical and reverse transcription polymerase chain reaction (RT-PCR) methods. The pituitary capsule and medial wall were identified as two different structures. The fibrous membrane, as the third membrane, was identified as staying whole in eight of ten specimens. Increased type IV collagen was determined in the pituitary gland, medial wall and pituitary capsule, respectively, in both RT-PCR and immunhistochemical studies. Immunhistochemical studies revealed that collagen I was strongly expressed in both the medial wall and pituitary gland. Increased type IV collagen was detected especially in pituitary tissue, the medial wall and the pituitary capsule by immune staining and RT-PCR. Type IV collagen was considered to be an important factor in the progression of adenoma and invasion.

  9. Extracellular Vesicles Derived from Adipose Mesenchymal Stem Cells Regulate the Phenotype of Smooth Muscle Cells to Limit Intimal Hyperplasia.

    Science.gov (United States)

    Liu, Rong; Shen, Hong; Ma, Jian; Sun, Leiqing; Wei, Meng

    2016-04-01

    Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) play important roles in the reduction of inflammation in multiple disease models. However, their role in vein graft (VG) remodeling is undefined. We aimed to investigate the effect of EVs from adipose MSCs (ADMSC-EVs) on VG intimal hyperplasia and to explore the possible mechanisms. After generation and characterization of control-EVs and ADMSC-EVs in vitro, we investigated their effect on the proliferation and migration of vascular smooth muscle cells (VSMCs) in vitro. Next, we established a mouse model of VG transplantation. Mice underwent surgery and received control-EVs or ADMSC-EVs by intraperitoneal injection every other day for 20 days. VG remodeling was evaluated after 4 weeks. We also assessed the effect of ADMSC-EVs on macrophage migration and inflammatory cytokine expression. Significant inhibitory effects of ADMSC-EVs on in vitro VSMC proliferation (p < 0.05) and migration (p < 0.05) were observed compared with control-EVs. The extent of intimal hyperplasia was significantly decreased in ADMSC-EV-treated mice compared with control-EV-treated mice (26 ± 8.4 vs. 45 ± 9.0 μm, p < 0.05). A reduced presence of macrophages was observed in ADMSC-EV-treated mice (p < 0.05). Significantly decreased expression of inflammatory cytokines interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) was also found in the ADMSC-EV-treated group (both p < 0.05). In addition, phosphorylation of Akt, Erk1/2, and p38 in VGs was decreased in the ADMSC-EV-treated group. We demonstrated that ADMSC-EVs exert an inhibitory effect on VG neointima formation by regulating VSMC proliferation and migration, macrophage migration, inflammatory cytokine expression, and the related signaling pathways.

  10. Topology of membrane proteins-predictions, limitations and variations.

    Science.gov (United States)

    Tsirigos, Konstantinos D; Govindarajan, Sudha; Bassot, Claudio; Västermark, Åke; Lamb, John; Shu, Nanjiang; Elofsson, Arne

    2017-10-26

    Transmembrane proteins perform a variety of important biological functions necessary for the survival and growth of the cells. Membrane proteins are built up by transmembrane segments that span the lipid bilayer. The segments can either be in the form of hydrophobic alpha-helices or beta-sheets which create a barrel. A fundamental aspect of the structure of transmembrane proteins is the membrane topology, that is, the number of transmembrane segments, their position in the protein sequence and their orientation in the membrane. Along these lines, many predictive algorithms for the prediction of the topology of alpha-helical and beta-barrel transmembrane proteins exist. The newest algorithms obtain an accuracy close to 80% both for alpha-helical and beta-barrel transmembrane proteins. However, lately it has been shown that the simplified picture presented when describing a protein family by its topology is limited. To demonstrate this, we highlight examples where the topology is either not conserved in a protein superfamily or where the structure cannot be described solely by the topology of a protein. The prediction of these non-standard features from sequence alone was not successful until the recent revolutionary progress in 3D-structure prediction of proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. 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...

  12. HEPARINS MODULATE EXTRACELLULAR-MATRIX AND PROTEIN-SYNTHESIS OF CULTURED RAT MESANGIAL CELLS

    NARCIS (Netherlands)

    WOLTHUIS, A; BOES, A; BERDEN, JHM

    Heparins blunt the development of glomerulosclerosis in several disease models in the rat and this protective effect may be related to suppression of glomerular cell proliferation. In this study the direct effect of heparins on another key event in glomerulosclerosis, extracellular matrix (ECM)

  13. Extracellular functions of glycolytic enzymes of parasites: unpredicted use of ancient proteins.

    Science.gov (United States)

    Gómez-Arreaza, Amaranta; Acosta, Hector; Quiñones, Wilfredo; Concepción, Juan Luis; Michels, Paul A M; Avilán, Luisana

    2014-02-01

    In addition of their usual intracellular localization where they are involved in catalyzing reactions of carbohydrate and energy metabolism by glycolysis, multiple studies have shown that glycolytic enzymes of many organisms, but notably pathogens, can also be present extracellularly. In the case of parasitic protists and helminths, they can be found either secreted or attached to the surface of the parasites. At these extracellular localizations, these enzymes have been shown to perform additional, very different so-called "moonlighting" functions, such as acting as ligands for a variety of components of the host. Due to this recognition, different extracellular glycolytic enzymes participate in various important parasite-host interactions such as adherence and invasion of parasites, modulation of the host's immune and haemostatic systems, promotion of angiogenesis, and acquisition of specific nutrients by the parasites. Accordingly, extracellular glycolytic enzymes are important for the invasion of the parasites and their establishment in the host, and in determining their virulence. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Radiation damage limits to XPCS studies of protein dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Vodnala, Preeti, E-mail: preeti.vodnala@gmail.com; Karunaratne, Nuwan; Lurio, Laurence [Department of Physics, Northern Illinois University, DeKalb IL, 60115 (United States); Bera, Sambhunath [Amity Center for Spintronic Materials, Amity University UP, Sector 125, NOIDA 201313 (India); Thurston, George M. [School of Physics and Astronomy, Rochester Institute of Technology, Rochester, NY 14623-5603 (United States); Karonis, Nick [Department of Computer Science, Northern Illinois University, DeKalb, IL 60115 (United States); Mathematics and Computer Science, Argonne National Laboratory, Argonne, IL 60495 (United States); Winans, John [Department of Computer Science, Northern Illinois University, DeKalb, IL 60115 (United States); Sandy, Alec; Narayanan, Suresh [Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60495 (United States); Yasui, Linda [Department of Biology, Northern Illinois University, DeKalb, IL 60115 (United States); Gaillard, Elizabeth; Karumanchi, Kalyan [Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb IL, 60115 (United States)

    2016-07-27

    The limitations to x-ray photon correlation spectroscopy (XPCS) imposed by radiation damage have been evaluated for suspensions of alpha crystallin. We find that the threshold for radiation damage to the measured protein diffusion rate is significantly lower than the threshold for damage to the protein structure. We provide damage thresholds beyond which the measured diffusion coeffcients have been modified using both XPCS and dynamic light scattering (DLS).

  15. Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering.

    Science.gov (United States)

    Low, Pearlie S J; Tjin, Monica S; Fong, Eileen

    2015-06-11

    Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

  16. Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review

    Directory of Open Access Journals (Sweden)

    Mina Ghahremani

    2016-09-01

    Full Text Available Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.

  17. Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review.

    Science.gov (United States)

    Ghahremani, Mina; Stigter, Kyla A; Plaxton, William

    2016-09-01

    Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs) of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.

  18. Interaction of erythromycin ethylsuccinate and acetaminophen with protein fraction of extracellular polymeric substances (EPS) from various bacterial aggregates.

    Science.gov (United States)

    Métivier, Romain; Bourven, Isabelle; Labanowski, Jérome; Guibaud, Gilles

    2013-10-01

    Extracellular polymeric substances (EPS) are, along with microbial cells, the main components of the biological sludges used in wastewater treatment and natural biofilms. EPS play a major role in removing pollutants from water by means of sorption. The ability of soluble EPS (S-EPS) and bound EPS (B-EPS) derived from various bacterial aggregates (flocs, granules, biofilms) to bind at pH 7.0 ± 0.1 to two pharmaceutical substances, acetaminophen (ACE) and erythromycin ethylsuccinate (ERY), has been investigated using the fluorescence quenching method. Two intense fluorescence peaks, A (Ex/Em range, 200-250/275-380 nm) and B (Ex/Em range, 260-320/275-360 nm), corresponding respectively to the aromatic protein region and soluble microbial by-product-like region, were identified in a three-dimensional excitation-emission matrix of EPS samples. The fluorescence peak, which corresponds to humic-like substances, was also identified though at low intensity. The ability of EPS to bind ACE was found to exceed that for ERY. The aromatic protein fraction of EPS displays a slightly higher affinity for drugs than that shown by the soluble microbial by-product-like fraction. The S-EPS and B-EPS present the same affinity for ACE and ERY. The effective quenching constants (log K) derived from the Stern-Volmer Equation equaled at peak A (with S-EPS): 3.7 ± 0.2 to 4.0 ± 0.1 for ACE and 2.1 ± 0.3 to 2.7 ± 0.1 for ERY. With B-EPS, these values were 3.9 ± 0.1 to 4.0 ± 0.1 for ACE and 2.0 ± 0.2 to 2.6 ± 0.1 for ERY. Our results suggest that the weaker EPS affinity for ERY than for ACE serves to partially explain why only about 50-80 % of ERY is removed from wastewater at the treatment plant. Moreover, this work demonstrates that EPS from natural river biofilms are able to bind drugs, which in turn may limit the mobility of drugs in natural waters.

  19. Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.

    Science.gov (United States)

    Kondo, Ayano; Yamamoto, Shogo; Nakaki, Ryo; Shimamura, Teppei; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Yoshida, Tetsuo; Aburatani, Hiroyuki; Osawa, Tsuyoshi

    2017-02-28

    Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles in cancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but not SREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Reciprocal regulation of two G protein-coupled receptors sensing extracellular concentrations of Ca2+ and H.

    Science.gov (United States)

    Wei, Wei-Chun; Jacobs, Benjamin; Becker, Esther B E; Glitsch, Maike D

    2015-08-25

    G protein-coupled receptors (GPCRs) are cell surface receptors that detect a wide range of extracellular messengers and convey this information to the inside of cells. Extracellular calcium-sensing receptor (CaSR) and ovarian cancer gene receptor 1 (OGR1) are two GPCRs that sense extracellular Ca(2+) and H(+), respectively. These two ions are key components of the interstitial fluid, and their concentrations change in an activity-dependent manner. Importantly, the interstitial fluid forms part of the microenvironment that influences cell function in health and disease; however, the exact mechanisms through which changes in the microenvironment influence cell function remain largely unknown. We show that CaSR and OGR1 reciprocally inhibit signaling through each other in central neurons, and that this is lost in their transformed counterparts. Furthermore, strong intracellular acidification impairs CaSR function, but potentiates OGR1 function. Thus, CaSR and OGR1 activities can be regulated in a seesaw manner, whereby conditions promoting signaling through one receptor simultaneously inhibit signaling through the other receptor, potentiating the difference in their relative signaling activity. Our results provide insight into how small but consistent changes in the ionic microenvironment of cells can significantly alter the balance between two signaling pathways, which may contribute to disease progression.

  1. Reciprocal regulation of two G protein-coupled receptors sensing extracellular concentrations of Ca2+ and H+

    Science.gov (United States)

    Wei, Wei-Chun; Jacobs, Benjamin; Becker, Esther B. E.; Glitsch, Maike D.

    2015-01-01

    G protein-coupled receptors (GPCRs) are cell surface receptors that detect a wide range of extracellular messengers and convey this information to the inside of cells. Extracellular calcium-sensing receptor (CaSR) and ovarian cancer gene receptor 1 (OGR1) are two GPCRs that sense extracellular Ca2+ and H+, respectively. These two ions are key components of the interstitial fluid, and their concentrations change in an activity-dependent manner. Importantly, the interstitial fluid forms part of the microenvironment that influences cell function in health and disease; however, the exact mechanisms through which changes in the microenvironment influence cell function remain largely unknown. We show that CaSR and OGR1 reciprocally inhibit signaling through each other in central neurons, and that this is lost in their transformed counterparts. Furthermore, strong intracellular acidification impairs CaSR function, but potentiates OGR1 function. Thus, CaSR and OGR1 activities can be regulated in a seesaw manner, whereby conditions promoting signaling through one receptor simultaneously inhibit signaling through the other receptor, potentiating the difference in their relative signaling activity. Our results provide insight into how small but consistent changes in the ionic microenvironment of cells can significantly alter the balance between two signaling pathways, which may contribute to disease progression. PMID:26261299

  2. Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer.

    Science.gov (United States)

    Hiratsuka, T; Sano, T; Kato, H; Komatsu, N; Imajo, M; Kamioka, Y; Sumiyama, K; Banno, F; Miyata, T; Matsuda, M

    2017-07-01

    Essentials Spatiotemporal regulation of protein kinases during thrombus formation remains elusive in vivo. Activities of protein kinases were live imaged in mouse platelets at laser-ablated arterioles. Protein kinase A was activated in the dislodging platelets at the downstream side of the thrombus. Extracellular signal-regulated kinase was activated at the core of contracting platelet aggregates. Background The dynamic features of thrombus formation have been visualized by conventional video widefield microscopy or confocal microscopy in live mice. However, owing to technical limitations, the precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives To visualize, by the use of two-photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles. Results When a core of densely packed platelets had developed, ERK activity was increased from the basal region close to the injured arterioles. PKA was activated at the downstream side of an unstable shell overlaying the core of platelets. Intravenous administration of a MEK inhibitor, PD0325901, suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog, dbcAMP, inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase. Conclusion In vivo imaging of transgenic mice expressing FRET biosensors will open a new opportunity to visualize the spatiotemporal changes in signaling molecule activities not only during thrombus formation but also in other hematologic disorders. © 2017 International

  3. The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz Cuesta, Elena; Olesen, Christina Wilkens

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...... the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D...... and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2....

  4. Intracellular and extracellular microtubule associated protein tau as a therapeutic target in Alzheimer disease and other tauopathies.

    Science.gov (United States)

    Avila, Jesús; Pallas, Noemí; Bolós, Marta; Sayas, C Laura; Hernandez, Felix

    2016-06-01

    Microtubule associated protein tau, a protein mainly expressed in neurons, plays an important role in several diseases related to dementia, named tauopathies. Alzheimer disease is the most relevant tauopathy. The role of tau protein in dementia is now a topic under discussion, and is the focus of this review. We have covered two major areas: tau pathology and tau as a therapeutic target. Tau pathology is mainly related to a gain of toxic function due to an abnormal accumulation, aberrant modifications (such as hyperphosphorylation and truncation, among others) and self-aggregation of tau into oligomers or larger structures. Also, tau can be found extracellularly in a toxic form. Tau-based therapy is mainly centered on avoiding the gain of these toxic functions of tau. Tau therapies are focused on lowering tau levels, mainly of modified tau species that could be toxic for neurons (phosphorylated, truncated or aggregated tau), in intracellular or extracellular form. Decreasing the levels of those toxic species is a possible therapeutic strategy.

  5. Analysis of AKT and ERK1/2 protein kinases in extracellular vesicles isolated from blood of patients with cancer

    Directory of Open Access Journals (Sweden)

    Johannes C. van der Mijn

    2014-12-01

    Full Text Available Background: Extracellular vesicles (EVs are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors. Methods: EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2 levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used. Results: In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR, AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067 compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12 shows a significant decrease in AKT levels among patients with a favourable treatment response (p<0.005. Conclusion: Phosphorylation of protein kinases in EVs reflects their phosphorylation in tumour cells. Total AKT protein levels may allow monitoring of kinase inhibitor responses in patients with cancer.

  6. Neovibsanin B increases extracellular matrix proteins in optic nerve head cells via activation of Smad signalling pathway.

    Science.gov (United States)

    Wang, Zhen; Xu, Wei; Rong, Ao; Lin, Yan; Qiu, Xu-Ling; Qu, Shen; Lan, Xian-Hai

    2015-01-01

    The present study demonstrates the effect of neovibsanin B on the synthesis and deposition of ECM proteins and the signalling pathways used in optic nerve head (ONH) astrocytes and lamina cribrosa (LC) cells. For investigation of the signalling pathway used by neovibsanin B, ONH cells were treated with neovibsanin B. Western blot and immunostaining analyses were used to examine the phosphorylation of proteins involved in Smad and non-Smad signalling pathway. The results revealed that ONH cells on treatment with neovibsanin B showed enhanced synthesis of extracellular matrix (ECM) proteins. Neovibsanin B induced phosphorylation of canonical signalling proteins, Smad2/3. However phosphorylation of non-canonical signalling proteins, extracellular signal-regulated kinases, p38, and c-Jun N-terminal kinases (JNK) 1/2 remained unaffected. There was also increase in co-localization of pSmad2/3 with Co-Smad4 in the nucleus of ONH astrocytes and LC cells indicating activation of the canonical Smad signalling pathway. Treatment of ONH cells with SIS3, inhibitor of Smad3 phosphorylation reversed the neovibsanin B stimulated ECM expression as well as activation of canonical pathway signalling molecules. In addition, inhibition of Smad2 or Smad3 using small interfering RNA (siRNA) also suppressed neovibsanin B stimulated ECM protein synthesis in ONH astrocytes and LC cells. Thus neovibsanin B utilizes the canonical Smad signalling pathway to stimulate ECM synthesis in human ONH cells. The neovibsanin B induced ECM synthesis and activation of the canonical Smad signalling pathway may be due to its effect on transforming growth factor-β2 (TGF-β2). However, further studies are under process to understand the mechanism.

  7. Effect of Limited Hydrolysis on Traditional Soy Protein Concentrate

    Directory of Open Access Journals (Sweden)

    Mirjana B. Pesic

    2006-09-01

    Full Text Available The influence of limited proteolysis of soy protein concentrate on proteinextractability, the composition of the extractable proteins, their emulsifying properties andsome nutritional properties were investigated. Traditional concentrate (alcohol leachedconcentrate was hydrolyzed using trypsin and pepsin as hydrolytic agents. Significantdifferences in extractable protein composition between traditional concentrate and theirhydrolysates were observed by polyacrylamide gel electrophoresis (PAGE and by SDSPAGE.All hydrolysates showed better extractability than the original protein concentrate,whereas significantly better emulsifying properties were noticed at modified concentratesobtained by trypsin induced hydrolysis. These improved properties are the result of twosimultaneous processes, dissociation and degradation of insoluble alcohol-induced proteinaggregates. Enzyme induced hydrolysis had no influence on trypsin-inibitor activity, andsignificantly reduced phytic acid content.

  8. Rate limit of protein elastic response is tether dependent

    Science.gov (United States)

    Berkovich, Ronen; Hermans, Rodolfo I.; Popa, Ionel; Stirnemann, Guillaume; Garcia-Manyes, Sergi; Berne, Bruce J.; Fernandez, Julio M.

    2012-01-01

    The elastic restoring force of tissues must be able to operate over the very wide range of loading rates experienced by living organisms. It is surprising that even the fastest events involving animal muscle tissues do not surpass a few hundred hertz. We propose that this limit is set in part by the elastic dynamics of tethered proteins extending and relaxing under a changing load. Here we study the elastic dynamics of tethered proteins using a fast force spectrometer with sub-millisecond time resolution, combined with Brownian and Molecular Dynamics simulations. We show that the act of tethering a polypeptide to an object, an inseparable part of protein elasticity in vivo and in experimental setups, greatly reduces the attempt frequency with which the protein samples its free energy. Indeed, our data shows that a tethered polypeptide can traverse its free-energy landscape with a surprisingly low effective diffusion coefficient Deff ∼ 1,200 nm2/s. By contrast, our Molecular Dynamics simulations show that diffusion of an isolated protein under force occurs at Deff ∼ 108 nm2/s. This discrepancy is attributed to the drag force caused by the tethering object. From the physiological time scales of tissue elasticity, we calculate that tethered elastic proteins equilibrate in vivo with Deff ∼ 104–106 nm2/s which is two to four orders magnitude smaller than the values measured for untethered proteins in bulk. PMID:22895787

  9. Conserved Ankyrin Repeat Proteins and Their NIMA Kinase Partners Regulate Extracellular Matrix Remodeling and Intracellular Trafficking in Caenorhabditis elegans.

    Science.gov (United States)

    Lažetić, Vladimir; Fay, David S

    2017-01-01

    Molting is an essential developmental process in nematodes during which the epidermal apical extracellular matrix, the cuticle, is remodeled to accommodate further growth. Using genetic approaches, we identified a requirement for three conserved ankyrin repeat-rich proteins, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, in Caenorhabditis elegans molting. Loss of mlt function resulted in severe defects in the ability of larvae to shed old cuticle and led to developmental arrest. Genetic analyses demonstrated that MLT proteins functionally cooperate with the conserved NIMA kinase family members NEKL-2/NEK8 and NEKL-3/NEK6/NEK7 to promote cuticle shedding. MLT and NEKL proteins were specifically required within the hyp7 epidermal syncytium, and fluorescently tagged mlt and nekl alleles were expressed in puncta within this tissue. Expression studies further showed that NEKL-2-MLT-2-MLT-4 and NEKL-3-MLT-3 colocalize within largely distinct assemblies of apical foci. MLT-2 and MLT-4 were required for the normal accumulation of NEKL-2 at the hyp7-seam cell boundary, and loss of mlt-2 caused abnormal nuclear accumulation of NEKL-2 Correspondingly, MLT-3, which bound directly to NEKL-3, prevented NEKL-3 nuclear localization, supporting the model that MLT proteins may serve as molecular scaffolds for NEKL kinases. Our studies additionally showed that the NEKL-MLT network regulates early steps in clathrin-mediated endocytosis at the apical surface of hyp7, which may in part account for molting defects observed in nekl and mlt mutants. This study has thus identified a conserved NEKL-MLT protein network that regulates remodeling of the apical extracellular matrix and intracellular trafficking, functions that may be conserved across species. Copyright © 2017 by the Genetics Society of America.

  10. Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury.

    Directory of Open Access Journals (Sweden)

    Young-Eun Cho

    Full Text Available Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with N-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or d-galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity.

  11. Long Polar Fimbriae of Enterohemorrhagic Escherichia coli O157:H7 Bind to Extracellular Matrix Proteins

    Science.gov (United States)

    Farfan, Mauricio J.; Cantero, Lidia; Vidal, Roberto; Botkin, Douglas J.; Torres, Alfredo G.

    2011-01-01

    Adherence to intestinal cells is a key process in infection caused by enterohemorrhagic Escherichia coli (EHEC). Several adhesion factors that mediate the binding of EHEC to intestinal cells have been described, but the receptors involved in their recognition are not fully characterized. Extracellular matrix (ECM) proteins might act as receptors involved in the recognition of enteric pathogens, including EHEC. In this study, we sought to characterize the binding of EHEC O157:H7 to ECM proteins commonly present in the intestine. We found that EHEC prototype strains as well as other clinical isolates adhered more abundantly to surfaces coated with fibronectin, laminin, and collagen IV. Further characterization of this phenotype, by using antiserum raised against the LpfA1 putative major fimbrial subunit and by addition of mannose, showed that a reduced binding of EHEC to ECM proteins was observed in a long polar fimbria (lpf) mutant. We also found that the two regulators, H-NS and Ler, had an effect in EHEC Lpf-mediated binding to ECM, supporting the roles of these tightly regulated fimbriae as adherence factors. Purified Lpf major subunit bound to all of the ECM proteins tested. Finally, increased bacterial adherence was observed when T84 cells, preincubated with ECM proteins, were infected with EHEC. Taken together, these findings suggest that the interaction of Lpf and ECM proteins contributes to the EHEC colonization of the gastrointestinal tract. PMID:21708988

  12. Candida albicans Modifies the Protein Composition and Size Distribution of THP-1 Macrophage-Derived Extracellular Vesicles.

    Science.gov (United States)

    Reales-Calderón, Jose Antonio; Vaz, Catarina; Monteoliva, Lucía; Molero, Gloria; Gil, Concha

    2017-01-06

    The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effective clearance of the pathogen. The secretion of proteins, mRNAs, noncoding RNAs and lipids through extracellular vesicles (EVs) is one of the mechanisms of communication between immune cells. EVs change their cargo to mediate different responses, and may play a role in the response against infections. Thus we have undertaken the first quantitative proteomic analysis on the protein composition of THP-1 macrophage-derived EVs during the interaction with Candida albicans. This study revealed changes in EVs sizes and in protein composition, and allowed the identification and quantification of 717 proteins. Of them, 133 proteins changed their abundance due to the interaction. The differentially abundant proteins were involved in functions relating to immune response, signaling, or cytoskeletal reorganization. THP-1-derived EVs, both from control and from Candida-infected macrophages, had similar effector functions on other THP-1-differenciated macrophages, activating ERK and p38 kinases, and increasing both the secretion of proinflammatory cytokines and the candidacidal activity; while in THP-1 nondifferenciated monocytes, only EVs from infected macrophages increased significantly the TNF-α secretion. Our findings provide new information on the role of macrophage-derived EVs in response to C. albicans infection and in macrophages communication.

  13. An extracellular Serine/Threonine-rich protein from Lactobacillus plantarum NCIMB 8826 is a novel aggregation-promoting factor with affinity to mucin.

    Science.gov (United States)

    Hevia, Arancha; Martínez, Noelia; Ladero, Víctor; Alvarez, Miguel A; Margolles, Abelardo; Sánchez, Borja

    2013-10-01

    Autoaggregation in lactic acid bacteria is directly related to the production of certain extracellular proteins, notably, aggregation-promoting factors (APFs). Production of aggregation-promoting factors confers beneficial traits to probiotic-producing strains, contributing to their fitness for the intestinal environment. Furthermore, coaggregation with pathogens has been proposed to be a beneficial mechanism in probiotic lactic acid bacteria. This mechanism would limit attachment of the pathogen to the gut mucosa, favoring its removal by the human immune system. In the present paper, we have characterized a novel aggregation-promoting factor in Lactobacillus plantarum. A mutant with a knockout of the D1 gene showed loss of its autoaggregative phenotype and a decreased ability to bind to mucin, indicating an adhesion role of this protein. In addition, heterologous production of the D1 protein or an internal fragment of the protein, characterized by its abundance in serine/threonine, strongly induced autoaggregation in Lactococcus lactis. This result strongly suggested that this internal fragment is responsible for the bioactivity of D1 as an APF. To our knowledge, this is the first report on a gene coding for an aggregation-promoting factor in Lb. plantarum.

  14. Diversity of extracellular proteins during the transition from the ‘proto-apicomplexan’ alveolates to the apicomplexan obligate parasites

    KAUST Repository

    Templeton, Thomas J.

    2015-11-20

    The recent completion of high-coverage draft genome sequences for several alveolate protozoans – namely, the chromerids, Chromera velia and Vitrella brassicaformis ; the perkinsid Perkinsus marinus ; the apicomplexan, Gregarina niphandrodes , as well as high coverage transcriptome sequence information for several colpodellids, allows for new genome-scale comparisons across a rich landscape of apicomplexans and other alveolates. Genome annotations can now be used to help interpret fine ultrastructure and cell biology, and guide new studies to describe a variety of alveolate life strategies, such as symbiosis or free living, predation, and obligate intracellular parasitism, as well to provide foundations to dissect the evolutionary transitions between these niches. This review focuses on the attempt to identify extracellular proteins which might mediate the physical interface of cell–cell interactions within the above life strategies, aided by annotation of the repertoires of predicted surface and secreted proteins encoded within alveolate genomes. In particular, we discuss what descriptions of the predicted extracellular proteomes reveal regarding a hypothetical last common ancestor of a pre-apicomplexan alveolate – guided by ultrastructure, life strategies and phylogenetic relationships – in an attempt to understand the evolution of obligate parasitism in apicomplexans.

  15. Characterization of Co-Cultivation of Cyanobacteria on Growth, Productions of Polysaccharides and Extracellular Proteins, Nitrogenase Activity, and Photosynthetic Activity.

    Science.gov (United States)

    Xue, Chuizhao; Wang, Libo; Wu, Tong; Zhang, Shiping; Tang, Tao; Wang, Liang; Zhao, Quanyu; Sun, Yuhan

    2017-01-01

    Cyanobacteria as biofertilizers are benefit to reduce the use of chemical fertilizers and reestablish the ecological system in soil. In general, several strains of cyanobacteria were involved in the biofertilizers. The co-cultivation of cyanobacteria were characterized on growth profile, production of polysaccharides and extracellular proteins, nitrogenase activity, and photosynthetic activity for three selected N 2 -fixing cyanobacteria, Anabaena cylindrica (B1611 and F243) and Nostoc sp. (F280). After eight-day culture, the highest dry weights were obtained in F280 pure culture and co-cultivation of B1611 and F280. Higher production of extracellular proteins and cell-bonding polysaccharides (CPS) were observed in co-cultivations compared with pure culture. The highest released polysaccharides (RPS) contents were obtained in pure culture of F280 and co-cultivation of F280 and F243. Galactose and glucose were major components of CPS and RPS in all samples. Trehalose was a specific component of RPS in F280 pure culture. Based on the monosaccharide contents of CPS and RPS, F280 was the dominant species in the related treatments of co-cultivation. The nitrogenase activities in all treatments exhibited a sharp rise at the late stage while a significant decrease existed when three cyanobacteria strains were mixed. Photosynthetic activities for all treatments were determined with rapid light curve, and the related parameters were estimated.

  16. Probing for protein-protein interactions during cell migration: limitations and challenges.

    Science.gov (United States)

    Chan, Jeremy Soon Kiat; Teo, Zi; Sng, Ming Keat; Tan, Nguan Soon

    2014-08-01

    Cellular migration is a fundamental biological process occurring as early as embryogenesis to the pathological state of cancer metastasis. Nearly all cellular migrations involve an extracellular signal that is transduced internally by members of a signalling cascade. These signal transduction events are driven by protein-protein interactions (PPIs) that coordinate intracellular activities to enable a cell to migrate. Understanding these PPIs will provide valuable insight into how cellular migration can be modulated perhaps towards a therapeutic end. Histologically, not many techniques are available to demonstrate PPIs. Contrasting agents only demonstrate the presence of a particular protein, and perhaps its co-localisation with another protein. Yet, co-localisation need not necessarily indicate physical interaction between the two proteins. In this review, we highlight four commonly used methods that continue to expand our understanding of PPIs underlying cell migration: co-immunoprecipitation, bimolecular fluorescence complementation, proximity ligation assay and surface plasmon resonance. The methods discussed herein allow for the study of PPIs in a wide variety of biological samples, including cell lysates, live cells, fixed cells and tissues, enabling the quantification of endogenous PPIs and exploration of the nature of PPIs. We also include a rudimentary framework for researchers to decide which experimental method best suits their research goals.

  17. Cloning from purified high endothelial venule cells of hevin, a close relative of the antiadhesive extracellular matrix protein SPARC.

    Science.gov (United States)

    Girard, J P; Springer, T A

    1995-01-01

    High endothelial venules (HEV) in lymphoid tissues support high levels of lymphocyte extravasion from the blood. We purified high endothelial cells from human tonsils by immunomagnetic selection with MECA-79 MAb to construct an HEV cDNA library. Differential screening of this library using cDNA probes from HEV (plus) or flat-walled vessel (minus) endothelial cells allowed us to characterize a novel human cDNA expressed to high levels in HEV. The cDNA encodes a secreted acidic calcium-binding glycoprotein of 664 aa residues, designated hevin, exhibiting 62% identity with the antiadhesive extracellular matrix protein SPARC, over a region of 232 aa spanning more than four fifths of the SPARC coding sequence. The primary structure and sequence of hevin and similar to SPARC-like proteins from rat and quail, called SC1 or QR1. Hevin could contribute to the induction or maintenance of features of the HEV endothelium that facilitate lymphocyte migration.

  18. 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 ...... decomposition of protein chloramines....

  19. Proteome of cell wall-extracts from pathogenic Paracoccidioides brasiliensis: Comparison among morphological phases, isolates, and reported fungal extracellular vesicle proteins

    Directory of Open Access Journals (Sweden)

    Larissa V.G. Longo

    2014-06-01

    Full Text Available We identified non-covalently linked cell wall proteins from Paracoccidioides brasiliensis yeasts and mycelia, with focus on the yeast pathogenic phase, and correlated them with reported fungal extracellular vesicle proteins. We studied isolates Pb3 and Pb18, which evoke distinct patterns of experimental paracoccidioidomycosis and represent two phylogenetic groups. Proteins were extracted mildly with dithiothreitol, trypsinized, and peptides analyzed by liquid chromatography coupled to high-resolution mass spectrometry. Among 132 yeast-exclusive sequences, 92 were Pb18-exclusive. About 80% of total proteins were classified as secretory, mostly showing non-conventional signals. Extracellular vesicular transportation could be involved, since 60% had orthologs reported in fungal extracellular vesicles.

  20. Recovery of gold from industrial wastewater by extracellular proteins obtained from a thermophilic bacterium Tepidimonas fonticaldi AT-A2.

    Science.gov (United States)

    Han, Yin-Lung; Wu, Jen-Hao; Cheng, Chieh-Lun; Nagarajan, Dillirani; Lee, Ching-Ray; Li, Yi-Heng; Lo, Yung-Chung; Chang, Jo-Shu

    2017-09-01

    Biosorption has emerged as a promising alternative approach for treating wastewater with dilute metal contents in a green and cost effective way. In this study, extracellular proteins of an isolated thermophilic bacterium (Tepidimonas fonticaldi AT-A2) were used as biosorbent to recover precious metal (i.e., Au) from wastewater. The Au (III) adsorption capacity on the T. fonticaldi AT-A2 proteins was the highest when the pH was set at about 4.0-5.0. The adsorption capacity increased with increasing temperature from 15 to 70°C. Adsorption isotherm studies show that both Langmuir and Freundrich models could describe the adsorption equilibrium. The maximum adsorption capacity of Au (III) at 50°C and pH 5 could reach 9.7mg Au/mg protein. The protein-based biosorbent was also used for the recovery of Au from a wastewater containing 15mg/L of Au, achieving a high adsorption capacity of 1.45mg Au/mg protein and a removal efficiency of 71%. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein.

    Science.gov (United States)

    Elliott, Candace; Müller, Judith; Miklis, Marco; Bhat, Riyaz A; Schulze-Lefert, Paul; Panstruga, Ralph

    2005-01-01

    We performed a structure-function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop-loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family.

  2. Conserved extracellular cysteine residues and cytoplasmic loop–loop interplay are required for functionality of the heptahelical MLO protein

    Science.gov (United States)

    Elliott, Candace; Müller, Judith; Miklis, Marco; Bhat, Riyaz A.; Schulze-Lefert, Paul; Panstruga, Ralph

    2004-01-01

    We performed a structure–function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop–loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family. PMID:15352871

  3. Proteomic analysis of temperature dependent extracellular proteins from Aspergillus fumigatus grown under solid-state culture condition.

    Science.gov (United States)

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2013-06-07

    Fungal species of the genus Aspergillus are filamentous ubiquitous saprophytes that play a major role in lignocellulosic biomass recycling and also are considered as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be a more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated a fungal strain from compost, identified as Aspergillus fumigatus, and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid-state fermentation of sawdust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40-50 °C, demonstrating the thermophilic nature of the isolate A. fumigatus LF9. Characterization of the thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application.

  4. Influence of subtilisin on the adhesion of a marine bacterium which produces mainly proteins as extracellular polymers.

    Science.gov (United States)

    Leroy, C; Delbarre, C; Ghillebaert, F; Compere, C; Combes, D

    2008-09-01

    The nature of exopolymers involved in the adhesion of a marine biofilm-forming bacterium Pseudoalteromonas sp. D41 was investigated to evaluate and understand the antifouling potential of subtilisin. The exopolymers of D41 produced by fermentation were analysed by FTIR and SDS-PAGE showing the presence of polysaccharides, glycoproteins and proteins. A high content of proteins was detected both in soluble and capsular fractions. The microscopic observations of fluorescamine and calcofluor stained adhered D41 indicated mainly the presence of proteins in exopolymers produced during adhesion. Subtilisin, the broad spectrum protease, tested in natural sea water and in polystyrene microplates showed that antifouling activity was higher in the prevention of bacterial adhesion than in the detachment of adhered D41 cells. Overall, these results demonstrate the involvement of proteins in Pseudoalteromonas sp. D41 adhesion and confirm the high antifouling potential of subtilisin. This study emphasizes the major role of proteins instead of polysaccharides, thus extending our knowledge regarding the nature of extracellular polymers involved in bacterial adhesion. Furthermore, the high antifouling potential of subtilisin evaluated in the very first stages of fouling, bacterial adhesion, could lead to less toxic compounds than organometallic compounds in antifouling paint.

  5. Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

    Science.gov (United States)

    Liao, Sumei; Klein, Marlise I.; Heim, Kyle P.; Fan, Yuwei; Bitoun, Jacob P.; Ahn, San-Joon; Burne, Robert A.; Koo, Hyun; Brady, L. Jeannine

    2014-01-01

    Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. PMID:24748612

  6. QRI, a retina-specific gene, encodes an extracellular matrix protein exclusively expressed during neural retina differentiation.

    Science.gov (United States)

    Casado, F J; Pouponnot, C; Jeanny, J C; Lecoq, O; Calothy, G; Pierani, A

    1996-02-01

    Neural retina development results from growth arrest of neuroectodermal precursors and differentiation of postmitotic cells. The QRI gene is specifically expressed in Müller retinal glial cells. Its expression coincides with the stage of withdrawal from the cell cycle and establishment of differentiation and is repressed upon induction of retinal cell proliferation by the v-src gene product. In this report, we show that the QR1 gene encodes several glycosylated proteins that are secreted and can either associate with the extracellular matrix or remain diffusible in the medium. By using pulse-chase experiments, the 100-103 kDa forms seem to appear first and are specifically incorporated into the extracellular matrix, whereas the 108 and 60 kDa polypeptides appear later and are detected as soluble forms in the culture medium. We also report that expression of the QR1 gene is developmentally regulated in the chicken. Its mRNA is first detectable at embryonic day 10, reaches a maximal level at embryonic day 15 and is no longer detected at embryonic day 18. Immunolocalization of the QR1 protein in chicken retina sections during development shows that expression of the protein parallels the differentiation pattern of post-miotic cells (in particular Müller cells and rods), corresponding to the two differentiation gradients in the retina: from the ganglion cell layer to the inner nuclear layer and outer nuclear layer, and from the optic nerve to the iris. At embryonic day 10, expression of the QR1 protein(s) is restricted to the optic nerve region and the inner nuclear layer, colocalizing with Müller cell bodies. As development proceeds, QR1 protein localization spreads towards the iris and towards the outer nuclear layer, following Müller cell elongations towards the photoreceptors. Between embryonic days 16 and 18, the QR1 protein is no longer detectable in the optic nerve region and is concentrated around the basal segment of the photoreceptors in the peripheral

  7. The feelgood mutation in zebrafish dysregulates COPII-dependent secretion of select extracellular matrix proteins in skeletal morphogenesis

    Directory of Open Access Journals (Sweden)

    David B. Melville

    2011-11-01

    Craniofacial and skeletal dysmorphologies account for the majority of birth defects. A number of the disease phenotypes have been attributed to abnormal synthesis, maintenance and composition of extracellular matrix (ECM, yet the molecular and cellular mechanisms causing these ECM defects remain poorly understood. The zebrafish feelgood mutant manifests a severely malformed head skeleton and shortened body length due to defects in the maturation stage of chondrocyte development. In vivo analyses reveal a backlog of type II and type IV collagens in rough endoplasmic reticulum (ER similar to those found in coat protein II complex (COPII-deficient cells. The feelgood mutation hinders collagen deposition in the ECM, but trafficking of small cargos and other large ECM proteins such as laminin to the extracellular space is unaffected. We demonstrate that the zebrafish feelgood mutation causes a single amino acid substitution within the DNA-binding domain of transcription factor Creb3l2. We show that Creb3l2 selectively regulates the expression of genes encoding distinct COPII proteins (sec23a, sec23b and sec24d but find no evidence for its regulation of sec24c expression. Moreover, we did not detect activation of ER stress response genes despite intracellular accumulation of collagen and prominent skeletal defects. Promoter trans-activation assays show that the Creb3l2 feelgood variant is a hypomorphic allele that retains approximately 50% of its transcriptional activity. Transgenic rescue experiments of the feelgood phenotype restore craniofacial development, illustrating that a precise level of Creb3l2 transcriptional activity is essential for skeletogenesis. Our results indicate that Creb3l2 modulates the availability of COPII machinery in a tissue- and cargo-specific manner. These findings could lead to a better understanding of the etiology of human craniofacial and skeletal birth defects as well as adult-onset diseases that are linked to dysregulated ECM deposition

  8. Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens.

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    B David Persson

    2010-09-01

    Full Text Available The human membrane cofactor protein (MCP, CD46 is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6, Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4 that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.

  9. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    Science.gov (United States)

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Switch-like genes populate cell communication pathways and are enriched for extracellular proteins

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2008-01-01

    Full Text Available Abstract Background Recent studies have placed gene expression in the context of distribution profiles including housekeeping, graded, and bimodal (switch-like. Single-gene studies have shown bimodal expression results from healthy cell signaling and complex diseases such as cancer, however developing a comprehensive list of human bimodal genes has remained a major challenge due to inherent noise in human microarray data. This study presents a two-component mixture analysis of mouse gene expression data for genes on the Affymetrix MG-U74Av2 array for the detection and annotation of switch-like genes. Two-component normal mixtures were fit to the data to identify bimodal genes and their potential roles in cell signaling and disease progression. Results Seventeen percent of the genes on the MG-U74Av2 array (1519 out of 9091 were identified as bimodal or switch-like. KEGG pathways significantly enriched for bimodal genes included ECM-receptor interaction, cell communication, and focal adhesion. Similarly, the GO biological process "cell adhesion" and cellular component "extracellular matrix" were significantly enriched. Switch-like genes were found to be associated with such diseases as congestive heart failure, Alzheimer's disease, arteriosclerosis, breast neoplasms, hypertension, myocardial infarction, obesity, rheumatoid arthritis, and type I and type II diabetes. In diabetes alone, over two hundred bimodal genes were in a different mode of expression compared to normal tissue. Conclusion This research identified and annotated bimodal or switch-like genes in the mouse genome using a large collection of microarray data. Genes with bimodal expression were enriched within the cell membrane and extracellular environment. Hundreds of bimodal genes demonstrated alternate modes of expression in diabetic muscle, pancreas, liver, heart, and adipose tissue. Bimodal genes comprise a candidate set of biomarkers for a large number of disease states because

  11. Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples

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    Larissa Belov

    2016-04-01

    Full Text Available Extracellular vesicles (EV are membranous particles (30–1,000 nm in diameter secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes and the microvesicles (MV; bud from plasma membranes. Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer and MEC1 (B-cell chronic lymphocytic leukaemia; CLL, were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326 and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19+ EV from the plasma of CLL patients. These EV expressed a subset (~40% of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.

  12. Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues

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    Éva Korpos

    2015-01-01

    Full Text Available The extracellular matrix (ECM performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic changes during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2 in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junction formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth.

  13. The Protective Effects of Erythropoietin on Rat Glomerular Podocytes in Culture are Modulated by Extracellular Matrix Proteins

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    Jan Krtil

    2014-03-01

    Full Text Available Background/Aims: Podocytes are typically cultured on collagen I; however, collagen I is absent from healthy glomerular basement membranes. Erythropoietin (EPO is thought to protect podocytes in vivo. Here, we studied how various types of extracellular matrix (ECM proteins and EPO affect podocytes in culture. Methods: Primary rat podocytes were replated on collagen I, collagen IV, whole ECM extract, laminin, or bare plastic. Cellular adhesion (8 hours after plating, proliferation (5 days, 10 % serum, and resistance to serum deprivation (3 days, 0.5 % serum were assessed. BrdU incorporation and expression of podocyte-specific markers were employed as measures of cellular proliferation and differentiation, respectively. qPCR was used to verify expression of EPO receptor in cultured podocytes. Results: Cellular adhesion was similar on all ECM proteins and unaffected by EPO. Proliferation was accelerated by laminin and the ECM extract, but the final cell density was similar on all ECM surfaces. Collagen IV supported the serum-deprived cells better than the other ECM proteins. EPO (2-20 ng/ml improved viability of serum-deprived podocytes on collagen I, collagen IV, and ECM, but not on laminin or bare plastic. The cells expressed mRNA for EPO receptor. Conclusion: The physiological ECM proteins are more supportive of primary podocytic cultures compared with collagen I. The protective effects of EPO during serum deprivation are modulated by the cultivation surface.

  14. AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

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    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.

  15. Amylase-Binding Protein B of Streptococcus gordonii Is an Extracellular Dipeptidyl-Peptidase▿

    OpenAIRE

    Chaudhuri, Biswendu; Paju, Susanna; Haase, Elaine M.; Vickerman, M. Margaret; Tanzer, Jason M.; Scannapieco, Frank A.

    2008-01-01

    The oral commensal bacterium Streptococcus gordonii interacts with salivary amylase via two amylase-binding proteins, AbpA and AbpB. Based on sequence analysis, the 20-kDa AbpA protein is unique to S. gordonii, whereas the 82-kDa AbpB protein appears to share sequence homology with other bacterial dipeptidases. The aim of this study was to verify the peptidase activity of AbpB and further explore its potential functions. The abpB gene was cloned, and histidine-tagged AbpB (His-AbpB) was expre...

  16. Heterotrimeric G protein alpha subunit controls growth, stress response, extracellular protease activity, and cyclopiazonic acid production in Penicillium camemberti.

    Science.gov (United States)

    García-Rico, Ramón O; Gil-Durán, Carlos; Rojas-Aedo, Juan F; Vaca, Inmaculada; Figueroa, Luis; Levicán, Gloria; Chávez, Renato

    2017-09-01

    The fungus Penicillium camemberti is widely used in the ripening of various bloomy-rind cheeses. Several properties of P. camemberti are important in cheese ripening, including conidiation, growth and enzyme production, among others. However, the production of mycotoxins such as cyclopiazonic acid during the ripening process by P. camemberti has raised concerns among consumers that demand food with minimal contamination. Here we show that overexpressing an α-subunit from the subgroup I of the heterotrimeric G protein (Gαi) influences several of these processes: it negatively affects growth in a media-dependent manner, triggers conidial germination, reduces the rate of sporulation, affects thermal and osmotic stress resistance, and also extracellular protease and cyclopiazonic acid production. Our results contribute to understanding the biological determinants underlying these biological processes in the economically important fungus P. camemberti. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  17. A protein involved in central nervous system myelination: localization in the extracellular matrix and induction in neuroblastoma cells.

    Science.gov (United States)

    Notterpek, L M; Rome, L H

    1994-01-01

    G21.3, a monoclonal antibody previously shown to block central nervous system (CNS) myelination, is now demonstrated to recognize an extracellular matrix (ECM) component. The antigen is present on the surface of neurons but not oligodendrocytes and is highly abundant in the white matter of the adult rat brain; however, it is not found in isolated myelin. Double immunostaining studies indicate a neuronal and ependymal cell source of the G21.3 antigen and a developmental expression pattern distinct from known markers of CNS myelination. The antigen is found at low levels in non-neuronal tissue and is mainly localized to basement membranes. G21.3 immunoreactive proteins are upregulated by retinoic acid-induced differentiation of SK-N-SHF neuroblastoma cells. These results suggest that the G21.3 antigen is an axolemma-associated ECM component with roles in postnatal CNS development and cell-matrix interactions during morphological differentiation of neuroblastoma cells.

  18. Development of a Sensitive Luciferase-Based Sandwich ELISA System for the Detection of Human Extracellular Matrix 1 Protein.

    Science.gov (United States)

    Li, Ya; Li, Yanqing; Zhao, Junli; Zheng, Xiaojing; Mao, Qinwen; Xia, Haibin

    2016-12-01

    Enzyme-linked immunosorbent assay (ELISA) has been one of the main methods for detecting an antigen in an aqueous sample for more than four decades. Nowadays, one of the biggest concerns for ELISA is still how to improve the sensitivity of the assay, and the luciferase-luciferin reaction system has been noticed as a new detection method with high sensitivity. In this study, a luciferin-luciferase reaction system was used as the detection method for a sandwich ELISA system. It was shown that this new system led to an increase in the detection sensitivity of at least two times when compared with the traditional horseradish peroxidase (HRP) detection method. Lastly, the serum levels of the human extracellular matrix 1 protein of breast cancer patients were determined by the new system, which were overall similar to the HRP chemiluminescent system. Furthermore, this new luciferase reporter can be implemented into other ELISA systems for the purpose of increasing the assay sensitivity.

  19. Combined antiretroviral therapy attenuates hepatic extracellular matrix remodeling in HIV patients assessed by novel protein fingerprint markers

    DEFF Research Database (Denmark)

    Leeming, Diana J; Anadol, Evrim; Schierwagen, Robert

    2014-01-01

    extracellular matrix (ECM) proteins into small soluble fragments, which reflect hepatic remodeling processes. This study used these novel biomarkers to investigate the effect of HIV and cART on hepatic fibrosis remodeling. DESIGN: In 249 patients with HIV monoinfection and 55 healthy controls, the serum levels......OBJECTIVES: Combined antiretroviral therapy (cART) attenuates hepatic fibrosis in hepatitis C virus and HIV coinfected patients. However, the role of HIV or cART on hepatic fibrosis in HIV monoinfection is discussed controversially. During liver fibrosis, matrix metalloproteinases (MMPs) degrade...... and fibrosis using transient elastography (Fibroscan). RESULTS: C3M, BGM, C4M and P4NP 7S were significantly elevated in HIV patients compared to controls and correlated to HIV viral loads and inversely to cART duration. C4M, P4NP 7S and ELM were lower in patients under cART therapy and in patients without HIV...

  20. The functions of grainy head-like proteins in animals and fungi and the evolution of apical extracellular barriers.

    Directory of Open Access Journals (Sweden)

    Adam Paré

    Full Text Available The Grainy head (GRH family of transcription factors are crucial for the development and repair of epidermal barriers in all animals in which they have been studied. This is a high-level functional conservation, as the known structural and enzymatic genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed. Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in animals, are also found in fungi--organisms that lack epidermal tissues. To shed light on CP2 protein function in fungi, we characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl. We show that Neurospora GRHL has a DNA-binding specificity similar to that of animal GRH proteins and dissimilar to that of animal LSF proteins. Neurospora grhl mutants are defective in conidial-spore dispersal due to an inability to remodel the cell wall, and we show that grhl mutants and the long-known conidial separation-2 (csp-2 mutants are allelic. We then characterized the transcriptomes of both Neurospora grhl mutants and Drosophila grh mutant embryos to look for similarities in the affected genes. Neurospora grhl appears to play a role in the development and remodeling of the cell wall, as well as in the activation of genes involved in defense and virulence. Drosophila GRH is required to activate the expression of many genes involved in cuticular/epidermal-barrier formation. We also present evidence that GRH plays a role in adult antimicrobial defense. These results, along with previous studies of animal GRH proteins, suggest the fascinating possibility that the apical extracellular barriers of some animals and fungi might share an evolutionary connection, and that the formation of physical barriers in the last common ancestor was under the control of a transcriptional code that included GRH-like proteins.

  1. BmECM25, from the silkworm Bombyx mori, is an extracellular matrix protein.

    Science.gov (United States)

    Zou, Ziliang; Xu, Yunmin; Ma, Bi; Xiang, Zhonghuai; He, Ningjia

    2015-10-01

    BmECM25 (previously reported as BmVMP25) was previously predicted as a gene encoding the vitelline membrane protein in silkworm, Bombyx mori. In this study, we investigated the detail temporal and spatial patterns of BmECM25 protein. Western blot results showed that BmECM25 was expressed in the follicular epithelium cells from stages -6 to +1, and was then secreted into the oocytes. However, the abundance of BmECM25 decreased during the subsequent oogenesis and finally disappeared in the mature follicles. Immunofluorescence detection showed that BmECM25 locates inside the VM layer and forms a discontinuous layer. These features of BmECM25 suggest that it is an oocyte membrane matrix protein, not a vitelline membrane protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Extracellular heat-shock protein 70 aggravates cerulein-induced pancreatitis through toll-like receptor-4 in mice

    Institute of Scientific and Technical Information of China (English)

    SONG Jun-min; WANG Rong; LIU Hong-xiang; LI Yuan; ZENG Yu-jian; ZHOU Zong-guang; LIU Hai-yi; XU Bing; WANG Ling; ZHOU Bin

    2008-01-01

    Background In patients suffering from acute pancreatitis, the pathogenesis is not completely understood, and several recent studies in vitro suggested that heat shock proteins might play an important role in cell signaling. To investigate the possible role of extracellular heat shock protein 70 (Hsp70) in pancreatitis, toll-like receptor-4 (TLR4)-deficient and wild-type mice were administered with exogenous Hsp70 during the course of cerulein-induced pancreatitis (CIP).Methods Acute pancreatitis was induced by 5 intraperitoneal injections of cerulein at hourly intervals, and then treated with recombinant Hsp70 through the caudal vein 4 hours after the start of cerulein injections. Subsequently serum amylase and serum cytokines levels were detected. Histologic alteration of the pancreas was evaluated. Tumor necrosis factor alpha (TNF-a) concentrations and myeloperoxidase (MPO) activity in both pancreas and lungs were analyzed. The nuclear factor kappa B (NF-KB) activation in pancreatic tissue was measured using a sensitive RelA enzyme-linked immunosorbent assay.Results Treatment with recombinant Hsp70 to wild-type mice in CIP resulted in significant aggravation of inflammation in pancreas, elevated levels of serum cytokines, up-regulation of pulmonary MPO activity and increase of lung tissues TNF-α concentrations. In contrast, treatment with Hsp70 to TLR4-deficient mice had little effect on serum cytokines levels, pancreatic inflammation, pulmonary MPO activity and TNF-a concentrations.Conclusions The results suggest that extracellular HspTO might induce systemic inflammatory response syndrome (SIRS)-Iike response in vivo and TLR4 might be involved in the Hsp70-mediated activation of inflammatory reaction in the progression of CIP without infection.

  3. DYS-REGULATION OF EXTRACELLULAR MATRIX PROTEINS TURNOVER BY HIGH GLUCOSE CONCENTRATIONS IN CULTURED HUMAN GLOMERULAR MESANGIAL CELLS

    Directory of Open Access Journals (Sweden)

    ROHOLLAH HOSSEINI

    1999-10-01

    Full Text Available Diabetic glomerulopathy is generally believed to be the major cause for the development of chronic renal failure in diabetes mellitus. Diabetic glomerulosclerosis is characterized by the accumulation of extracellular matrix proteins such as fibronectin and type IV collagen in the mesangium. In this study, the effect of high glucose (33.6 mM on fibronectin and type IV collagen concentrations in the supernatant of human glomerular mesangial cell culture was studied. The concentrations of fibronection and type IV collagen in tissue culture supernatant, assayed by ELISA techniques increased significantly (P<0.01 by the high glucose level (33.6 mM after 6 days incubation. Direct cell counting and thymidine incorporation methods showed that high glucose concentrations (33.6 mM, 56 mM and 112 mM inhibit the mesangial cell (MC proliferation in concentration-dependent manner. To study the osmotic effect of high glucose concentrations, the mesangial cells were also cultured in the presence of manitol and it was found that manitol did not have effect on cellular proliferation but increased fibronectin and type IV collagen concentrations significantly (P<0.05 in the supernatants. These results indicate that the increase of synthesis and/or decrease degradation of fibronectin and type IV collagen by MCs may, in part, result from changes in osmolarity induced by high glucose concentration. These results suggest that elevation of fibronectin and type IV collagen production and/or decrease their degradation by the mesangial cells may play an important role in the accumulation of these extracellular matrix proteins which is common to diabetic glomerulosclerosis.

  4. Impact of cyclic mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff fibroblasts.

    Science.gov (United States)

    Lohberger, Birgit; Kaltenegger, Heike; Stuendl, Nicole; Rinner, Beate; Leithner, Andreas; Sadoghi, Patrick

    2016-12-01

    Mechanical stimulation plays an important role in the development and remodelling of tendons. The aim of the study was to evaluate the effects of mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff (RC) fibroblasts. RC fibroblasts were isolated from patients with degenerative RC tears and characterized using flow cytometry and immunohistochemistry. Cells were stimulated using the Flexcell FX5K™ Tension System. The stimulation regime was a uniaxial sinusoidal waveform with 10 % elongation and a frequency of 0.5 Hz, whereby each cycle consists of 10-s strain and 30-s relaxation. Data were normalized to mechanically unstimulated control groups for every experimental condition. RT-qPCR was performed to determine relative mRNA levels, and collagen production was measured by a colorimetric assay. The positive expression of CD91 and CD10, and negativity for CD45 and CD4 confirmed the fibroblast phenotype of RC primary cells. RT-qPCR revealed that 10 % continuous cyclic strain for 7 and 14 days induced a significant increase in the mRNA expression both on the matrix metalloproteinases MMP1, MMP3, MMP13, and MMP14 and on the extracellular matrix proteins decorin, tenascin-C, and scleraxis. Furthermore, mechanically stimulated groups produced significantly higher amounts of total collagen. These results may contribute to a better understanding of strain-induced tendon remodelling and will form the basis for the correct choice of applied force in rehabilitation after orthopaedic surgery. These findings underline the fact that early passive motion of the joint in order to induce remodelling of the tendon should be included within a rehabilitation protocol for rotator cuff repair.

  5. Cooperative Regulation of Substrate Stiffness and Extracellular Matrix Proteins in Skin Wound Healing of Axolotls

    Directory of Open Access Journals (Sweden)

    Ting-Yu Huang

    2015-01-01

    Full Text Available Urodele amphibians (Ambystoma mexicanum, unique among vertebrates, can regenerate appendages and other body parts entirely and functionally through a scar-free healing process. The wound epithelium covering the amputated or damaged site forms early and is essential for initiating the subsequent regenerative steps. However, the molecular mechanism through which the wound reepithelializes during regeneration remains unclear. In this study, we developed an in vitro culture system that mimics an in vivo wound healing process; the biomechanical properties in the system were precisely defined and manipulated. Skin explants that were cultured on 2 to 50 kPa collagen-coated substrates rapidly reepithelialized within 10 to 15 h; however, in harder (1 GPa and other extracellular matrices (tenascin-, fibronectin-, and laminin-coated environments, the wound epithelium moved slowly. Furthermore, the reepithelialization rate of skin explants from metamorphic axolotls cultured on a polystyrene plate (1 GPa increased substantially. These findings afford new insights and can facilitate investigating wound epithelium formation during early regeneration using biochemical and mechanical techniques.

  6. 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.

  7. Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization.

    Science.gov (United States)

    Heiss, Silvia; Puxbaum, Verena; Gruber, Clemens; Altmann, Friedrich; Mattanovich, Diethard; Gasser, Brigitte

    2015-07-01

    Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.

  8. Colonization of the meat extracellular matrix proteins by O157 and non-O157 enterohemorrhagic Escherichia coli.

    Science.gov (United States)

    Chagnot, Caroline; Caccia, Nelly; Loukiadis, Estelle; Ganet, Sarah; Durand, Alexandra; Bertin, Yolande; Talon, Régine; Astruc, Thierry; Desvaux, Mickaël

    2014-10-01

    Enterohemorrhagic Escherichia coli (EHEC) are anthropozoonotic agents that range third among food-borne pathogens respective to their incidence and dangerousness in the European Union. EHEC are Shiga-toxin producing E. coli (STEC) responsible for foodborne poisoning mainly incriminated to the consumption of contaminated beef meat. Among the hundreds of STEC serotypes identified, EHEC mainly belong to O157:H7 but non-O157 can represent 20 to 70% of EHEC infections per year. Seven of those serogroups are especially of high-risk for human health, i.e. O26, O45, O103, O111, O121, O145 and O104. While meat can be contaminated all along the food processing chain, EHEC contamination essentially occurs at the dehiding stage of slaughtering. Investigating bacterial colonization to the skeletal-muscle extracellular matrix (ECM) proteins, it appeared that environmental factors influenced specific and non-specific bacterial adhesion of O157 and non-O157 EHEC as well as biofilm formation. Importantly, mechanical treatment (i.e. shaking, centrifugation, pipetting and vortexing) inhibited and biased the results of bacterial adhesion assay. Besides stressing the importance of the protocol to investigate bacterial adhesion to ECM proteins, this study demonstrated that the colonization abilities to ECM proteins vary among EHEC serogroups and should ultimately be taken into consideration to evaluate the risk of contamination for different types of food matrices. Copyright © 2014. Published by Elsevier B.V.

  9. Hepatitis B virus X protein inhibits extracellular IFN-α-mediated signal transduction by downregulation of type I IFN receptor.

    Science.gov (United States)

    Cho, Il-Rae; Oh, Myungju; Koh, Sang Seok; Malilas, Waraporn; Srisuttee, Ratakorn; Jhun, Byung Hak; Pellegrini, Sandra; Fuchs, Serge Y; Chung, Young-Hwa

    2012-04-01

    We have previously shown that hepatitis B virus (HBV) protein X (HBX), a regulatory protein of HBV, activates Stat1, leading to type I interferon (IFN) production. Type I IFN secreted from HBX-expressing hepatic cells enforces antiviral signals through its binding to the cognate type I IFN receptor. We therefore investigated how cells handle this detrimental situation. Interestingly, compared to Chang cells stably expressing an empty vector (Chang-Vec), Chang cells stably expressing HBX (Chang-HBX) showed lower levels of IFN-α receptor 1 (IFNAR1) protein, a subunit of type I IFN receptor. The levels of IFNAR1 transcripts detected in Chang-HBX cells were lower than the levels in Chang-Vec cells, indicating that HBX regulates IFNAR1 at the transcriptional level. Moreover, we observed that HBX induced the translocation of IFNAR1 to the cytoplasm. Consistent with these observations, HBX also downregulated Tyk2, which is required for the stable expression of IFNAR1 on the cell surface. Eventually, Chang-HBX cells consistently maintained a lower level of IFNAR1 expression and displayed no proper response to IFN-α, while Chang-Vec cells exhibited a proper response to IFN-α treatment. Taken together, we propose that HBX downregulates IFNAR1, leading to the avoidance of extracellular IFN-α signal transduction.

  10. Characterization ofTrichuris murissecreted proteins and extracellular vesicles provides new insights into host-parasite communication.

    Science.gov (United States)

    Eichenberger, Ramon M; Talukder, Md Hasanuzzaman; Field, Matthew A; Wangchuk, Phurpa; Giacomin, Paul; Loukas, Alex; Sotillo, Javier

    2018-01-01

    Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material, the mouse whipworm Trichuris muris has been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products of T. muris . We identify 148 proteins secreted by T. muris and show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep® gradient to purify the EVs, highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs, identifying >350 proteins, 56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping to T. muris gene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation, implying a role in parasite-driven immunomodulation. In addition, for the first time to our knowledge, colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted by T. muris provides important information on whipworm-host communication and forms the basis for future studies.

  11. 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

  12. CUB domain-containing protein 1, a prognostic factor for human pancreatic cancers, promotes cell migration and extracellular matrix degradation.

    Science.gov (United States)

    Miyazawa, Yuri; Uekita, Takamasa; Hiraoka, Nobuyoshi; Fujii, Satoko; Kosuge, Tomoo; Kanai, Yae; Nojima, Yoshihisa; Sakai, Ryuichi

    2010-06-15

    CUB domain-containing protein 1 (CDCP1) is a membrane protein that is highly expressed in several solid cancers. We reported previously that CDCP1 regulates anoikis resistance as well as cancer cell migration and invasion, although the underlying mechanisms have not been elucidated. In this study, we found that expression of CDCP1 in pancreatic cancer tissue was significantly correlated with overall survival and that CDCP1 expression in pancreatic cancer cell lines was relatively high among solid tumor cell lines. Reduction of CDCP1 expression in these cells suppressed extracellular matrix (ECM) degradation by inhibiting matrix metalloproteinase-9 secretion. Using the Y734F mutant of CDCP1, which lacks the tyrosine phosphorylation site, we showed that CDCP1 regulates cell migration, invasion, and ECM degradation in a tyrosine phosphorylation-dependent manner and that these CDCP1-associated characteristics were inhibited by blocking the association of CDCP1 and protein kinase Cdelta (PKCdelta). CDCP1 modulates the enzymatic activity of PKCdelta through the tyrosine phosphorylation of PKCdelta by recruiting PKCdelta to Src family kinases. Cortactin, which was detected as a CDCP1-dependent binding partner of PKCdelta, played a significant role in migration and invasion but not in ECM degradation of pancreatic cells. These results suggest that CDCP1 expression might play a crucial role in poor outcome of pancreatic cancer through promotion of invasion and metastasis and that molecules blocking the expression, phosphorylation, or the PKCdelta-binding site of CDCP1 are potential therapeutic candidates.

  13. Resting extracellular signal-regulated protein kinase 1/2 expression following a continuum of chronic resistance exercise training paradigms.

    Science.gov (United States)

    Galpin, Andrew J; Fry, Andrew C; Nicoll, Justin X; Moore, Christopher A; Schilling, Brian K; Thomason, Donald B

    2016-01-01

    Extracellular signal-regulated protein kinase 1/2 (ERK1/2) moderates skeletal muscle growth; however, chronic responses of this protein to unique resistance exercise (RE) paradigms are yet to be explored. The purpose of this investigation was to describe the long-term response of ERK1/2 following circuit weight training (CWT), recreationally weight training (WT), powerlifting (PL) and weightlifting (WL). Independent t-tests were used to determine differences in trained groups compared to sedentary controls. Total ERK1/2 content was lower in PL and WL compared to their controls (p ≤ 0.05). Specific trained groups displayed large (WL: pERK/total-ERK; d = 1.25) and moderate (CWT: total ERK1/2; d = 0.54) effect sizes for altered kinase expression compared to controls. The results indicate ERK1/2 expression is down-regulated after chronic RE in well-trained weightlifters and powerlifters. Lower expression of this protein may be a method in which anabolism is tightly regulated after many years of high-intensity RE.

  14. Synaptic release and extracellular actions of Zn2+ limit propagation of spreading depression and related events in vitro and in vivo

    Science.gov (United States)

    Aiba, Isamu; Carlson, Andrew P.; Sheline, Christian T.

    2012-01-01

    Cortical spreading depression (CSD) is a consequence of a slowly propagating wave of neuronal and glial depolarization (spreading depolarization; SD). Massive release of glutamate contributes to SD propagation, and it was recently shown that Zn2+ is also released from synaptic vesicles during SD. The present study examined consequences of extracellular Zn2+ accumulation on the propagation of SD. SD mechanisms were studied first in murine brain slices, using focal KCl applications as stimuli and making electrical and optical recordings in hippocampal area CA1. Elevating extracellular Zn2+ concentrations with exogenous ZnCl2 reduced SD propagation rates. Selective chelation of endogenous Zn2+ (using TPEN or CaEDTA) increased SD propagation rates, and these effects appeared due to chelation of Zn2+ derived from synaptic vesicles. Thus, in tissues where synaptic Zn2+ release was absent [knockout (KO) of vesicular Zn2+ transporter ZnT-3], SD propagation rates were increased, and no additional increase was observed following chelation of endogenous Zn2+ in these tissues. The role of synaptic Zn2+ was then examined on CSD in vivo. ZnT-3 KO animals had higher susceptibility to CSD than wild-type controls as evidenced by significantly higher propagation rates and frequencies. Studies of candidate mechanisms excluded changes in neuronal excitability, presynaptic release, and GABA receptors but left open a possible contribution of N-methyl-d-aspartate (NMDA) receptor inhibition. These results suggest the extracellular accumulation of synaptically released Zn2+ can serve as an intrinsic inhibitor to limit SD events. The inhibitory action of extracellular Zn2+ on SD may counteract to some extent the neurotoxic effects of intracellular Zn2+ accumulation in acute brain injury models. PMID:22131381

  15. Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery.

    Science.gov (United States)

    Wang, Ming; Altinoglu, Sarah; Takeda, Yuji S; Xu, Qiaobing

    2015-01-01

    Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes.

  16. An improved and robust DNA immunization method to develop antibodies against extra-cellular loops of multi-transmembrane proteins

    Science.gov (United States)

    Hazen, Meredith; Bhakta, Sunil; Vij, Rajesh; Randle, Steven; Kallop, Dara; Chiang, Vicki; Hötzel, Isidro; Jaiswal, Bijay S; Ervin, Karen E; Li, Bing; Weimer, Robby M; Polakis, Paul; Scheller, Richard H; Junutula, Jagath R; Hongo, Jo-Anne S

    2014-01-01

    Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation. PMID:24121517

  17. Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

    Science.gov (United States)

    Durmaz, Evelyn; Hu, Yan; Aroian, Raffi V.

    2015-01-01

    The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe. PMID:26682852

  18. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    Energy Technology Data Exchange (ETDEWEB)

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  19. 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.

  20. Immunophenotyping of congenital myopathies: disorganization of sarcomeric, cytoskeletal and extracellular matrix proteins

    NARCIS (Netherlands)

    van der Ven, P. F.; Jap, P. H.; ter Laak, H. J.; Nonaka, I.; Barth, P. G.; Sengers, R. C.; Stadhouders, A. M.; Ramaekers, F. C.

    1995-01-01

    We have studied the expression and distribution patterns of the intermediate filament proteins desmin and vimentin, the sarcomere components titin, nebulin and myosin, the basement membrane constituents collagen type IV and laminin, and the reticular layer component collagen type VI in skeletal

  1. Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia

    Science.gov (United States)

    McKee, Marc D.; Hoac, Betty; Addison, William N.; Barros, Nilana M.T.; Millán, José Luis; Chaussain, Catherine

    2013-01-01

    As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth – where calcium phosphate crystals are deposited and grow within an extracellular matrix – is essential to dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition such that teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibres (Sharpey's fibres) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here with clinical examples given, namely tissue-nonspecific alkaline phosphatase (TNAP) and phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX). Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH), respectively, where levels of local and systemic circulating mineralization determinants are perturbed. In XLH, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating SIBLING proteins such as matrix extracellular phosphoglycoprotein (MEPE) and osteopontin (OPN), and the phosphorylated peptides proteolytically released from them such as the acidic serine- and aspartate-rich motif (ASARM) peptide, may accumulate locally to impair mineralization in this disease. PMID:23931057

  2. Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells.

    Science.gov (United States)

    Mathews, Smitha; Bhonde, Ramesh; Gupta, Pawan Kumar; Totey, Satish

    2012-09-01

    The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications. Copyright © 2012 International

  3. Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner.

    Science.gov (United States)

    Polanco, Juan Carlos; Scicluna, Benjamin James; Hill, Andrew Francis; Götz, Jürgen

    2016-06-10

    The microtubule-associated protein tau has a critical role in Alzheimer disease and related tauopathies. There is accumulating evidence that tau aggregates spread and replicate in a prion-like manner, with the uptake of pathological tau seeds causing misfolding and aggregation of monomeric tau in recipient cells. Here we focused on small extracellular vesicles enriched for exosomes that were isolated from the brains of tau transgenic rTg4510 and control mice. We found that these extracellular vesicles contained tau, although the levels were significantly higher in transgenic mice that have a pronounced tau pathology. Tau in the vesicles was differentially phosphorylated, although to a lower degree than in the brain cells from which they were derived. Several phospho-epitopes (AT8, AT100, and AT180) thought to be critical for tau pathology were undetected in extracellular vesicles. Despite this, when assayed with FRET tau biosensor cells, extracellular vesicles derived from transgenic mice were capable of seeding tau aggregation in a threshold-dependent manner. We also observed that the dye used to label extracellular vesicle membranes was still present during nucleation and formation of tau inclusions, suggesting either a role for membranes in the seeding or in the process of degradation. Together, we clearly demonstrate that extracellular vesicles can transmit tau pathology. This indicates a role for extracellular vesicles in the transmission and spreading of tau pathology. The characteristics of tau in extracellular vesicles and the seeding threshold we identified may explain why tau pathology develops very slowly in neurodegenerative diseases such as Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine

    NARCIS (Netherlands)

    Wisselink, H.J.; Vecht, U.; Stockhofe Zurwieden, N.; Smith, H.E.

    2001-01-01

    The efficacy of a muramidase-released protein (MRP) and extracellular factor (EF) vaccine in preventing infection and disease in pigs challenged either with a homologous or a heterologous Streptococcus suis serotype 2 strain (MRP EF ) was compared with the efficacy of a vaccine containing

  5. The effect of stromelysin-1 (MMP-3) on non-collagenous extracellular matrix proteins of demineralized dentin and the adhesive properties of restorative resins

    NARCIS (Netherlands)

    Boukpessi, T.; Menashi, S.; Camoin, L.; ten Cate, J.M.; Goldberg, M.; Chaussain-Miller, C.

    2008-01-01

    Dentin non-collagenous matrix components (NCPs) are structural proteins involved in the formation, the architecture and the mineralization of the extracellular matrix (ECM). We investigated here how recombinant metalloproteinase stromelysin-1, also termed MMP-3, initiates the release of ECM

  6. Glioma progression through the prism of heat shock protein mediated extracellular matrix remodeling and epithelial to mesenchymal transition.

    Science.gov (United States)

    Rajesh, Y; Biswas, Angana; Mandal, Mahitosh

    2017-10-15

    Glial tumor is one of the intrinsic brain tumors with high migratory and infiltrative potential. This essentially contributes to the overall poor prognosis by circumvention of conventional treatment regimen in glioma. The underlying mechanism in gliomagenesis is bestowed by two processes- Extracellular matrix (ECM) Remodeling and Epithelial to mesenchymal transition (EMT). Heat Shock Family of proteins (HSPs), commonly known as "molecular chaperons" are documented to be upregulated in glioma. A positive correlation also exists between elevated expression of HSPs and invasive capacity of glial tumor. HSPs overexpression leads to mutational changes in glioma, which ultimately drive cells towards EMT, ECM modification, malignancy and invasion. Differential expression of HSPs - a factor providing cytoprotection to glioma cells, also contributes towards its radioresistance /chemoresistance. Various evidences also display upregulation of EMT and ECM markers by various heat shock inducing proteins e.g. HSF-1. The aim of this review is to study in detail the role of HSPs in EMT and ECM leading to radioresistance/chemoresistance of glioma cells. The existing treatment regimen for glioma could be enhanced by targeting HSPs through immunotherapy, miRNA and exosome mediated strategies. This could be envisaged by better understanding of molecular mechanisms underlying glial tumorigenesis in relation to EMT and ECM remodeling under HSPs influence. Our review might showcase fresh potential for the development of next generation therapeutics for effective glioma management. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Comparative adherence of Candida albicans and Candida dubliniensis to human buccal epithelial cells and extracellular matrix proteins.

    Science.gov (United States)

    Jordan, Rachael P C; Williams, David W; Moran, Gary P; Coleman, David C; Sullivan, Derek J

    2014-04-01

    Candida albicans and Candida dubliniensis are very closely related pathogenic yeast species. Despite their close relationship, C. albicans is a far more successful colonizer and pathogen of humans. The purpose of this study was to determine if the disparity in the virulence of the two species is attributed to differences in their ability to adhere to human buccal epithelial cells (BECs) and/or extracellular matrix proteins. When grown overnight at 30°C in yeast extract peptone dextrose, genotype 1 C. dubliniensis isolates were found to be significantly more adherent to human BECs than C. albicans or C. dubliniensis genotypes 2-4 (P albicans to human BECs was observed, and C. dubliniensis genotype 1 and C. albicans adhered to BECs in significantly greater numbers than the other C. dubliniensis genotypes (P albicans to type I and IV collagen, fibronectin, laminin, vitronectin, and proline-rich peptides. These data suggest that C. albicans is not more adherent to epithelial cells or matrix proteins than C. dubliniensis and therefore other factors must contribute to the greater levels of virulence exhibited by C. albicans.

  8. Alternative methods for characterization of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Fatemeh eMomen-Heravi

    2012-09-01

    Full Text Available Extracellular vesicles are nano-sized vesicles released by all cells in vitro as well as in vivo. Their role has been implicated mainly in cell-cell communication, but also in disease biomarkers and more recently in gene delivery. They represent a snapshot of the cell status at the moment of release and carry bioreactive macromolecules such as nucleic acids, proteins and lipids. A major limitation in this emerging new field is the availability/awareness of techniques to isolate and properly characterize Extracellular vesicles. The lack of gold standards makes comparing different studies very difficult and may potentially hinder some Extracellular vesicles -specific evidence. Characterization of Extracellular vesicles has also recently seen many advances with the use of Nanoparticle Tracking Analysis (NTA, flow cytometry, cryo-EM instruments and proteomic technologies. In this review, we discuss the latest developments in translational technologies involving characterization methods including the facts in their support and the challenges they face.

  9. 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...... 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...

  10. Proteases decode the extracellular matrix cryptome.

    Science.gov (United States)

    Ricard-Blum, Sylvie; Vallet, Sylvain D

    2016-03-01

    The extracellular matrix is comprised of 1100 core-matrisome and matrisome-associated proteins and of glycosaminoglycans. This structural scaffold contributes to the organization and mechanical properties of tissues and modulates cell behavior. The extracellular matrix is dynamic and undergoes constant remodeling, which leads to diseases if uncontrolled. Bioactive fragments, called matricryptins, are released from the extracellular proteins by limited proteolysis and have biological activities on their own. They regulate numerous physiological and pathological processes such as angiogenesis, cancer, diabetes, wound healing, fibrosis and infectious diseases and either improve or worsen the course of diseases depending on the matricryptins and on the molecular and biological contexts. Several protease families release matricryptins from core-matrisome and matrisome-associated proteins both in vitro and in vivo. The major proteases, which decrypt the extracellular matrix, are zinc metalloproteinases of the metzincin superfamily (matrixins, adamalysins and astacins), cysteine proteinases and serine proteases. Some matricryptins act as enzyme inhibitors, further connecting protease and matricryptin fates and providing intricate regulation of major physiopathological processes such as angiogenesis and tumorigenesis. They strengthen the role of the extracellular matrix as a key player in tissue failure and core-matrisome and matrisome-associated proteins as important therapeutic targets. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  11. Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles.

    Science.gov (United States)

    Mota, Rita; Pereira, Sara B; Meazzini, Marianna; Fernandes, Rui; Santos, Arlete; Evans, Caroline A; De Philippis, Roberto; Wright, Phillip C; Tamagnini, Paula

    2015-04-29

    The effects of several heavy metals on the growth/survival, EPS production, ultrastructure and protein profiles of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110 were evaluated. Our results clearly show that each heavy metal affects the cells in a particular manner, triggering distinctive responses. Concerning chronic exposure, cells were more affected by Cu(2+) followed by Pb(2+), Cd(2+), and Li(+). The presence of metal leads to remarkable ultrastructural changes, mainly at the thylakoid level. The comparison of the proteomes (iTRAQ) allowed to follow the stress responses and to distinguish specific effects related to the time of exposure and/or the concentration of an essential (Cu(2+)) and a non-essential (Cd(2+)) metal. The majority of the proteins identified and with fold changes were associated with photosynthesis, CO2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism. Moreover, our results indicate that during chronic exposure to sub-lethal concentrations of Cu(2+), the cells tune down their metabolic rate to invest energy in the activation of detoxification mechanisms, which eventually result in a remarkable recovery. In contrast, the toxic effects of Cd(2+) are cumulative. Unexpectedly, the amount of released polysaccharides (RPS) was not enhanced by the presence of heavy metals. This work shows the holistic effects of different heavy metals on the cells of the highly efficient EPS-producer the cyanobacterium Cyanothece sp. CCY 0110. The growth/survival, EPS production, ultrastructure, protein profiles and stress response were evaluated. The knowledge generated by this study will contribute to the implementation of heavy-metal removal systems based on cyanobacteria EPS or their isolated polymers. Copyright © 2015. Published by Elsevier B.V.

  12. Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

    Science.gov (United States)

    Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A; Marciano-Cabral, Francine

    2012-03-01

    Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

  13. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

    Energy Technology Data Exchange (ETDEWEB)

    Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Asaoka, Yoichi; Nishina, Hiroshi [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Kaga Itabashi-Ku, Tokyo 173-8605 (Japan); Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.

  14. Vps15p regulates the distribution of cup-shaped organelles containing the major eisosome protein Pil1p to the extracellular fraction required for endocytosis of extracellular vesicles carrying metabolic enzymes.

    Science.gov (United States)

    Stein, Kathryn; Winters, Chelsea; Chiang, Hui-Ling

    2017-05-01

    Exosomes are small vesicles secreted from virtually every cell from bacteria to humans. Saccharomyces cerevisiae is a model system to study trafficking of small vesicles in response to changes in the environment. When yeast cells are grown in low glucose, vesicles carrying gluconeogenic enzymes are present as free vesicles and aggregated clusters in the cytoplasm. These vesicles are also secreted into the periplasm and account for more than 90% of total extracellular organelles, while less than 10% are larger 100-300 nm structures with unknown functions. When glucose is added to glucose-starved cells, secreted vesicles are endocytosed and then targeted to the vacuole. Recent secretomic studies indicated that more than 300 proteins involved in diverse biological functions are secreted during glucose starvation and endocytosed during glucose re-feeding. We hypothesised that extracellular vesicles are internalised using novel mechanisms independent of clathrin-mediated endocytosis. Our results showed that vesicles carrying metabolic enzymes were endocytosed at a fast rate, whereas vesicles carrying the heat shock protein Ssa1p were endocytosed at a slow rate. The PI3K regulator Vps15p is critical for the fast internalisation of extracellular vesicles. VPS15 regulates the distribution of the 100-300 nm organelles that contain the major eisosome protein Pil1p to the extracellular fraction. These Pil1p-containing structures were purified and showed unique cup-shape with their centres deeper than the peripheries. In the absence of VPS15, PIL1 or when PIL1 was mutated, the 100-300 nm structures were not observed in the extracellular fraction and the rapid internalisation of vesicles was impaired. We conclude that VPS15 regulates the distribution of the 100-300 nm Pil1p-containing organelles to the extracellular fraction required for fast endocytosis of vesicles carrying metabolic enzymes. This work provides the first evidence showing that Pil1p displayed unique

  15. Neuronal nitric oxide contributes to neuroplasticity-associated protein expression through cGMP, protein kinase G, and extracellular signal-regulated kinase.

    Science.gov (United States)

    Gallo, Eduardo F; Iadecola, Costantino

    2011-05-11

    Nitric oxide (NO) synthesized by neuronal NO synthase (nNOS) has long been implicated in brain plasticity. However, it is unclear how this short-lived mediator contributes to the long-term molecular changes underlying neuroplasticity, which typically require activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway and gene expression. To address this issue, we used a neuroplasticity model based on treatment of neuronal cultures with bicuculline and a model of experience-dependent plasticity in the barrel cortex. In neuronal cultures, NOS inhibition attenuated the bicuculline-induced activation of ERK and the expression of c-Fos, Egr-1, Arc, and brain-derived neurotrophic factor (BDNF), proteins essential for neuroplasticity. Furthermore, inhibition of the NO target soluble guanylyl cyclase or of the cGMP effector kinase protein kinase G (PKG) reduced both ERK activation and plasticity-related protein expression. NOS inhibition did not affect phosphorylation of cAMP response element-binding protein (CREB), a well-established ERK nuclear target, but it attenuated the nuclear accumulation of the CREB coactivator TORC1 and suppressed the activation of Elk-1, another transcription factor target of ERK. Consistent with these in vitro observations, induction of c-Fos, Egr-1, and BDNF was attenuated in the D1 cortical barrel of nNOS(-/-) mice subjected to single whisker experience. These results establish nNOS-derived NO as a key factor in the expression of proteins involved in neuroplasticity, an effect mediated through cGMP, PKG, and ERK signaling. These actions of NO do not depend on CREB phosphorylation but may involve TORC1 and Elk-1. Our data unveil a previously unrecognized link between neuronal NO and the molecular machinery responsible for the sustained synaptic changes underlying neuroplasticity.

  16. 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

    Pulmonary surfactant protein A (SP-A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro-organisms, and mediates microbial lysis and clearance. SP-A also modulates multiple immune-related functions including cytokine production and chemotaxis...... 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...... with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium-dependent binding between MFAP4 and human SP-A1 and SP-A2. No binding was seen to recombinant SP-A...

  17. Extracellular adherence protein of Staphylococcus aureus suppresses disease by inhibiting T-cell recruitment in a mouse model of psoriasis.

    Science.gov (United States)

    Wang, Honglin; von Rohrscheidt, Julia; Roehrbein, Jan; Peters, Thorsten; Sindrilaru, Anca; Kess, Daniel; Preissner, Klaus T; Scharffetter-Kochanek, Karin

    2010-03-01

    Psoriasis is a T-cell-mediated inflammatory disease. Previous studies focused on lymphocyte function-associated antigen 1 (LFA-1)-expressing T cells as a molecular target for therapeutic intervention. By contrast, information on therapeutic effects and the underlying mechanism of blocking the LFA-1 counter receptor, ICAM-1 is scarce. Here, we used the CD18 (beta2-integrin) hypomorphic (CD18hypo) mouse model of psoriasis to investigate the therapeutic role of extracellular adherence protein (Eap) of Staphylococcus aureus, which exerts antiinflammatory activities by interacting with the ICAM-1 function. We show that ICAM-1 is predominantly upregulated on endothelial cells in lesional skin of CD18hypo mice. In vitro Eap was found to disrupt cell-cell contacts between T cells and dendritic cells, and inhibit T-cell proliferation. By contrast, in vivo Eap rather blocked transmigration of T cells from vessels to inflamed skin of CD18hypo mice, but did not inhibit their proliferation and activation. Most importantly, Eap successfully suppressed the disease by blocking T-cell extravasation into the inflamed skin. Together, these data indicate that interaction between LFA-1 and ICAM-1 is causally involved in the pathogenesis of psoriasiform skin inflammation, and targeting ICAM-1 to selectively block T-cell extravasation by Eap without immune suppression may represent a potential therapeutic strategy for psoriasis.

  18. Role of extracellular matrix protein CabA in resistance of Vibrio vulnificus biofilms to decontamination strategies.

    Science.gov (United States)

    Park, Jin Hwan; Lee, Byungho; Jo, Youmi; Choi, Sang Ho

    2016-11-07

    Biofilms are recalcitrant and raise safety problems in the food industry. In this study, the role of CabA, an extracellular matrix protein, in the resistance of the biofilms of Vibrio vulnificus, a foodborne pathogen, to decontamination strategies was investigated. Biofilms of the cabA mutant revealed reduced resistance to detachment by vibration and disinfection by sodium hypochlorite compared to the biofilms of the parental wild type in vitro. The reduced resistance of the cabA mutant biofilms was complemented by introducing a recombinant cabA, indicating that the reduced resistance of the cabA mutant biofilms is caused by the inactivation of cabA. The expression of cabA was induced in cells bound to oyster, the primary vehicle of the pathogen. The cabA mutant biofilms on oyster are defective in biomass and resistance to detachment and disinfection. The bacterial cells in the wild-type biofilms are clustered by filaments which are not apparent in the cabA mutant biofilms. The combined results indicated that CabA contributes to the structural integrity of V. vulnificus biofilms possibly by forming filaments in the matrix and thus rendering the biofilms robust, suggesting that CabA could be a target to control V. vulnificus biofilms on oyster. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. 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.

  20. Apelin increases cardiac contractility via protein kinase Cε- and extracellular signal-regulated kinase-dependent mechanisms.

    Directory of Open Access Journals (Sweden)

    Ábel Perjés

    Full Text Available BACKGROUND: Apelin, the endogenous ligand for the G protein-coupled apelin receptor, is an important regulator of the cardiovascular homoeostasis. We previously demonstrated that apelin is one of the most potent endogenous stimulators of cardiac contractility; however, its underlying signaling mechanisms remain largely elusive. In this study we characterized the contribution of protein kinase C (PKC, extracellular signal-regulated kinase 1/2 (ERK1/2 and myosin light chain kinase (MLCK to the positive inotropic effect of apelin. METHODS AND RESULTS: In isolated perfused rat hearts, apelin increased contractility in association with activation of prosurvival kinases PKC and ERK1/2. Apelin induced a transient increase in the translocation of PKCε, but not PKCα, from the cytosol to the particulate fraction, and a sustained increase in the phosphorylation of ERK1/2 in the left ventricle. Suppression of ERK1/2 activation diminished the apelin-induced increase in contractility. Although pharmacological inhibition of PKC attenuated the inotropic response to apelin, it had no effect on ERK1/2 phosphorylation. Moreover, the apelin-induced positive inotropic effect was significantly decreased by inhibition of MLCK, a kinase that increases myofilament Ca2+ sensitivity. CONCLUSIONS: Apelin increases cardiac contractility through parallel and independent activation of PKCε and ERK1/2 signaling in the adult rat heart. Additionally MLCK activation represents a downstream mechanism in apelin signaling. Our data suggest that, in addition to their role in cytoprotection, modest activation of PKCε and ERK1/2 signaling improve contractile function, therefore these pathways represent attractive possible targets in the treatment of heart failure.

  1. Soluble human leukocyte antigen-g5 activates extracellular signal-regulated protein kinase signaling and stimulates trophoblast invasion.

    Science.gov (United States)

    Guo, YiFan; Lee, Cheuk-Lun; So, Kam-Hei; Gao, Jing; Yeung, William S B; Yao, YuanQing; Lee, Kai-Fai

    2013-01-01

    Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), in primary trophoblasts and trophoblastic (JAr and JEG-3) cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (ptrophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059) nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway.

  2. Soluble human leukocyte antigen-g5 activates extracellular signal-regulated protein kinase signaling and stimulates trophoblast invasion.

    Directory of Open Access Journals (Sweden)

    YiFan Guo

    Full Text Available Soluble human leukocyte antigen-G (HLA-G is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1 and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4, in primary trophoblasts and trophoblastic (JAr and JEG-3 cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (p<0.05 and increased urokinase (uPA and matrix metalloproteinases (MMPs transcripts and their activity (p<0.05 in trophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059 nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway.

  3. Parathyroid hormone inhibits TGF-β/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells.

    Science.gov (United States)

    Peng, Fang-Fang; Xiao, Ze-Ling; Chen, Hong-Min; Chen, Yan; Zhou, Jian; Yu, Hong; Zhang, Bai-Fang

    2016-09-23

    Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. TGF-β1 is a major cytokine mediating the production of various extracellular matrix (ECM) proteins. The aim of this study is to elucidate the effect of parathyroid hormone (PTH) on TGF-β1 and high glucose-induced upregulation of ECM proteins in primary mesangial cells from Sprague-Dawley rat. The results showed that PTH pretreatment prevented TGF-β1 and high glucose-induced Smad2/3 phosphorylation and consequent upregulation of fibronectin and type IV collagen within 4 h. The inhibitory effect of PTH is due to PTH1R activation, because knocking down PTH 1 receptor (PTH1R) by RNA interference reversed the inhibitory effect of PTH on TGF-β1 and high glucose-induced Smad2/3 phosphorylation and ECM upregulation. Furthermore, it is found that PTH1R associated with TGF-β type II receptor (TβR II) and both receptors internalized into the cytoplasm when mesangial cells were stimulated with PTH alone. The internalization of TβR II might reduce the amount of membrane TβR II, attenuate the sensitivity of mesangial cells to TGF-β1, and therefore inhibit Smad activation and ECM upregulation induced by TGF-β1 and high glucose. Further studies are needed to know whether the endocytic receptors are to be degraded or recycled, and evaluate the role of PTH in TGF-β1 signaling more comprehensively. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Down regulation of NO signaling in Trypanosoma cruzi upon parasite-extracellular matrix interaction: changes in protein modification by nitrosylation and nitration.

    Directory of Open Access Journals (Sweden)

    Milton Pereira

    2015-04-01

    Full Text Available Adhesion of the Trypanosoma cruzi trypomastigotes, the causative agent of Chagas' disease in humans, to components of the extracellular matrix (ECM is an important step in host cell invasion. The signaling events triggered in the parasite upon binding to ECM are less explored and, to our knowledge, there is no data available regarding •NO signaling.Trypomastigotes were incubated with ECM for different periods of time. Nitrated and S-nitrosylated proteins were analyzed by Western blotting using anti-nitrotyrosine and S-nitrosyl cysteine antibodies. At 2 h incubation time, a decrease in NO synthase activity, •NO, citrulline, arginine and cGMP concentrations, as well as the protein modifications levels have been observed in the parasite. The modified proteins were enriched by immunoprecipitation with anti-nitrotyrosine antibodies (nitrated proteins or by the biotin switch method (S-nitrosylated proteins and identified by MS/MS. The presence of both modifications was confirmed in proteins of interest by immunoblotting or immunoprecipitation.For the first time it was shown that T. cruzi proteins are amenable to modifications by S-nitrosylation and nitration. When T. cruzi trypomastigotes are incubated with the extracellular matrix there is a general down regulation of these reactions, including a decrease in both NOS activity and cGMP concentration. Notwithstanding, some specific proteins, such as enolase or histones had, at least, their nitration levels increased. This suggests that post-translational modifications of T. cruzi proteins are not only a reflex of NOS activity, implying other mechanisms that circumvent a relatively low synthesis of •NO. In conclusion, the extracellular matrix, a cell surrounding layer of macromolecules that have to be trespassed by the parasite in order to be internalized into host cells, contributes to the modification of •NO signaling in the parasite, probably an essential move for the ensuing invasion step.

  5. Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

    Science.gov (United States)

    Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

    2016-12-21

    Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

  6. 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.

  7. Sub-inhibitory tigecycline concentrations induce extracellular matrix binding protein Embp dependent Staphylococcus epidermidis biofilm formation and immune evasion.

    Science.gov (United States)

    Weiser, Julian; Henke, Hanae A; Hector, Nina; Both, Anna; Christner, Martin; Büttner, Henning; Kaplan, Jeffery B; Rohde, Holger

    2016-09-01

    Biofilm-associated Staphylococcus epidermidis implant infections are notoriously reluctant to antibiotic treatment. Here we studied the effect of sub-inhibitory concentrations of penicillin, oxacillin, vancomycin, daptomycin, linezolid and tigecycline on S. epidermidis 1585 biofilm formation, expression of extracellular matrix binding protein (Embp) and potential implications for S. epidermidis - macrophage interactions. Penicillin, vancomycin, daptomycin, and linezolid had no biofilm augmenting effect at any of the concentrations tested. In contrast, at sub-inhibitory concentrations tigecycline and oxacillin exhibited significant biofilm inducing activity. In S. epidermidis 1585, SarA is a negative regulator of giant 1 MDa Embp, and down regulation of sarA induces Embp-dependent assembly of a multi-layered biofilm architecture. Dot blot immune assays, confocal laser scanning microscopy, and qPCR showed that under biofilm inducing conditions, tigecycline augmented embp expression compared to the control grown without antibiotics. Conversely, expression of regulator sarA was suppressed, suggesting that tigecycline exerts its effects on embp expression through SarA. Tigecycline failed to induce biofilm formation in embp transposon mutant 1585-M135, proving that under these conditions Embp up-regulation is necessary for biofilm accumulation. As a functional consequence, tigecycline induced biofilm formation significantly impaired the up-take of S. epidermidis by mouse macrophage-like cell line J774A.1. Our data provide novel evidence for the molecular basis of antibiotic induced biofilm formation, a phenotype associated with inherently increased antimicrobial tolerance. While this could explain failure of antimicrobial therapies, persistence of S. epidermidis infections in the presence of sub-inhibitory antimicrobials is additionally propelled by biofilm-related impairment of macrophage-mediated pathogen eradication. Copyright © 2016 Elsevier GmbH. All rights

  8. Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

    OpenAIRE

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H. Eric

    2011-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism...

  9. The physical limits of metal reduction by long-range extracellular electron transfer, and the role of cytochrome-bound flavins

    Science.gov (United States)

    Michelson, K.; Sanford, R. A.; Valocchi, A. J.; Werth, C. J.

    2015-12-01

    Microbial reduction of metals and radionuclides in the subsurface plays an essential role in the biogeochemical cycling of micronutrients and the remediation of contaminated groundwater. While recent advances in the field have improved our ability to understand and predict bioreduction in these environments, the contribution of long-range extracellular electron transfer (EET) by electron shuttling or reduction along conductive pili remains elusive. Long-range EET is implicated in the reduction of radionuclides like uranium that are reversibly sorbed in clay nanopores and exist as persistant sources of contamination. In regions of low hydraulic conductivity, electron shuttles and conductive pili may increase physical mixing beyond what is possible by advection and diffusion, resulting in reduction over a larger area than predicted by current models. We present a novel microfluidic platform that allows us to study long-range EET to the exclusion of other mechanisms, directly observe these phenomena under a controlled environment representative of groundwater conditions, monitor the metabolic activity and redox state of bacteria, and determine the presence of reduced products in-situ. Using Geobacter sulfurreducens as a model metal-reducing bacteria, insoluble manganese dioxide as an electron acceptor, and Escherichia coli K-12 as a reductant and redox buffer, we demonstate that 1) long-range EET by conductive pili requires the presence of flavins 2) Reduction by direct contact only requires the presence of a lowered electric potential 3) The limit of reduction by conductive pili is on the order of 15-20 microns. We are actively exploring the influence of hydrological conditions on the expression of different mechanisms of long-range EET, and the importance of extracellular cytochromes and pili conductivity on metal reduction.

  10. Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles.

    Science.gov (United States)

    Mørk, Morten; Handberg, Aase; Pedersen, Shona; Jørgensen, Malene M; Bæk, Rikke; Nielsen, Morten K; Kristensen, Søren R

    2017-01-01

    Introduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA). Methods: Ten platelet-free plasma (PFP) samples from healthy fasting subjects were incubated with magnetic beads coated with antibodies against apolipoprotein B-48 and B-100 (ApoB). Plasma samples were analysed with NTA before and after application of the bead procedure. Four fasting PFP samples were analysed with an ELISA specific for human ApoB to estimate the degree of removal of lipoproteins and EV array analysis was used for identification of possible EV loss. Results: The magnetic bead separation procedure resulted in a median reduction of the particle concentration in plasma by 62% (interquartile range 32-72%). The mean size of the remaining particles generally increased. ApoB concentration was reduced to a level close to the background signal, whereas a median reduction of the EV content by 21% (range 8-43%) was observed. Conclusion: Anti-ApoB antibody coated magnetic beads may hold potential for removal of lipoproteins from human PFP prior to EV measurement by NTA but some artefactual effect and EV loss may have to be endured.

  11. The Small GTPase MoSec4 Is Involved in Vegetative Development and Pathogenicity by Regulating the Extracellular Protein Secretion in Magnaporthe oryzae

    Directory of Open Access Journals (Sweden)

    Huakun Zheng

    2016-09-01

    Full Text Available The Rab GTPase proteins play important roles in the membrane trafficking, and consequently protein secretion and development of eukaryotic organisms. However, little is known about the function of Rab GTPases in Magnaporthe oryzae. To further explore the function of Rab GTPases, we deleted the ortholog of the yeast Sec4p protein in M. oryzae, namely MoSEC4. The Mosec4 mutant is defective in polarized growth and conidiation, and it displays decreased appressorium turgor pressure and attenuated pathogenicity. Notably, the biotrophic invasive hyphae produced in rice cells are more bulbous and compressed in the Mosec4 mutant. Further studies showed that deletion of the MoSEC4 gene resulted in decreased secretion of extracellular enzymes and mislocalization of the cytoplasmic effector PWL2-mCherry-NLS. In accordance with its role in secretion, the GFP-MoSec4 fusion protein mainly accumulates at tips of growing vegetative hyphae. Our results suggest that the MoSec4 protein plays important roles in the secretion of extracellular proteins and consequently hyphal development and pathogenicity in the rice blast fungus.

  12. 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.

  13. Adenovirus-induced extracellular signal-regulated kinase phosphorylation during the late phase of infection enhances viral protein levels and virus progeny

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    The Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling cascade enhances tumor cell proliferation in many cases. Here, we show that adenovirus type 5, a small DNA tumor virus used in experimental cancer therapy, strongly induces ERK phosphorylation...... during the late phase of infection. Pharmacologic inhibition of ERK phosphorylation reduced virus recovery by >100-fold. Blocking MEK/ERK signaling affected virus DNA replication and mRNA levels only weakly but strongly reduced the amount of viral proteins, independently of the kinases MNK1 and PKR...

  14. Growth-limiting Proteins in Relation to Auxin-induced Elongation in Lupin Hypocotyls

    Science.gov (United States)

    Penny, Pauline

    1971-01-01

    The role of protein synthesis in auxin-induced cell elongation in lupin hypocotyl segments was studied using cycloheximide. Cycloheximide inhibited protein synthesis by 9 minutes. Experiments adding cycloheximide at various times before and after indolyl-3-acetic acid are reported. Estimates of the relative amounts of growth-limiting protein(s), and a first order rate constant for the apparent turnover of the growth-limiting protein(s) were made. It was shown that there is a sizeable growth promotion by auxin after protein synthesis has essentially ceased. It is concluded that the initial phases of auxin action do not require protein synthesis but that its action depends on the existing pool of growth-limiting proteins which is rapidly depleted, and protein synthesis is then required for continued elongation. PMID:16657867

  15. Carboxy-terminal modulator protein attenuated extracellular matrix deposit by inhibiting phospho-Akt, TGF-β1 and α-SMA in kidneys of diabetic mice.

    Science.gov (United States)

    Chen, Ning; Hao, Jun; Li, Lisha; Li, Fan; Liu, Shuxia; Duan, Huijun

    2016-06-10

    Glomerulosclerosis and tubular interstitial extracellular matrix deposit and fibrosis are the main features of diabetic nephropathy, which are mediated by activation of PI3K/Akt signal pathway. Carboxy-terminal modulator protein (CTMP) is known as a negative regulator of PI3K/Akt pathway. Whether CTMP regulates renal extracellular matrix metabolism of diabetic nephropathy is still not known. Here, renal decreased CTMP, enhanced phospho-Akt (Ser 473), TGF-β1, α-SMA and extracellular matrix deposit are found in diabetic mice. Furthermore, high glucose decreases CTMP expression accompanied by enhanced phospho-Akt (Ser 473), TGF-β1 and α-SMA in cultured human renal proximal tubular epithelial cells (HKC), which are effectively prevented by transfection of pYr-ads-4-musCTMP vector. Moreover, delivery of pYr-ads-4-musCTMP vector into kidneys via tail vein of diabetic mice increases CTMP expression by 8.84 times followed by 60.00%, 76.50% and 24.37% decreases of phospho-Akt (Ser 473), TGF-β1 and α-SMA compared with diabetic mice receiving pYr-adshuttle-4 vector. Again, increased renal extracellular matrix accumulation of diabetic mice is also inhibited with delivery of pYr-ads-4-musCTMP vector. Our results indicate that CTMP attenuates renal extracellular matrix deposit by regulating the phosphorylation of Akt, TGF-β1 and α-SMA expression in diabetic mice. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. High ω-3:ω-6 fatty acids ratio increases fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in human ectopic endometrial cells

    Science.gov (United States)

    Khanaki, Korosh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mehdi; Darabi, Masoud; Mehdizadeh, Amir; Shabani, Mahdi; Rahimipour, Ali; Nouri, Mohammad

    2014-01-01

    Background: Endometriosis, a common chronic inflammatory disorder, is defined by the atypical growth of endometrium- like tissue outside of the uterus. Secretory phospholipase A2 group IIa (sPLA2-IIa) and fatty acid binding protein4 (FABP4) play several important roles in the inflammatory diseases. Objective: Due to reported potential anti-inflammatory effects of ω-3 and ω-6 fatty acids, the purpose of the present study was to investigate the effects of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) on fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in cultured endometrial cells. Materials and Methods: Ectopic and eutopic endometrial tissues obtained from 15 women were snap frozen. After thawing and tissue digestion, primary mixed stromal and endometrial epithelial cell culture was performed for 8 days in culture mediums supplemented with normal and high ratios of ω-3 and ω-6 PUFA. sPLA2-IIa in the culture medium and FABP4 level was determined using enzyme immuno assay (EIA) technique. Results: Within ectopic endometrial cells group, the level of cellular FABP4 and extracellular sPLA2-IIa were remarkably increased under high ω-3 PUFA exposure compared with control condition (p=0.014 and p=0.04 respectively). Conclusion: ω-3 PUFAs may increase the level of cellular FABP4 and extracellular sPLA2-IIa in ectopic endometrial cells, since sPLAIIa and FABP4 may affect endometriosis via several mechanisms, more relevant studies are encouraged to know the potential effect of increased cellular FABP4 and extracellular sPLA2-IIa on endometriosis. PMID:25709631

  17. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou (NCSU)

    2016-10-26

    Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses inArabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein,Camelina sativalectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space groupC222 orC2221, with unit-cell parametersa= 94.7,b= 191.5,c= 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.

  18. Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer.

    Science.gov (United States)

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Izaguirre, Maitane; Hernández-Lizoain, José Luis; Baixauli, Jorge; Martí, Pablo; Valentí, Víctor; Moncada, Rafael; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

    2016-01-01

    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 (Pobesity (Pobese 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.

  19. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa.

    Science.gov (United States)

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou

    2016-10-01

    Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses in Arabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein, Camelina sativa lectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space group C222 or C2221, with unit-cell parameters a = 94.7, b = 191.5, c = 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.

  20. Brain metabolism and the acquisition of new behaviors. III. Evidence for secretion of two proteins into the brain extracellular fluid after training.

    Science.gov (United States)

    Shashoua, V E

    1979-04-27

    Immunochemical and double labeling experiments were used to demonstrate that at least two out of three brain cytoplasmic proteins, whose metabolism is markedly influenced by behavior, are products which are secreted into the extracellular fluid (ECF) of goldfish brain. Even after 1 h of labeling, the extracts of goldfish brains with 0.32 M sucrose were found to contain highly labeled proteins. Electrophoretic analyses of the proteins, on SDS polyacrylamide gels, indicated that an increased incorporation of [3H]valine occurs for trained animals as compared to untrained controls ([14C]valine) at specific protein bands migrating at 32,000 and 26,000 daltons. The proteins in the ECF gave identical precipitin bands to goldfish brain proteins whose metabolism was responsive to the acquisition of new patterns of behavior and to proteins in CSF. These results are consistant with the hypothesis that the cells which contain the proteins and whose location in the ependymal zone was determined by immunohistochemistry can secrete the products into ECF. It is therefore quite possible that the functional sites of the proteins are away from the locus of their synthesis.

  1. Characterizing the range of extracellular protein post-translational modifications in a cellulose-degrading bacteria using a multiple proteolyic digestion/peptide fragmentation approach.

    Science.gov (United States)

    Dykstra, Andrew B; Rodriguez, Miguel; Raman, Babu; Cook, Kelsey D; Hettich, Robert L

    2013-03-19

    Post-translational modifications (PTMs) are known to play a significant role in many biological functions. The focus of this study is to optimize an integrated experimental/informatics approach to more confidently characterize the range of post-translational modifications of the cellulosome protein complex used by the bacterium Clostridium thermocellum to better understand how this protein machine is tuned for enzymatic cellulose solubilization. To enhance comprehensive characterization, the extracellular cellulosome proteins were analyzed using multiple proteolytic digests (trypsin, Lys-C, Glu-C) and multiple fragmentation techniques (collisionally activated dissociation, electron transfer dissociation, decision tree). As expected, peptide and protein identifications were increased by utilizing alternate proteases and fragmentation methods, in addition to the increase in protein sequence coverage. The complementarity of these experiments also allowed for a global exploration of PTMs associated with the cellulosome based upon a set of defined PTMs that included methylation, oxidation, acetylation, phosphorylation, and signal peptide cleavage. In these experiments, 85 modified peptides corresponding to 28 cellulosome proteins were identified. Many of these modifications were located in active cellulolytic or structural domains of the cellulosome proteins, suggesting a level of possible regulatory control of protein function in various cellulotyic conditions. The use of complementary proteolytic digestion/peptide fragmentation processes allowed for independent verification of PTMs in different experiments, thus leading to increased confidence in PTM identifications.

  2. Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach

    Energy Technology Data Exchange (ETDEWEB)

    Dykstra, Andrew B [ORNL; Rodriguez, Jr., Miguel [ORNL; Raman, Babu [Dow Chemical Company, The; Cook, Kelsey [ORNL; Hettich, Robert {Bob} L [ORNL

    2013-01-01

    Post-translational modifications (PTMs) are known to play a significant role in many biological functions. The focus of this study is to characterize the post-translational modifications of the cellulosome protein complex used by the bacterium Clostridium thermocellum to better understand how this protein machine is tuned for enzymatic cellulose solubilization. To enhance comprehensive characterization, the extracellular cellulosome proteins were analyzed using multiple proteolytic digests (trypsin, Lys-C, Glu-C) and multiple fragmentation techniques (collisionally-activated dissociation, electron transfer dissociation, decision tree). As expected, peptide and protein identifications were increased by utilizing alternate proteases and fragmentation methods, in addition to the increase in protein sequence coverage. The complementarity of these experiments also allowed for a global exploration of PTMs associated with the cellulosome based upon a set of defined PTMs that included methylation, oxidation, acetylation, phosphorylation, and signal peptide cleavage. In these experiments, 85 modified peptides corresponding to 28 cellulosome proteins were identified. Many of these modifications were located in active cellulolytic or structural domains of the cellulosome proteins, suggesting a level of possible regulatory control of protein function in various cellulotyic conditions. The use of multiple enzymes and fragmentation technologies allowed for independent verification of PTMs in different experiments, thus leading to increased confidence in PTM identifications.

  3. Cytoglobin expression in the hepatic stellate cell line HSC-T6 is regulated by extracellular matrix proteins dependent on FAK-signalling.

    Science.gov (United States)

    Stone, Louise Catherine; Thorne, Lorna Susan; Weston, Christopher John; Graham, Mark; Hodges, Nikolas John

    2015-01-01

    Fibrosis is a physiological response to cellular injury in the liver and is mediated by the activation of hepatic stellate cells resulting in the replacement of hepatocytes with extracellular matrix comprised principally of collagen 1 to form a hepatic scar. Although the novel hexaco-ordinated globin cytoglobin was identified in activated hepatic stellate cells more than 10 years ago, its role in stellate cell biology and liver fibrosis remains enigmatic. In the current study, we investigated the role of different extracellular matrix proteins in stellate cell proliferation, activation (alpha smooth muscle actin expression and retinoic acid uptake) and cytoglobin expression. Our results demonstrate that cytoglobin expression is correlated with a more quiescent phenotype of stellate cells in culture and that cytoglobin is regulated by the extracellular matrix through integrin signalling dependent on activation of focal adhesion kinase. Although further studies are required, we provide evidence that cytoglobin is a negative regulator of stellate cell activation and therefore may represent a novel target for anti-fibrotic treatments in the future.

  4. Escherichia coli-derived and Staphylococcus aureus-derived extracellular vesicles induce MUC5AC expression via extracellular signal related kinase 1/2 and p38 mitogen-activated protein kinase in human airway epithelial cells.

    Science.gov (United States)

    Bae, Chang Hoon; Choi, Yoon Seok; Song, Si-Youn; Kim, Yoon-Keun; Kim, Yong-Dae

    2017-01-01

    Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) release extracellular vesicles (EVs). E. coli-derived and S. aureus-derived EVs are associated with neutrophilic respiratory inflammation. In neutrophilic respiratory inflammation of human, expression of mucin is increased in airway epithelial cells and is associated with increased morbidity and mortality of the affected patients. However, no study on the effects of EVs on expression of mucin genes has been reported in airway epithelial cells. Therefore, this study was conducted in order to examine the effects and the brief signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression in human airway epithelial cells. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effects and signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression were examined using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). E. coli-derived and S. aureus-derived EVs induced MUC5AC expression. E. coli-derived and S. aureus-derived EVs significantly activated phosphorylation of extracellular signal related kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) and p38 MAPK. ERK1/2 MAPK inhibitor, p38 MAPK inhibitor, ERK1/2 MAPK siRNA, and p38 MAPK siRNA significantly blocked E. coli-derived and S. aureus-derived EVs induced MUC5AC messenger RNA (mRNA) expression. The results of this study suggest that E. coli-derived and S. aureus-derived EVs induced MUC5AC expression via ERK1/2 and p38 MAPK signaling pathways in human airway epithelial cells. © 2016 ARS-AAOA, LLC.

  5. CD73 protein as a source of extracellular precursors for sustained NAD+ biosynthesis in FK866-treated tumor cells.

    Science.gov (United States)

    Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

    2013-09-06

    NAD(+) is mainly synthesized in human cells via the "salvage" pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the "salvage" pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD(+) or NAD(+) precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD(+) precursors for NAD(+) biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD(+) biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.

  6. Analysis of morphological characteristics and expression levels of extracellular matrix proteins in skin wounds to determine wound age in living subjects in forensic medicine.

    Science.gov (United States)

    Fronczek, Judith; Lulf, Ronald; Korkmaz, H Ibrahim; Witte, Birgit I; van de Goot, Franklin R W; Begieneman, Mark P V; Krijnen, Paul A J; Rozendaal, Lawrence; Niessen, Hans W M; Reijnders, Udo J L

    2015-01-01

    Wound age determination in living subjects is important in routine diagnostics in forensic medicine. Macroscopical description of a wound to determine wound age however is inadequate. The aim of this study was to assess whether it would be feasible to determine wound age via analysis of morphological characteristics and extracellular matrix proteins in skin biopsies of living subjects referred to a forensic outpatient clinic. Skin biopsies (n=101), representing the border area of the wound, were taken from skin injuries of known wound age (range: 4.5h-25 days) in living subjects. All biopsies were analyzed for 3 morphological features (ulceration, parakeratosis and hemorrhage) and 3 extracellular matrix markers (collagen III, collagen IV and α-SMA). For quantification, biopsies were subdivided in 4 different timeframes: 0.2-2 days, 2-4 days, 4-10 days and 10-25 days old wounds. Subsequently, a probability scoring system was developed. For hemorrhage, collagen III, collagen IV and α-SMA expression no relation with wound age was found. Ulceration was only found in wounds of 0.2-2, 2-4 and 4-10 days old, implying that the probability that a wound was more than 10 days old in case of ulceration is equal to 0%. Also parakeratosis was almost exclusively found in wounds of 0.2-2, 2-4 and 4-10 days old, except for one case with a wound age of 15 days old. The probability scoring system of all analyzed markers, as depicted above, however can be used to calculate individual wound age probabilities in biopsies of skin wounds of living subjects. We have developed a probability scoring system, analysing morphological characteristics and extracellular matrix proteins in superficial skin biopsies of wounds in living subjects that can be applied in forensic medicine for wound age determination. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. Characterisation of extracellular vesicle-subsets derived from brain endothelial cells and analysis of their protein cargo modulation after TNF exposure.

    Science.gov (United States)

    Dozio, Vito; Sanchez, Jean-Charles

    2017-01-01

    Little is known about the composition and functional differences between extracellular vesicle (EV) subsets, such as microvesicles (MVs) and exosomes (EXOs), nor to what extent their cargo reflects the phenotypic state of the cell of origin. Brain endothelial cells are the constitutive part of the blood-brain barrier (BBB), a selective barrier that maintains brain homeostasis. BBB impairment is associated with several neuroinflammatory diseases with the pro-inflammatory cytokine tumour necrosis factor (TNF) often playing a key role. In the present study, shotgun proteomics and parallel reaction monitoring (PRM)-based targeted mass spectrometry were used to characterise brain endothelial cell-released EVs, and to study how TNF exposure modulated EV protein cargoes. MVs were found to be enriched in mitochondrial and cytoskeletal proteins, whereas EXOs were enriched in adhesion, histone and ribosomal proteins. After stimulation with TNF, several proteins involved in TNF and NF-κB signalling pathways, that were found to be differentially expressed in cells, were also differentially expressed in both MVs and EXOs. Thus, our results revealed some novel proteins as potentially useful candidates for discriminating between MVs and EXOs, together with additional evidence that cells "package" proteins in EVs systematically and according to their phenotypic state.

  8. Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis.

    Science.gov (United States)

    Oostendorp, Corien; Meyer, Sarah; Sobrio, Monia; van Arendonk, Joyce; Reichmann, Ernst; Daamen, Willeke F; van Kuppevelt, Toin H

    2017-05-01

    Treatment of full-thickness skin defects with split-thickness skin grafts is generally associated with contraction and scar formation and cellular skin substitutes have been developed to improve skin regeneration. The evaluation of cultured skin substitutes is generally based on qualitative parameters focusing on histology. In this study we focused on quantitative evaluation to provide a template for comparison of human bio-engineered skin substitutes between clinical and/or research centers, and to supplement histological data. We focused on extracellular matrix proteins since these components play an important role in skin regeneration. As a model we analyzed the human dermal substitute denovoDerm and the dermo-epidermal skin substitute denovoSkin. The quantification of the extracellular matrix proteins type III collagen and laminin 5 in tissue homogenates using western blotting analysis and ELISA was not successful. The same was true for assaying lysyl oxidase, an enzyme involved in crosslinking of matrix molecules. As an alternative, gene expression levels were measured using qPCR. Various RNA isolation procedures were probed. The gene expression profile for specific dermal and epidermal genes could be measured reliably and reproducibly. Differences caused by changes in the cell culture conditions could easily be detected. The number of cells in the skin substitutes was measured using the PicoGreen dsDNA assay, which was found highly quantitative and reproducible. The (dis) advantages of assays used for quantitative evaluation of skin substitutes are discussed. Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.

  9. The Role of Extracellular Binding Proteins in the Cellular Uptake of Drugs: Impact on Quantitative In Vitro-to-In Vivo Extrapolations of Toxicity and Efficacy in Physiologically Based Pharmacokinetic-Pharmacodynamic Research.

    Science.gov (United States)

    Poulin, Patrick; Burczynski, Frank J; Haddad, Sami

    2016-02-01

    A critical component in the development of physiologically based pharmacokinetic-pharmacodynamic (PBPK/PD) models for estimating target organ dosimetry in pharmacology and toxicology studies is the understanding of the uptake kinetics and accumulation of drugs and chemicals at the cellular level. Therefore, predicting free drug concentrations in intracellular fluid will contribute to our understanding of concentrations at the site of action in cells in PBPK/PD research. Some investigators believe that uptake of drugs in cells is solely driven by the unbound fraction; conversely, others argue that the protein-bound fraction contributes a significant portion of the total amount delivered to cells. Accordingly, the current literature suggests the existence of a so-called albumin-mediated uptake mechanism(s) for the protein-bound fraction (i.e., extracellular protein-facilitated uptake mechanisms) at least in hepatocytes and cardiac myocytes; however, such mechanism(s) and cells from other organs deserve further exploration. Therefore, the main objective of this present study was to discuss further the implication of potential protein-facilitated uptake mechanism(s) on drug distribution in cells under in vivo conditions. The interplay between the protein-facilitated uptake mechanism(s) and the effects of a pH gradient, metabolism, transport, and permeation limitation potentially occurring in cells was also discussed, as this should violate the basic assumption on similar free drug concentration in cells and plasma. This was made because the published equations used to calculate drug concentrations in cells in a PBPK/PD model did not consider potential protein-facilitated uptake mechanism(s). Consequently, we corrected some published equations for calculating the free drug concentrations in cells compared with plasma in PBPK/PD modeling studies, and we proposed a refined strategy for potentially performing more accurate quantitative in vitro-to-in vivo extrapolations

  10. Increased Obesity-Associated Circulating Levels of the Extracellular Matrix Proteins Osteopontin, Chitinase-3 Like-1 and Tenascin C Are Associated with Colon Cancer

    Science.gov (United States)

    Catalán, Victoria; Gómez-Ambrosi, Javier; Rodríguez, Amaia; Ramírez, Beatriz; Izaguirre, Maitane; Hernández-Lizoain, José Luis; Baixauli, Jorge; Martí, Pablo; Valentí, Víctor; Moncada, Rafael; Silva, Camilo; Salvador, Javier; Frühbeck, Gema

    2016-01-01

    Background 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. Methods 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. Results 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. Conclusions 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. PMID:27612200

  11. 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.

  12. Equilibrium-phase MR angiography: Comparison of unspecific extracellular and protein-binding gadolinium-based contrast media with respect to image quality.

    Science.gov (United States)

    Erb-Eigner, Katharina; Taupitz, Matthias; Asbach, Patrick

    2016-01-01

    The purpose of this study was to compare contrast and image quality of whole-body equilibrium-phase high-spatial-resolution MR angiography using a non-protein-binding unspecific extracellular gadolinium-based contrast medium with that of two contrast media with different protein-binding properties. 45 patients were examined using either 15 mL of gadobutrol (non-protein-binding, n = 15), 32 mL of gadobenate dimeglumine (weakly protein binding, n = 15) or 11 mL gadofosveset trisodium (protein binding, n = 15) followed by equilibrium-phase high-spatial-resolution MR-angiography of four consecutive anatomic regions. The time elapsed between the contrast injection and the beginning of the equilibrium-phase image acquisition in the respective region was measured and was up to 21 min. Signal intensity was measured in two vessels per region and in muscle tissue. Relative contrast (RC) values were calculated. Vessel contrast, artifacts and image quality were rated by two radiologists in consensus on a five-point scale. Compared with gadobutrol, gadofosveset trisodium revealed significantly higher RC values only when acquired later than 15 min after bolus injection. Otherwise, no significant differences between the three contrast media were found regarding vascular contrast and image quality. Equilibrium-phase high-spatial-resolution MR-angiography using a weakly protein-binding or even non-protein-binding contrast medium is equivalent to using a stronger protein-binding contrast medium when image acquisition is within the first 15 min after contrast injection, and allows depiction of the vasculature with high contrast and image quality. The protein-binding contrast medium was superior for imaging only later than 15 min after contrast medium injection. Copyright © 2015 John Wiley & Sons, Ltd.

  13. 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.

  14. Extracellular polymeric substances act as transient media in extracellular electron transfer of Shewanella oneidensis MR-1

    DEFF Research Database (Denmark)

    Xiao, Yong; Zhang, Jingdong; Ulstrup, Jens

    It is well known that microorganism is surrounded by extracellular polymeric substances (EPS) which include polysaccharides, proteins, glycoproteins, nucleic acids, phospholipids, and humic acids. However, previous studies on microbial extracellular electron transfer (EET) are conducted on cells...

  15. Induction of trans-resveratrol and extracellular pathogenesis-related proteins in elicited suspension cultured cells of Vitis vinifera cv Monastrell.

    Science.gov (United States)

    Belchí-Navarro, Sarai; Almagro, Lorena; Sabater-Jara, Ana Belén; Fernández-Pérez, Francisco; Bru, Roque; Pedreño, Maria Angeles

    2013-02-15

    Suspension-cultured cells of Vitis vinifera cv Monastrell were used to investigate the effects of methyljasmonate, ethylene and salicylic acid separately or in combination with cyclodextrins on both trans-resveratrol production and the induction of defense responses. The results showed that the addition of methyljasmonate or ethylene to suspension-cultured cells jointly treated with cyclodextrins and salicylic acid provoked a decrease of trans-resveratrol levels suggesting that salicylic acid has a negative and antagonistic effect with methyljasmonate or ethylene on trans-resveratrol production. Likewise, the exogenous application of these compounds induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to an specific β-1,3-glucanase, class III peroxidases and a β-1,4-mannanase, which suggests that these signal molecules could play a role in mediating defense-related gene product expression in V. vinifera cv Monastrell. Apart from these inducible proteins, other proteins were found in both the control and elicited cell cultures of V. vinifera. These included class IV chitinase, polygalacturonase inhibitor protein and reticuline oxidase-like protein, suggesting that their expression is constitutive being involved in the modification of the cell wall architecture during cell culture growth and in the prevention of pathogen attack. Copyright © 2012 Elsevier GmbH. All rights reserved.

  16. A role for oxygen delivery and extracellular magnesium in limiting cold tolerance of the sub-antarctic stone crab Paralomis granulosa?

    Science.gov (United States)

    Wittmann, Astrid C; Pörtner, Hans O; Sartoris, Franz J

    2012-01-01

    A low capacity for regulation of extracellular Mg(2+) has been proposed to exclude reptant marine decapod crustaceans from temperatures below 0°C and thus to exclude them from the high Antarctic. To test this hypothesis and to elaborate the underlying mechanisms in the most cold-tolerant reptant decapod family of the sub-Antarctic, the Lithodidae, thermal tolerance was determined in the crab Paralomis granulosa (Decapoda, Anomura, Lithodidae) using an acute stepwise temperature protocol (-1°, 1°, 4°, 7°, 10°, and 13°C). Arterial and venous oxygen partial pressures (Po(2)) in hemolymph, heartbeat and ventilation beat frequencies, and hemolymph cation composition were measured at rest and after a forced activity (righting) trial. Scopes for heartbeat and ventilation beat frequencies and intermittent heartbeat and scaphognathite beat rates at rest were evaluated. Hemolymph [Mg(2+)] was experimentally reduced from 30 mmol L(-1) to a level naturally observed in Antarctic caridean shrimps (12 mmol L(-1)) to investigate whether the animals remain more active and tolerant to cold (-1°, 1°, and 4°C). In natural seawater, righting speed was significantly slower at -1° and 13°C, compared with acclimation temperature (4°C). Arterial and venous hemolymph Po(2) increased in response to cooling even though heartbeat and ventilation beat frequencies as well as scopes decreased. At rest, ionic composition of the hemolymph was not affected by temperature. Activity induced a significant increase in hemolymph [K(+)] at -1° and 1°C. Reduction of hemolymph [Mg(2+)] did not result in an increase in activity, an increase in heartbeat and ventilation beat frequencies, or a shift in thermal tolerance to lower temperatures. In conclusion, oxygen delivery in this cold-water crustacean was not acutely limiting cold tolerance, and animals may have been constrained more by their functional capacity and motility. In contrast to earlier findings in temperate and subpolar brachyuran

  17. Mitogen-activated protein kinase/extracellular signal-regulated kinase attenuates 3-hydroxykynurenine-induced neuronal cell death.

    Science.gov (United States)

    Lee, Hyun Jung; Bach, Jae-Hyung; Chae, Hee-Sun; Lee, Sang Hyung; Joo, Wan Seok; Choi, Se Hoon; Kim, Kyung Yong; Lee, Won Bok; Kim, Sung Su

    2004-02-01

    3-Hydroxykynurenine (3-HK), an endogenous tryptophan metabolite, is known to have toxic effects in brain. However, the molecular mechanism of the toxicity has not been well identified. In this study, we investigated the involvement of MAPK/extracellular signal-regulated kinase (ERK) in the 3-HK-induced neuronal cell damage. Our results showed that 3-HK induced apoptotic neuronal cell death and ERK phosphorylation occurred during cell death. Inhibition of ERK activation using PD98059 considerably increased cell death. Furthermore, cell death was preceded by mitochondrial malfunction including collapse of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release from mitochondria to the cytosol. Interestingly, inhibition of ERK dramatically increased mitochondrial malfunction, and enhanced caspase activation, resulting in enhanced neuronal cell death. Thus, our results show that ERK plays a protective role by maintaining mitochondrial function and regulating caspase activity under conditions of cellular stress.

  18. Extracellular matrix type modulates cell migration on mechanical gradients.

    Science.gov (United States)

    Hartman, Christopher D; Isenberg, Brett C; Chua, Samantha G; Wong, Joyce Y

    2017-10-15

    Extracellular matrix composition and stiffness are known to be critical determinants of cell behavior, modulating processes including differentiation, traction generation, and migration. Recent studies have demonstrated that the ECM composition can modulate how cells migrate in response to gradients in environmental stiffness, altering a cell's ability to undergo durotaxis. These observations were limited to single varieties of extracellular matrix, but typically cells are exposed to environments containing complex mixtures of extracellular matrix proteins. Here, we investigate migration of NIH 3T3 fibroblasts on mechanical gradients coated with one or more type of extracellular matrix protein. Our results show that NIH 3T3 fibroblasts exhibit durotaxis on fibronectin-coated mechanical gradients but not on those coated with laminin, demonstrating that extracellular matrix type can act as a regulator of cell response to mechanical gradients. Interestingly, NIH 3T3 fibroblasts were also observed to migrate randomly on gradients coated with a mixture of both fibronectin and laminin, suggesting that there may be a complex interplay in the cellular response to mechanical gradients in the presence of multiple extracellular matrix signals. These findings indicate that specific composition of available adhesion ligands is a critical determinant of a cell's migratory response to mechanical gradients. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. An extracellular disulfide bond forming protein (DsbF) from Mycobacterium tuberculosis: Structural, biochemical and gene expression analysis

    OpenAIRE

    Chim, Nicholas; Riley, Robert; The, Juliana; Im, Soyeon; Segelke, Brent; Lekin, Tim; Yu, Minmin; Hung, Li Wei; Terwilliger, Tom; Whitelegge, Julian P.; Goulding, Celia W.

    2010-01-01

    Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here we present structural, biochemical and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold althou...

  20. 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.

  1. Changes in dietary zinc and copper affect zinc-status indicators of postmenopausal women, notably, extracellular superoxide dismutase and amyloid precursor proteins.

    Science.gov (United States)

    Davis, C D; Milne, D B; Nielsen, F H

    2000-03-01

    Zinc is an essential trace element for human health and well-being; however, methods currently available for the assessment of zinc status in humans are unsatisfactory. The objective was to critically evaluate the use of various indicators of zinc status in humans in a controlled metabolic ward study. Indicators of zinc status were measured in 25 healthy postmenopausal women aged 64.9 +/- 6.7 y. After a 10-d equilibration period, volunteers consumed a diet with either a low (1 mg/d; n = 12) or a high (3 mg/d; n = 13) copper content based on a total energy content of 8.4 MJ. They received the same amount of copper throughout the study. Both groups were fed the basal diet (3 mg Zn/d) with no zinc supplement for one 90-d period, and the diet supplemented with 50 mg Zn/d for another 90-d period. Zinc supplementation significantly increased (P < 0.0001) extracellular but not erythrocyte superoxide dismutase activity. This increase was more apparent when subjects were fed the low-copper diet. Zinc supplementation in combination with the low-copper diet significantly decreased (P < 0.01) amyloid precursor protein expression in platelets. Other indicators of zinc status that were significantly elevated after zinc supplementation were as follows: plasma zinc and free thyroxine concentrations and mononuclear 5'-nucleotidase activity. The measurement of serum extracellular superoxide dismutase activity may be useful as a marker for the functional assessment of zinc status in humans.

  2. 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 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.

  3. Activation of Yes-Associated Protein in Low-Grade Meningiomas Is Regulated by Merlin, Cell Density, and Extracellular Matrix Stiffness.

    Science.gov (United States)

    Tanahashi, Kuniaki; Natsume, Atsushi; Ohka, Fumiharu; Motomura, Kazuya; Alim, Adiljan; Tanaka, Ichidai; Senga, Takeshi; Harada, Ichiro; Fukuyama, Ryuichi; Sumiyoshi, Naoyuki; Sekido, Yoshitaka; Wakabayashi, Toshihiko

    2015-07-01

    The NF2 gene product Merlin is a protein containing ezrin, radixin, and moesin domains; it is a member of the 4.1 protein superfamily associated with the membrane cytoskeleton and also interacts with cell surface molecules. The mammalian Hippo cascade, a downstream signaling cascade of merlin, inactivates the Yes-associated protein (YAP). Yes-associated protein is activated by loss of the NF2 gene and functions as an oncogene in meningioma cells; however, the factors controlling YAP expression, phosphorylation, and subcellular localization in meningiomas have not been fully elucidated. Here, we demonstrate that merlin expression is heterogeneous in 1 NF2 gene-negative and 3 NF2 gene-positive World Health Organization grade I meningiomas. In the NF2 gene-positive meningiomas, regions with low levels of merlin (tumor rims) had greater numbers of cells with nuclear YAP versus regions with high merlin levels (tumor cores). Merlin expression and YAP phosphorylation were also affected by cell density in the IOMM-Lee and HKBMM human meningioma cell lines; nuclear localization of YAP was regulated by cell density and extracellular matrix (ECM) stiffness in IOMM-Lee cells. These results suggest that cell density and ECM stiffness may contribute to the heterogeneous loss of merlin and increased nuclear YAP expression in human meningiomas.

  4. Ultrastructural localization of extracellular matrix proteins of the lymph node cortex: evidence supporting the reticular network as a pathway for lymphocyte migration

    Directory of Open Access Journals (Sweden)

    Sobocinski Gregg P

    2010-08-01

    Full Text Available Abstract Background The lymph node (LN is a crossroads of blood and lymphatic vessels allowing circulating lymphocytes to efficiently recognize foreign molecules displayed on antigen presenting cells. Increasing evidence indicates that after crossing high endothelial venules, lymphocytes migrate within the node along the reticular network (RN, a scaffold of fibers enwrapped by fibroblastic reticular cells (FRC. Light microscopy has shown that the RN contains specific extracellular matrix (ECM proteins, which are putative molecular "footholds" for migration, and are known ligands for lymphocyte integrin adhesion receptors. Results To investigate whether ECM proteins of the RN are present on the outer surface of the FRC and are thus accessible to migrating lymphocytes, ultrastructural immunohistochemical staining of cynomolgus monkey LN was performed using antibodies to human ECM proteins that were successfully employed at the light microscopic level. The fibrillar collagens I and III were observed primarily within the reticular network fibers themselves. In contrast, the matrix proteins laminin, fibronectin, collagen IV, and tenascin were observed within the reticular fibers and also on the outer membrane surface of the FRC. Conclusions These findings suggest a molecular basis for how the RN functions as a pathway for lymphocyte migration within the lymph node.

  5. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Science.gov (United States)

    Chitin-binding proteins (CBPs) existed in various species and involved in different biology processes. In the present study, we cloned a full length cDNA of chitin-binding protein-like (PpCBP-like) from Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. PpCBP-like encoded a 96 putative amin...

  6. Cysteine cathepsins and extracellular matrix degradation.

    Science.gov (United States)

    Fonović, Marko; Turk, Boris

    2014-08-01

    Cysteine cathepsins are normally found in the lysosomes where they are involved in intracellular protein turnover. Their ability to degrade the components of the extracellular matrix in vitro was first reported more than 25years ago. However, cathepsins were for a long time not considered to be among the major players in ECM degradation in vivo. During the last decade it has, however, become evident that abundant secretion of cysteine cathepsins into extracellular milieu is accompanying numerous physiological and disease conditions, enabling the cathepsins to degrade extracellular proteins. In this review we will focus on cysteine cathepsins and their extracellular functions linked with ECM degradation, including regulation of their activity, which is often enhanced by acidification of the extracellular microenvironment, such as found in the bone resorption lacunae or tumor microenvironment. We will further discuss the ECM substrates of cathepsins with a focus on collagen and elastin, including the importance of that for pathologies. Finally, we will overview the current status of cathepsin inhibitors in clinical development for treatment of ECM-linked diseases, in particular osteoporosis. Cysteine cathepsins are among the major proteases involved in ECM remodeling, and their role is not limited to degradation only. Deregulation of their activity is linked with numerous ECM-linked diseases and they are now validated targets in a number of them. Cathepsins S and K are the most attractive targets, especially cathepsin K as a major therapeutic target for osteoporosis with drugs targeting it in advanced clinical trials. Due to their major role in ECM remodeling cysteine cathepsins have emerged as an important group of therapeutic targets for a number of ECM-related diseases, including, osteoporosis, cancer and cardiovascular diseases. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All

  7. Emergent properties of extracellular vesicles: a holistic approach to decode the complexity of intercellular communication networks.

    Science.gov (United States)

    Gho, Yong Song; Lee, Changjin

    2017-06-27

    Shedding of nano-sized bilayered extracellular vesicles and extracellular vesicle-mediated intercellular communication are evolutionarily conserved biological processes. Communication between cells and the environment is an essential process in living organisms and dysregulation of intercellular communication leads to various diseases. Thus, systematic studies on extracellular vesicles, also known as exosomes, microvesicles, and outer membrane vesicles, are critical for a deeper understanding of intercellular communication networks that are crucial for decoding the exact causes of various difficult-to-cure diseases. Recent progress in this emerging field reveals that extracellular vesicles are endogenous carriers of specific subsets of proteins, mRNAs, miRNAs, and other bioactive materials, as well as play diverse pathophysiological roles. However, certain issues regarding diverse subtypes and the complex pathophysiological roles of extracellular vesicles are not yet clearly elucidated. In this review, we first briefly introduce the complexity of extracellular vesicles in terms of their vesicular cargos and protein-protein interaction networks, their diverse subtypes, and multifaceted pathophysiological functions. Then, we introduce the limitation of reductionist approaches in understanding the complexity of extracellular vesicles. We finally suggest that molecular systems biology approaches based on the concept of emergent properties are essential for a comprehensive understanding of the complex pathophysiological functions of heterogeneous extracellular vesicles, either at the single vesicle level or at a systems level as a whole.

  8. Fungus-mediated preferential bioleaching of waste material such as fly - ash as a means of producing extracellular, protein capped, fluorescent and water soluble silica nanoparticles.

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    Shadab Ali Khan

    Full Text Available In this paper, we for the first time show the ability of the mesophilic fungus Fusarium oxysporum in the bioleaching of waste material such as Fly-ash for the extracellular production of highly crystalline and highly stable, protein capped, fluorescent and water soluble silica nanoparticles at ambient conditions. When the fungus Fusarium oxysporum is exposed to Fly-ash, it is capable of selectively leaching out silica nanoparticles of quasi-spherical morphology within 24 h of reaction. These silica nanoparticles have been completely characterized by UV-vis spectroscopy, Photoluminescence (PL, Transmission electron microscopy (TEM, X-ray diffraction (XRD, Fourier transform infrared spectroscopy (FTIR and Energy dispersive analysis of X-rays (EDAX.

  9. Limitations of gene duplication models: evolution of modules in protein interaction networks.

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    Frank Emmert-Streib

    Full Text Available It has been generally acknowledged that the module structure of protein interaction networks plays a crucial role with respect to the functional understanding of these networks. In this paper, we study evolutionary aspects of the module structure of protein interaction networks, which forms a mesoscopic level of description with respect to the architectural principles of networks. The purpose of this paper is to investigate limitations of well known gene duplication models by showing that these models are lacking crucial structural features present in protein interaction networks on a mesoscopic scale. This observation reveals our incomplete understanding of the structural evolution of protein networks on the module level.

  10. Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania amazonensis.

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    Fernando Almeida-Souza

    2016-08-01

    Full Text Available The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni against Leishmania (Leishmania amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography-mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L. amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L. amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.

  11. Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis.

    Science.gov (United States)

    Almeida-Souza, Fernando; Cardoso, Flávia de Oliveira; Souza, Bruno Vinicius da Conceição; do Valle, Tânia Zaverucha; de Sá, Joicy Cortez; Oliveira, Iara Dos Santos da Silva; de Souza, Celeste da Silva Freitas; Moragas Tellis, Carla Junqueira; Chagas, Maria do Socorro Dos Santos; Behrens, Maria Dutra; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

    2016-08-01

    The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography-mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.

  12. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

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    Yu Zhu

    2015-11-01

    Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

  13. Homology modelling of protein-protein complexes: a simple method and its possibilities and limitations

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    Simonson Thomas

    2008-10-01

    Full Text Available Abstract Background Structure-based computational methods are needed to help identify and characterize protein-protein complexes and their function. For individual proteins, the most successful technique is homology modelling. We investigate a simple extension of this technique to protein-protein complexes. We consider a large set of complexes of known structures, involving pairs of single-domain proteins. The complexes are compared with each other to establish their sequence and structural similarities and the relation between the two. Compared to earlier studies, a simpler dataset, a simpler structural alignment procedure, and an additional energy criterion are used. Next, we compare the Xray structures to models obtained by threading the native sequence onto other, homologous complexes. An elementary requirement for a successful energy function is to rank the native structure above any threaded structure. We use the DFIREβ energy function, whose quality and complexity are typical of the models used today. Finally, we compare near-native models to distinctly non-native models. Results If weakly stable complexes are excluded (defined by a binding energy cutoff, as well as a few unusual complexes, a simple homology principle holds: complexes that share more than 35% sequence identity share similar structures and interaction modes; this principle was less clearcut in earlier studies. The energy function was then tested for its ability to identify experimental structures among sets of decoys, produced by a simple threading procedure. On average, the experimental structure is ranked above 92% of the alternate structures. Thus, discrimination of the native structure is good but not perfect. The discrimination of near-native structures is fair. Typically, a single, alternate, non-native binding mode exists that has a native-like energy. Some of the associated failures may correspond to genuine, alternate binding modes and/or native complexes that

  14. Endogenous 5-HT2C Receptors Phosphorylate the cAMP Response Element Binding Protein via Protein Kinase C-Promoted Activation of Extracellular-Regulated Kinases-1/2 in Hypothalamic mHypoA-2/10 Cells.

    Science.gov (United States)

    Lauffer, Lisa; Glas, Evi; Gudermann, Thomas; Breit, Andreas

    2016-07-01

    Serotonin 5-HT2C receptors (5-HT2CR) activate Gq proteins and are expressed in the central nervous system (CNS). 5-HT2CR regulate emotion, feeding, reward, or cognition and may serve as promising drug targets to treat psychiatric disorders or obesity. Owing to technical difficulties in isolating cells from the CNS and the lack of suitable cell lines endogenously expressing 5-HT2CR, our knowledge about this receptor subtype in native environments is rather limited. The hypothalamic mHypoA-2/10 cell line was recently established and resembles appetite-regulating hypothalamic neurons of the paraventricular nucleus (PVN), where 5-HT2CR have been detected in vivo. Therefore, we tested mHypoA-2/10 cells for endogenous 5-HT2CR expression. Serotonin or the 5-HT2CR preferential agonist WAY-161,503 initiated cAMP response element (CRE)-dependent gene transcription with EC50 values of 15.5 ± 9.8 and 1.1 ± 0.9 nM, respectively. Both responses were blocked by two unrelated 5-HT2CR-selective antagonists (SB-242,084, RS-102,221) but not by a 5-HT2AR (EMD-281,014) or 5-HT2BR (RS-127,455) antagonists. By single-cell calcium imaging, we found that serotonin and WAY-161,503 induced robust calcium transients, which were also blunted by both 5-HT2CR antagonists. Additionally we revealed, first, that 5-HT2CR induced CRE activation via protein kinase C (PKC)-mediated engagement of extracellular-regulated kinases-1/2 and, second, that intrinsic activity of WAY-161,503 was in the range of 0.3-0.5 compared with serotonin, defining the frequently used 5-HT2CR agonist as a partial agonist of endogenous 5-HT2CR. In conclusion, we have shown that hypothalamic mHypoA-2/10 cells endogenously express 5-HT2CR and thus are the first cell line in which to analyze 5-HT2CR pharmacology, signaling, and regulation in its natural environment. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

    Science.gov (United States)

    Chan, Chien-Pin; Tsai, Yao-Ting; Chen, Yao-Li; Hsu, Yu-Wen; Tseng, Joseph T; Chuang, Hung-Yi; Shiurba, Robert; Lee, Mei-Hsien; Wang, Jaw-Yuan; Chang, Wei-Chiao

    2015-02-01

    Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 μM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture. © 2013 Wiley Periodicals, Inc.

  16. 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

  17. Random amino acid mutations and protein misfolding lead to Shannon limit in sequence-structure communication.

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    Andreas Martin Lisewski

    2008-09-01

    Full Text Available The transmission of genomic information from coding sequence to protein structure during protein synthesis is subject to stochastic errors. To analyze transmission limits in the presence of spurious errors, Shannon's noisy channel theorem is applied to a communication channel between amino acid sequences and their structures established from a large-scale statistical analysis of protein atomic coordinates. While Shannon's theorem confirms that in close to native conformations information is transmitted with limited error probability, additional random errors in sequence (amino acid substitutions and in structure (structural defects trigger a decrease in communication capacity toward a Shannon limit at 0.010 bits per amino acid symbol at which communication breaks down. In several controls, simulated error rates above a critical threshold and models of unfolded structures always produce capacities below this limiting value. Thus an essential biological system can be realistically modeled as a digital communication channel that is (a sensitive to random errors and (b restricted by a Shannon error limit. This forms a novel basis for predictions consistent with observed rates of defective ribosomal products during protein synthesis, and with the estimated excess of mutual information in protein contact potentials.

  18. Random amino acid mutations and protein misfolding lead to Shannon limit in sequence-structure communication.

    Science.gov (United States)

    Lisewski, Andreas Martin

    2008-09-01

    The transmission of genomic information from coding sequence to protein structure during protein synthesis is subject to stochastic errors. To analyze transmission limits in the presence of spurious errors, Shannon's noisy channel theorem is applied to a communication channel between amino acid sequences and their structures established from a large-scale statistical analysis of protein atomic coordinates. While Shannon's theorem confirms that in close to native conformations information is transmitted with limited error probability, additional random errors in sequence (amino acid substitutions) and in structure (structural defects) trigger a decrease in communication capacity toward a Shannon limit at 0.010 bits per amino acid symbol at which communication breaks down. In several controls, simulated error rates above a critical threshold and models of unfolded structures always produce capacities below this limiting value. Thus an essential biological system can be realistically modeled as a digital communication channel that is (a) sensitive to random errors and (b) restricted by a Shannon error limit. This forms a novel basis for predictions consistent with observed rates of defective ribosomal products during protein synthesis, and with the estimated excess of mutual information in protein contact potentials.

  19. Extracellular receptor kinase and cAMP response element binding protein activation in the neonatal rat heart after perinatal cocaine exposure.

    Science.gov (United States)

    Sun, Lena S; Quamina, Aaron

    2004-12-01

    Prenatal exposure to cocaine has been shown to induce an increase in the myocardial expression and activation of the cAMP response binding protein (CREB), a transcriptional factor that has been shown to regulate gene expression. Several different kinases, including protein kinase A, calcium calmodulin kinase II, and mitogen-activated protein kinase can induce phosphorylation of CREB at serine 133, a necessary step for CREB activation. We examined whether the mitogen-activated protein kinase-extracellular receptor kinase (ERK) pathway may be involved in mediating the serine 133 CREB phosphorylation in cardiac nuclei after perinatal cocaine exposure. Pregnant rats were treated daily with saline or cocaine at 60 mg/kg (C60) by intragastric administration during the entire gestational period, and treatment was continued in the nursing dams after delivery until the time of the study. Nuclear extracts were isolated from hearts of 1-d- and 7-d-old neonatal rats. We performed immunoblotting experiments using an antibody that recognized CREB with phosphorylation specifically at the serine 133 site and an antibody that recognized both the phosphorylated and the unphosphorylated forms of CREB, as well as antibodies for total ERK, phospho-ERK, total ribosomal S6 kinase 1 (RSK1), RSK2, and phospho-RSK. We assessed the interaction of RSK with CREB or CREB-binding protein by performing co-immunoprecipitation experiments. We found that perinatal cocaine exposure increased both phospho-ERK and phospho-RSK expression, indicative of an increased activity of these two enzymes. Furthermore, we demonstrated that phospho-RSK was immunoprecipitated with CREB in all neonatal cardiac nuclei and that the greatest interaction was found in day 7 hearts after perinatal cocaine exposure. Our results thus illustrate that the ERK-RSK pathway was active in the postnatal rat heart at 1 and 7 d of age and that this pathway may mediate the increase in myocardial CREB activation after perinatal cocaine

  20. Cytocompatibility of novel extracellular matrix protein analogs of biodegradable polyester polymers derived from α-hydroxy amino acids.

    Science.gov (United States)

    Lecht, Shimon; Cohen-Arazi, Naomi; Cohen, Gadi; Ettinger, Keren; Momic, Tatjana; Kolitz, Michal; Naamneh, Majdi; Katzhendler, Jehoshua; Domb, Abraham J; Lazarovici, Philip; Lelkes, Peter I

    2014-01-01

    One of the challenges in regenerative medicine is the development of novel biodegradable materials to build scaffolds that will support multiple cell types for tissue engineering. Here we describe the preparation, characterization, and cytocompatibility of homo- and hetero-polyesters of α-hydroxy amino acid derivatives with or without lactic acid conjugation. The polymers were prepared by a direct condensation method and characterized using gel permeation chromatography, (1)H-nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, optical activity, and solubility. The surface charge of the polymers was evaluated using zeta potential measurements. The polymers were coated onto glass cover slips followed by characterization using nano-surface profiler, thin film reflectometry, and atomic force microscopy (AFM). Their interaction with endothelial and neuronal cells was assessed using adhesion, proliferation, and differentiation assays. Of the characterized polymers, Poly-HOVal-LA, but not Poly-(D)HOPhe, significantly augmented nerve growth factor (NGF)-induced neuronal differentiation of the PC12 pheochromcytoma cells. In contrast, Poly-HOLeu increased by 20% the adhesion of endothelial cells, but did not affect PC12 cell differentiation. NGF-induced Erk1/2 phosphorylation in PC12 cells grown on the different polymers was similar to the effect observed for cells cultured on collagen type I. While no significant association could be established between charge and the differentiative/proliferative properties of the polymers, AFM analysis indicated augmentation of NGF-induced neuronal differentiation on smooth polymer surfaces. We conclude that overall selective cytocompatibility and bioactivity might render α-hydroxy amino acid polymers useful as extracellular matrix-mimicking materials for tissue engineering.

  1. Venom allergen-like proteins in secretions of plant-parasitic nematodes activate and suppress extracellular plant immune receptors

    NARCIS (Netherlands)

    Lozano Torres, J.L.

    2014-01-01

      Parasitic worms threaten human, animal and plant health by infecting people, livestock and crops worldwide. Animals and plants share an anciently evolved innate immune system. Parasites modulate this immune system by secreting proteins to maintain their parasitic lifestyle. This thesis

  2. Estrogens increase the expression of fibulin-1, an extracellular matrix protein secreted by human ovarian cancer cells

    NARCIS (Netherlands)

    Clinton, GM; Rougeot, C; Derancourt, J; Roger, P; Defrenne, A; Godyna, S; Argraves, WS; Rochefort, H

    1996-01-01

    Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens, In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins, This study,vas aimed at

  3. S100A4 and bone morphogenetic protein-2 codependently induce vascular smooth muscle cell migration via phospho-extracellular signal-regulated kinase and chloride intracellular channel 4.

    Science.gov (United States)

    Spiekerkoetter, Edda; Guignabert, Christophe; de Jesus Perez, Vinicio; Alastalo, Tero-Pekka; Powers, Janine M; Wang, Lingli; Lawrie, Allan; Ambartsumian, Noona; Schmidt, Ann-Marie; Berryman, Mark; Ashley, Richard H; Rabinovitch, Marlene

    2009-09-25

    S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMCs), through an interaction with the RAGE (receptor for advanced glycation end products). We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (BMPR)II, observed in pulmonary arterial hypertension. Both S100A4/Mts1 (500 ng/mL) and BMP-2 (10 ng/mL) induce migration of hPASMCs in a novel codependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII short interference (si)RNA. Phosphorylation of extracellular signal-regulated kinase is induced by both ligands and is required for motility by inducing matrix metalloproteinase 2 activity, but phospho-extracellular signal-regulated kinase 1/2 is blocked by anti-RAGE and not by BMPRII short interference RNA. In contrast, BMPRII short interference RNA, but not anti-RAGE, reduces expression of intracellular chloride channel (CLIC)4, a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA, but does alter alignment of myosin heavy chain IIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodia in motile cells. Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, ie, cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease.

  4. Latent transforming growth factor β-binding protein-3 and fibulin-1C interact with the extracellular domain of the heparin-binding EGF-like growth factor precursor

    Directory of Open Access Journals (Sweden)

    Eidels Leon

    2002-01-01

    Full Text Available Abstract Background The membrane-bound cell-surface precursor and soluble forms of heparin-binding epidermal growth factor-like growth factor (HB-EGF contribute to many cellular developmental processes. The widespread occurrence of HB-EGF in cell and tissue types has led to observations of its role in such cellular and tissue events as tumor formation, cell migration, extracellular matrix formation, wound healing, and cell adherence. Several studies have reported the involvement of such extracellular matrix proteins as latent transforming growth factor β-binding protein, TGF-β, and fibulin-1 in some of these processes. To determine whether HB-EGF interacts with extracellular matrix proteins we used the extracellular domain of proHB-EGF in a yeast two-hybrid system to screen a monkey kidney cDNA library. cDNA clones containing nucleotide sequences encoding domains of two proteins were obtained and their derived amino acid sequences were evaluated. Results From ≈ 3 × 106 screened monkey cDNA clones, cDNA clones were recovered that contained nucleotide sequences encoding domains of the monkey latent transforming growth factor-β binding protein-3 (MkLTBP-3 and fibulin-1C protein. The amino acid sequence derived from the MkLTBP-3 gene shared 98.6% identity with human LTBP-3 and 86.7% identity with mouse LTBP-3 amino acid sequences. The amino acid sequence derived from the monkey fibulin-1C gene shared 97.2% identity with human fibulin-1C. Yeast two-hybrid screens indicate that LTBP-3 and fibulin-1C interact with proHB-EGF through their calcium-binding EGF-like modules. Conclusions The interactions of the extracellular domain of proHB-EGF with LTBP-3 and fibulin-1C suggest novel functions for HB-EGF between cell and tissue surfaces.

  5. 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

  6. Redox-sensitive structure and function of the first extracellular loop of the cell-cell contact protein claudin-1: lessons from molecular structure to animals.

    Science.gov (United States)

    Dabrowski, Sebastian; Staat, Christian; Zwanziger, Denise; Sauer, Reine-Solange; Bellmann, Christian; Günther, Ramona; Krause, Eberhard; Haseloff, Reiner Fritz; Rittner, Heike; Blasig, Ingolf Ernst

    2015-01-01

    The paracellular cleft within epithelia/endothelia is sealed by tight junction (TJ) proteins. Their extracellular loops (ECLs) are assumed to control paracellular permeability and are targets of pathogenes. We demonstrated that claudin-1 is crucial for paracellular tightening. Its ECL1 is essential for the sealing and contains two cysteines conserved throughout all claudins. We prove the hypothesis that this cysteine motif forms a redox-sensitive intramolecular disulfide bridge and, hence, the claudin-1-ECL1 constitutes a functional structure which is associated to ECLs of this and other TJ proteins. The structure and function of claudin-1-ECL1 was elucidated by investigating sequences of this ECL as synthetic peptides, C1C2, and as recombinant proteins, and exhibited a β-sheet binding surface flanked by an α-helix. These sequences bound to different claudins, their ECL1, and peptides with nanomolar binding constants. C-terminally truncated C1C2 (-4aaC) opened cellular barriers and the perineurium. Recombinant ECL1 formed oligomers, and bound to claudin-1 expressing cells. Oligomerization and claudin association were abolished by reducing agents, indicating intraloop disulfide bridging and redox sensitivity. The structural and functional model based on our in vitro and in vivo investigations suggested that claudin-1-ECL1 constitutes a functional and ECL-binding β-sheet, stabilized by a shielded and redox-sensitive disulfide bond. Since the β-sheet represents a consensus sequence of claudins and further junctional proteins, a general structural feature is implied. Therefore, our model is of general relevance for the TJ assembly in normal and pathological conditions. C1C2-4aaC is a new drug enhancer that is used to improve pharmacological treatment through tissue barriers.

  7. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

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    Haroldo Cid da Silva Junior

    2016-08-01

    Full Text Available The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV. The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS. The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

  8. A bacterial-biofilm-induced oral osteolytic infection can be successfully treated by immuno-targeting an extracellular nucleoid-associated protein.

    Science.gov (United States)

    Freire, M O; Devaraj, A; Young, A; Navarro, J B; Downey, J S; Chen, C; Bakaletz, L O; Zadeh, H H; Goodman, S D

    2017-02-01

    Periodontal disease exemplifies a chronic and recurrent infection with a necessary biofilm component. Mucosal inflammation is a hallmark response of the host seen in chronic diseases, such as colitis, gingivitis, and periodontitis (and the related disorder peri-implantitis). We have taken advantage of our recently developed rat model of human peri-implantitis that recapitulates osteolysis, the requirement of biofilm formation, and the perpetuation of the bona fide disease state, to test a new therapeutic modality with two novel components. First we used hyperimmune antiserum directed against the DNABII family of proteins, now known to be a critical component of the extracellular matrix of bacterial biofilms. Second we delivered the antiserum as cargo in biodegradable microspheres to the site of the biofilm infection. We demonstrated that delivery of a single dose of anti-DNABII in poly(lactic-co-glycolic acid) (PLGA) microspheres induced significant resolution of experimental peri-implantitis, including marked reduction of inflammation. These data support the continued development of a DNABII protein-targeted therapeutic for peri-implantitis and other chronic inflammatory pathologies of the oral cavity in animals and humans. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Dynamic regulation of extracellular signal-regulated kinase (ERK by protein phosphatase 2A regulatory subunit B56γ1 in nuclei induces cell migration.

    Directory of Open Access Journals (Sweden)

    Ei Kawahara

    Full Text Available Extracellular signal-regulated kinase (ERK signalling plays a central role in various biological processes, including cell migration, but it remains unknown what factors directly regulate the strength and duration of ERK activation. We found that, among the B56 family of protein phosphatase 2A (PP2A regulatory subunits, B56γ1 suppressed EGF-induced cell migration on collagen, bound to phosphorylated-ERK, and dephosphorylated ERK, whereas B56α1 and B56β1 did not. B56γ1 was immunolocalized in nuclei. The IER3 protein was immediately highly expressed in response to costimulation of cells with EGF and collagen. Knockdown of IER3 inhibited cell migration and enhanced dephosphorylation of ERK. Analysis of the time course of PP2A-B56γ1 activity following the costimulation showed an immediate loss of phosphatase activity, followed by a rapid increase in activity, and this activity then remained at a stable level that was lower than the original level. Our results indicate that the strength and duration of the nuclear ERK activation signal that is initially induced by ERK kinase (MEK are determined at least in part by modulation of the phosphatase activity of PP2A-B56γ1 through two independent pathways.

  10. Extracellular Signal-Regulated Kinase (ERK Activation and Mitogen-Activated Protein Kinase Phosphatase 1 Induction by Pulsatile Gonadotropin-Releasing Hormone in Pituitary Gonadotrophs

    Directory of Open Access Journals (Sweden)

    Haruhiko Kanasaki

    2012-01-01

    Full Text Available The frequency of gonadotropin-releasing hormone (GnRH pulse secreted from the hypothalamus differently regulates the expressions of gonadotropin subunit genes, luteinizing hormone β (LHβ and follicle-stimulating hormone β (FSHβ, in the pituitary gonadotrophs. FSHβ is preferentially stimulated at slower GnRH pulse frequencies, whereas LHβ is preferentially stimulated at more rapid pulse frequencies. Several signaling pathways are activated, including mitogen-activated protein kinase (MAPK, protein kinase C, calcium influx, and calcium-calmodulin kinases, and these may be preferentially regulated under certain conditions. Previous studies demonstrated that MAPK pathways, especially the extracellular signal-regulated kinase (ERK, play an essential role for induction of gonadotropin subunit gene expression by GnRH, whereas, MAPK phosphatases (MKPs inactivate MAPKs through dephosphorylation of threonine and/or tyrosine residues. MKPs are also induced by GnRH, and potential feedback regulation between MAPK signaling and MKPs within the GnRH signaling pathway is evident in gonadotrophs. In this paper, we reviewed and mainly focused on our observations of the pattern of ERK activation and the induction of MKP by different frequencies of GnRH stimulation.

  11. A Novel Function for Arabidopsis CYCLASE1 in Programmed Cell Death Revealed by Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Analysis of Extracellular Matrix Proteins.

    Science.gov (United States)

    Smith, Sarah J; Kroon, Johan T M; Simon, William J; Slabas, Antoni R; Chivasa, Stephen

    2015-06-01

    Programmed cell death is essential for plant development and stress adaptation. A detailed understanding of the signal transduction pathways that regulate plant programmed cell death requires identification of the underpinning protein networks. Here, we have used a protagonist and antagonist of programmed cell death triggered by fumonisin B1 as probes to identify key cell death regulatory proteins in Arabidopsis. Our hypothesis was that changes in the abundance of cell death-regulatory proteins induced by the protagonist should be blocked or attenuated by concurrent treatment with the antagonist. We focused on proteins present in the mobile phase of the extracellular matrix on the basis that they are important for cell-cell communications during growth and stress-adaptive responses. Salicylic acid, a plant hormone that promotes programmed cell death, and exogenous ATP, which can block fumonisin B1-induced cell death, were used to treat Arabidopsis cell suspension cultures prior to isobaric-tagged relative and absolute quantitation analysis of secreted proteins. A total of 33 proteins, whose response to salicylic acid was suppressed by ATP, were identified as putative cell death-regulatory proteins. Among these was CYCLASE1, which was selected for further analysis using reverse genetics. Plants in which CYCLASE1 gene expression was knocked out by insertion of a transfer-DNA sequence manifested dramatically increased cell death when exposed to fumonisin B1 or a bacterial pathogen that triggers the defensive hypersensitive cell death. Although pathogen inoculation altered CYCLASE1 gene expression, multiplication of bacterial pathogens was indistinguishable between wild type and CYCLASE1 knockout plants. However, remarkably severe chlorosis symptoms developed on gene knockout plants in response to inoculation with either a virulent bacterial pathogen or a disabled mutant that is incapable of causing disease in wild type plants. These results show that CYCLASE1, which

  12. Protein Intake and Mobility Limitation in Community-Dwelling Older Adults: the Health ABC Study.

    Science.gov (United States)

    Houston, Denise K; Tooze, Janet A; Garcia, Katelyn; Visser, Marjolein; Rubin, Susan; Harris, Tamara B; Newman, Anne B; Kritchevsky, Stephen B

    2017-08-01

    The current Recommended Dietary Allowance (RDA) for protein is based on short-term nitrogen balance studies in young adults and may underestimate the amount needed to optimally preserve physical function in older adults. We examined the association between protein intake and the onset of mobility limitation over 6 years of follow-up in older adults in the Health ABC study. Prospective cohort study. Memphis, Tennessee and Pittsburgh, Pennsylvania. Community-dwelling, initially well-functioning adults aged 70-79 years (n = 1998). Protein intake (g/kg body weight/d) was calculated using an interviewer-administered 108-item food frequency questionnaire at baseline. Mobility limitation was assessed semi-annually and defined as reporting any difficulty walking one-quarter of a mile or climbing 10 steps on 2 consecutive 6-month contacts. The association between protein intake and incident mobility limitation was examined using Cox proportional hazard regression models adjusting for demographics, behavioral characteristics, chronic conditions, total energy intake, and height. Mean (SD) protein intake was 0.91 (0.38) g/kg body weight/d, with 43% reporting intakes less than the RDA (0.8 g/kg body weight/d). During 6 years of follow-up, 705 participants (35.3%) developed mobility limitations. Compared to participants in the upper tertile of protein intake (≥1.0 g/kg body weight/d), participants in the lower two tertiles of protein intake (mobility limitation over 6 years of follow-up (RR (95% CI): 1.86 (1.41-2.44) and 1.49 (1.20-1.84), respectively). Lower protein intake was associated with increased risk of mobility limitation in community-dwelling, initially well-functioning older adults. These results suggest that protein intakes of ≥1.0 g/kg body weight/d may be optimal for maintaining physical function in older adults. © 2017, Copyright the Authors Journal compilation © 2017, The American Geriatrics Society.

  13. 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...... resistance and immunological analysis. It was highly modified by N- and O-glycosylation but not by glycosaminoglycans. Ultracentrifugation showed non-covalent association into oligomers with molar masses of 1000-1500 kDa. Electron microscopy showed ring-like shapes with diameters of 30-40 nm. M2BP bound...... in the extracellular matrix of several mouse tissues....

  14. The Fibronectin-Binding Protein Fnm Contributes to Adherence to Extracellular Matrix Components and Virulence of Enterococcus faecium

    Science.gov (United States)

    Somarajan, Sudha R.; La Rosa, Sabina Leanti; Singh, Kavindra V.; Roh, Jung H.; Höök, Magnus

    2015-01-01

    The interaction between bacteria and fibronectin is believed to play an important role in the pathogenicity of clinically important Gram-positive cocci. In the present study, we identified a gene encoding a predicted fibronectin-binding protein of Enterococcus faecium (fnm), a homologue of Streptococcus pneumoniae pavA, in the genomes of E. faecium strain TX82 and all other sequenced E. faecium isolates. Full-length recombinant Fnm from strain TX82 bound to immobilized fibronectin in a concentration-dependent manner and also appeared to bind collagen type V and laminin, but not other proteins, such as transferrin, heparin, bovine serum albumin, mucin, or collagen IV. We demonstrated that the N-terminal fragment of Fnm is required for full fibronectin binding, since truncation of this region caused a 2.4-fold decrease (P faecium TX82 to fibronectin. Deletion of fnm resulted in a significant reduction (P faecium to fibronectin and is important in the pathogenesis of experimental endocarditis. PMID:26371130

  15. Extracellular guanosine regulates extracellular adenosine levels

    Science.gov (United States)

    Cheng, Dongmei; Jackson, Travis C.; Verrier, Jonathan D.; Gillespie, Delbert G.

    2013-01-01

    The aim of this investigation was to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels. Rat preglomerular vascular smooth muscle cells were incubated with adenosine, guanosine, or both. Guanosine (30 μmol/l) per se had little effect on extracellular adenosine levels. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) were 0.125 ± 0.020 μmol/l, indicating rapid disposition of extracellular adenosine. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) plus guanosine (30 μmol/l) were 1.173 ± 0.061 μmol/l, indicating slow disposition of extracellular adenosine. Cell injury increased extracellular levels of endogenous adenosine and guanosine, and the effects of cell injury on endogenous extracellular adenosine were modulated by altering the levels of endogenous extracellular guanosine with exogenous purine nucleoside phosphorylase (converts guanosine to guanine) or 8-aminoguanosine (inhibits purine nucleoside phosphorylase). Extracellular guanosine also slowed the disposition of extracellular adenosine in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts, and kidney epithelial cells and in human aortic and coronary artery vascular smooth muscle cells and coronary artery endothelial cells. The effects of guanosine on adenosine levels were not mimicked or attenuated by 5-iodotubericidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)-adenine (adenosine deaminase inhibitor), 5-aminoimidazole-4-carboxamide (guanine deaminase inhibitor), aristeromycin (S-adenosylhomocysteine hydrolase inhibitor), low sodium (inhibits concentrative nucleoside transporters), S-(4-nitrobenzyl)−6-thioinosine [inhibits equilibrative nucleoside transporter (ENT) type 1], zidovudine (inhibits ENT type 2), or acadesine (known modulator of adenosine levels). Guanosine also increases extracellular inosine, uridine, thymidine, and cytidine, yet decreases

  16. Protein Characterization of Extracellular Microvesicles/Exosomes Released from Cytotoxin-Challenged Rat Cerebrocortical Mixed Culture and Mouse N2a Cells.

    Science.gov (United States)

    Kumar, Dhwani; Manek, Rachna; Raghavan, Vijaya; Wang, Kevin K

    2017-03-10

    A number of neuronal and glial proteins were previously found to be released in free-standing soluble form from cultured brain cells into cell-conditioned media. Here, we sought to examine if similar proteins are also contained in neural and astroglial cell-released extracellular microvesicles/exosomes (MV/E). In this study, MV/E were isolated from cell-conditioned media from control and cytotoxin-challenged rat cerebrocortical mixed culture (CTX) and mouse neuroblastoma N2a cells. Cytotoxin challenges included pro-necrosis calcium ionophore A23187, pro-apoptosis staurosporine (STS), and excitotoxin N-methyl-D-aspartate. Based on established nanoparticle characterization method (dynamic light scattering, NanoTracker, and transmission electron microscopy), we confirmed that these released vesicles are in fact characteristic representation of MV/E by morphology (lipid bilayered vesicles) and by particle size (132-142 nm for CTX and 49-77 nm for N2a cells). We indeed identified neural cell body protein UCH-L1, axonal injury marker αII-spectrin and its breakdown products (SBDPs), astroglial markers GFAP and its breakdown products (GFAP-BDP), dendritic protein BIII-tubulin, synaptic protein synaptophysin, and exosome marker Alix in microvesicles from CTX and/or N2a cells. Furthermore, SBDPs, GFAP-BDP, UCH-L1, and synaptophysin are especially dominant in MV/E isolated from cytotoxin-treated CTX cells. Similarly, SBDPs, βIII-tubulin, and UCH-L1 are more prominently observed in cytotoxin-challenged N2a cells. Lastly, when isolated MV/E from A23187- or STS-challenged N2a cells were introduced to healthy N2a culture, they are capable of evoking cytotoxicity in the latter. Taken together, our study identified that microvesicles/exosomes isolated form healthy and injured brain cells contain certain neural and astroglial proteins, as well as possibly other cytotoxic factors that are capable of propagating cytotoxic effects.

  17. Extracellular vesicles in cardiovascular homeostasis and disease.

    Science.gov (United States)

    Hutcheson, Joshua D; Aikawa, Elena

    2018-02-19

    Extracellular vesicles have emerged as one of the most important means through which cells interact with each other and the extracellular environment, but extracellular vesicle research remains challenging due to their small size, limited amount of material required for traditional molecular biology assays and inconsistency in the methods of their isolation. The advent of new technologies and standards in the field, however, have led to increased mechanistic insight into extracellular vesicle function. Herein, the latest studies on the role of extracellular vesicles in cardiovascular physiology and disease are discussed. Extracellular vesicles help control cardiovascular homeostasis and remodelling by mediating communication between cells and directing alterations in the extracellular matrix to respond to changes in the environment. The message carried from the parent cell to extracellular space can be intended for both local (within the same tissue) and distal (downstream of blood flow) targets. Pathological cargo loaded within extracellular vesicles could further result in various diseases. On the contrary, new studies indicate that injection of extracellular vesicles obtained from cultured cells into diseased tissues can promote restoration of normal tissue function. Extracellular vesicles are an integral part of cell and tissue function, and harnessing the properties inherent to extracellular vesicles may provide a therapeutic strategy to promote tissue regeneration.

  18. Experiences from occupational exposure limits set on aerosols containing allergenic proteins

    DEFF Research Database (Denmark)

    Nielsen, Gunnar; Larsen, Søren; Hansen, Jitka S

    2012-01-01

    Occupational exposure limits (OELs) together with determined airborne exposures are used in risk assessment based managements of occupational exposures to prevent occupational diseases. In most countries, OELs have only been set for few protein-containing aerosols causing IgE-mediated allergies...... for setting OELs. Our aim is to analyse prerequisites for setting OELs for the allergenic protein-containing aerosols. Opposite to the key effect of toxicological reactions, two thresholds, one for the sensitization phase and one for elicitation of IgE-mediated symptoms in sensitized individuals, are used...... is available for setting OELs for proteins and protein-containing aerosols where the critical effect is IgE sensitization and IgE-mediated airway diseases....

  19. Micropatterning Extracellular Matrix Proteins on Electrospun Fibrous Substrate Promote Human Mesenchymal Stem Cell Differentiation Toward Neurogenic Lineage.

    Science.gov (United States)

    Li, Huaqiong; Wen, Feng; Chen, Huizhi; Pal, Mintu; Lai, Yuekun; Zhao, Allan Zijian; Tan, Lay Poh

    2016-01-13

    In this study, hybrid micropatterned grafts constructed via a combination of microcontact printing and electrospinning techniques process were utilized to investigate the influencing of patterning directions on human mesenchymal stem cells (hMSCs) differentiation to desired phenotypes. We found that the stem cells could align and elongate along the direction of the micropattern, where they randomly distributed on nonmicropatterned surfaces. Concomitant with patterning effect of component on stem cell alignment, a commensurate increase on the expression of neural lineage commitment markers, such as microtubule associated protein 2 (MAP2), Nestin, NeuroD1, and Class III β-Tubulin, were revealed from mRNA expression by quantitative Real Time PCR (qRT-PCR) and MAP2 expression by immunostaining. In addition, the effect of electrospun fiber orientation on cell behaviors was further examined. An angle of 45° between the direction of micropatterning and orientation of aligned fibers was verified to greatly prompt the outgrowth of filopodia and neurogenesis of hMSCs. This study demonstrates that the significance of hybrid components and electrospun fiber alignment in modulating cellular behavior and neurogenic lineage commitment of hMSCs, suggesting promising application of porous scaffolds with smart component and topography engineering in clinical regenerative medicine.

  20. cse, a Chimeric and variable gene, encodes an extracellular protein involved in cellular segregation in Streptococcus thermophilus.

    Science.gov (United States)

    Borges, Frédéric; Layec, Séverine; Thibessard, Annabelle; Fernandez, Annabelle; Gintz, Brigitte; Hols, Pascal; Decaris, Bernard; Leblond-Bourget, Nathalie

    2005-04-01

    The isolation of a Streptococcus thermophilus CNRZ368 mutant displaying a long-chain phenotype allowed us to identify the cse gene (for cellular segregation). The N terminus of Cse exhibits high similarity to Streptococcus agalactiae surface immunogenic protein (SIP), while its C terminus exhibits high similarity to S. thermophilus PcsB. In CNRZ368, deletion of the entire cse open reading frame leads to drastic lengthening of cell chains and altered colony morphology. Complementation of the Deltacse mutation with a wild-type allele restored both wild-type phenotypes. The central part of Cse is a repeat-rich region with low sequence complexity. Comparison of cse from CNRZ368 and LMG18311 strains reveals high variability of this repeat-rich region. To assess the impact of this central region variability, the central region of LMG18311 cse was exchanged with that of CNRZ368 cse. This replacement did not affect chain length, showing that divergence of the central part does not modify cell segregation activity of Cse. The structure of the cse locus suggests that the chimeric organization of cse results from insertion of a duplicated sequence deriving from the pcsB 3' end into an ancestral sip gene. Thus, the cse locus illustrates the module-shuffling mechanism of bacterial gene evolution.

  1. Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H. Eric (Van Andel); (NU Singapore)

    2012-02-21

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 {angstrom} crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  2. Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

    Science.gov (United States)

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H Eric

    2011-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  3. Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein.

    Science.gov (United States)

    Schepens, Bert; Sedeyn, Koen; Vande Ginste, Liesbeth; De Baets, Sarah; Schotsaert, Michael; Roose, Kenny; Houspie, Lieselot; Van Ranst, Marc; Gilbert, Brian; van Rooijen, Nico; Fiers, Walter; Piedra, Pedro; Saelens, Xavier

    2014-11-01

    Infections with human respiratory syncytial virus (HRSV) occur globally in all age groups and can have devastating consequences in young infants. We demonstrate that a vaccine based on the extracellular domain (SHe) of the small hydrophobic (SH) protein of HRSV, reduced viral replication in challenged laboratory mice and in cotton rats. We show that this suppression of viral replication can be transferred by serum and depends on a functional IgG receptor compartment with a major contribution of FcγRI and FcγRIII. Using a conditional cell depletion method, we provide evidence that alveolar macrophages are involved in the protection by SHe-specific antibodies. HRSV-infected cells abundantly express SH on the cell surface and are likely the prime target of the humoral immune response elicited by SHe-based vaccination. Finally, natural infection of humans and experimental infection of mice or cotton rats does not induce a strong immune response against HRSV SHe. Using SHe as a vaccine antigen induces immune protection against HRSV by a mechanism that differs from the natural immune response and from other HRSV vaccination strategies explored to date. Hence, HRSV vaccine candidates that aim at inducing protective neutralizing antibodies or T-cell responses could be complemented with a SHe-based antigen to further improve immune protection. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  4. A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

    Directory of Open Access Journals (Sweden)

    Rocha H.A.O.

    2001-01-01

    Full Text Available Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1 and the mutant type deficient in xylosyltransferase (CHO-745. The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5(and CHO-745 (2 x 10(5 and 5 x 10(5 cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

  5. Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Shiva Kumar

    Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR. Crystal structures of a number of Class B GPCR extracellular domains (ECD bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

  6. Heterotrimeric G Protein-Regulated Ca2+ Influx and PIN2 Asymmetric Distribution Are Involved in Arabidopsis thaliana Roots' Avoidance Response to Extracellular ATP

    Directory of Open Access Journals (Sweden)

    Ruojia Zhu

    2017-09-01

    Full Text Available Extracellular ATP (eATP has been reported to be involved in plant growth as a primary messenger in the apoplast. Here, roots of Arabidopsis thaliana seedlings growing in jointed medium bent upon contact with ATP-containing medium to keep away from eATP, showing a marked avoidance response. Roots responded similarly to ADP and bz-ATP but did not respond to AMP and GTP. The eATP avoidance response was reduced in loss-of-function mutants of heterotrimeric G protein α subunit (Gα (gpa1-1 and gpa1-2 and enhanced in Gα-over-expression (OE lines (wGα and cGα. Ethylenebis(oxyethylenenitrilo tetraacetic acid (EGTA and Gd3+ remarkably suppressed eATP-induced root bending. ATP-stimulated Ca2+ influx was impaired in Gα null mutants and increased in its OE lines. DR5-GFP and PIN2 were asymmetrically distributed in ATP-stimulated root tips, this effect was strongly suppressed by EGTA and diminished in Gα null mutants. In addition, some eATP-induced genes' expression was also impaired in Gα null mutants. Based on these results, we propose that heterotrimeric Gα-regulated Ca2+ influx and PIN2 distribution may be key signaling events in eATP sensing and avoidance response in Arabidopsis thaliana roots.

  7. Evidence against Extracellular Exposure of a Highly Immunogenic Region in the C-Terminal Domain of the Simian Immunodeficiency Virus gp41 Transmembrane Protein

    Science.gov (United States)

    Postler, Thomas S.; Martinez-Navio, José M.; Yuste, Eloísa

    2012-01-01

    transmembrane protein with a single membrane-spanning domain and without any extracellular loops. PMID:22072749

  8. 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.

  9. Modification of EGF-like module 1 of thrombospondin-1, an animal extracellular protein, by O-linked N-acetylglucosamine.

    Directory of Open Access Journals (Sweden)

    Brian R Hoffmann

    Full Text Available Thrombospondin-1 (TSP-1 is known to be subject to three unusual carbohydrate modifications: C-mannosylation, O-fucosylation, and O-glucosylation. We now describe a fourth: O-β-N-acetylglucosaminylation. Previously, O-β-N-acetylglucosamine (O-β-GlcNAc was found on a threonine in the loop between the fifth and sixth cysteines of the 20(th epidermal growth factor (EGF-like module of Drosophila Notch. A BLAST search based on the Drosophila Notch loop sequence identified a number of human EGF-like modules that contain a similar sequence, including EGF-like module 1 of TSP-1 and its homolog, TSP-2. TSP-1, which has a potentially modifiable serine in the loop, reacted in immuno-blots with the CTD110.6 anti-O-GlcNAc antibody. Antibody reactivity was diminished by treatment of TSP-1 with β-N-acetylhexosaminidase. TSP-2, which lacks a potentially modifiable serine/threonine in the loop, did not react with CTD110.6. Analysis of tandem modules of TSP-1 localized reactivity of CTD110.6 to EGF-like module 1. Top-down mass spectrometric analysis of EGF-like module 1 demonstrated the expected modifications with glucose (+162 Da and xylose (+132 Da separately from modification with N-acetyl hexosamine (+203 Da. Mass spectrometric sequence analysis localized the +203-Da modification to Ser580 in the sequence (575CPPGYSGNGIQC(586. These results demonstrate that O-β-N-acetylglucosaminylation can occur on secreted extracellular matrix proteins as well as on cell surface proteins.

  10. Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells

    Energy Technology Data Exchange (ETDEWEB)

    Tada, Hiroyuki [Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Nemoto, Eiji, E-mail: e-nemoto@umin.ac.jp [Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Kanaya, Sousuke; Hamaji, Nozomu; Sato, Hisae; Shimauchi, Hidetoshi [Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan)

    2010-04-16

    Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca{sup 2+}Ca{sub o}{sup 2+} has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca{sub o}{sup 2+} signaling in odontogenesis remains unclear. We found that elevated Ca{sub o}{sup 2+} increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca{sup 2+} increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca{sup 2+} channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca{sup 2+}, suggesting that the Ca{sup 2+} influx from Ca{sup 2+} channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca{sup 2+}-sensing receptors (CaSR) and only respond slightly to other cations such as Sr{sup 2+} and spermine, suggesting that dental pulp cells respond to Ca{sub o}{sup 2+} to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca{sub o}{sup 2+} among cations.

  11. Urinary extracellular vesicles: biomarkers and beyond

    NARCIS (Netherlands)

    M. Salih (Mahdi)

    2017-01-01

    markdownabstractExtracellular vesicles have been isolated in various body fluids including urine. The cargo of urinary extracellular vesicles (uEVs) is composed of proteins and nucleic acids reflecting the physiological and possibly the pathophysiological state of cells lining the nephron. Because

  12. Extracellular Molecules Involved in Cancer Cell Invasion

    Energy Technology Data Exchange (ETDEWEB)

    Stivarou, Theodora; Patsavoudi, Evangelia, E-mail: epatsavoudi@pasteur.gr [Department of Biochemistry, Hellenic Pasteur Institute, Athens 11521 (Greece); Technological Educational Institute of Athens, Egaleo, Athens 12210 (Greece)

    2015-01-26

    Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion.

  13. A Perspective on Extracellular Vesicles Proteomics

    Directory of Open Access Journals (Sweden)

    Livia Rosa-Fernandes

    2017-11-01

    Full Text Available Increasing attention has been given to secreted extracellular vesicles (EVs in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieved from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

  14. A Perspective on Extracellular Vesicles Proteomics.

    Science.gov (United States)

    Rosa-Fernandes, Livia; Rocha, Victória Bombarda; Carregari, Victor Corasolla; Urbani, Andrea; Palmisano, Giuseppe

    2017-01-01

    Increasing attention has been given to secreted extracellular vesicles (EVs) in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieved from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

  15. Serodiagnosis efficacy and immunogenicity of the fusion protein of Mycobacterium tuberculosis composed of the 10-kilodalton culture filtrate protein, ESAT-6, and the extracellular domain fragment of PPE68.

    Science.gov (United States)

    Xu, Jia-Nan; Chen, Jian-Ping; Chen, Da-Li

    2012-04-01

    In order to identify immunodominant antigens of Mycobacterium tuberculosis that may be used in the serodiagnosis of active tuberculosis (TB), we designed an M. tuberculosis fusion protein consisting of CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secreted antigenic target), and the extracellular domain fragment of PPE68 (PPE68'). Then, the coding sequences of the three proteins were inserted into a prokaryotic expression vector, pET-32a(+). To enhance the immunological response, the proteins were linked together. The fusion proteins with a 6 × His tag were successfully overexpressed in Escherichia coli BL21 and purified. The purified proteins were applied for detection of the total IgG titer by using an enzyme-linked immunosorbent assay (ELISA) with human sera from well-characterized TB cases and the control cases, and results were compared to those with purified protein derivative tuberculin (PPD). The ELISA results showed that among 140 cases of confirmed active TB and 70 control cases, CFP-10-ESAT-6-PPE68' had a sensitivity of 73.3% and specificity of 94.3%, compared to a sensitivity of 66.7% and specificity of 74.3% for PPD and a sensitivity of 65% and specificity of 91.4% for CFP-10-ESAT-6. In addition, the fusion protein CFP-10-ESAT-6-PPE68' stimulated a higher level of antigen-specific gamma interferon (IFN-γ) release for active-TB patients than PPD and CFP-10-ESAT-6. After immunization of C57BL/6 mice, the findings indicated that the total IgG titers and the concentrations of IFN-γ in mice immunized by CFP-10-ESAT-6-PPE68' were high and induced strong, long-term humoral immunity compared to results with PPD and CFP-10-ESAT-6. Thus, our study indicates that the fusion protein CFP-10-ESAT-6-PPE68' may be useful as an immunodominant antigen for the serodiagnosis of active TB.

  16. Extracellular matrix in tooth cementum and mantle dentin: localization of osteopontin and other noncollagenous proteins, plasma proteins, and glycoconjugates by electron microscopy.

    Science.gov (United States)

    McKee, M D; Zalzal, S; Nanci, A

    1996-06-01

    Noncollagenous proteins (NCPs) are considered to have multiple functions related to the formation, turnover, and repair of the collagen-based mineralized tissues. Collectively, they comprise a class of generally acidic, mineral-binding proteins showing extensive posttranslational modifications, including glycosylation, phosphorylation, and sulfation. METHODS. We have used colloidal-gold immunocytochemistry and lectin-gold cytochemistry, together with transmission electron microscopy, to examine the organic matrix composition of tooth cementum and the subjacent mantle dentin in rodent molar teeth. Molars were processed for immunocytochemistry using antibodies against osteopontin (OPN), osteocalcin (OC), bone sialoprotein (BSP), bone acidic glycoprotein-75 (BAG-75), albumin (ALB), and alpha 2HS-glycoprotein (alpha 2HS-GP), or for glycoconjugate cytochemistry using lectin-gold complexes. Ultrastructurally, at the advancing root edge in developing molars, OPN and BSP initially were associated with small calcification foci in the mantle dentin. With progressing mineralization, OC and alpha 2HS-GP appeared diffusely distributed throughout the calcified mantle dentin, and diminished as a gradient toward the circumpulpal dentin. Immediately following disruption of Hertwig's epithelial root sheath, cementum deposition commenced at the root surface occasionally with the appearance of a cement line rich in OPN. Cementum matrix proper contained abundant OPN, BSP, OC, and alpha 2HS-GP, but no or little BAG-75 or ALB. Protein immunolabeling, as well as lectin labeling for beta-D-galactose and N-acetyl-neuraminic acid and/or N-glycolyl-neuraminic acid, both being prominent sugars of certain NCPs, was primarily concentrated between, and at the surface of, collagen fibrils in acellular extrinsic fiber cementum. OPN, BSP, OC, and alpha 2HS-GP were also prominent components of cellular cementum and of Sharpey's fibers. In cellular cementum, laminae limitantes sometimes present

  17. Cystamine-mediated inhibition of protein disulfide isomerase triggers aggregation of misfolded orexin-A in the Golgi apparatus and prevents extracellular secretion of orexin-A.

    Science.gov (United States)

    Fujita, Issei; Nobunaga, Mizuki; Seki, Takahiro; Kurauchi, Yuki; Hisatsune, Akinori; Katsuki, Hiroshi

    2017-07-22

    Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy. Copyright © 2017. Published by Elsevier Inc.

  18. Interactions of calcium/calmodulin-dependent protein kinases (CaMK) and extracellular-regulated kinase (ERK) in monocyte adherence and TNFalpha production.

    Science.gov (United States)

    Rosengart, M R; Arbabi, S; Garcia, I; Maier, R V

    2000-03-01

    The circulating monocyte possesses a markedly different functional phenotype relative to the macrophage (Mphi). The adhesive interactions encountered by the monocyte, en route to the inflammatory focus, generate signals that culminate in the expression of a pro-inflammatory Mphi phenotype, marked by enhanced cytokine production. Previously, we demonstrated that calcium and calmodulin are essential for maximal Mphi activation and, in particular, TNFalpha production. These effects are likely to be mediated through signal transduction kinases that require the calcium/calmodulin complex. Here, we investigated the effect of adherence on calcium/calmodulin-dependent protein kinase (CaMK) II and IV activation of the extracellular-signal regulated kinase (ERK) 1/2 cascade and on lipopolysaccharide (LPS)-induced TNFalpha production by human monocytes. Adherence activated ERK 1/2 and led to an 8-fold potentiation in LPS-induced TNFalpha production over similarly stimulated non-adherent cells. Inhibition of CaMK II prior to adherence prevented ERK 1/2 activation and attenuated by up to 40%, the TNFalpha response to subsequent LPS stimulation. CaMK II inhibition after adherence, however, failed to modify cytokine release. Inhibition of CaMK IV, both after adherence and in non-adherent monocytes, significantly inhibited LPS-induced ERK 1/2 activation and abrogated TNFalpha production by up to 75%. These data suggest that the function of CaMK II in TNFalpha production by adherent monocytes occurs during adhesion, is mediated in part by activation of ERK 1/2, and appears to "prime" the monocyte for enhanced cytokine production. CaMK IV, through activation of ERK 1/2, appears to have a direct role in the LPS signal transduction for TNFalpha production.

  19. Anesthetics inhibit extracellular signal-regulated Kinase1/2 phosphorylation via NMDA receptor, phospholipase C and protein kinase C in mouse hippocampal slices.

    Science.gov (United States)

    Haiying, Gao; Mingjie, Han; Lingyu, Zhang; Qingxiang, Wang; Haisong, Wang; Bingxi, Zhang

    2017-02-01

    Extracellular signal-regulated kinase 1/2 (ERK1/2) has been implicated in learning and memory; however, whether intravenous anesthetics modulate ERK1/2 remains unknown. The aim of this study was to examine the effect of several intravenous anesthetics on the phosphorylation of ERK1/2 in the hippocampus of adult mice. Western blotting was used to examine cellular levels of phosphorylated and unphosphorylated ERK1/2 in mouse hippocampus slices, which were incubated with or without anesthetics including propofol, etomidate, ketamine and midazolam, a protein kinase C (PKC) activator or inhibitor, or phospholipase C (PLC) activator or inhibitor. Propofol, etomidate, ketamine and midazolam reduced phosphorylation of ERK1/2 in a time-dependent manner. Washing out propofol after 5 min increased ERK1/2 phosphorylation. The anesthetic-induced depression of ERK1/2 phosphorylation was blocked by 0.1 μM phorbol-12-myristate 13-acetate (an activator of PKC), 50 μM U73122 (an inhibitor of PLC). The anesthetic-induced depression of ERK1 phosphorylation was blocked by 1 mMN-methyl-d-aspartate (NMDA). Whereas 100 μM chelerythrine (an inhibitor of PKC) and 100 μM carbachol (an activator of PLC) and 20 μM PD-98059 (an inhibitor of MEK) had additive effects on propofol-induced inhibition of ERK1/2 phosphorylation. In contrast, 10 μM MK801 (a NMDA receptor antagonist) did not block anesthetic-induced inhibition of ERK1/2 phosphorylation. Intravenous anesthetics markedly decreased phosphorylation of ERK1/2 in mouse hippocampal slices, most likely via the NMDA receptor, and PLC- and PKC-dependent pathways. Thus, ERK1/2 represents a target for anesthetics in the brain. Copyright © 2016. Published by Elsevier Ltd.

  20. Association of apoptosis with the inhibition of extracellular signal-regulated protein kinase activity in the tumor necrosis factor alpha-resistant ovarian carcinoma cell line UCI 101.

    Science.gov (United States)

    Yazlovitskaya, E M; Pelling, J C; Persons, D L

    1999-05-01

    Tumor necrosis factor-alpha (TNF alpha) can function as both an autocrine and a paracrine growth factor and may therefore play a role in ovarian tumor progression. TNF alpha initiates multiple cellular responses, many of which are mediated through the mitogen-activated protein kinase pathways, which transduce signals from the TNF alpha receptors through the cytoplasm to the nucleus, resulting in regulation of gene expression. We examined the role of c-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated protein kinase (ERK) 1 and 2 in the cellular growth response to TNF alpha in the ovarian carcinoma cell line UCI 101. JNK1 activity was increased to a maximum level ninefold above the basal level after 10-20 min of treatment with 10 ng/mL TNF alpha. A maximum threefold induction of ERK1/2 activity was observed after 1 min of treatment. At concentrations up to 100 ng/mL, TNF alpha had neither a stimulatory nor an inhibitory effect on growth of UCI 101 cells. However, inhibition of TNF alpha-induced ERK1/2 activity by the MAP/ERK kinase 1 inhibitor PD 98059 resulted in 60% inhibition of cell growth in TNF alpha-treated UCI 101 cells. This decrease in cell growth was accompanied by apoptosis, as demonstrated by the presence of a 180-bp DNA ladder. Thus, the inhibition of TNF alpha-induced ERK1/2 activity was associated with induction of apoptosis in the TNF alpha-resistant cell line UCI 101. Inhibition of TNF alpha-induced ERK1/2 activity was accompanied by a subsequent transient increase in TNF alpha-induced JNK1 activity. The significance of this increase with respect to apoptosis induction remains to be determined. These findings demonstrated that ERK1/2 activity can modulate cellular sensitivity to TNF alpha and suggested that the balance between the levels of ERK1/2 and JNK1 activation may be critical in the cellular growth response to TNF alpha.

  1. Effect of ceramic membrane channel diameter on limiting retentate protein concentration during skim milk microfiltration.

    Science.gov (United States)

    Adams, Michael C; Barbano, David M

    2016-01-01

    Our objective was to determine the effect of retentate flow channel diameter (4 or 6mm) of nongraded permeability 100-nm pore size ceramic membranes operated in nonuniform transmembrane pressure mode on the limiting retentate protein concentration (LRPC) while microfiltering (MF) skim milk at a temperature of 50°C, a flux of 55 kg · m(-2) · h(-1), and an average cross-flow velocity of 7 m · s(-1). At the above conditions, the retentate true protein concentration was incrementally increased from 7 to 11.5%. When temperature, flux, and average cross-flow velocity were controlled, ceramic membrane retentate flow channel diameter did not affect the LRPC. This indicates that LRPC is not a function of the Reynolds number. Computational fluid dynamics data, which indicated that both membranes had similar radial velocity profiles within their retentate flow channels, supported this finding. Membranes with 6-mm flow channels can be operated at a lower pressure decrease from membrane inlet to membrane outlet (ΔP) or at a higher cross-flow velocity, depending on which is controlled, than membranes with 4-mm flow channels. This implies that 6-mm membranes could achieve a higher LRPC than 4-mm membranes at the same ΔP due to an increase in cross-flow velocity. In theory, the higher LRPC of the 6-mm membranes could facilitate 95% serum protein removal in 2 MF stages with diafiltration between stages if no serum protein were rejected by the membrane. At the same flux, retentate protein concentration, and average cross-flow velocity, 4-mm membranes require 21% more energy to remove a given amount of permeate than 6-mm membranes, despite the lower surface area of the 6-mm membranes. Equations to predict skim milk MF retentate viscosity as a function of protein concentration and temperature are provided. Retentate viscosity, retentate recirculation pump frequency required to maintain a given cross-flow velocity at a given retentate viscosity, and retentate protein

  2. Infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b

    NARCIS (Netherlands)

    Kint, Joeri; Langereis, Martijn A.; Maier, Helena J.; Britton, Paul; Kuppeveld, van Frank J.; Koumans, Joseph; Wiegertjes, Geert F.; Forlenza, Maria

    2016-01-01

    During infection of their host cells, viruses often inhibit the production of host proteins, a process that is referred to as host shutoff. By doing this, viruses limit the production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the genera

  3. Infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b

    NARCIS (Netherlands)

    Kint, Joeri; Langereis, Martijn A|info:eu-repo/dai/nl/304823597; Maier, Helena J; Britton, Paul; van Kuppeveld, Frank J|info:eu-repo/dai/nl/156614723; Koumans, Joseph; Wiegertjes, Geert F; Forlenza, Maria

    2016-01-01

    During infection of their host cell, viruses often inhibit production of host proteins, a process which is referred to as host shutoff. By doing this, viruses limit production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the Alpha- and Betacoronavirus

  4. Targeting pH regulating proteins for cancer therapy-Progress and limitations.

    Science.gov (United States)

    Parks, Scott K; Pouysségur, Jacques

    2017-04-01

    Tumour acidity induced by metabolic alterations and incomplete vascularisation sets cancer cells apart from normal cellular physiology. This distinguishing tumour characteristic has been an area of intense study, as cellular pH (pH i ) disturbances disrupt protein function and therefore multiple cellular processes. Tumour cells effectively utilise pH i regulating machinery present in normal cells with enhancements provided by additional oncogenic or hypoxia induced protein modifications. This overall improvement of pH regulation enables maintenance of an alkaline pH i in the continued presence of external acidification (pH e ). Considerable experimentation has revealed targets that successfully disrupt tumour pH i regulation in efforts to develop novel means to weaken or kill tumour cells. However, redundancy in these pH-regulating proteins, which include Na + /H + exchangers (NHEs), carbonic anhydrases (CAs), Na + /HCO 3 - co-transporters (NBCs) and monocarboxylate transporters (MCTs) has prevented effective disruption of tumour pH i when individual protein targeting is performed. Here we synthesise recent advances in understanding both normoxic and hypoxic pH regulating mechanisms in tumour cells with an ultimate focus on the disruption of tumour growth, survival and metastasis. Interactions between tumour acidity and other cell types are also proving to be important in understanding therapeutic applications such as immune therapy. Promising therapeutic developments regarding pH manipulation along with current limitations are highlighted to provide a framework for future research directives. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

    Science.gov (United States)

    Georgiadou, Maria; Jacquemet, Guillaume; De Franceschi, Nicola; Peuhu, Emilia; Hamidi, Hellyeh; Pouwels, Jeroen; Martens, Victoria; Nia, Fatemeh Hassani; Beifuss, Malte; Boeckers, Tobias; Kreienkamp, Hans-Juergen; Barsukov, Igor L; Ivaska, Johanna

    2017-01-01

    SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN-domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN-domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN-domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1/RIAM/talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes. PMID:28263956

  6. SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras.

    Science.gov (United States)

    Lilja, Johanna; Zacharchenko, Thomas; Georgiadou, Maria; Jacquemet, Guillaume; De Franceschi, Nicola; Peuhu, Emilia; Hamidi, Hellyeh; Pouwels, Jeroen; Martens, Victoria; Nia, Fatemeh Hassani; Beifuss, Malte; Boeckers, Tobias; Kreienkamp, Hans-Juergen; Barsukov, Igor L; Ivaska, Johanna

    2017-04-01

    SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes.

  7. Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake.

    Science.gov (United States)

    Abels, Erik R; Breakefield, Xandra O

    2016-04-01

    Extracellular vesicles are a heterogeneous group of membrane-limited vesicles loaded with various proteins, lipids, and nucleic acids. Release of extracellular vesicles from its cell of origin occurs either through the outward budding of the plasma membrane or through the inward budding of the endosomal membrane, resulting in the formation of multivesicular bodies, which release vesicles upon fusion with the plasma membrane. The release of vesicles can facilitate intercellular communication by contact with or by internalization of contents, either by fusion with the plasma membrane or by endocytosis into "recipient" cells. Although the interest in extracellular vesicle research is increasing, there are still no real standards in place to separate or classify the different types of vesicles. This review provides an introduction into this expanding and complex field of research focusing on the biogenesis, nucleic acid cargo loading, content, release, and uptake of extracellular vesicles.

  8. OASIS/CREB3L1 is induced by endoplasmic reticulum stress in human glioma cell lines and contributes to the unfolded protein response, extracellular matrix production and cell migration.

    Directory of Open Access Journals (Sweden)

    Ravi N Vellanki

    Full Text Available OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines (U373, A172 and U87 and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells.

  9. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region

    DEFF Research Database (Denmark)

    Jiang, Y P; Wang, H; D'Eustachio, P

    1993-01-01

    We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology......, separated from the transmembrane segment by an uncharacteristically large juxta-membrane region. The extracellular domain of the R-PTP-kappa precursor protein contains an immunoglobulin-like domain and four fibronectin type III-like repeats, preceded by a signal peptide and a region of about 150 amino acids...... processing, following which both cleavage products remain associated. By site-directed mutagenesis, the likely cleavage site was shown to be a consensus sequence for cleavage by the processing endopeptidase furin, located in the fourth fibronectin type III-like repeat. In situ hybridization analysis...

  10. Brain Extracellular Space: The Final Frontier of Neuroscience.

    Science.gov (United States)

    Nicholson, Charles; Hrabětová, Sabina

    2017-11-21

    Brain extracellular space is the narrow microenvironment that surrounds every cell of the central nervous system. It contains a solution that closely resembles cerebrospinal fluid with the addition of extracellular matrix molecules. The space provides a reservoir for ions essential to the electrical activity of neurons and forms an intercellular chemical communication channel. Attempts to reveal the size and structure of the extracellular space using electron microscopy have had limited success; however, a biophysical approach based on diffusion of selected probe molecules has proved useful. A point-source paradigm, realized in the real-time iontophoresis method using tetramethylammonium, as well as earlier radiotracer methods, have shown that the extracellular space occupies ∼20% of brain tissue and small molecules have an effective diffusion coefficient that is two-fifths that in a free solution. Monte Carlo modeling indicates that geometrical constraints, including dead-space microdomains, contribute to the hindrance to diffusion. Imaging the spread of macromolecules shows them increasingly hindered as a function of size and suggests that the gaps between cells are predominantly ∼40 nm with wider local expansions that may represent dead-spaces. Diffusion measurements also characterize interactions of ions and proteins with the chondroitin and heparan sulfate components of the extracellular matrix; however, the many roles of the matrix are only starting to become apparent. The existence and magnitude of bulk flow and the so-called glymphatic system are topics of current interest and controversy. The extracellular space is an exciting area for research that will be propelled by emerging technologies. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  11. A novel multiplex bead-based platform highlights the diversity of extracellular vesicles

    OpenAIRE

    Wild, Stefan; Koliha, Nina; Wiencek, Yvonne; Heider, Ute; Jüngst, Christian; Kladt, Nikolay; Krauthäuser, Susanne; Ian C. D. Johnston; Bosio, Andreas; Schauss, Astrid

    2016-01-01

    The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the an...

  12. Comparative Proteomic Profiling of Human Osteoblast-Derived Extracellular Matrices Identifies Proteins Involved in Mesenchymal Stromal Cell Osteogenic Differentiation and Mineralization.

    NARCIS (Netherlands)

    M. Baroncelli (Marta); B.C.J. van der Eerden (Bram); Kan, Y.Y. (Yik-Yang); R.D.A.M. Alves (Rodrigo); J.A.A. Demmers (Jeroen); J. van de Peppel (Jeroen); J.P.T.M. van Leeuwen (Hans)

    2017-01-01

    textabstractThe extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell-secreted ECMs have become of interest as they reproduce tissue-architecture and modulate the

  13. Immunotherapeutic Potential of Extracellular Vesicles

    OpenAIRE

    Zhang, Bin; Yin, Yijun; Lai, Ruenn Chai; Lim, Sai Kiang

    2014-01-01

    Extracellular vesicle or EV is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes, the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized...

  14. Extracellular vesicles: new players in cardiovascular diseases.

    Science.gov (United States)

    Gaceb, Abderahim; Martinez, Maria Carmen; Andriantsitohaina, Ramaroson

    2014-05-01

    Extracellular vesicles, particles released by all cell types, represent a new way to convey information between cells such as proteins, second messengers, and genetic information to modify the phenotype and function of the target cells. Recent data suggest that extracellular vesicles play a crucial role in both physiology and pathology, including coagulation, angiogenesis, cell survival, modulation of the immune response, and inflammation. Thus extracellular vesicles participate in the processes of cardiovascular diseases from atherosclerosis, myocardial infarction to heart failure. Consequently, extracellular vesicles can potentially be exploited for therapy, prognosis, and biomarkers for health and disease. This review focuses on the role of extracellular vesicles in the development of cardiovascular diseases, as well as the deleterious and beneficial effects that they may provide in vascular cells and myocardium. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. The Bloom syndrome protein limits the lethality associated with RAD51 deficiency.

    Science.gov (United States)

    Lahkim Bennani-Belhaj, Kenza; Rouzeau, Sébastien; Buhagiar-Labarchède, Géraldine; Chabosseau, Pauline; Onclercq-Delic, Rosine; Bayart, Emilie; Cordelières, Fabrice; Couturier, Jérôme; Amor-Guéret, Mounira

    2010-03-01

    Little is known about the functional interaction between the Bloom's syndrome protein (BLM) and the recombinase RAD51 within cells. Using RNA interference technology, we provide the first demonstration that RAD51 acts upstream from BLM to prevent anaphase bridge formation. RAD51 downregulation was associated with an increase in the frequency of BLM-positive anaphase bridges, but not of BLM-associated ultrafine bridges. Time-lapse live microscopy analysis of anaphase bridge cells revealed that BLM promoted cell survival in the absence of Rad51. Our results directly implicate BLM in limiting the lethality associated with RAD51 deficiency through the processing of anaphase bridges resulting from the RAD51 defect. These findings provide insight into the molecular basis of some cancers possibly associated with variants of the RAD51 gene family.

  16. Extracellular-signal regulated kinase (Erk1/2), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and tristetraprolin (TTP) comprehensively regulate injury-induced immediate early gene (IEG) response in in vitro liver organ culture.

    Science.gov (United States)

    Tran, Doan Duy Hai; Koch, Alexandra; Saran, Shashank; Armbrecht, Marcel; Ewald, Florian; Koch, Martina; Wahlicht, Tom; Wirth, Dagmar; Braun, Armin; Nashan, Björn; Gaestel, Matthias; Tamura, Teruko

    2016-05-01

    Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15 min and continued to be phosphorylated for more than 2h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2 h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24 h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MK2 that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TTP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver. Copyright © 2016 Elsevier

  17. Extracellular Vesicles in Renal Pathophysiology.

    Science.gov (United States)

    Pomatto, Margherita A C; Gai, Chiara; Bussolati, Benedetta; Camussi, Giovanni

    2017-01-01

    Extracellular vesicles are a heterogeneous population of microparticles released by virtually all living cells which have been recently widely investigated in different biological fields. They are typically composed of two primary types (exosomes and microvesicles) and are recently commanding increasing attention as mediators of cellular signaling. Indeed, these vesicles can affect recipient cells by carrying and delivering complex cargos of biomolecules (including proteins, lipids and nucleic acids), protected from enzymatic degradation in the environment. Their importance has been demonstrated in the pathophysiology of several organs, in particular in kidney, where different cell types secrete extracellular vesicles that mediate their communication with downstream urinary tract cells. Over the past few years, evidence has been shown that vesicles participate in kidney development and normal physiology. Moreover, EVs are widely demonstrated to be implicated in cellular signaling during renal regenerative and pathological processes. Although many EV mechanisms are still poorly understood, in particular in kidney, the discovery of their role could help to shed light on renal biological processes which are so far elusive. Lastly, extracellular vesicles secreted by renal cells gather in urine, thus becoming a great resource for disease or recovery markers and a promising non-invasive diagnostic instrument for renal disease. In the present review, we discuss the most recent findings on the role of extracellular vesicles in renal physiopathology and their potential implication in diagnosis and therapy.

  18. Extracellular matrix and wound healing.

    Science.gov (United States)

    Maquart, F X; Monboisse, J C

    2014-04-01

    Extracellular matrix has been known for a long time as an architectural support for the tissues. Many recent data, however, have shown that extracellular matrix macromolecules (collagens, elastin, glycosaminoglycans, proteoglycans and connective tissue glycoproteins) are able to regulate many important cell functions, such as proliferation, migration, protein synthesis or degradation, apoptosis, etc., making them able to play an important role in the wound repair process. Not only the intact macromolecules but some of their specific domains, that we called "Matrikines", are also able to regulate many cell activities. In this article, we will summarize main findings showing the effects of extracellular matrix macromolecules and matrikines on connective tissue and epithelial cells, particularly in skin, and their potential implication in the wound healing process. These examples show that extracellular matrix macromolecules or some of their specific domains may play a major role in wound healing. Better knowledge of these interactions may suggest new therapeutic targets in wound healing defects. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  19. Melanoma affects the composition of blood cell-derived extracellular vesicles

    OpenAIRE

    Nina Koliha; Ute Heider; Tobias Ozimkowski; Martin Wiemann; Andreas Bosio; Stefan Wild

    2016-01-01

    Extracellular vesicles are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of extracellular vesicles reflects the type and status of the originating cell and extracellular vesicles in melanoma patient’s plasma could be indicative for the tumor. Likewise, extracellular vesicles might influence tumor progression by regulating immune responses. We performed a broad protein characterization of extracellular vesicles from plasma of...

  20. First demonstration of transmissible spongiform encephalopathy-associated prion protein (PrPTSE) in extracellular vesicles from plasma of mice infected with mouse-adapted variant Creutzfeldt-Jakob disease by in vitro amplification.

    Science.gov (United States)

    Saá, Paula; Yakovleva, Oksana; de Castro, Jorge; Vasilyeva, Irina; De Paoli, Silvia H; Simak, Jan; Cervenakova, Larisa

    2014-10-17

    The development of variant Creutzfeldt-Jakob disease (vCJD) in three recipients of non-leukoreduced red blood cells from asymptomatic donors who subsequently developed the disease has confirmed existing concerns about the possible spread of transmissible spongiform encephalopathies (TSEs) via blood products. In addition, the presence of disease-associated misfolded prion protein (PrP(TSE)), generally associated with infectivity, has been demonstrated in the blood of vCJD patients. However, its origin and distribution in this biological fluid are still unknown. Various studies have identified cellular prion protein (PrP(C)) among the protein cargo in human blood-circulating extracellular vesicles released from endothelial cells and platelets, and exosomes isolated from the conditioned media of TSE-infected cells have caused the disease when injected into experimental mice. In this study, we demonstrate the detection of PrP(TSE) in extracellular vesicles isolated from plasma samples collected during the preclinical and clinical phases of the disease from mice infected with mouse-adapted vCJD and confirm the presence of the exosomal marker Hsp70 in these preparations. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Extracellular vesicles as emerging intercellular communicasomes.

    Science.gov (United States)

    Yoon, Yae Jin; Kim, Oh Youn; Gho, Yong Song

    2014-10-01

    All living cells release extracellular vesicles having pleiotropic functions in intercellular communication. Mammalian extracellular vesicles, also known as exosomes and microvesicles, are spherical bilayered proteolipids composed of various bioactive molecules, including RNAs, DNAs, proteins, and lipids. Extracellular vesicles directly and indirectly control a diverse range of biological processes by transferring membrane proteins, signaling molecules, mRNAs, and miRNAs, and activating receptors of recipient cells. The active interaction of extracellular vesicles with other cells regulates various physiological and pathological conditions, including cancer, infectious diseases, and neurodegenerative disorders. Recent developments in high-throughput proteomics, transcriptomics, and lipidomics tools have provided ample data on the common and specific components of various types of extracellular vesicles. These studies may contribute to the understanding of the molecular mechanism involved in vesicular cargo sorting and the biogenesis of extracellular vesicles, and, further, to the identification of disease-specific biomarkers. This review focuses on the components, functions, and therapeutic and diagnostic potential of extracellular vesicles under various pathophysiological conditions.

  2. Platelet-derived Extracellular Vesicles: An Emerging Therapeutic Approach.

    Science.gov (United States)

    Tao, Shi-Cong; Guo, Shang-Chun; Zhang, Chang-Qing

    2017-01-01

    Extracellular vesicles (EVs) are a newly-discovered way by which cells communicate with their neighbors, as well as transporting cargos which once were considered to be limited by membrane barriers, including membrane proteins, cytosolic proteins and RNA. The discovery of platelet-derived EVs (P-EVs), the most abundant EVs in human blood, has been a very tortuous process. At first, P-EVs were identified as nothing but 'platelet dust', and subsequent research did not progress smoothly because of the limited research techniques to study EVs. Following leaps and bounds of technical progress in studying EVs, more and more attractive features of P-EVs were revealed and they began to be further researched. The aim of this review is to present the latest knowledge about the role of P-EVs in tissue repair and tumor progression. The potential mechanism of P-EVs is emphasized. Then the limitations of the present study and future research directions are discussed.

  3. Erratum: Hayward IP, Bridle KR, Campbell GR, Underwood PA, Campbell JH (1995) Effect of Extracellular Matrix Proteins on Vascular Smooth Muscle Cell Phenotype. Cell Biology International 19: 839-846. doi: 10.1006/cbir.1995.1019.

    Science.gov (United States)

    2016-02-01

    The above article, published in print in Cell Biology International in September 1995 and online on 2 January 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1006/cbir.1995.1019/abstract), is an exact duplicate of the article 'Effect of Extracellular Matrix Proteins on Vascular Smooth Muscle Cell Phenotype' (Hayward et al., ), which was published in the previous issue of Cell Biology International (Hayward et al., ). The duplicate publication is the result of an administrative error. The publisher apologises for any inconvenience. © 2015 International Federation for Cell Biology.

  4. 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

  5. A genetic upper limit to whole-body protein deposition in a strain of growing pigs.

    Science.gov (United States)

    Moughan, P J; Jacobson, L H; Morel, P C H

    2006-12-01

    The genetic upper limit to daily whole-body protein deposition (Pdmax) is an important constraint on pig growth. The Pdmax was determined for a specified pig genotype using N balance and serial slaughter techniques. A traditional N-balance study, involving 36 and 90 kg of BW Large White x (Landrace x Large White) entire male pigs, was first conducted to demonstrate that a highly digestible, nutrient-dense diet (1.54% Lys; 18 MJ of DE/kg, air-dried basis) was able to support the attainment of Pdmax within the constraints of pig appetite. Animals were allocated to set levels of feed intake [set proportions of ad libitum DE intake (DEi), 50 to 100%]. Nitrogen retention increased linearly with DEi up to 25.3 and 35.2 MJ of DE/d for the 36 and 90 kg of BW pigs, respectively, then showed a departure (P body protein was determined at slaughter. Growth data were analyzed by differentiating and combining continuous mathematical functions for BW and body composition. The ad libitum DEi were 27.4 and 50.7 MJ/d at 36 and 90 kg of BW for the entire males and were assumed, based on the N-balance results, sufficiently high to allow expression of Pdmax. There was an effect (P < 0.05) of sex on Pdmax vs. time (days on trial). Over the BW range of 25 to 85 kg, Pdmax was constant for the entire male and female pigs at 170 and 147 g/d, respectively. Above 85 kg of BW, Pdmax was no longer constant for either sex.

  6. RNA in extracellular vesicles.

    Science.gov (United States)

    Kim, Kyoung Mi; Abdelmohsen, Kotb; Mustapic, Maja; Kapogiannis, Dimitrios; Gorospe, Myriam

    2017-07-01

    Cells release a range of membrane-enclosed extracellular vesicles (EVs) into the environment. Among them, exosomes and microvesicles (collectively measuring 40-1000 nm in diameter) carry proteins, signaling lipids, and nucleic acids from donor cells to recipient cells, and thus have been proposed to serve as intercellular mediators of communication. EVs transport cellular materials in many physiologic processes, including differentiation, stem cell homeostasis, immune responses, and neuronal signaling. EVs are also increasingly recognized as having a direct role in pathologies such as cancer and neurodegeneration. Accordingly, EVs have been the focus of intense investigation as biomarkers of disease, prognostic indicators, and even therapeutic tools. Here, we review the classes of RNAs present in EVs, both coding RNAs (messenger RNAs) and noncoding RNAs (long noncoding RNAs, microRNAs, and circular RNAs). The rising attention to EV-resident RNAs as biomarkers stems from the fact that RNAs can be detected at extremely low quantities using a number of methods. To illustrate the interest in EV biology, we discuss EV RNAs in cancer and neurodegeneration, two major age-associated disease processes. WIREs RNA 2017, 8:e1413. doi: 10.1002/wrna.1413 For further resources related to this article, please visit the WIREs website. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  7. Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication.

    Science.gov (United States)

    Peel, Nina; Iyer, Jyoti; Naik, Anar; Dougherty, Michael P; Decker, Markus; O'Connell, Kevin F

    2017-01-01

    In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.

  8. Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication.

    Directory of Open Access Journals (Sweden)

    Nina Peel

    2017-01-01

    Full Text Available In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.

  9. Systemic protein delivery by muscle-gene transfer is limited by a local immune response.

    Science.gov (United States)

    Wang, Lixin; Dobrzynski, Eric; Schlachterman, Alexander; Cao, Ou; Herzog, Roland W

    2005-06-01

    Adeno-associated viral (AAV) vectors have been successfully used for therapeutic expression of systemic transgene products (such as factor IX or erythropoietin) following in vivo administration to skeletal muscle of animal models of inherited hematologic disorders. However, an immune response may be initiated if the transgene product represents a neoantigen. Here, we use ovalbumin (OVA) as a model antigen and demonstrate immune-mediated elimination of expression on muscle-directed AAV-2 gene transfer. Administration to immune competent mice resulted in transient systemic OVA expression. Within 10 days, OVA-specific T-helper cells had been activated in draining lymph nodes, an inflammatory immune response ensued, and OVA-expressing muscle fibers were destroyed by a cytotoxic CD8(+) T-cell response. Use of a muscle-specific promoter did not prevent this immune response. Adoptively transferred CD4(+) cells transgenic for a T-cell receptor specific to OVA peptide-major histocompatibility complex class II showed antigen-specific, vector dose-dependent proliferation confined to the draining lymph nodes of AAV-OVA-transduced muscle within 5 days after gene transfer and subsequently participated in lymphocytic infiltration of transduced muscle. This study documents that a local immune response limits sustained expression of a secreted protein in muscle gene transfer, a finding that may have consequences for design of clinical protocols.

  10. The Speed Limit of Protein Folding: Alpha-Helix Initiation Modeled and Observed

    Science.gov (United States)

    Lin, Milo; Mohammed, Omar; Zewail, Ahmed

    2011-03-01

    As a primary event of protein folding, alpha-helix initiation is the starting point of macromolecular complexity. In this work, an analytic coarse-grained model which predicts the initiation rate as a function of temperature, is presented. Helix initiation was measured via ultrafast temperature-jump fluorescence refolding experiments on two penta-peptides, and the measured rates agreed well with those of the model. In addition, the temporal separation of rate-limiting diffusion from fast annealing stipulated by the model was confirmed via ensemble-converging all-atom molecular dynamics simulations, which reproduced both the diffusion and the picosecond annealing processes and rates observed experimentally. Some of these results were published in: Mohammed OF, Jas GS, Lin MM, Ma H, Zewail AH (2009) Primary peptide folding dynamics observed with ultrafast temperature jump. Angew Chem 48: 5628-5632. We are grateful to the National Science Foundation and the National Institutes of Health for funding of this research at Caltech. M.M.L. acknowledges financial support from the Krell Institute and the US Department of Energy for a graduate fellowship.

  11. Dietary protein supplementation in the elderly for limiting muscle mass loss.

    Science.gov (United States)

    Lancha, Antonio Herbert; Zanella, Rudyard; Tanabe, Stefan Gleissner Ohara; Andriamihaja, Mireille; Blachier, Francois

    2017-01-01

    Supplementation with whey and other dietary protein, mainly associated with exercise training, has been proposed to be beneficial for the elderly to gain and maintain lean body mass and improve health parameters. The main objective of this review is to examine the evidence provided by the scientific literature indicating benefit from such supplementation and to define the likely best strategy of protein uptake for optimal objectified results in the elderly. Overall, it appears that an intake of approximately 0.4 g protein/kg BW per meal thus representing 1.2-1.6 g protein/kg BW/day may be recommended taking into account potential anabolic resistance. The losses of the skeletal muscle mass contribute to lower the capacity to perform activities in daily living, emphasizing that an optimal protein consumption may represent an important parameter to preserve independence and contribute to health status. However, it is worth noting that the maximal intake of protein with no adverse effect is not known, and that high levels of protein intake is associated with increased transfer of protein to the colon with potential deleterious effects. Thus, it is important to examine in each individual case the benefit that can be expected from supplementation with whey protein, taking into account the usual protein dietary intake.

  12. The Protein Content of Extracellular Vesicles Derived from Expanded Human Umbilical Cord Blood-Derived CD133+and Human Bone Marrow-Derived Mesenchymal Stem Cells Partially Explains Why both Sources are Advantageous for Regenerative Medicine.

    Science.gov (United States)

    Angulski, Addeli B B; Capriglione, Luiz G; Batista, Michel; Marcon, Bruna H; Senegaglia, Alexandra C; Stimamiglio, Marco A; Correa, Alejandro

    2017-04-01

    Adult stem cells have beneficial effects when exposed to damaged tissue due, at least in part, to their paracrine activity, which includes soluble factors and extracellular vesicles (EVs). Given the multiplicity of signals carried by these vesicles through the horizontal transfer of functional molecules, human mesenchymal stem cell (hMSCs) and CD133 + cell-derived EVs have been tested in various disease models and shown to recover damaged tissues. In this study, we profiled the protein content of EVs derived from expanded human CD133 + cells and bone marrow-derived hMSCs with the intention of better understanding the functions performed by these vesicles/cells and delineating the most appropriate use of each EV in future therapeutic procedures. Using LC-MS/MS analysis, we identified 623 proteins for expanded CD133 + -EVs and 797 proteins for hMSCs-EVs. Although the EVs from both origins were qualitatively similar, when protein abundance was considered, hMSCs-EVs and CD133 + -EVs were different. Gene Ontology (GO) enrichment analysis in CD133 + -EVs revealed proteins involved in a variety of angiogenesis-related functions as well proteins related to the cytoskeleton and highly implicated in cell motility and cellular activation. In contrast, when overrepresented proteins in hMSCs-EVs were analyzed, a GO cluster of immune response-related genes involved with immune response-regulating factors acting on phagocytosis and innate immunity was identified. Together our data demonstrate that from the point of view of protein content, expanded CD133 + -EVs and hMSCs-EVs are in part similar but also sufficiently different to reflect the main beneficial paracrine effects widely reported in pre-clinical studies using expanded CD133 + cells and/or hBM-MSCs.

  13. Extracellular Vesicles in Lung Disease.

    Science.gov (United States)

    Kubo, Hiroshi

    2018-01-01

    Accumulating evidence suggests that extracellular vesicles (EVs) play a role in the pathogenesis of lung diseases. These vesicles include exosomes, ectosomes (ie, microparticles, extracellular vesicles, microvesicles, and shedding vesicles), and apoptotic bodies. Exosomes are generated by inward budding of the membrane (endocytosis), subsequent forming of multivesicular bodies, and release by exocytosis. Ectosomes are formed by outward blebbing from the plasma membrane and are then released by proteolytic cleavage from the cell surface. Apoptotic bodies are generated on apoptotic cell shrinkage and death. Extracellular vesicles are released when the cells are activated or undergo apoptosis under inflammatory conditions. The number and types of released EVs are different according to the pathophysiological status of the disease. Therefore, EVs can be novel biomarkers for various lung diseases. EVs contain several molecules, including proteins, mRNA, microRNA, and DNA; they transfer these molecules to distant recipient cells. Circulating EVs modify the targeted cells and influence the microenvironment of the lungs. For this unique capability, EVs are expected to be a new drug delivery system and a novel therapeutic target. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  14. The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.

    Science.gov (United States)

    Signer, Robert A J; Qi, Le; Zhao, Zhiyu; Thompson, David; Sigova, Alla A; Fan, Zi Peng; DeMartino, George N; Young, Richard A; Sonenberg, Nahum; Morrison, Sean J

    2016-08-01

    Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis. © 2016 Signer et al.; Published by Cold Spring Harbor Laboratory Press.

  15. Assembly of Fibronectin Extracellular Matrix

    Science.gov (United States)

    Singh, Purva; Carraher, Cara; Schwarzbauer, Jean E.

    2013-01-01

    In the process of matrix assembly, multivalent extracellular matrix (ECM) proteins are induced to self-associate and to interact with other ECM proteins to form fibrillar networks. Matrix assembly is usually initiated by ECM glycoproteins binding to cell surface receptors, such as fibronectin (FN) dimers binding to α5β1 integrin. Receptor binding stimulates FN self-association mediated by the N-terminal assembly domain and organizes the actin cytoskeleton to promote cell contractility. FN conformational changes expose additional binding sites that participate in fibril formation and in conversion of fibrils into a stabilized, insoluble form. Once assembled, the FN matrix impacts tissue organization by contributing to the assembly of other ECM proteins. Here, we describe the major steps, molecular interactions, and cellular mechanisms involved in assembling FN dimers into fibrillar matrix while highlighting important issues and major questions that require further investigation. PMID:20690820

  16. Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program

    Science.gov (United States)

    Yablonka-Reuveni, Zipora; Christ, Bodo; Benson, Janice M.

    2014-01-01

    Whereas the understanding of the mechanisms underlying skeletal and cardiac muscle development has been increased dramatically in recent years, the understanding of smooth muscle development is still in its infancy. This paper summarizes studies on the ontogeny of chicken smooth muscle cells in the wall of the aorta and aortic arch-derived arteries. Employing immunocyto-chemistry with antibodies against smooth muscle contractile and extracellular matrix proteins we trace smooth muscle cell patterning from early development throughout adulthood. Comparing late stage embryos to young and adult chickens we demonstrate, for all the stages analyzed, that the cells in the media of aortic arch-derived arteries and of the thoracic aorta are organized in alternating lamellae. The lamellar cells, but not the interlamellar cells, express smooth muscle specific contractile proteins and are surrounded by basement membrane proteins. This smooth muscle cell organization of lamellar and interlamellar cells is fully acquired by embryonic day 11 (ED11). We further show that, during earlier stages of embryogenesis (ED3 through ED7), cells expressing smooth muscle proteins appear only in the peri-endothelial region of the aortic and aortic arch wall and are organized as a narrow band of cells that does not demonstrate the lamellar-interlamellar pattern. On ED9, infrequent cells organized in lamellar-interlamellar organization can be detected and their frequency increases by ED10. In addition to changes in cell organization, we show that there is a characteristic sequence of contractile and extracellular matrix protein expression during development of the aortic wall. At ED3 the peri-endothelial band of differentiated smooth muscle cells is already positive for smooth muscle alpha actin (αSM-actin) and fibronectin. By the next embryonic day the peri-endothelial cell layer is also positive for smooth muscle myosin light chain kinase (SM-MLCK). Subsequently, by ED5 this peri

  17. Binding of host extracellular matrix proteins to Mycoplasma fermentans and its effect on adherence to, and invasion of HeLa cells.

    Science.gov (United States)

    Yavlovich, Amichai; Rottem, Shlomo

    2007-01-01

    In the present study, we show that intact Mycoplasma fermentans cells have a wealth of adhesive interactions with components of the extracellular matrix. Mycoplasma fermentans intensively bind plasminogen, and to a lesser extent, fibronectin, heparin, and laminin. The binding of collagen type III, IV, or V was low. The binding of plasminogen, collagen type III, or collagen type V markedly enhanced the adherence of M. fermentans to HeLa cells, whereas the binding of fibronectin, heparin, laminin, or collagen IV induced only a small effect on mycoplasma adherence. Utilizing plasminogen-treated M. fermentans preparations, we detected microorganisms within host HeLa cells by the gentamicin protection assay or by confocal laser scanning microscopy of immunofluorescent preparations. However, no intracellular M. fermentans was detected when M. fermentans preparations treated with fibronectin, heparin, laminin, or collagen type III, IV, or V were utilized.

  18. Surface glycosylation profiles of urine extracellular vesicles.

    Directory of Open Access Journals (Sweden)

    Jared Q Gerlach

    Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

  19. Are There Any Differences in the Neural and Extracellular Matrix Proteins Density Between Children and Adults with Intrinsic Ureteropelvic Junction Obstruction?

    Directory of Open Access Journals (Sweden)

    Bahadır Şahin

    2017-12-01

    Full Text Available Objective: To compare changes in connective, neural and muscle tissues, and extracellular matrix in child and adult patients so that we can create a more objective view on the timing of surgery. Materials and Methods: Twenty-six patients, who were operated for ureteropelvic junction (UPJ obstruction in our clinic between September 2014 and May 2016, were included in the study. For the evaluation of connective tissue, Masson’s trichrome staining was used. Muscle, extracellular matrix elements and neural tissue were evaluated with immunohistochemistry using alpha-smooth muscle actin (α-SMA, Tenascin C and S100, respectively. Microscopically, the tissues were scored according to their staining density (0: No staining; 1: Minor; 2: Moderate, 3: Dense. Results: There were 12 children and 14 adults in the study groups. The initial evaluation showed no statistically significant difference between studied tissue types with respect to staining density for all parameters (Masson: p=0.414, α-SMA: p=0.204, Tenascin-C: p=0.264, S100: p=0.534. There was no statistically significant correlation between staining density and renal function percentage of the affected kidney (Masson: r=0.454, p=0.051 - α-SMA: r=-0.323, p=0.177 - Tenascin-C: r=0.290, p=0.229 - S100: r=-0.080, p=0.744. Conclusion: Our preliminary study showed some structural changes between adult and child patients but there is no statistically significant difference between the groups with respect to staining density scores. These results state that although UPJ obstruction is an ongoing process, there is no correlation between the histological deterioration degree of the UPJ segment and the loss on renal function for both children and adults.

  20. The complex extracellular biology of Streptomyces.

    Science.gov (United States)

    Chater, Keith F; Biró, Sandor; Lee, Kye Joon; Palmer, Tracy; Schrempf, Hildgund

    2010-03-01

    Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

  1. Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

    Science.gov (United States)

    Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

    2018-02-15

    Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this

  2. Limited coalescence and Ostwald ripening in emulsions stabilized by hydrophobin HFBII and milk proteins

    NARCIS (Netherlands)

    Dimitrova, Lydia M.; Boneva, Mariana P.; Danov, Krassimir D.; Kralchevsky, Peter A.; Basheva, Elka S.; Marinova, Krastanka G.; Petkov, Jordan T.; Stoyanov, Simeon D.

    2016-01-01

    Hydrophobins are proteins isolated from filamentous fungi, which are excellent foam stabilizers, unlike most of the proteins. In the present study, we demonstrate that hydrophobin HFBII can also serve as excellent emulsion stabilizer. The HFBII adsorption layers at the oil/water interface

  3. Studies on middle silkgland proteins of cocoon colour sex-limited ...

    Indian Academy of Sciences (India)

    Qualitative and quantitative differences in proteins expressed in the middle silkglands of male and female silkworm larvae that differ in silk colour were investigated by high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer assisted image analysis. About 1000 protein spots ...

  4. Limiting factors in Escherichia colifed-batch production of recombinant proteins

    DEFF Research Database (Denmark)

    Sanden, A.M.; Prytz, I.; Tubelekas, I.

    2003-01-01

    recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation......recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation...

  5. Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth) families.

    Science.gov (United States)

    Böttger, Angelika; Doxey, Andrew C; Hess, Michael W; Pfaller, Kristian; Salvenmoser, Willi; Deutzmann, Rainer; Geissner, Andreas; Pauly, Barbara; Altstätter, Johannes; Münder, Sandra; Heim, Astrid; Gabius, Hans-Joachim; McConkey, Brendan J; David, Charles N

    2012-01-01

    The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.

  6. Horizontal gene transfer contributed to the evolution of extracellular surface structures: the freshwater polyp Hydra is covered by a complex fibrous cuticle containing glycosaminoglycans and proteins of the PPOD and SWT (sweet tooth families.

    Directory of Open Access Journals (Sweden)

    Angelika Böttger

    Full Text Available The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.

  7. The lateral membrane organization and dynamics of myelin proteins PLP and MBP are dictated by distinct galactolipids and the extracellular matrix

    NARCIS (Netherlands)

    Ozgen, Hande; Schrimpf, Waldemar; Hendrix, Jelle; de Jonge, Jenny C.; Lamb, Don C.; Hoekstra, Dick; Kahya, Nicoletta; Baron, Wia

    2014-01-01

    In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we

  8. Peptidomics of Peptic Digest of Selected Potato Tuber Proteins: Post-Translational Modifications and Limited Cleavage Specificity.

    Science.gov (United States)

    C K Rajendran, Subin R; Mason, Beth; Udenigwe, Chibuike C

    2016-03-23

    Bioinformatic tools are useful in predicting bioactive peptides from food proteins. This study was focused on using bioinformatics and peptidomics to evaluate the specificity of peptide release and post-translational modifications (PTMs) in a peptic digest of potato protein isolate. Peptides in the protein hydrolysate were identified by LC-MS/MS and subsequently aligned to their parent potato tuber proteins. Five major proteins were selected for further analysis, namely, lipoxygenase, α-1,4-glucan phosphorylase, annexin, patatin, and polyubiquitin, based on protein coverage, abundance, confidence levels, and function. Comparison of the in silico peptide profile generated with ExPASy PeptideCutter and experimental peptidomics data revealed several differences. The experimental peptic cleavage sites were found to vary in number and specificity from PeptideCutter predictions. Average peptide chain length was also found to be higher than predicted with hexapeptides as the smallest detected peptides. Moreover, PTMs, particularly Met oxidation and Glu/Asp deamidation, were observed in some peptides, and these were unaccounted for during in silico analysis. PTMs can be formed during aging of potato tubers, or as a result of processing conditions during protein isolation and hydrolysis. The findings provide insights on the limitations of current bioinformatics tools for predicting bioactive peptide release from proteins, and on the existence of structural modifications that can alter the peptide bioactivity and functionality.

  9. Exchange Protein Activated by Cyclic AMP 2 (Epac2) Plays a Specific and Time-Limited Role in Memory Retrieval

    NARCIS (Netherlands)

    Ostroveanu, Anghelus; van der Zee, Eddy A.; Eisel, Ulrich L. M.; Schmidt, Martina; Nijholt, Ingrid M.

    2010-01-01

    Knowledge on the molecular mechanisms involved in memory retrieval is limited due to the lack of tools to study this stage of the memory process. Here we report that exchange proteins activated by cAMP (Epac) play a surprisingly specific role in memory retrieval. Intrahippocampal injection of the

  10. Using in situ X-ray reflectivity to study protein adsorption on hydrophilic and hydrophobic surfaces: benefits and limitations.

    Science.gov (United States)

    Richter, Andrew G; Kuzmenko, Ivan

    2013-04-30

    We have employed in situ X-ray reflectivity (IXRR) to study the adsorption of a variety of proteins (lysozyme, cytochrome c, myoglobin, hemoglobin, serum albumin, and immunoglobulin G) on model hydrophilic (silicon oxide) and hydrophobic surfaces (octadecyltrichlorosilane self-assembled monolayers), evaluating this recently developed technique for its applicability in the area of biomolecular studies. We report herein the highest resolution depiction of adsorbed protein films, greatly improving on the precision of previous neutron reflectivity (NR) results and previous IXRR studies. We were able to perform complete scans in 5 min or less with the maximum momentum transfer of at least 0.52 Å(-1), allowing for some time-resolved information about the evolution of the protein film structure. The three smallest proteins (lysozyme, cytochrome c, and myoglobin) were seen to deposit as fully hydrated, nondenatured molecules onto hydrophilic surfaces, with indications of particular preferential orientations. Time evolution was observed for both lysozyme and myoglobin films. The larger proteins were not observed to deposit on the hydrophilic substrates, perhaps because of contrast limitations. On hydrophobic surfaces, all proteins were seen to denature extensively in a qualitatively similar way but with a rough trend that the larger proteins resulted in lower coverage. We have generated high-resolution electron density profiles of these denatured films, including capturing the growth of a lysozyme film. Because the solution interface of these denatured films is diffuse, IXRR cannot unambiguously determine the film extent and coverage, a drawback compared to NR. X-ray radiation damage was systematically evaluated, including the controlled exposure of protein films to high-intensity X-rays and exposure of the hydrophobic surface to X-rays before adsorption. Our analysis showed that standard measuring procedures used for XRR studies may lead to altered protein films

  11. Limited variation in vaccine candidate Plasmodium falciparum Merozoite Surface Protein-6 over multiple transmission seasons

    Directory of Open Access Journals (Sweden)

    Branch OraLee H

    2010-05-01

    Full Text Available Abstract Background Plasmodium falciparum Merozoite Surface Protein-6 (PfMSP6 is a component of the complex proteinacious coat that surrounds P. falciparum merozoites. This location, and the presence of anti-PfMSP6 antibodies in P. falciparum-exposed individuals, makes PfMSP6 a potential blood stage vaccine target. However, genetic diversity has proven to be a major hurdle for vaccines targeting other blood stage P. falciparum antigens, and few endemic field studies assessing PfMSP6 gene diversity have been conducted. This study follows PfMSP6 diversity in the Peruvian Amazon from 2003 to 2006 and is the first longitudinal assessment of PfMSP6 sequence dynamics. Methods Parasite DNA was extracted from 506 distinct P. falciparum infections spanning the transmission seasons from 2003 to 2006 as part of the Malaria Immunology and Genetics in the Amazon (MIGIA cohort study near Iquitos, Peru. PfMSP6 was amplified from each sample using a nested PCR protocol, genotyped for allele class by agarose gel electrophoresis, and sequenced to detect diversity. Allele frequencies were analysed using JMP v.8.0.1.0 and correlated with clinical and epidemiological data collected as part of the MIGIA project. Results Both PfMSP6 allele classes, K1-like and 3D7-like, were detected at the study site, confirming that both are globally distributed. Allele frequencies varied significantly between transmission seasons, with 3D7-class alleles dominating and K1-class alleles nearly disappearing in 2005 and 2006. There was a significant association between allele class and village location (p-value = 0.0008, but no statistically significant association between allele class and age, sex, or symptom status. No intra-allele class sequence diversity was detected. Conclusions Both PfMSP6 allele classes are globally distributed, and this study shows that allele frequencies can fluctuate significantly between communities separated by only a few kilometres, and over time in the

  12. Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

    Directory of Open Access Journals (Sweden)

    Noda M

    2012-11-01

    Full Text Available Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand; 5Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 6Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanBackground: Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4 are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and results: To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the

  13. [EXTRACELLULAR VESICLES: INTERCELLULAR INFORMATION FLOW AND MEDICAL APPLICATIONS].

    Science.gov (United States)

    Pupyshev, A B

    2015-01-01

    The major features of extracellular vesicles secreted by mammalian cells are considered. Cell activation caused by formation of pathology stimulates the secretion acutely. The vesicles (exosomes, microvesicles) are enriched with annexin V, tetraspanin, miRNA. Exosomes are enriched especially by integrins, heat shock proteins. Microvesicles contain elevated amounts of tissue factors, phosphatidylserine, mRNA. The vesicles carry information about the pathological process, and microvesicles contain more proteins characteristic of inflammation and death than exosomes. They are important mediators of inflammation and infection in the body, have different effects on the immune system and the processes of carcinogenesis and neurodegeneration. However, antigenic profiles of extracellular vesicles differ not profoundly in various pathologies and so far they help diagnostics limitedly. The vesicles carry signals of genetic reprogramming of the cells and epigenetic stimulation, connected with both protein factors and mRNA and miRNA. Profiles of miRNA vesicles produced by the various pathological sources are studied actively and are useful as indicators of source and stage of cancer. Some ways of therapeutic use of the vesicles are also considered.

  14. Preliminary research of recombinant matrix extracellular ...

    African Journals Online (AJOL)

    ... and predentin, but not by dental pulp cells. Furthermore, we used von kossa staining and the results suggested that, MEPE could induce mineralization and we propose that this protein had a potential effect on dental rehabilitation. Key words: Matrix extracellular phosphoglycoprotein (MEPE), mineralization Von kossa.

  15. Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases

    Energy Technology Data Exchange (ETDEWEB)

    Miyakawa, Takuya; Sawano, Yoriko; Miyazono, Ken-ichi [Department of Applied Biochemical Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan); Hatano, Ken-ichi [Department of Chemistry and Chemical Biology, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biochemical Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2007-09-01

    Purification and crystallization of ginkbilobin-2 and its selenomethionine derivative allowed the collection of complete data to 2.38 Å resolution and multiwavelength anomalous diffraction data sets, respectively. The antifungal protein ginkbilobin-2 (Gnk2) from Ginkgo biloba seeds does not show homology to other pathogenesis-related proteins, but does show homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Native Gnk2 purified from ginkgo nuts and the selenomethionine derivative of recombinant Gnk2 (SeMet-rGnk2) were crystallized by the sitting-drop vapour-diffusion method using different precipitants. X-ray diffraction data were collected from Gnk2 at 2.38 Å resolution and from SeMet-rGnk2 at 2.79 Å resolution using a synchrotron-radiation source. The crystals of both proteins belonged to the primitive cubic space group P2{sub 1}3, with unit-cell parameters a = b = c = 143.2 Å.

  16. Limited tryptic proteolysis of the benzodiazepine binding proteins in different species reveals structural homologies.

    Science.gov (United States)

    Friedl, W; Lentes, K U; Schmitz, E; Propping, P; Hebebrand, J

    1988-12-01

    Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.

  17. Combined nitrogen limitation and cadmium stress stimulate total carbohydrates, lipids, protein and amino acid accumulation in Chlorella vulgaris (Trebouxiophyceae)

    Energy Technology Data Exchange (ETDEWEB)

    Chia, Mathias Ahii, E-mail: chia28us@yahoo.com [Department of Botany, Federal University of São Carlos, Rodovia Washington Luis km 235, São Carlos, SP Cep 13565905 (Brazil); Lombardi, Ana Teresa [Department of Botany, Federal University of São Carlos, Rodovia Washington Luis km 235, São Carlos, SP Cep 13565905 (Brazil); Graça Gama Melão, Maria da [Department of Hydrobiology, Federal University of São Carlos, Rodovia Washington Luis km 235, São Carlos, SP Cep 13565905 (Brazil); Parrish, Christopher C. [Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, Newfoundland A1C 5S7 (Canada)

    2015-03-15

    Highlights: • Chlorella vulgaris was exposed to Cd under varying N concentrations. • Growth rate and cell density decreased with increasing Cd stress and N limitation. • Dry weight, chlorophyll a, total lipid, carbohydrate and protein were accumulated. • Amino acids like proline and glutamine were accumulated under N and Cd stress. • Changes in amino acid composition are sensitive biomarkers for Cd and N stress. - Abstract: Metals have interactive effects on the uptake and metabolism of nutrients in microalgae. However, the effect of trace metal toxicity on amino acid composition of Chlorella vulgaris as a function of varying nitrogen concentrations is not known. In this research, C. vulgaris was used to investigate the influence of cadmium (10{sup −7} and 2.0 × 10{sup −8} mol L{sup −1} Cd) under varying nitrogen (2.9 × 10{sup −6}, 1.1 × 10{sup −5} and 1.1 × 10{sup −3} mol L{sup −1} N) concentrations on its growth rate, biomass and biochemical composition. Total carbohydrates, total proteins, total lipids, as well as individual amino acid proportions were determined. The combination of Cd stress and N limitation significantly inhibited growth rate and cell density of C. vulgaris. However, increasing N limitation and Cd stress stimulated higher dry weight and chlorophyll a production per cell. Furthermore, biomolecules like total proteins, carbohydrates and lipids increased with increasing N limitation and Cd stress. Ketogenic and glucogenic amino acids were accumulated under the stress conditions investigated in the present study. Amino acids involved in metal chelation like proline, histidine and glutamine were significantly increased after exposure to combined Cd stress and N limitation. We conclude that N limitation and Cd stress affects the physiology of C. vulgaris by not only decreasing its growth but also stimulating biomolecule production.

  18. Functional properties of a cross-linked soy protein-gelatin composite towards limited tryptic digestion of two extents.

    Science.gov (United States)

    Sheng, Wen-Wen; Zhao, Xin-Huai

    2013-12-01

    Both enzymatic cross-linking and hydrolysis are used to treat food proteins for functionality modification. The present study aimed to reveal the impact of limited tryptic digestion on some functional properties of a cross-linked composite generated from soy protein isolate and gelatin by transglutaminase. The composite was digested by trypsin for 1.5 and 4 h to produce two hydrolyzed composites with degrees of hydrolysis of 1% and 2%, respectively. Electrophoretic and chemical analysis showed that only part of the composite was degraded, and the gelatin fraction in the composite was more sensitive to tryptic digestion than the soy protein fraction. The hydrolyzed composites exhibited higher protein dispersion index at pH 4.5 or 7.0 and surface hydrophobicity, better digestibility in vitro, emulsifying property and oil absorption capacity, but lower water holding capacity and rheological properties (apparent viscosity, storage and loss modulus) than the composite. Compared to soy protein isolate, the hydrolyzed composites had better rheological properties, and higher water-holding capacity and emulsion stability index. The applied tryptic digestion conferred some improved properties on the hydrolyzed composites compared with the composite or soy protein isolate, and has potential for obtaining a new functional ingredient for processed foods. © 2013 Society of Chemical Industry.

  19. Combined nitrogen limitation and cadmium stress stimulate total carbohydrates, lipids, protein and amino acid accumulation in Chlorella vulgaris (Trebouxiophyceae).

    Science.gov (United States)

    Chia, Mathias Ahii; Lombardi, Ana Teresa; da Graça Gama Melão, Maria; Parrish, Christopher C

    2015-03-01

    Metals have interactive effects on the uptake and metabolism of nutrients in microalgae. However, the effect of trace metal toxicity on amino acid composition of Chlorella vulgaris as a function of varying nitrogen concentrations is not known. In this research, C. vulgaris was used to investigate the influence of cadmium (10(-7) and 2.0×10(-8)molL(-1) Cd) under varying nitrogen (2.9×10(-6), 1.1×10(-5) and 1.1×10(-3)molL(-1)N) concentrations on its growth rate, biomass and biochemical composition. Total carbohydrates, total proteins, total lipids, as well as individual amino acid proportions were determined. The combination of Cd stress and N limitation significantly inhibited growth rate and cell density of C. vulgaris. However, increasing N limitation and Cd stress stimulated higher dry weight and chlorophyll a production per cell. Furthermore, biomolecules like total proteins, carbohydrates and lipids increased with increasing N limitation and Cd stress. Ketogenic and glucogenic amino acids were accumulated under the stress conditions investigated in the present study. Amino acids involved in metal chelation like proline, histidine and glutamine were significantly increased after exposure to combined Cd stress and N limitation. We conclude that N limitation and Cd stress affects the physiology of C. vulgaris by not only decreasing its growth but also stimulating biomolecule production. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Chaperone role for proteins p618 and p892 in the extracellular tail development of Acidianus two-tailed virus

    DEFF Research Database (Denmark)

    Scheele, Urte; Erdmann, Susanne; Ungewickell, Ernst J.

    2011-01-01

    , is involved in this process. We propose that an additional chaperone system is required, consisting of a MoxR-type AAA ATPase (p618) and a von Willebrand domain A (VWA)-containing cochaperone, p892. Both proteins are absent from the other known bicaudavirus, STSV1, which develops a single tail intracellularly...

  1. Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity

    NARCIS (Netherlands)

    Takahara, Hiroyuki; Hacquard, Stéphane; Kombrink, Anja; Hughes, H.B.; Halder, Vivek; Robin, Guillaume P.; Hiruma, Kei; Neumann, Ulla; Shinya, Tomonori; Kombrink, Erich; Shibuya, Naoto; Thomma, Bart P.H.J.; O'Connell, Richard J.

    2016-01-01

    The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of

  2. Uses and limitations of green fluorescent protein as a viability marker in Enterococcus faecalis: An observational investigation.

    Science.gov (United States)

    Hoogenkamp, Michel A; Crielaard, Wim; Krom, Bastiaan P

    2015-08-01

    Enterococci are capable of producing biofilms that are notoriously difficult to treat and remove, for instance in root canal infections. The tenacious nature of these organisms makes screening of known and novel antimicrobial compounds necessary. While traditionally growth and fluorescence-based screening methods have proven useful, these methods have their limitations when applied to enterococci (e.g. time consuming, no kinetic data, diffusion properties of the fluorescent dyes). The aim of this study was to develop and validate a GFP-based high-throughput screening system to assess the bactericidal activity of a broad range of antimicrobial agents on Enterococcus faecalis and its biofilms. The effect of antimicrobial compounds on cell viability and GFP fluorescence of enterococcal planktonic and biofilm cells was determined using colony forming unit counts, fluorescence spectrophotometry and real-time imaging devices. There was a linear correlation between cell viability and GFP fluorescence. The intensity of the GFP signal was effected by the extracellular pH. For a range of antimicrobials however, there was no correlation between these two parameters. In contrast, for oxidizing agents such as sodium hypochlorite, the antimicrobial of choice for root canal disinfection, there was a correlation between loss of fluorescence and loss of viability. To conclude, the use of a GFP-based system to monitor the antimicrobial activity of compounds on E. faecalis is possible despite significant limitations. This approach is useful for analysis of susceptibility to oxidizing agents. Using real-time measuring devices to follow GFP fluorescence it should be possible to investigate the mode of action and rate of diffusion of oxidizing agents in E. faecalis biofilm. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Physiological proteins in resource-limited herbivores experiencing a population die-off

    Science.gov (United States)

    Garnier, R.; Bento, A. I.; Hansen, C.; Pilkington, J. G.; Pemberton, J. M.; Graham, A. L.

    2017-08-01

    Nutrient availability is predicted to interact with herbivore population densities. Competition for low quality food at high density may reduce summer food intake, and in turn winter survival. Conversely, low population density may favor physiological recovery through better access to better quality spring forage. Here, we take advantage of the long-term study of the Soay sheep population of St. Kilda (Scotland) to measure plasma protein markers and immunity in two consecutive summers with contrasting population densities. We show that, following a winter die-off resulting in a shift to low population density, albumin and total proteins increased, but only in adult sheep. The effect was not solely attributable to selective disappearance of malnourished sheep. Similarly, the concentration of antibodies was higher following the die-off, potentially indicating recovery of immune function. Overall, our results are consistent with the physiological recovery of surviving individuals after a harsh winter.

  4. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

    Science.gov (United States)

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

    2014-01-01

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  5. TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions: EVIDENCE FOR DISTINCT BINDING MECHANISMS FOR DIFFERENT TspanC8 PROTEINS.

    Science.gov (United States)

    Noy, Peter J; Yang, Jing; Reyat, Jasmeet S; Matthews, Alexandra L; Charlton, Alice E; Furmston, Joanna; Rogers, David A; Rainger, G Ed; Tomlinson, Michael G

    2016-02-12

    A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15 was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Extracellular granzymes in inflammation

    NARCIS (Netherlands)

    Wensink, A.C.

    2014-01-01

    It has been well established that granzymes released by cytotoxic lymphocytes induce cell death in virus-infected cells and tumor cells. Next to this intracellular role of granzymes in triggering apoptosis, granzymes also exist extracellularly in the circulation of patients with autoimmune diseases

  7. High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein β-galactosidase expression.

    Science.gov (United States)

    Lin, Hsiao-Hsien; Lee, Tsung-Yih; Liu, Ting-Wei; Tseng, Ching-Ping

    2017-07-01

    Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (∼300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27 kip by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PKA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERK phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the abovementioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce β-galactosidase (β-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the β-Gal production (from 2.8- to 4.5-fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. Copyright © 2017. Published by Elsevier B.V.

  8. Exercise training protects against atherosclerotic risk factors through vascular NADPH oxidase, extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun N-terminal kinase downregulation in obese rats.

    Science.gov (United States)

    Touati, Sabeur; Montezano, Augusto C I; Meziri, Fayçal; Riva, Catherine; Touyz, Rhian M; Laurant, Pascal

    2015-02-01

    Exercise training reverses atherosclerotic risk factors associated with metabolic syndrome and obesity. The aim of the present study was to determine the molecular anti-inflammatory, anti-oxidative and anti-atherogenic effects in aorta from rats with high-fat diet-induced obesity. Male Sprague-Dawley rats were placed on a high-fat (HFD) or control (CD) diet for 12 weeks. The HFD rats were then divided into four groups: (i) sedentary HFD-fed rats (HFD-S); (ii) exercise trained (motor treadmill 5 days/week, 60 min/day, 12 weeks) HFD-fed rats (HFD-Ex); (iii) modified diet (HFD to CD) sedentary rats (HF/CD-S); and (iv) an exercise-trained modified diet group (HF/CD-Ex). Tissue levels of NADPH oxidase (activity and expression), NADPH oxidase (Nox) 1, Nox2, Nox4, p47(phox) , superoxide dismutase (SOD)-1, angiotensin AT1 and AT2 receptors, phosphorylated mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the aorta. Plasma cytokines (tumour necrosis factor (TNF)-α and interleukin (IL)-6) levels were also measured. Obesity was accompanied by increases in NADPH oxidase activity, p47(phox) translocation, Nox4 and VCAM-1 protein expression, MAPK (ERK1/2, SAPK/JNK) phosphorylation and plasma TNF-α and IL-6 levels. Exercise training and switching from the HFD to CD reversed almost all these molecular changes. In addition, training increased aortic SOD-1 protein expression and decreased ERK1/2 phosphorylation. These findings suggest that protective effects of exercise training on atherosclerotic risk factors induced by obesity are associated with downregulation of NADPH oxidase, ERK1/2 and SAPK/JNK activity and increased SOD-1 expression. © 2014 Wiley Publishing Asia Pty Ltd.

  9. Studies on middle silkgland proteins of cocoon colour sex-limited ...

    Indian Academy of Sciences (India)

    Unknown

    Generally, bivoltine silkworm varieties produce a white cocoon. Some varieties, however, can pro- duce coloured cocoons, and are sex-limited (colour co- coons are females and white cocoons are males). The molecular mechanism of cocoon colour form, and the pig- ments transfer in the silkworm body is not clear. Zeinab.

  10. Ligustrazine attenuates the platelet-derived growth factor-BB-induced proliferation and migration of vascular smooth muscle cells by interrupting extracellular signal-regulated kinase and P38 mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Yu, Lifei; Huang, Xiaojing; Huang, Kai; Gui, Chun; Huang, Qiaojuan; Wei, Bin

    2015-07-01

    The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) leads to intimal thickening of the aorta and is, therefore, important in the development of arteriosclerosis. As a result, the use of antiproliferative and antimigratory agents for VSMCs offers promise for the treatment of vascular disorders. Although several studies have demonstrated that ligustrazine may be used to treat heart and blood vessel diseases, the detailed mechanism underlying its actions remain to be elucidated. In the present study, the inhibitory effect of ligustrazine on platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation and migration, and the underlying mechanisms were investigated. The findings demonstrated that ligustrazine significantly inhibited PDGF-BB-stimulated VSMC proliferation. VSMCs dedifferentiated into a proliferative phenotype under PDGF-BB stimulation, which was effectively reversed by the administration of ligustrazine. In addition, ligustrazine also downregulated the production of nitric oxide and cyclic guanine monophosphate, induced by PDGF-BB. Additionally, ligustrazine significantly inhibited PDGF-BB-stimulated VSMC migration. Mechanistic investigation indicated that the upregulation of cell cycle-associated proteins and the activation of the extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (MAPK) signaling induced by PDGF-BB was suppressed by the administration of ligustrazine. In conclusion, the present study, demonstrated for the first time, to the best of our knowledge, that ligustrazine downregulated PDGF-BB-induced VSMC proliferation and migration partly, at least, through inhibiting the activation of the ERK and P38 MAPK signaling.

  11. Biological properties of extracellular vesicles and their physiological functions

    NARCIS (Netherlands)

    Yáñez-Mó, María; Siljander, Pia R-M; Andreu, Zoraida; Zavec, Apolonija Bedina; Borràs, Francesc E; Buzas, Edit I; Buzas, Krisztina; Casal, Enriqueta; Cappello, Francesco; Carvalho, Joana; Colás, Eva; Cordeiro-da Silva, Anabela; Fais, Stefano; Falcon-Perez, Juan M; Ghobrial, Irene M; Giebel, Bernd; Gimona, Mario; Graner, Michael; Gursel, Ihsan; Gursel, Mayda; Heegaard, Niels H H; Hendrix, An; Kierulf, Peter; Kokubun, Katsutoshi; Kosanovic, Maja; Kralj-Iglic, Veronika; Krämer-Albers, Eva-Maria; Laitinen, Saara; Lässer, Cecilia; Lener, Thomas; Ligeti, Erzsébet; Linē, Aija; Lipps, Georg; Llorente, Alicia; Lötvall, Jan; Manček-Keber, Mateja; Marcilla, Antonio; Mittelbrunn, Maria; Nazarenko, Irina; Nolte-'t Hoen, Esther N M; Nyman, Tuula A; O'Driscoll, Lorraine; Olivan, Mireia; Oliveira, Carla; Pállinger, Éva; Del Portillo, Hernando A; Reventós, Jaume; Rigau, Marina; Rohde, Eva; Sammar, Marei; Sánchez-Madrid, Francisco; Santarém, N; Schallmoser, Katharina; Ostenfeld, Marie Stampe; Stoorvogel, Willem|info:eu-repo/dai/nl/074352385; Stukelj, Roman; Van der Grein, Susanne G|info:eu-repo/dai/nl/412755211; Vasconcelos, M Helena; Wauben, Marca H M|info:eu-repo/dai/nl/112675735; De Wever, Olivier

    2015-01-01

    In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological

  12. Extracellular nucleotide signaling in plants

    Energy Technology Data Exchange (ETDEWEB)

    Stacey, Gary [Univ. of Missouri, Columbia, MO (United States)

    2016-09-08

    Over the life of this funded project, our research group identified and characterized two key receptor proteins in plants; one mediating the innate immunity response to chitin and the other elucidating the key receptor for extracellular ATP. In the case of chitin recognition, we recently described the quaternary structure of this receptor, shedding light on how the receptor functions. Perhaps more importantly, we demonstrated that all plants have the ability to recognize both chitin oligomers and lipochitooligosacchardes, fundamentally changing how the community views the evolution of these systems and strategies that might be used, for example, to extend symbiotic nitrogen fixation to non-legumes. Our discovery of DORN1 opens a new chapter in plant physiology documenting conclusively that eATP is an important extracellular signal in plants, as it is in animals. At this point, we cannot predict just how far reaching this discovery may prove to be but we are convinced that eATP signaling is fundamental to plant growth and development and, hence, we believe that the future will be very exciting for the study of DORN1 and its overall function in plants.

  13. On the Power and Limits of Sequence Similarity Based Clustering of Proteins Into Families

    DEFF Research Database (Denmark)

    Wiwie, Christian; Röttger, Richard

    2017-01-01

    Over the last decades, we have observed an ongoing tremendous growth of available sequencing data fueled by the advancements in wet-lab technology. The sequencing information is only the beginning of the actual understanding of how organisms survive and prosper. It is, for instance, equally...... important to also unravel the proteomic repertoire of an organism. A classical computational approach for detecting protein families is a sequence-based similarity calculation coupled with a subsequent cluster analysis. In this work we have intensively analyzed various clustering tools on a large scale. We...

  14. Expression of Integrins and Extracellular Matrix Proteins in Non-small Cell Lung Cancer:Correlation with Tumor Metastasis and Prognosis

    Directory of Open Access Journals (Sweden)

    Zhidong LIU

    2009-05-01

    Full Text Available Background and objective As invasion and metastasis of cancer seriously threaten human's life, it is of more important clinical significance to understand and evaluate the biologic behavior of cancer correctly. Abnormal expression of integrin and ECM proteins play an important role in invasion and metastasis of cancer. The objective of this study is to analyze the expression of the integrin α5, β1 and ECM proteins in patients with NSCLC and its correlationwith lymph node (LN metastasis as well as prognosis, and to evaluate its roles in the metastasis of tumor. Methods The expression of integrin α5, β1 and collagen type Ⅳ, fibronectin, tenascin was determined by immunohistochemistry. The relationship was analysized that between integrin α5, β1 and ECM proteins expression and clinico-pathologic parameters especially LN metastasis and prognosis. Results The expression of collagen type Ⅳ was negatively correlated with LN metastasis, and positively to prognosis of patients; The expression of integrin α5, β1 was positively correlated with LN metastasis, and integrin α5 was negatively to prognosis of patients. The expression of integrin α5 was positively correlated with the expression of integrin β1, and negatively with the expression of collagen type Ⅳ. The expressionof collagen type Ⅳ in stage Ⅰ disease P-TNM stage was remarkably higher than that in stage Ⅲ disease and poordifferentiated disease (P <0.001, but integrin α5, β1 was significantly higher than that in stage Ⅲ disease and poordifferentiated disease. Conclusion Increased expression of integrin α5, β1 and decreased expression of ECM significantly correlated with LN metastasis of NSCLC. Integrin α5 and collagen type Ⅳ are prognosic factors in patients with NSCLC.

  15. 'Green mice' display limitations in enhanced green fluorescent protein expression in retina and optic nerve cells.

    Science.gov (United States)

    Caminos, Elena; Vaquero, Cecilia F; García-Olmo, Dolores C

    2014-12-01

    Characterization of retinal cells, cell transplants and gene therapies may be helped by pre-labeled retinal cells, such as those transfected with vectors for green fluorescent protein expression. The aim of this study was to analyze retinal cells and optic nerve components from transgenic green mice (GM) with the 'enhanced' green fluorescent protein (EGFP) gene under the control of the CAG promoter (a chicken β-actin promoter and a cytomegalovirus enhancer). The structural analysis and electroretinography recordings showed a normal, healthy retina. Surprisingly, EGFP expression was not ubiquitously located in the retina and optic nerve. Epithelial cells, photoreceptors and bipolar cells presented high green fluorescence levels. In contrast, horizontal cells, specific amacrine cells and ganglion cells exhibited a null EGFP expression level. The synaptic terminals of rod bipolar cells displayed a high green fluorescence level when animals were kept in the dark. Immature retinas exhibited different EGFP expression patterns to those noted in adults. Axons and glial cells in the optic nerve revealed a specific regional EGFP expression pattern, which correlated with the presence of myelin. These results suggest that EGFP expression might be related to the activity of both the CAG promoter and β-actin in mature retinal neurons and oligodendrocytes. Moreover, EGFP expression might be regulated by light in both immature and adult animals. Since GM are used in numerous retina bioassays, it is essential to know the differential EGFP expression in order to select cells of interest for each study.

  16. Functional proteins involved in regulation of intracellular Ca(2+) for drug development: the extracellular calcium receptor and an innovative medical approach to control secondary hyperparathyroidism by calcimimetics.

    Science.gov (United States)

    Nagano, Nobuo; Nemeth, Edward F

    2005-03-01

    Circulating levels of calcium ion (Ca(2+)) are maintained within a narrow physiological range mainly by the action of parathyroid hormone (PTH) secreted from parathyroid cells. Parathyroid cells can sense small fluctuations in plasma Ca(2+) levels by virtue of a cell surface Ca(2+) receptor (CaR) that belongs to the superfamily of G-protein-coupled receptors. Calcimimetics are positive allosteric modulators that activate the CaR on parathyroid cells and thereby immediately suppress PTH secretion. Pre-clinical studies with NPS R-568, a first generation calcimimetic compound, have demonstrated that daily oral administration inhibits the elevation of plasma PTH levels and parathyroid gland hyperplasia and ameliorates impaired bone qualities in rats with chronic renal insufficiency. The results of clinical trials with cinacalcet hydrochloride, a second generation calcimimetic compound, have shown that calcimimetics possess lowering effects not only on serum PTH levels but also on serum calcium x phosphorus product levels, a hallmark of an increased risk for cardiovascular death in dialysis patients with end-stage renal disease (ESRD). Thus, calcimimetics have considerable potential as an innovative medical approach to manage secondary hyperparathyroidism associated with ESRD. Indeed, cinacalcet hydrochloride has been approved in several countries and is the first positive allosteric modulator of any G protein-coupled receptor to reach the market.

  17. Extracellular vesicles and blood diseases.

    Science.gov (United States)

    Nomura, Shosaku

    2017-04-01

    Extracellular vesicles (EVs) are small membrane vesicles released from many different cell types by the exocytic budding of the plasma membrane in response to cellular activation or apoptosis. EVs disseminate various bioactive effectors originating from the parent cells and transfer functional RNA and protein between cells, enabling them to alter vascular function and induce biological responses involved in vascular homeostasis. Although most EVs in human blood originate from platelets, EVs are also released from leukocytes, erythrocytes, endothelial cells, smooth muscle cells, and cancer cells. EVs were initially thought to be small particles with procoagulant activity; however, they can also evoke cellular responses in the immediate microenvironments and transport microRNAs (miRNA) into target cells. In this review, we summarize the recent literature relevant to EVs, including a growing list of clinical disorders that are associated with elevated EV levels. These studies suggest that EVs play roles in various blood diseases.

  18. Effect of the deletion of genes encoding proteins of the extracellular virion form of vaccinia virus on vaccine immunogenicity and protective effectiveness in the mouse model.

    Directory of Open Access Journals (Sweden)

    Clement A Meseda

    Full Text Available Antibodies to both infectious forms of vaccinia virus, the mature virion (MV and the enveloped virion (EV, as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.

  19. Effect of soluble calcium and lactose on limiting flux and serum protein removal during skim milk microfiltration.

    Science.gov (United States)

    Adams, Michael C; Hurt, Emily E; Barbano, David M

    2015-11-01

    The tendency of calcium to promote microfiltration (MF) membrane fouling is well documented, but the role of lactose has not been studied. Milk protein concentrate that is 85% protein on a dry basis (MPC85) contains less calcium and lactose than skim milk. Our objectives were to determine the effects of skim milk soluble calcium and lactose concentrations on the limiting fluxes (LF) and serum protein (SP) removal factors of 0.1-µm ceramic graded permeability membranes. The MF was fed with 3 different milks: skim milk, liquid MPC85 that had been standardized to the protein content of skim milk with reverse osmosis water (MPC), and liquid MPC85 that had been standardized to the protein and lactose contents of skim milk with reverse osmosis water and lactose monohydrate (MPC+L). Retentate and permeate were continuously recycled to the feed tank. The LF for each feed was determined by increasing flux once per hour from 55 kg·m(-2)·h(-1) until flux did not increase with increasing transmembrane pressure. Temperature, pressure drop across the membrane length, and protein concentration in the retentate recirculation loop were maintained at 50°C, 220 kPa, and 8.77 ± 0.2%, respectively. Experiments were replicated 3 times and the Proc GLM procedure of SAS was used for statistical analysis. An increase in LF between skim milk (91 kg·m(-2)·h(-1)) and MPC+L (124 kg·m(-2)·h(-1)) was associated with a reduction in soluble calcium. The LF of MPC+L was lower than the LF of MPC (137 kg·m(-2)·h(-1)) due to the higher viscosity contributed by lactose. Permeates produced from the MPC and MPC+L contained more protein than the skim milk permeate due to the transfer of caseins from the micelles into the reduced-calcium sera of the MPC and MPC+L. A SP removal factor was calculated by dividing true protein in the permeate by SP in the permeate portion of the feed to describe the ease of SP passage through the membrane. No differences in SP removal factors were detected among the

  20. Tendon functional extracellular matrix.

    Science.gov (United States)

    Screen, Hazel R C; Berk, David E; Kadler, Karl E; Ramirez, Francesco; Young, Marian F

    2015-06-01

    This article is one of a series, summarizing views expressed at the Orthopaedic Research Society New Frontiers in Tendon Research Conference. This particular article reviews the three workshops held under the "Functional Extracellular Matrix" stream. The workshops focused on the roles of the tendon extracellular matrix, such as performing the mechanical functions of tendon, creating the local cell environment, and providing cellular cues. Tendon is a complex network of matrix and cells, and its biological functions are influenced by widely varying extrinsic and intrinsic factors such as age, nutrition, exercise levels, and biomechanics. Consequently, tendon adapts dynamically during development, aging, and injury. The workshop discussions identified research directions associated with understanding cell-matrix interactions to be of prime importance for developing novel strategies to target tendon healing or repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  1. Extracellular vesicles: fundamentals and clinical relevance

    Directory of Open Access Journals (Sweden)

    Wael Nassar

    2015-01-01

    Full Text Available All types of cells of eukaryotic organisms produce and release small nanovesicles into their extracellular environment. Early studies have described these vesicles as ′garbage bags′ only to remove obsolete cellular molecules. Valadi and colleagues, in 2007, were the first to discover the capability of circulating extracellular vesicles (EVs to horizontally transfer functioning gene information between cells. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, chemoresistance, genetic exchange, and signaling pathway activation through growth factor/receptor transfer. EVs represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, signaling proteins, and RNAs. They contribute to physiology and pathology, and they have a myriad of potential clinical applications in health and disease. Moreover, vesicles can pass the blood-brain barrier and may perhaps even be considered as naturally occurring liposomes. These cell-derived EVs not only represent a central mediator of the disease microenvironment, but their presence in the peripheral circulation may serve as a surrogate for disease biopsies, enabling real-time diagnosis and disease monitoring. In this review, we′ll be addressing the characteristics of different types of extracellular EVs, as well as their clinical relevance and potential as diagnostic markers, and also define therapeutic options.

  2. Endothelial Extracellular Vesicles-Promises and Challenges.

    Science.gov (United States)

    Hromada, Carina; Mühleder, Severin; Grillari, Johannes; Redl, Heinz; Holnthoner, Wolfgang

    2017-01-01

    Extracellular vesicles, including exosomes, microparticles, and apoptotic bodies, are phospholipid bilayer-enclosed vesicles that have once been considered as cell debris lacking biological functions. However, they have recently gained immense interest in the scientific community due to their role in intercellular communication, immunity, tissue regeneration as well as in the onset, and progression of various pathologic conditions. Extracellular vesicles of endothelial origin have been found to play a versatile role in the human body, since they are on the one hand known to contribute to cardiovascular diseases, but on the other hand have also been reported to promote endothelial cell survival. Hence, endothelial extracellular vesicles hold promising therapeutic potential to be used as a new tool to detect as well as treat a great number of diseases. This calls for clinically approved, standardized, and efficient isolation and characterization protocols to harvest and purify endothelial extracellular vesicles. However, such methods and techniques to fulfill stringent requirements for clinical trials have yet to be developed or are not harmonized internationally. In this review, recent advances and challenges in the field of endothelial extracellular vesicle research are discussed and current problems and limitations regarding isolation and characterization are pointed out.

  3. Compiled data set of exact NOE distance limits, residual dipolar couplings and scalar couplings for the protein GB3

    Directory of Open Access Journals (Sweden)

    Beat Vögeli

    2015-12-01

    Full Text Available We compiled an NMR data set consisting of exact nuclear Overhauser enhancement (eNOE distance limits, residual dipolar couplings (RDCs and scalar (J couplings for GB3, which forms one of the largest and most diverse data set for structural characterization of a protein to date. All data have small experimental errors, which are carefully estimated. We use the data in the research article Vogeli et al., 2015, Complementarity and congruence between exact NOEs and traditional NMR probes for spatial decoding of protein dynamics, J. Struct. Biol., 191, 3, 306–317, doi:10.1016/j.jsb.2015.07.008 [1] for cross-validation in multiple-state structural ensemble calculation. We advocate this set to be an ideal test case for molecular dynamics simulations and structure calculations.

  4. Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration.

    Science.gov (United States)

    Xiao, Bo; Rao, Feng; Guo, Zhi-Yuan; Sun, Xun; Wang, Yi-Guo; Liu, Shu-Yun; Wang, Ai-Yuan; Guo, Quan-Yi; Meng, Hao-Ye; Zhao, Qing; Peng, Jiang; Wang, Yu; Lu, Shi-Bi

    2016-07-01

    The extracellular matrix, which includes collagens, laminin, or fibronectin, plays an important role in peripheral nerve regeneration. Recently, a Schwann cell-derived extracellular matrix with classical biomaterial was used to mimic the neural niche. However, extensive clinical use of Schwann cells remains limited because of the limited origin, loss of an autologous nerve, and extended in vitro culture times. In the present study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs), which are easily accessible and more proliferative than Schwann cells, were used to prepare an extracellular matrix. We identified the morphology and function of hUCMSCs and investigated their effect on peripheral nerve regeneration. Compared with a non-coated dish tissue culture, the hUCMSC-derived extracellular matrix enhanced Schwann cell proliferation, upregulated gene and protein expression levels of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor in Schwann cells, and enhanced neurite outgrowth from dorsal root ganglion neurons. These findings suggest that the hUCMSC-derived extracellular matrix promotes peripheral nerve repair and can be used as a basis for the rational design of engineered neural niches.

  5. Protein phosphatase 1ß limits ring canal constriction during Drosophila germline cyst formation.

    Directory of Open Access Journals (Sweden)

    Shinya Yamamoto

    Full Text Available Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw, a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT. This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well.

  6. The WRN and MUS81 proteins limit cell death and genome instability following oncogene activation.

    Science.gov (United States)

    Murfuni, I; Nicolai, S; Baldari, S; Crescenzi, M; Bignami, M; Franchitto, A; Pichierri, P

    2013-01-31

    Oncogene-induced replication stress is recognized as the primary cause of accumulation of DNA damage and genome instability in precancerous cells. Although the molecular mechanisms responding to such type of replication perturbation are not fully characterized, it has been speculated that their dysfunction may enhance genome instability and accelerate tumor progression. Here, we show that the WRN protein, a member of the human RecQ helicases, is necessary to sustain replication fork progression in response to oncogene-induced replication stress. Loss of WRN affects cell cycle progression and results in enhanced accumulation of double-strand breaks and instability at common fragile sites in cells experiencing oncogene-induced replication stress. Moreover, we demonstrate that double-strand breaks, observed upon oncogene over-expression, depend on the MUS81 endonuclease, which represents a parallel pathway collaborating with WRN to prevent cell death. Overall, our findings give insights into the mechanisms protecting replication forks in cells experiencing oncogene-induced replication stress, and identify factors that, when mutated or dysfunctional, may enhance genome instability in precancerous cells. In addition, because concomitant depletion of WRN and MUS81 causes synthetic sickness in cells growing under oncogene-induced replication stress, our results support the possibility of targeting cancer cells with an impaired replication fork recovery pathway by a specific inactivation of the other parallel pathway.

  7. Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA.

    Directory of Open Access Journals (Sweden)

    Martin Löwer

    Full Text Available Exported proteases of Helicobacter pylori (H. pylori are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI. Among these, we found the predicted serine protease HtrA (Hp1019, which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies.

  8. Engineering vesicle trafficking improves the extracellular activity and surface display efficiency of cellulases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tang, Hongting; Song, Meihui; He, Yao; Wang, Jiajing; Wang, Shenghuan; Shen, Yu; Hou, Jin; Bao, Xiaoming

    2017-01-01

    Cellulase expression via extracellular secretion or surface display in Saccharomyces cerevisiae is one of the most frequently used strategies for a consolidated bioprocess (CBP) of cellulosic ethanol production. However, the inefficiency of the yeast secretory pathway often results in low production of heterologous proteins, which largely limits cellulase secretion or display. In this study, the components of the vesicle trafficking from the endoplasmic reticulum (ER) to the Golgi and from the Golgi to the plasma membrane, involved in vesicle budding, tethering and fusion, were over-expressed in Clostridium thermocellum endoglucanase (CelA)- and Sacchromycopsis fibuligera β-glucosidase (BGL1)-secreting or -displaying strains. Engineering the targeted components in the ER to Golgi vesicle trafficking, including Sec12p, Sec13p, Erv25p and Bos1p, enhanced the extracellular activity of CelA. However, only Sec13p over-expression increased BGL1 secretion. By contrast, over-expression of the components in the Golgi to plasma membrane vesicle trafficking, including Sso1p, Snc2p, Sec1p, Exo70p, Ypt32p and Sec4p, showed better performance in increasing BGL1 secretion compared to CelA secretion, and the over-expression of these components all increased BGL1 extracellular activity. These results revealed that various cellulases showed different limitations in protein transport, and engineering vesicle trafficking has protein-specific effects. Importantly, we found that engineering the above vesicle trafficking components, particularly from the ER to the Golgi, also improved the display efficiency of CelA and BGL1 when a-agglutinin was used as surface display system. Further analyses illustrated that the display efficiency of a-agglutinin was increased by engineering vesicle trafficking, and the trend was consistent with displayed CelA and BGL1. These results indicated that fusion with a-agglutinin may affect the proteins' properties and alter the rate-limiting step in the

  9. Ultrafast mid-IR laser scalpel: protein signals of the fundamental limits to minimally invasive surgery.

    Science.gov (United States)

    Amini-Nik, Saeid; Kraemer, Darren; Cowan, Michael L; Gunaratne, Keith; Nadesan, Puviindran; Alman, Benjamin A; Miller, R J Dwayne

    2010-09-28

    Lasers have in principle the capability to cut at the level of a single cell, the fundamental limit to minimally invasive procedures and restructuring biological tissues. To date, this limit has not been achieved due to collateral damage on the macroscale that arises from thermal and shock wave induced collateral damage of surrounding tissue. Here, we report on a novel concept using a specifically designed Picosecond IR Laser (PIRL) that selectively energizes water molecules in the tissue to drive ablation or cutting process faster than thermal exchange of energy and shock wave propagation, without plasma formation or ionizing radiation effects. The targeted laser process imparts the least amount of energy in the remaining tissue without any of the deleterious photochemical or photothermal effects that accompanies other laser wavelengths and pulse parameters. Full thickness incisional and excisional wounds were generated in CD1 mice using the Picosecond IR Laser, a conventional surgical laser (DELight Er:YAG) or mechanical surgical tools. Transmission and scanning electron microscopy showed that the PIRL laser produced minimal tissue ablation with less damage of surrounding tissues than wounds formed using the other modalities. The width of scars formed by wounds made by the PIRL laser were half that of the scars produced using either a conventional surgical laser or a scalpel. Aniline blue staining showed higher levels of collagen in the early stage of the wounds produced using the PIRL laser, suggesting that these wounds mature faster. There were more viable cells extracted from skin using the PIRL laser, suggesting less cellular damage. β-catenin and TGF-β signalling, which are activated during the proliferative phase of wound healing, and whose level of activation correlates with the size of wounds was lower in wounds generated by the PIRL system. Wounds created with the PIRL systsem also showed a lower rate of cell proliferation. Direct comparison of wound

  10. Ultrafast mid-IR laser scalpel: protein signals of the fundamental limits to minimally invasive surgery.

    Directory of Open Access Journals (Sweden)

    Saeid Amini-Nik

    Full Text Available Lasers have in principle the capability to cut at the level of a single cell, the fundamental limit to minimally invasive procedures and restructuring biological tissues. To date, this limit has not been achieved due to collateral damage on the macroscale that arises from thermal and shock wave induced collateral damage of surrounding tissue. Here, we report on a novel concept using a specifically designed Picosecond IR Laser (PIRL that selectively energizes water molecules in the tissue to drive ablation or cutting process faster than thermal exchange of energy and shock wave propagation, without plasma formation or ionizing radiation effects. The targeted laser process imparts the least amount of energy in the remaining tissue without any of the deleterious photochemical or photothermal effects that accompanies other laser wavelengths and pulse parameters. Full thickness incisional and excisional wounds were generated in CD1 mice using the Picosecond IR Laser, a conventional surgical laser (DELight Er:YAG or mechanical surgical tools. Transmission and scanning electron microscopy showed that the PIRL laser produced minimal tissue ablation with less damage of surrounding tissues than wounds formed using the other modalities. The width of scars formed by wounds made by the PIRL laser were half that of the scars produced using either a conventional surgical laser or a scalpel. Aniline blue staining showed higher levels of collagen in the early stage of the wounds produced using the PIRL laser, suggesting that these wounds mature faster. There were more viable cells extracted from skin using the PIRL laser, suggesting less cellular damage. β-catenin and TGF-β signalling, which are activated during the proliferative phase of wound healing, and whose level of activation correlates with the size of wounds was lower in wounds generated by the PIRL system. Wounds created with the PIRL systsem also showed a lower rate of cell proliferation. Direct

  11. Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

    Science.gov (United States)

    Silvas, Jesus A.; Popov, Vsevolod L.; Paulucci-Holthauzen, Adriana

    2015-01-01

    ABSTRACT Severe fever with thrombocytopenia syndrome (SFTS) virus is a newly recognized member of the genus Phlebovirus in the family Bunyaviridae. The virus was isolated from patients presenting with hemorrhagic manifestations and an initial case fatality rate of 12 to 30% was reported. Due to the recent emergence of this pathogen, there is limited knowledge on the molecular virology of SFTS virus. Recently, we reported that the SFTS virus NSs protein inhibited the activation of the beta interferon (IFN-β) promoter. Furthermore, we also found that SFTS virus NSs relocalizes key components of the IFN response into NSs-induced cytoplasmic structures. Due to the important role these structures play during SFTS virus replication, we conducted live cell imaging studies to gain further insight into the role and trafficking of these cytoplasmic structures during virus infection. We found that some of the SFTS virus NSs-positive cytoplasmic structures were secreted to the extracellular space and endocytosed by neighboring cells. We also found that these secreted structures isolated from NSs-expressing cells and SFTS virus-infected cells were positive for the viral protein NSs and the host protein CD63, a protein associated with extracellular vesicles. Electron microscopy studies also revealed that the isolated CD63-immunoprecipitated extracellular vesicles produced during SFTS virus infection contained virions. The virions harbored within these structures were efficiently delivered to uninfected cells and were able to sustain SFTS virus replication. Altogether, these results suggest that SFTS virus exploits extracellular vesicles to mediate virus receptor-independent transmission to host cells and open the avenue for novel therapeutic strategies against SFTS virus and related pathogens. IMPORTANCE SFTS virus is novel bunyavirus associated with hemorrhagic fever illness. Currently, limited information is available about SFTS virus. In the present study, we demonstrated

  12. Aflatoxin B1 Suppressed T-Cell Response to Anti-pig-CD3 Monoclonal Antibody Stimulation in Primary Porcine Splenocytes: A Role for the Extracellular Regulated Protein Kinase (ERK1/2) MAPK Signaling Pathway.

    Science.gov (United States)

    Hao, Shu; Pan, Shengchi; Hu, Junfa; Qian, Gang; Gan, Fang; Huang, Kehe

    2015-07-08

    The aim of the present study is to investigate whether aflatoxin B1 (AFB1)-induced immunotoxicity is associated with oxidative stress and the expression of extracellular regulated protein kinases (ERK) 1/2. The primary splenocytes isolated from healthy pigs were activated and proliferated by anti-pig-CD3 monoclonal antibodies (mAb) in the present experiment, which is an antigen-specific stimulant. Results indicated that cell proliferation and interleukin-2 (IL-2) production were significantly suppressed by AFB1 from 4 to 8 μg/mL in a dose-dependent manner compared to the control group. Furthermore, AFB1 significantly increased malondialdehyde (MDA) levels, decreased reduced glutathione (GSH) and total superoxide dismutase levels, and up-regulated p-ERK1/2 expression in the activated splenocytes. N-Acetyl-l-cysteine blocked anti-CD3-induced T-cell suppression by AFB1 through increasing intracellular concentrations of GSH levels, decreasing MDA levels, and down-regulated p-ERK1/2 expression, respectively. Inhibition of the ERK1/2 expression by ERK-specific iRNA attenuated the decrease of T-cell proliferation and IL-2 production induced by AFB1. It was concluded that AFB1 inhibits anti-CD3-induced lymphocyte proliferation and IL-2 production by the oxidative stress mediated ERK1/2 MAPK signaling pathway.

  13. MiR-21-5p in urinary extracellular vesicles is a novel biomarker of urothelial carcinoma

    OpenAIRE

    Matsuzaki, Kyosuke; Fujita, Kazutoshi; Jingushi, Kentaro; Kawashima, Atsunari; Ujike, Takeshi; Nagahara, Akira; Ueda, Yuko; Tanigawa, Go; Yoshioka, Iwao; Ueda, Koji; Hanayama, Rikinari; Uemura, Motohide; Miyagawa, Yasushi; Tsujikawa, Kazutake; Nonomura, Norio

    2017-01-01

    Background Extracellular vesicles are lipid bilayer vesicles containing protein, messengerRNA and microRNA. Cancer cell-derived extracellular vesicles may be diagnostic and therapeutic targets. We extracted extracellular vesicles from urine of urothelial carcinoma patients and the control group to identify cancer-specific microRNAs in urinary extracellular vesicles as new biomarkers. Materials and methods microRNA from urinary extracellular vesicles extracted from 6 urothelial carcinoma patie...

  14. JNK mitogen-activated protein kinase limits calcium-dependent chloride secretion across colonic epithelial cells.

    LENUS (Irish Health Repository)

    Donnellan, Fergal

    2010-01-01

    Neuroimmune agonists induce epithelial Cl(-) secretion through elevations in intracellular Ca2+ or cAMP. Previously, we demonstrated that epidermal growth factor receptor (EGFR) transactivation and subsequent ERK MAPK activation limits secretory responses to Ca2+-dependent, but not cAMP-dependent, agonists. Although JNK MAPKs are also expressed in epithelial cells, their role in regulating transport function is unknown. Here, we investigated the potential role for JNK in regulating Cl(-) secretion in T(84) colonic epithelial cells. Western blot analysis revealed that a prototypical Ca2+-dependent secretagogue, carbachol (CCh; 100 microM), induced phosphorylation of both the 46-kDa and 54-kDa isoforms of JNK. This effect was mimicked by thapsigargin (TG), which specifically elevates intracellular Ca2+, but not by forskolin (FSK; 10 microM), which elevates cAMP. CCh-induced JNK phosphorylation was attenuated by the EGFR inhibitor, tyrphostin-AG1478 (1 microM). Pretreatment of voltage-clamped T(84) cells with SP600125 (2 microM), a specific JNK inhibitor, potentiated secretory responses to both CCh and TG but not to FSK. The effects of SP600125 on CCh-induced secretion were not additive with those of the ERK inhibitor, PD98059. Finally, in apically permeabilized T(84) cell monolayers, SP600125 potentiated CCh-induced K+ conductances but not Na+\\/K+ATPase activity. These data demonstrate a novel role for JNK MAPK in regulating Ca2+ but not cAMP-dependent epithelial Cl(-) secretion. JNK activation is mediated by EGFR transactivation and exerts its antisecretory effects through inhibition of basolateral K+ channels. These data further our understanding of mechanisms regulating epithelial secretion and underscore the potential for exploitation of MAPK-dependent signaling in treatment of intestinal transport disorders.

  15. Eosinophils enhance WNT-5a and TGF-β1 genes expression in airway smooth muscle cells and promote their proliferation by increased extracellular matrix proteins production in asthma.

    Science.gov (United States)

    Januskevicius, Andrius; Vaitkiene, Simona; Gosens, Reinoud; Janulaityte, Ieva; Hoppenot, Deimante; Sakalauskas, Raimundas; Malakauskas, Kestutis

    2016-06-13

    Recent studies have suggested that eosinophils may have a direct effect on airway smooth muscle cells (ASMC), causing their proliferation in patients with asthma, but the precise mechanism of the interaction between these cells remains unknown. We propose that changes in Wnt signaling activity and extracellular matrix (ECM) production may help explain these findings. Therefore, the aim of this study was to investigate the effect of eosinophils from asthmatic and non-asthmatic subjects on Wnt-5a, transforming growth factor β1 (TGF-β1), and ECM protein (fibronectin and collagen) gene expression and ASMC proliferation. A total of 18 subjects were involved in the study: 8 steroid-free asthma patients and 10 healthy subjects. Peripheral blood eosinophils were isolated using centrifugation and magnetic separation. An individual co-culture of eosinophils with human ASMC was prepared for each study subject. Adhesion of eosinophils to ASMC (evaluated by assaying eosinophil peroxidase activity) was determined following various incubation periods (30, 45, 60, 120, and 240 min). The expression of Wnt-5a, TGF-β1, and ECM protein genes in ASMC was measured using quantitative real-time polymerase chain reaction (PCR) after 24 h of co-culture. Proliferation of ASMC was measured using the Alamar blue method after 48 h and 72 h of co-culture with eosinophils. Eosinophils from asthmatic subjects demonstrated increased adhesion to ASMC compared with eosinophils from healthy subjects (p proliferation was augmented after co-culture with eosinophils from asthma patients compared with co-culture with eosinophils from healthy subjects (p proliferation of these cells in asthma. ClinicalTrials.gov Identifier: NCT02648074 .

  16. Staphylococcal Superantigen-Like Protein 1 and 5 (SSL1 & SSL5 Limit Neutrophil Chemotaxis and Migration through MMP-Inhibition

    Directory of Open Access Journals (Sweden)

    Kirsten J. Koymans

    2016-07-01

    Full Text Available Matrix metalloproteinases (MMPs are endopeptidases that degrade components of the extracellular matrix, but also modulate inflammation. During bacterial infections, MMPs are important in the recruitment and migration of inflammatory cells. Besides facilitating cell migration by degrading extracellular matrix components, they potentiate the action of several inflammatory molecules, including cytokines, chemokines, and antimicrobial peptides. Staphylococcus aureus secretes an arsenal of immune evasion molecules that interfere with immune cell functioning and hamper proper immune responses. An earlier study identified staphylococcal superantigen-like protein 5 (SSL5 as an MMP9 inhibitor. Since multiple MMPs are involved in neutrophil recruitment, we set up an in-depth search for additional MMP inhibitors by testing a panel of over 70 secreted staphylococcal proteins on the inhibition of the two main neutrophil MMPs: MMP8 (neutrophil collagenase and MMP9 (neutrophil gelatinase B. We identified SSL1 and SSL5 as potent inhibitors of both neutrophil MMPs and show that they are actually broad range MMP inhibitors. SSL1 and SSL5 prevent MMP-induced cleavage and potentiation of IL-8 and inhibit the migration of neutrophils through collagen. Thus, through MMP-inhibition, SSL1 and SSL5 interfere with neutrophil activation, chemotaxis, and migration, all vital neutrophil functions in bacterial clearance. Studies on MMP-SSL interactions can have therapeutic potential and SSL based derivatives might prove useful in treatment of cancer and destructive inflammatory diseases.

  17. Extracellular Alix regulates integrin-mediated cell adhesions and extracellular matrix assembly

    OpenAIRE

    Pan, Shujuan; Wang, Ruoning; Zhou, Xi; Corvera, Joe; Kloc, Malgorzata; Sifers, Richard; Gallick, Gary E; Lin, Sue-Hwa; Kuang, Jian

    2008-01-01

    Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to e...

  18. Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

    Science.gov (United States)

    Couch, Yvonne; Akbar, Naveed; Davis, Simon; Fischer, Roman; Dickens, Alex M; Neuhaus, Ain A; Burgess, Annette I; Rothwell, Peter M; Buchan, Alastair M

    2017-08-01

    Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs. EVs were quantified and analyzed in the sera of patients after an acute stroke (inflammation in immune cells. © 2017 American Heart Association, Inc.

  19. Gram-negative and Gram-positive bacterial extracellular vesicles.

    Science.gov (United States)

    Kim, Ji Hyun; Lee, Jaewook; Park, Jaesung; Gho, Yong Song

    2015-04-01

    Like mammalian cells, Gram-negative and Gram-positive bacteria release nano-sized membrane vesicles into the extracellular environment either in a constitutive manner or in a regulated manner. These bacterial extracellular vesicles are spherical bilayered proteolipids enriched with bioactive proteins, lipids, nucleic acids, and virulence factors. Recent progress in this field supports the critical pathophysiological functions of these vesicles in both bacteria-bacteria and bacteria-host interactions. This review provides an overview of the current understanding on Gram-negative and Gram-positive bacterial extracellular vesicles, especially regarding the biogenesis, components, and functions in poly-species communities. We hope that this review will stimulate additional research in this emerging field of bacterial extracellular vesicles and contribute to the development of extracellular vesicle-based diagnostic tools and effective vaccines against pathogenic Gram-negative and Gram-positive bacteria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Interaction of chondrocytes, extracellular matrix and growth factors: relevance for articular cartilage tissue engineering.

    NARCIS (Netherlands)

    Kraan, P.M. van der; Buma, P.; Kuppevelt, A.H.M.S.M. van; Berg, W.B. van den

    2002-01-01

    The abundant extracellular matrix of articular cartilage has to be maintained by a limited number of chondrocytes. Vice versa, the extracellular matrix has an important role in the regulation of chondrocyte function. OBJECTIVE: In this review we discuss the role of the extracellular matrix in the

  1. Extracellular enzyme kinetics scale with resource availability

    Science.gov (United States)

    Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

    2014-01-01

    Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

  2. Digestion of isolated legume cells in a stomach-duodenum model: three mechanisms limit starch and protein hydrolysis.

    Science.gov (United States)

    Bhattarai, Rewati R; Dhital, Sushil; Wu, Peng; Chen, Xiao Dong; Gidley, Michael J

    2017-07-19

    Retention of intact plant cells to the end of the small intestine leads to transport of entrapped macronutrients such as starch and protein for colonic microbial fermentation, and is a promising mechanism to increase the content of resistant starch in diets. However, the effect of gastro-intestinal bio-mechanical processing on the intactness of plant cells and the subsequent resistance to enzymatic digestion of intracellular starch and protein are not well understood. In this study, intact cells isolated from legume cotyledons are digested in a laboratory model which mimics the mechanical and biochemical conditions of the rat stomach and duodenum. The resulting digesta are characterised in terms of cell (wall) integrity as well as intracellular starch and protein hydrolysis. The cells remained essentially intact in the model with negligible (ca. 2-3%) starch or protein digestion; however when the cells were mechanically broken and digested in the model, the hydrolysis was increased to 45-50% suggesting that intact cellular structures could survive the mixing regimes in the model stomach and duodenum sufficiently to prevent digestive enzyme access. Apart from intact cell walls providing effective barrier properties, they also limit digestibility by restricting starch gelatinisation during cooking, and significant non-specific binding of α-amylase is observed to both intact and broken cell wall components, providing a third mechanism hindering starch hydrolysis. The study suggests that the preservation of intactness of plant cells, such as from legumes, could be a viable approach to achieve the targeted delivery of resistant starch to the colon.

  3. Sub-diffraction limit localization of proteins in volumetric space using Bayesian restoration of fluorescence images from ultrathin specimens.

    Directory of Open Access Journals (Sweden)

    Gordon Wang

    Full Text Available Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective and at the axial plane to 1.4nλ/NA(2 (n = refractive index of the imaging medium, 1.51 for oil immersion, which with visible wavelengths and a 1.4NA oil immersion objective is -220 nm and -600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT, with its native 50-100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF, a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.

  4. Extracellular vesicles: small bricks for tissue repair/regeneration.

    Science.gov (United States)

    Taverna, Simona; Pucci, Marzia; Alessandro, Riccardo

    2017-02-01

    Extracellular vesicles (EVs) are nano-sized membrane vesicles involved in intercellular communication. EVs have pleiotropic actions in physiological and pathological conditions. The ability of EVs to transports proteins, drugs and nucleic acid, to target specific cells and to increase the stability of therapeutic cargo, make EVs interesting as new devices for the treatment of human disease. In a recently published issue of European journal of pharmaceutical sciences, Silva and colleagues reviewed the ability of EVs to modulate tissue repair and regeneration, focusing on their roles and therapeutic potential as immunomodulatory messengers. In this perspective, we discussed the open questions regarding the dual role of EVs in immune system, as well as the technical limitation of the procedure for EVs isolation and administration in clinical practices. EV-based therapies require further studies to consider EVs as promising candidate for a novel cell-free therapy in the context of regeneration medicine.

  5. Extracellular Matrix and Liver Disease

    Science.gov (United States)

    Arriazu, Elena; Ruiz de Galarreta, Marina; Cubero, Francisco Javier; Varela-Rey, Marta; Pérez de Obanos, María Pilar; Leung, Tung Ming; Lopategi, Aritz; Benedicto, Aitor; Abraham-Enachescu, Ioana

    2014-01-01

    Abstract Significance: The extracellular matrix (ECM) is a dynamic microenvironment that undergoes continuous remodeling, particularly during injury and wound healing. Chronic liver injury of many different etiologies such as viral hepatitis, alcohol abuse, drug-induced liver injury, obesity and insulin resistance, metabolic disorders, and autoimmune disease is characterized by excessive deposition of ECM proteins in response to persistent liver damage. Critical Issues: This review describes the main collagenous and noncollagenous components from the ECM that play a significant role in pathological matrix deposition during liver disease. We define how increased myofibroblasts (MF) from different origins are at the forefront of liver fibrosis and how liver cell-specific regulation of the complex scarring process occurs. Recent Advances: Particular attention is paid to the role of cytokines, growth factors, reactive oxygen species, and newly identified matricellular proteins in the regulation of fibrillar type I collagen, a field to which our laboratory has significantly contributed over the years. We compile data from recent literature on the potential mechanisms driving fibrosis resolution such as MF’ apoptosis, senescence, and reversal to quiescence. Future Directions: We conclude with a brief description of how epigenetics, an evolving field, can regulate the behavior of MF and of how new “omics” tools may advance our understanding of the mechanisms by which the fibrogenic response to liver injury occurs. Antioxid. Redox Signal. 21, 1078–1097. PMID:24219114

  6. A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space.

    Science.gov (United States)

    Pérez-González, Rocío; Gauthier, Sebastien A; Kumar, Asok; Saito, Mitsuo; Saito, Mariko; Levy, Efrat

    2017-01-01

    Extracellular vesicles (EV), including exosomes, secreted vesicles of endocytic origin, and microvesicles derived from the plasma membrane, have been widely isolated and characterized from conditioned culture media and bodily fluids. The difficulty in isolating EV from tissues, however, has hindered their study in vivo. Here, we describe a novel method designed to isolate EV and characterize exosomes from the extracellular space of brain tissues. The purification of EV is achieved by gentle dissociation of the tissue to free the brain extracellular space, followed by sequential low-speed centrifugations, filtration, and ultracentrifugations. To further purify EV from other extracellular components, they are separated on a sucrose step gradient. Characterization of the sucrose step gradient fractions by electron microscopy demonstrates that this method yields pure EV preparations free of large vesicles, subcellular organelles, or debris. The level of EV secretion and content are determined by assays for acetylcholinesterase activity and total protein estimation, and exosomal identification and protein content are analyzed by Western blot and immuno-electron microscopy. Additionally, we present here a method to delipidate EV in order to improve the resolution of downstream electrophoretic analysis of EV proteins.

  7. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers?

    Science.gov (United States)

    Erdbrügger, Uta; Le, Thu H

    2016-01-01

    Extracellular vesicles from the urine and circulation have gained significant interest as potential diagnostic biomarkers in renal diseases. Urinary extracellular vesicles contain proteins from all sections of the nephron, whereas most studied circulating extracellular vesicles are derived from platelets, immune cells, and the endothelium. In addition to their diagnostic role as markers of kidney and vascular damage, extracellular vesicles may have functional significance in renal health and disease by facilitating communication between cells and protecting against kidney injury and bacterial infection in the urinary tract. However, the current understanding of extracellular vesicles has derived mostly from studies with very small numbers of patients or in vitro data. Moreover, accurate assessment of these vesicles remains a challenge, in part because of a lack of consensus in the methodologies to measure extracellular vesicles and the inability of most techniques to capture the entire size range of these vesicles. However, newer techniques and standardized protocols to improve the detection of extracellular vesicles are in development. A clearer understanding of the composition and biology of extracellular vesicles will provide insights into their pathophysiologic, diagnostic, and therapeutic roles. Copyright © 2016 by the American Society of Nephrology.

  8. Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis.

    Science.gov (United States)

    Lee, Jaewook; Kim, Si-Hyun; Choi, Dong-Sic; Lee, Jong Seok; Kim, Dae-Kyum; Go, Gyeongyun; Park, Seon-Min; Kim, Si Hyun; Shin, Jeong Hwan; Chang, Chulhun L; Gho, Yong Song

    2015-10-01

    The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Melanoma affects the composition of blood cell-derived extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Nina Koliha

    2016-07-01

    Full Text Available Extracellular vesicles are specifically loaded with nucleic acids, lipids, and proteins from their parental cell. Therefore, the constitution of extracellular vesicles reflects the type and status of the originating cell and extracellular vesicles in melanoma patient’s plasma could be indicative for the tumor. Likewise, extracellular vesicles might influence tumor progression by regulating immune responses. We performed a broad protein characterization of extracellular vesicles from plasma of melanoma patients and healthy donors as well as from T cells, B cells, natural killer cells, monocytes, monocyte-derived dendritic cells and platelets using a multiplex bead-based platform. Using this method, we succeeded in analyzing 58 proteins that were differentially displayed on extracellular vesicles. Hierarchal clustering of protein intensity patterns grouped extracellular vesicles according to their originating cell type. The analysis of extracellular vesicles from stimulated B cells and monocyte-derived dendritic cells revealed the transfer of surface proteins to vesicles depending on the cell status. The protein profiles of plasma vesicles resembled the protein profiles of extracellular vesicles from platelets, antigen presenting cells and natural cells as shown by platelet markers, costimulatory proteins, and a natural killer cell subpopulation marker. In comparison to healthy plasma vesicles, melanoma plasma vesicles showed altered signals for platelet markers indicating a changed vesicle secretion or protein loading of extracellular vesicles by platelets and a lower CD8 signal that might be associated with a diminished activity of natural killer cells or T cells. As we hardly detected melanoma-derived vesicles in patient’s plasma, we concluded that blood cells induced the observed differences. In summary, our results question a direct effect of melanoma cells on the composition of extracellular vesicles in melanoma plasma, but rather argue

  10. [Effect of mild and strong manual acupuncture stimulation of "Huantiao" (GB 30) on mechanical pain thresholds and extracellular signal-regulated kinase protein expression in spinal dorsal horns in rats with neuropathic mirror-image pain].

    Science.gov (United States)

    Shen, Zui; Shao, Xiao-Mei; Fang, Fang; Sun, Jing; Fang, Jun-Fan; Fang, Jian-Qiao

    2014-04-01

    To observe the effect of different intensities of manual acupuncture (MA) stimulation on mechanical pain thresholds (PTs) and the expression of phosphorylated extracellular signal-regulated kinases (p-ERK) in lumbar spinal dorsal horn regions in rats with neuropathic mirror-image pain, so as to explore its mechanisms underlying analgesia. Forty male SD rats were equally and randomly divided into control, spinal nerve ligation (SNL) model, mild MA-stimulation, and strong MA-stimulation groups. Neuropathological pain model was established by ligature of the spinal nerve (L 5). Three days after the SNL, bilateral "Huantiao" (GB 30) were stimulated by rotating the thin (0.22 mm x 13 mm) or thick (0.3 mm x 13 mm) filiform needles at a frequencies of 60 times/min or 180 times/min and at an angle of 180 degrees or 360 degrees for 2 min for rats in the mild and strong MA-stimulation groups, respectively, followed by remaining the needle in place for 30 min. The mechanical PTs were measured before and after SNL. The expression of p-ERK protein in bilateral dorsal horn regions of the lumbar spinal cord (L4- L 6) was detected by Western blot. In comparison with the control group, the mechanical PTs were significantly decreased beginning from the 3rd day on after SNL on the affected side and from the 7th day on after SNL on the healthy hindpaw (P horn regions on both sides of the spinal cord were considerably up-regulated on the 12th day (P horn regions were obviously down-regulated in the strong MA-stimulation group (P horn regions (P > 0.05) except the PTs of the healthy hindpaw on 7th day (P effect in down-regulating the expression of p-ERK in the bilateral spinal dorsal horn regions.

  11. Epstein-Barr virus-negative aggressive natural killer-cell leukaemia with high P-glycoprotein activity and phosphorylated extracellular signal-regulated protein kinases 1 and 2

    Directory of Open Access Journals (Sweden)

    Sanja Perkovic

    2012-09-01

    Full Text Available Aggressive natural killer-cell leukaemia (ANKL is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV and P-glycoprotein (P-gp expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56+ NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2 MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.

  12. Activation of extracellular signal-regulated kinase (ERK) and induction of mitogen-activated protein kinase phosphatase 1 (MKP-1) by perifused thyrotropin-releasing hormone (TRH) stimulation in rat pituitary GH3 cells.

    Science.gov (United States)

    Oride, Aki; Kanasaki, Haruhiko; Mutiara, Sandra; Purwana, Indri Nuryani; Miyazaki, Kohji

    2008-12-16

    We investigated the pattern of extracellular signal-regulated kinase (ERK) phosphorylation and the induction of mitogen-activated protein kinase phosphatase 1 (MKP-1) by thyrotropin-releasing hormone (TRH) under various stimulation conditions in pituitary GH3 cells. In static culture, ERK activation by continuous TRH was maximal at 10 min and persisted for up to 60 min, with a return to the basal level by 2h. Stimulation with continuous TRH in perifused cells resulted in a similar level of ERK phosphorylation. MKP-1 was expressed 60 min following either static or perifused, continuous TRH stimulation. When cells were stimulated with pulsatile TRH every 30 min, ERK activation was maximal at 10 min and returned to its baseline level by 30 min. ERK was phosphorylated again with each subsequent pulse. Pulsatile TRH did not induce MKP-1. Prolactin promoter activity following continuous, static TRH stimulation was higher than that following perifused TRH stimulation. TRH at a frequency of one pulse every 30 min increased prolactin promoter activity similar to that of perifused, continuous TRH stimulation. Additionally, changes in pulse frequency resulted in alterations in the level of prolactin promoter. Following static stimulation, a 10 min exposure to TRH was sufficient to obtain full activation of the prolactin promoter. Additionally, a 5-10 min exposure of TRH was sufficient to maintain ERK activation. A single 5-min pulse of TRH stimulation resulted in low activation of the prolactin promoter. ERK activation was necessary for prolactin gene transcription; however, prolactin gene transcription is not entirely determined by the strength or duration of TRH-induced ERK activation.

  13. Role of reactive oxygen species in extracellular signal-regulated ...

    Indian Academy of Sciences (India)

    Keywords. 6-hydroxydopamine; mitogen activated protein kinase; Parkinson's disease; redox signalling. Abstract. A number of reports indicate the potential for redox signalling via extracellular signal-regulated protein kinases (ERK) during neuronal injury. We have previously found that sustained ERK activation contributes ...

  14. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

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    Chad J Johnson

    2016-09-01

    Full Text Available Neutrophils release extracellular traps (NETs in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA and was associated with suppression of neutrophil reactive oxygen species (ROS production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

  15. The Limiting Sequence and Proper Ratio of Lysine, Methionine and Threonine for Calves Fed Milk Replacers Containing Soy Protein

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    Jianhong Wang

    2012-02-01

    Full Text Available The limiting sequence and relative ratio of lysine (Lys, methionine (Met, and threonine (Thr for calves about 2 mo of age fed milk replacers (MR containing soy protein are not clearly defined. The objective of the study was to investigate the effect of supplementing MR containing 22% CP, half from soy protein concentrate (SPC, 40.56% CP, flour and half from whey proteins, with Lys, Met, and Thr to estimate amino acid (AA sequence and their relative ratio for calves about 2 mo of age. A method of partial deduction of AA was adopted. Twenty-four newborn calves (half males and half females, 40.7±0.9 kg of BW were fed 1 of 4 MR diets for 56 d (n = 6/diet. The diets were supplemented with all (positive control or with 2 of the 3 AAs: Lys, Met and Thr, (i.e., PC (22% CP, 2.34% Lys, 0.72% Met and 1.80% Thr, PC-Lys (22% CP, 1.64% Lys, 0.72% Met and 1.80% Thr, PC-Met (22% CP, 2.34% Lys, 0.50% Met and 1.80% Thr, and PC-Thr (22% CP, 2.34% Lys, 0.72% Met and 1.26% Thr. Calves were fed thrice daily; starter (20% CP, 1.03% Lys, 0.30% Met and 0.69% Thr, hay (3.23% CP, 0.29% Lys, 0.12% Met and 0.23% Thr and water were offered free choice. Starter and hay were only offered beginning on d 36 (after 5 wk and d 43 (after 6 wk, respectively. BW, body size and blood samples measures were taken every two weeks. Three-day total collection of feed refusals, feces, and urine were recorded starting at d 33 and d 54 of age, respectively. From the results, the limiting sequence and relative ratio between the 3 AAs in calves with different diet structures were calculated. The limiting sequence of the 3 AAs were ranked as Lys, Met and Thr; the proper ratio was 100:29:70 for MR-only diet and 100:30:60 for diets consisted of MR, starter and hay. Nitrogen digestion and utilization and nutrient digestibility were negatively affected by AA deletion treatments. From the evidence of this experiment, it did not appear that the AA limiting sequence was selectively altered by

  16. Proteomic analysis of cerebrospinal fluid extracellular vesicles: a comprehensive dataset.

    Science.gov (United States)

    Chiasserini, Davide; van Weering, Jan R T; Piersma, Sander R; Pham, Thang V; Malekzadeh, Arjan; Teunissen, Charlotte E; de Wit, Heidi; Jiménez, Connie R

    2014-06-25

    Extracellular vesicles (EVs) are present in human cerebrospinal fluid (CSF), yet little is known about their protein composition. The aim of this study is to provide a comprehensive analysis of the proteome of CSF EVs by electron microscopy and high resolution tandem mass spectrometry (MS/MS) in conjunction with bioinformatics. We report an extensive catalog of 1315 proteins identified in EVs isolated from two different CSF pools by ultracentrifugation, including 230 novel EV proteins. Out of 1315 proteins, 760 were identified in both CSF pools and about 30% of those were also quantitatively enriched in the EV fraction versus the soluble CSF fraction. The proteome of CSF EVs was enriched in exosomal markers such as alix and syntenin-1, heat shock proteins and tetraspanins and contained a high proportion of brain-derived proteins (n=373). Interestingly, several known biomarkers for neurodegenerative diseases such as the amyloid precursor protein, the prion protein and DJ-1 were identified in the EV fractions. Our dataset represents the first comprehensive inventory of the EV proteome in CSF, underscoring the biomarker potential of this organelle. Further comparative studies on CSF EVs isolated from patients diagnosed with neurological disorders are warranted. Data are available via ProteomeXchange with identifier PXD000608. Biological significance In this study we analyzed the protein composition of extracellular vesicles isolated from pooled samples of human cerebrospinal fluid (CSF). CSF is a colorless fluid surrounding the brain and the spinal cord, important for the physiology of the central nervous system, ensuing mechanical protection, regulation of brain blood flow and elimination of byproducts of the brain. Since brain (patho)physiology is reflected in CSF, this biological fluid represents an ideal source of soluble and vesicle-based biomarkers for neurological diseases. Here we confirm the presence of exosome-like extracellular vesicles in CSF, underscoring

  17. Methods to isolate extracellular vesicles for diagnosis

    Science.gov (United States)

    Kang, Hyejin; Kim, Jiyoon; Park, Jaesung

    2017-12-01

    Extracellular vesicles (EVs) are small membrane-bound bodies that are released into extracellular space by diverse cells, and are found in body fluids like blood, urine and saliva. EVs contain RNA, DNA and proteins, which can be biomarkers for diagnosis. EVs can be obtained by minimally-invasive biopsy, so they are useful in disease diagnosis. High yield and purity contribute to precise diagnosis of disease, but damaged EVs and impurities can cause confu sed results. However, EV isolation methods have different yields and purities. Furthermore, the isolation method that is most suitable to maximize EV recovery efficiency depends on the experimental conditions. This review focuses on merits and demerits of several types of EV isolation methods, and provides examples of how to diagnose disease by exploiting information obtained by analysis of EVs.

  18. Transport-Limited Adsorption of Plasma Proteins on Bimodal Amphiphilic Polymer Co-Networks: Real-Time Studies by Spectroscopic Ellipsometry.

    Science.gov (United States)

    Guzman, Gustavo; Bhaway, Sarang M; Nugay, Turgut; Vogt, Bryan D; Cakmak, Mukerrem

    2017-03-21

    Traditional hydrogels are commonly limited by poor mechanical properties and low oxygen permeability. Bimodal amphiphilic co-networks (β-APCNs) are a new class of materials that can overcome these limitations by combining hydrophilic and hydrophobic polymer chains within a network of co-continuous morphology. Applications that can benefit from these improved properties include therapeutic contact lenses, enzymatic catalysis supports, and immunoisolation membranes. The continuous hydrophobic phase could potentially increase the adsorption of plasma proteins in blood-contacting medical applications and compromise in vivo material performance, so it is critical to understand the surface characteristics of β-APCNs and adsorption of plasma proteins on β-APCNs. From real-time spectroscopic visible (Vis) ellipsometry measurements, plasma protein adsorption on β-APCNs is shown to be transport-limited. The adsorption of proteins on the β-APCNs is a multistep process with adsorption to the hydrophilic surface initially, followed by diffusion into the material to the internal hydrophilic/hydrophobic interfaces. Increasing the cross-linking of the PDMS phase reduced the protein intake by limiting the transport of large proteins. Moreover, the internalization of the proteins is confirmed by the difference between the surface-adsorbed protein layer determined from XPS and bulk thickness change from Vis ellipsometry, which can differ up to 20-fold. Desorption kinetics depend on the adsorption history with rapid desorption for slow adsorption rates (i.e., slow-diffusing proteins within the network), whereas proteins with fast adsorption kinetics do not readily desorb. This behavior can be directly related to the ability of the protein to spread or reorient, which affects the binding energy required to bind to the internal hydrophobic interfaces.

  19. Immunotherapeutic potential of extracellular vesicles

    Directory of Open Access Journals (Sweden)

    Bin eZhang

    2014-10-01

    Full Text Available Extracellular vesicles or EVs is a term that encompasses all classes of secreted lipid membrane vesicles. Despite being scientific novelties, EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo of protein and RNA between cells. In particular, exosomes the currently best characterized EVs have been notable for their in vitro and in vivo immunomodulatory activities. Exosomes are nanometer-sized endosome-derived vesicles secreted by many cell types and their immunomodulatory potential is independent of their cell source. Besides immune cells such as dendritic cells, macrophages and T cells, cancer and stem cells also secrete immunologically active exosomes that could influence both physiological and pathological processes. The immunological activities of exosomes affect both innate and adaptive immunity and include antigen presentation, T cell activation, T cell polarisation to Tregs, immune suppression and anti-inflammation. As such, exosomes carry much immunotherapeutic potential as a therapeutic agent and a therapeutic target.

  20. Extracellular Vesicles in Luminal Fluid of the Ovine Uterus

    Science.gov (United States)

    Burns, Gregory; Brooks, Kelsey; Wildung, Mark; Navakanitworakul, Raphatphorn; Christenson, Lane K.; Spencer, Thomas E.

    2014-01-01

    Microvesicles and exosomes are nanoparticles released from cells and can contain small RNAs, mRNA and proteins that affect cells at distant sites. In sheep, endogenous beta retroviruses (enJSRVs) are expressed in the endometrial epithelia of the uterus and can be transferred to the conceptus trophectoderm. One potential mechanism of enJSRVs transfer from the uterus to the conceptus is via exosomes/microvesicles. Therefore, studies were conducted to evaluate exosomes in the uterine luminal fluid (ULF) of sheep. Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Transmission electron microscopy and nanoparticle tracking analysis found the isolates contained vesicles that ranged from 50 to 200 nm in diameter. The isolated extracellular vesicles were positive for two common markers of exosomes (CD63 and HSP70) by Western blot analysis. Proteins in the extracellular vesicles were determined by mass spectrometry and Western blot analysis. Extracellular vesicle RNA was analyzed for small RNAs by sequencing and enJSRVs RNA by RT-PCR. The ULF extracellular vesicles contained a large number of small RNAs and miRNAs including 81 conserved mature miRNAs. Cyclic and pregnant ULF extracellular vesicles contained enJSRVs env and gag RNAs that could be delivered to heterologous cells in vitro. These studies support the hypothesis that ULF extracellular vesicles can deliver enJSRVs RNA to the conceptus, which is important as enJSRVs regulate conceptus trophectoderm development. Importantly, these studies support the idea that extracellular vesicles containing select miRNAs, RNAs and proteins are present in the ULF and likely have a biological role in conceptus-endometrial interactions important for the establishment and maintenance of pregnancy. PMID:24614226

  1. Extracellular enzyme activity and biogeochemical cycling in restored prairies

    Science.gov (United States)

    Lynch, L.; Hernandez, D.; Schade, J. D.

    2011-12-01

    Winter microbial activity in mid-latitude prairie ecosystems is thermally sensitive and significantly influenced by snow depth. Snow insulates the soil column facilitating microbial processing of complex organic substrates. Previous studies in forests and tundra ecosystems suggest patterns of substrate utilization and limitation are seasonal; above freezing, soil microbes access fresh litter inputs and sugar exudates from plant roots, while under frozen condition they recycle nutrients incorporated in microbial biomass. In order to liberate nutrients required for carbon degradation, soil microbes invest energy in the production of extracellular enzymes that cleave monomers from polymer bonds. The inverse relationship between relative enzyme abundance and substrate availability makes enzyme assays a useful proxy to assess changes in resources over time. Our objective in this study was to assess patterns in microbial biomass, nutrient availability, and extracellular enzyme activity in four snow exclosure sites over a seven-month period. Over the past three years, we have maintained a snow removal experiment on two restored prairies in central Minnesota. In each prairie, snow was continuously removed annually from two 4 x 4 m plots by shoveling after each snow event. Extractable C, N and P, and microbial C, N and P in soil samples were measured in samples collected from these snow removal plots, as well as in adjacent unmanipulated prairie control plots. Pools of C, N, and P were estimated using standard extraction protocols, and microbial pools were estimated using chloroform fumigation direct extraction (CFDE). We conducted fluorometric extracellular enzyme assays (EEA) to assess how the degradation potential of cellulose (cellobiohydrolase, CBH), protein (leucine aminopeptidase, LAP), and phosphate esters (phosphatase, PHOS) changed seasonally. Microbial C and N declined between October and June, while microbial P declined during the fall and winter, but increased

  2. Part I: Characterization of the Extracellular Proteome of the Extreme Thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS2

    Science.gov (United States)

    Muddiman, David; Andrews, Genna; Lewis, Derrick; Notey, Jaspreet; Kelly, Robert

    2013-01-01

    The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms no doubt harbors technologically important enzymes and other thermostable biomolecules that to date have been characterized only to a limited extent. In the first of a two part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS2 analysis and prediction servers suggest probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments all including the C18 bead technique demonstrated that two levels of sample purification were necessary to effectively de-salt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described as enzymatic and putative uncharacterized proteins anticipating consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion and 11 contain transmembrane domain stretches suggested by Phobius and TMHMM. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study involves acetone precipitation followed by the C18 bead method in which 2.4% (63 proteins) of the predicted proteome was indentified including proteins suggested to have secretion and transmembrane moieties. PMID:20623222

  3. Mass-spectrometry-based molecular characterization of extracellular vesicles: lipidomics and proteomics.

    Science.gov (United States)

    Kreimer, Simion; Belov, Arseniy M; Ghiran, Ionita; Murthy, Shashi K; Frank, David A; Ivanov, Alexander R

    2015-06-05

    This review discusses extracellular vesicles (EVs), which are submicron-scale, anuclear, phospholipid bilayer membrane enclosed vesicles that contain lipids, metabolites, proteins, and RNA (micro and messenger). They are shed from many, if not all, cell types and are present in biological fluids and conditioned cell culture media. The term EV, as coined by the International Society of Extracellular Vesicles (ISEV), encompasses exosomes (30-100 nm in diameter), microparticles (100-1000 nm), apoptotic blebs, and other EV subsets. EVs have been implicated in cell-cell communication, coagulation, inflammation, immune response modulation, and disease progression. Multiple studies report that EV secretion from disease-affected cells contributes to disease progression, e.g., tumor niche formation and cancer metastasis. EVs are attractive sources of biomarkers due to their biological relevance and relatively noninvasive accessibility from a range of physiological fluids. This review is focused on the molecular profiling of the protein and lipid constituents of EVs, with emphasis on mass-spectrometry-based "omic" analytical techniques. The challenges in the purification and molecular characterization of EVs, including contamination of isolates and limitations in sample quantities, are discussed along with possible solutions. Finally, the review discusses the limited but growing investigation of post-translational modifications of EV proteins and potential strategies for future in-depth molecular characterization of EVs.

  4. Oxidation of cytochrome c peroxidase to compound I by peroxyacids: evidence for rate-limiting diffusion through the protein matrix.

    Science.gov (United States)

    Palamakumbura, A H; Foshay, M C; Vitello, L B; Erman, J E

    1999-11-23

    The rate of the reaction between p-nitroperoxybenzoic acid and cytochrome c peroxidase (CcP) has been investigated as a function of pH and ionic strength. The pH dependence of the reaction between CcP and peracetic acid has also been determined. The rate of the reactions are influenced by two heme-linked ionizations in the protein. The enzyme is active when His-52 (pK(a) 3.8 +/- 0.1) is unprotonated and an unknown group with a pK(a) of 9.8 +/- 0.1 is protonated. The bimolecular rate constant for the reaction between peracetic acid and CcP and between p-nitroperoxybenzoic acid and CcP are (1.8 +/- 0.1) x 10(7) and (1.6 +/- 0.2) x 10(7) M(-)(1) s(-)(1), respectively. These rates are about 60% slower than the reaction between hydrogen peroxide and CcP. A critical comparison of the pH dependence of the reactions of hydrogen peroxide, peracetic acid, and p-nitroperoxybenzoic acid with CcP provides evidence that both the neutral and anionic forms of the two peroxyacids react directly with the enzyme. The peracetate and p-nitroperoxybenzoate anions react with CcP with rates of (1.5 +/- 0.1) x 10(6) and (1.6 +/- 0.2) x 10(6) M(-)(1) s(-)(1), respectively, about 10 times slower than the neutral peroxyacids. These data indicate that CcP discriminates between the neutral peroxyacids and their negatively charged ions. However, the apparent bimolecular rate constant for reaction between p-nitroperoxybenzoate and CcP is independent of ionic strength in the range of 0.01-1.0 M, suggesting that electrostatic repulsion between the anion and CcP is not the cause of the lower reactivity for the peroxybenzoate anion. The data are consistent with the hypothesis that the rate-limiting step for the oxidation of CcP to compound I by both neutral peroxyacid and the negatively charged peroxide ion is diffusion of the reactants through the protein matrix, from the surface of the protein to the distal heme pocket.

  5. Insertion of tetracysteine motifs into dopamine transporter extracellular domains.

    Directory of Open Access Journals (Sweden)

    Deanna M Navaroli

    Full Text Available The neuronal dopamine transporter (DAT is a major determinant of extracellular dopamine (DA levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.

  6. Extracellular polymeric substances play roles in extracellular electron transfer of Shewanella oneidensis MR-1

    DEFF Research Database (Denmark)

    Xiao, Yong; Zhang, En-Hua; Christensen, Hans Erik Mølager

    It is well known that microorganism is surrounded by extracellular polymeric substances (EPS) which include polysaccharides, proteins, glycoproteins, nucleic acids, phospholipids, and humic acids. However, previous studies on microbial extracellular electron transfer (EET) are conducted on cells......, such as DNA, humic acids and some proteins, are electrochemically active or semiconductive. Herein, we report experimental evidences of EPS role on EET for Shewanella oneidensis MR-1. Atomic force microscopy clearly showed that the cell surface was cleaned and few EPS could be observed on MR-1 after...... without extracting EPS or cells collected from log stage or early-steady stage cultures with little EPS. Therefore, microbial cells are believed in contact directly with each other or electrode. Such attempt apparently ignored the role of EPS in microbial EET, even though many components of EPS...

  7. Preeclampsia and Extracellular Vesicles.

    Science.gov (United States)

    Gilani, Sarwat I; Weissgerber, Tracey L; Garovic, Vesna D; Jayachandran, Muthuvel

    2016-09-01

    Preeclampsia is a hypertensive pregnancy disorder characterized by development of hypertension and proteinuria after 20 weeks of gestation that remains a leading cause of maternal and neonatal morbidity and mortality. While preeclampsia is believed to result from complex interactions between maternal and placental factors, the proximate pathophysiology of this syndrome remains elusive. Cell-to-cell communication is a critical signaling mechanism for feto-placental development in normal pregnancies. One mechanism of cellular communication relates to activated cell-derived sealed membrane vesicles called extracellular vesicles (EVs). The concentrations and contents of EVs in biological fluids depend upon their cells of origin and the stimuli which trigger their production. Research on EVs in preeclampsia has focused on EVs derived from the maternal vasculature (endothelium, vascular smooth muscle) and blood (erythrocytes, leukocytes, and platelets), as well as placental syncytiotrophoblasts. Changes in the concentrations and contents of these EVs may contribute to the pathophysiology of preeclampsia by accentuating the pro-inflammatory and pro-coagulatory states of pregnancy. This review focuses on possible interactions among placental- and maternal-derived EVs and their contents in the initiation and progression of the pathogenesis of preeclampsia. Understanding the contributions of EVs in the pathogenesis of preeclampsia may facilitate their use as diagnostic and prognostic biomarkers.

  8. Extracellular matrix structure.

    Science.gov (United States)

    Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

    2016-02-01

    Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce