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Sample records for cell wall proteome

  1. Cell Wall Proteome

    OpenAIRE

    Boudart, Georges; Minic, Zoran; Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth; Pont-Lezica, Rafael F

    2007-01-01

    In this chapter, we will focus on the contribution of proteomics to the identification and determination of the structure and function of CWPs as well as discussing new perspectives in this area. The great variety of proteins found in the plant cell wall is described. Some families, such as glycoside hydrolases, proteases, lectins, and inhibitors of cell wall modifying enzymes, are discussed in detail. Examples of the use of proteomic techniques to elucidate the structure of various cell wall...

  2. Recent advances in plant cell wall proteomics.

    Science.gov (United States)

    Jamet, Elisabeth; Albenne, Cécile; Boudart, Georges; Irshad, Muhammad; Canut, Hervé; Pont-Lezica, Rafael

    2008-02-01

    The plant extracellular matrix contains typical polysaccharides such as cellulose, hemicelluloses, and pectins that interact to form dense interwoven networks. Plant cell walls play crucial roles during development and constitute the first barrier of defense against invading pathogens. Cell wall proteomics has greatly contributed to the description of the protein content of a compartment specific to plants. Around 400 cell wall proteins (CWPs) of Arabidopsis, representing about one fourth of its estimated cell wall proteome, have been described. The main points to note are that: (i) the diversity of enzymes acting on polysaccharides suggests a great plasticity of cell walls; (ii) CWPs such as proteases, polysaccharide hydrolytic enzymes, and lipases may contribute to the generation of signals; (iii) proteins of unknown functions were identified, suggesting new roles for cell walls. Recently, the characterization of PTMs such as N- and O-glycosylations improved our knowledge of CWP structure. The presence of many glycoside hydrolases and proteases suggests a complex regulation of CWPs involving various types of post-translational events. The first 3-D structures to be resolved gave clues about the interactions between CWPs, or between CWPs and polysaccharides. Future work should include: extracting and identifying CWPs still recalcitrant to proteomics, describing the cell wall interactome, improving quantification, and unraveling the roles of each of the CWPs.

  3. Cell wall proteins: a new insight through proteomics.

    Science.gov (United States)

    Jamet, Elisabeth; Canut, Hervé; Boudart, Georges; Pont-Lezica, Rafael F

    2006-01-01

    Cell wall proteins are essential constituents of plant cell walls; they are involved in modifications of cell wall components, wall structure, signaling and interactions with plasma membrane proteins at the cell surface. The application of proteomic approaches to the cell wall compartment raises important questions: are there technical problems specific to cell wall proteomics? What kinds of proteins can be found in Arabidopsis walls? Are some of them unexpected? What sort of post-translational modifications have been characterized in cell wall proteins to date? The purpose of this review is to discuss the experimental results obtained to date using proteomics, as well as some of the new questions challenging future research.

  4. Plant cell wall proteomics: the leadership of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Cécile eALBENNE

    2013-05-01

    Full Text Available Plant cell wall proteins (CWPs progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cells walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last ten years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii the main protein families identified and the still missing peptides; (iii the persistent issue of the non-canonical CWPs; (iv the present challenges to overcome technological bottlenecks; and (v the perspectives beyond cell wall proteomics to understand CWP functions.

  5. Plant cell wall proteomics: the leadership of Arabidopsis thaliana.

    Science.gov (United States)

    Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth

    2013-01-01

    Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last 10 years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i) a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii) the main protein families identified and the still missing peptides; (iii) the persistent issue of the non-canonical CWPs; (iv) the present challenges to overcome technological bottlenecks; and (v) the perspectives beyond cell wall proteomics to understand CWP functions.

  6. Analysis of the soluble cell wall proteome of gymnosperms.

    Science.gov (United States)

    Uzal, Esther Novo; Gómez-Ros, Laura V; Hernández, Jose A; Pedreño, María A; Cuello, Juan; Ros Barceló, Alfonso

    2009-05-15

    We analyzed the cell wall proteome of lignifying suspension cell cultures (SCCs) from four gymnosperms that differ in evolution degree. This analysis showed the presence of "peptide sequence tags" (PSTs) corresponding to glucan endo-1,3-beta-D-glucosidase, xyloglucan-endotrans-glucosylase/hydrolase, chitinases, thaumatin-like proteins and proteins involved in lignin/lignan biosynthesis, such as dirigent-like proteins and peroxidases. Surprisingly, and given the abundance of peroxidases in the cell wall proteome of these gymnosperms, PSTs corresponding to peroxidases were only detected in tryptic fragments of the cell wall proteome of Cycas revoluta. The current lack of knowledge regarding C. revoluta peroxidases led us to purify, characterize and partially sequence the peroxidases responsible for lignin biosynthesis in this species. This yielded three peroxidase-enriched fractions: CrPrx 1, CrPrx 2 and CrPrx 3. Analyses of tryptic peptides of CrPrx 2 (32kDa) and CrPrx 3 (26kDa) suggest that CrPrx 3 arises from CrPrx 2 by protein truncation, and that CrPrx 3 apparently constitutes a post-translational modification of CrPrx 2. That CrPrx 2 and CrPrx 3 are apparently the same enzyme was also deduced from the similarity between the k(cat) shown by both peroxidases for the three monolignols. These results emphasize the analogies between the cell wall proteome of gymnosperms and angiosperms, the complexity of the peroxidase proteome, and the difficulties involved in establishing fine structure-function relationships.

  7. Progress Towards the Tomato Fruit Cell Wall Proteome

    Directory of Open Access Journals (Sweden)

    Eliel eRuiz May

    2013-05-01

    Full Text Available The plant cell wall (CW compartment, or apoplast, is host to a highly dynamic proteome, comprising large numbers of both enzymatic and structural proteins. This reflects its importance as the interface between adjacent cells and the external environment, the presence of numerous extracellular metabolic and signaling pathways, and the complex nature of wall structural assembly and remodeling during cell growth and differentiation. Tomato fruit ontogeny, with its distinct phases of rapid growth and ripening, provides a valuable experimental model system for CW proteomic studies, in that it involves substantial wall assembly, remodeling and coordinated disassembly. Moreover, diverse populations of secreted proteins must be deployed to resist microbial infection and protect against abiotic stresses. Tomato fruits also provide substantial amounts of biological material, which is a significant advantage for many types of biochemical analyses, and facilitates the detection of lower abundance proteins. In this review we describe a variety of orthogonal techniques that have been applied to identify CW localized proteins from tomato fruit, including approaches that: target the proteome of the CW and the overlying cuticle; functional ‘secretome’ screens; lectin affinity chromatography; and computational analyses to predict proteins that enter the secretory pathway. Each has its merits and limitations, but collectively they are providing important insights into CW proteome composition and dynamics, as well as some potentially controversial issues, such as the prevalence of non-canonical protein secretion.

  8. Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls

    Directory of Open Access Journals (Sweden)

    Canut Hervé

    2006-05-01

    Full Text Available Abstract Background The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Reaching such a goal closely depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific difficulties: (i the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP during the isolation procedure, (ii polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50% of predicted intracellular proteins. Since isolated cell walls should hold secreted proteins, one can imagine alternative procedures to prepare cell walls containing a lower proportion of contaminant proteins. Results The rationales of several published procedures to isolate cell walls for proteomics were analyzed, with regard to the bioinformatic-predicted subcellular localization of the identified proteins. Critical steps were revealed: (i homogenization in low ionic strength acid buffer to retain CWP, (ii purification through increasing density cushions, (iii extensive washes with a low ionic strength acid buffer to retain CWP while removing as many cytosolic proteins as possible, and (iv absence of detergents. A new procedure was developed to prepare cell walls from etiolated hypocotyls of Arabidopsis thaliana. After salt extraction, a high proportion of proteins predicted to be secreted was released (73%, belonging to the same functional classes as proteins identified using previously described protocols. Finally, removal of intracellular proteins was obtained using detergents, but their amount represented less than 3% in mass of the total protein extract, based on protein quantification. Conclusion The

  9. Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

    Directory of Open Access Journals (Sweden)

    Jackson Champer

    2016-01-01

    Full Text Available We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy. Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4, Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here.

  10. Post-translational modifications of plant cell wall proteins and peptides: A survey from a proteomics point of view.

    Science.gov (United States)

    Canut, Hervé; Albenne, Cécile; Jamet, Elisabeth

    2016-08-01

    Plant cell wall proteins (CWPs) and peptides are important players in cell walls contributing to their assembly and their remodeling during development and in response to environmental constraints. Since the rise of proteomics technologies at the beginning of the 2000's, the knowledge of CWPs has greatly increased leading to the discovery of new CWP families and to the description of the cell wall proteomes of different organs of many plants. Conversely, cell wall peptidomics data are still lacking. In addition to the identification of CWPs and peptides by mass spectrometry (MS) and bioinformatics, proteomics has allowed to describe their post-translational modifications (PTMs). At present, the best known PTMs consist in proteolytic cleavage, N-glycosylation, hydroxylation of P residues into hydroxyproline residues (O), O-glycosylation and glypiation. In this review, the methods allowing the capture of the modified proteins based on the specific properties of their PTMs as well as the MS technologies used for their characterization are briefly described. A focus is done on proteolytic cleavage leading to protein maturation or release of signaling peptides and on O-glycosylation. Some new technologies, like top-down proteomics and terminomics, are described. They aim at a finer description of proteoforms resulting from PTMs or degradation mechanisms. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

  11. Combining proteomics and transcriptome sequencing to identify active plant-cell-wall-degrading enzymes in a leaf beetle

    Directory of Open Access Journals (Sweden)

    Kirsch Roy

    2012-11-01

    Full Text Available Abstract Background The primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs. The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae. Results Using a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH families: GH11 (xylanases, GH28 (polygalacturonases or pectinases, and GH45 (β-1,4-glucanases or cellulases. Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs families as well as polygalacturonase-inhibiting proteins (PGIPs were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome

  12. Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein.

    Science.gov (United States)

    Micallef, Giulia; Cash, Phillip; Fernandes, Jorge M O; Rajan, Binoy; Tinsley, John W; Bickerdike, Ralph; Martin, Samuel A M; Bowman, Alan S

    2017-01-01

    In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.

  13. Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein

    Science.gov (United States)

    Micallef, Giulia; Cash, Phillip; Fernandes, Jorge M. O.; Rajan, Binoy; Tinsley, John W.; Bickerdike, Ralph

    2017-01-01

    In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts. PMID:28046109

  14. Plant Cell Wall Proteomics: Mass Spectrometry Data, a Trove for Research on Protein Structure/Function Relationships

    Institute of Scientific and Technical Information of China (English)

    Cécile Albenne; Hervé Canut; Georges Boudart; Yu Zhang; Héléne San Clemente; Rafael Pont-Lezica; Elisabeth Jamet

    2009-01-01

    Proteomics allows the large-scale study of protein expression either in whole organisms or in purified organ-elles. In particular, mass spectrometry (MS) analysis of gel-separated proteins produces data not only for protein identi-fication, but for protein structure, location, and processing as well. An in-depth analysis was performed on MS data from etiolated hypocotyl cell wall proteomics of Arabidopsis thaliana. These analyses show that highly homologous members of multigene families can be differentiated. Two lectins presenting 93% amino acid identity were identified using peptide mass fingerprinting. Although the identification of structural proteins such as extensins or hydroxyproline/proline-rich proteins (H/PRPs) is arduous, different types of MS spectra were exploited to identify and characterize an H/PRR Matu-ration events in a couple of cell wall proteins (CWPs) were analyzed using site mapping. N-glycosylation of CWPs as well as the hydroxylation or oxidation of amino acids were also explored, adding information to improve our understanding of CWP structure/function relationships. A bioinformatic tool was developed to locate by means of MS the N-terminus of mature secreted proteins and N-glycosylation.

  15. Plant cell wall proteomics: mass spectrometry data, a trove for research on protein structure/function relationships.

    Science.gov (United States)

    Albenne, Cécile; Canut, Hervé; Boudart, Georges; Zhang, Yu; San Clemente, Hélène; Pont-Lezica, Rafael; Jamet, Elisabeth

    2009-09-01

    Proteomics allows the large-scale study of protein expression either in whole organisms or in purified organelles. In particular, mass spectrometry (MS) analysis of gel-separated proteins produces data not only for protein identification, but for protein structure, location, and processing as well. An in-depth analysis was performed on MS data from etiolated hypocotyl cell wall proteomics of Arabidopsis thaliana. These analyses show that highly homologous members of multigene families can be differentiated. Two lectins presenting 93% amino acid identity were identified using peptide mass fingerprinting. Although the identification of structural proteins such as extensins or hydroxyproline/proline-rich proteins (H/PRPs) is arduous, different types of MS spectra were exploited to identify and characterize an H/PRP. Maturation events in a couple of cell wall proteins (CWPs) were analyzed using site mapping. N-glycosylation of CWPs as well as the hydroxylation or oxidation of amino acids were also explored, adding information to improve our understanding of CWP structure/function relationships. A bioinformatic tool was developed to locate by means of MS the N-terminus of mature secreted proteins and N-glycosylation.

  16. Physiological and proteomic changes suggest an important role of cell walls in the high tolerance to metals of Elodea nuttallii.

    Science.gov (United States)

    Larras, Floriane; Regier, Nicole; Planchon, Sébastien; Poté, John; Renaut, Jenny; Cosio, Claudia

    2013-12-15

    Macrophytes bioaccumulate metals, the suggestion being made that they be considered for phytoremediation. However, a thorough understanding of the mechanisms of metal tolerance in these plants is necessary to allow full optimization of this approach. The present study was undertaken to gain insight into Hg and Cd accumulation and their effects in a representative macrophyte, Elodea nuttallii. Exposure to methyl-Hg (23 ng dm(-3)) had no significant effect while inorganic Hg (70 ng dm(-3)) and Cd (281 μg dm(-3)) affected root growth but did not affect shoots growth, photosynthesis, or antioxidant enzymes. Phytochelatins were confirmed as having a role in Cd tolerance in this plant while Hg tolerance seems to rely on different mechanisms. Histology and subcellular distribution revealed a localized increase in lignification, and an increased proportion of metal accumulation in cell wall over time. Proteomics further suggested that E. nuttallii was able to efficiently adapt its energy sources and the structure of its cells during Hg and Cd exposure. Storage in cell walls to protect cellular machinery is certainly predominant at environmental concentrations of metals in this plant resulting in a high tolerance highlighted by the absence of toxicity symptoms in shoots despite the significant accumulation of metals.

  17. Proteomic analysis of cellular response induced by multi-walled carbon nanotubes exposure in A549 cells.

    Directory of Open Access Journals (Sweden)

    Li Ju

    Full Text Available The wide application of multi-walled carbon nanotubes (MWCNT has raised serious concerns about their safety on human health and the environment. However, the potential harmful effects of MWCNT remain unclear and contradictory. To clarify the potentially toxic effects of MWCNT and to elucidate the associated underlying mechanisms, the effects of MWCNT on human lung adenocarcinoma A549 cells were examined at both the cellular and the protein level. Cytotoxicity and genotoxicity were examined, followed by a proteomic analysis (2-DE coupled with LC-MS/MS of the cellular response to MWCNT. Our results demonstrate that MWCNT induces cytotoxicity in A549 cells only at relatively high concentrations and longer exposure time. Within a relatively low dosage range (30 µg/ml and short time period (24 h, MWCNT treatment does not induce significant cytotoxicity, cell cycle changes, apoptosis, or DNA damage. However, at these low doses and times, MWCNT treatment causes significant changes in protein expression. A total of 106 proteins show altered expression at various time points and dosages, and of these, 52 proteins were further identified by MS. Identified proteins are involved in several cellular processes including proliferation, stress, and cellular skeleton organization. In particular, MWCNT treatment causes increases in actin expression. This increase has the potential to contribute to increased migration capacity and may be mediated by reactive oxygen species (ROS.

  18. STEM CELLS AND PROTEOMICS

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yong-ming; GUO Tian-nan; HUANG Shi-ang

    2006-01-01

    The distinctive features of proteomics are large-scale and high throughput. The key techniques of proteomics are two-dimensional gel electrophoresis, mass spectrometry and bioinformatics. Stem cell can differentiate into all kinds of cells, tissues and organs. There are many proteins and cytokines involved in the process of differentiation. Applying proteomics techniques to the research of the complex process of stem cell differentiation is of great importance to study the mechanism and applications of stem cell differentiation.

  19. Global Proteomic Profiling of the Secretome of Candida albicans ecm33 Cell Wall Mutant Reveals the Involvement of Ecm33 in Sap2 Secretion.

    Science.gov (United States)

    Gil-Bona, Ana; Monteoliva, Lucía; Gil, Concha

    2015-10-02

    Candida albicans secretes numerous proteins related to cell wall remodeling, adhesion, nutrient acquisition and host interactions. Also, extracellular vesicles containing cytoplasmic proteins are secreted into the medium. The C. albicans ecm33/ecm33 mutant (RML2U) presents an altered cell wall and is avirulent. The proteomic analysis of proteins secreted by RML2U cells identified a total of 170 proteins: 114 and 154 of which correspond to the vesicle-free secretome and extracellular vesicles, respectively. Notably, 98 proteins were common to both samples, and the groups most represented were metabolic and cell wall-related proteins. The results of this study showed that RML2U had an altered pattern of proteins secreted by the classical secretion pathway as well as the formation of extracellular vesicles, including their size, quantity, and protein composition. Specifically, the secretion of aspartic protease 2 (Sap2) was compromised but not its intracellular expression, with bovine serum albumin (BSA) degradation by RML2U being altered when BSA was used as the sole nitrogen source. Furthermore, as recent research links the expression of Sap2 to the TOR (Target Of Rapamycin) signaling pathway, the sensitivity of RML2U to rapamycin (the inhibitor of TOR kinase) was tested and found to be enhanced, connecting Ecm33 with this pathway.

  20. Proteomic Investigation of Rhizoctonia solani AG 4 Identifies Secretome and Mycelial Proteins with roles in Plant Cell Wall Degradation and Virulence

    KAUST Repository

    Lakshman, Dilip

    2016-03-28

    Rhizoctonia solani AG 4 is a soilborne necrotrophic fungal plant pathogen that causes economically important diseases on agronomic crops worldwide. Here we used a proteomics approach to characterize both intracellular proteins and the secretome of R. solani AG 4 isolate Rs23A under several growth conditions; the secretome being highly important in pathogenesis. From over 500 total secretome and soluble intracellular protein spots from 2-D gels, 457 protein spots were analyzed and 318 proteins positively matched with fungal proteins of known function by comparison with available R. solani genome databases specific for anastomosis groups 1-IA, 1-IB, and 3. These proteins were categorized to possible cellular locations and functional groups; and for some proteins their putative roles in plant cell wall degradation and virulence. The majority of the secreted proteins were grouped to extracellular regions and contain hydrolase activity.

  1. Isolation of plant cell wall proteins

    OpenAIRE

    Jamet, Elisabeth; Boudart, Georges; Borderies, Gisèle; Charmont, Stéphane; Lafitte, Claude; Rossignol, Michel; Canut, Hervé; Pont-Lezica, Rafael F

    2007-01-01

    The quality of a proteomic analysis of a cell compartment strongly depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific drawbacks: (i) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure; (ii) polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins; (iii) the presence of proteins ...

  2. Isolation of plant cell wall proteins.

    Science.gov (United States)

    Jamet, Elisabeth; Boudart, Georges; Borderies, Giséle; Charmont, Stephane; Lafitte, Claude; Rossignol, Michel; Canut, Herve; Pont-Lezica, Rafael

    2008-01-01

    The quality of a proteomic analysis of a cell compartment strongly depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific drawbacks: (1) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure; (2) polysaccharide networks of cellulose, hemicelluloses, and pectins form potential traps for contaminants such as intracellular proteins; (3) the presence of proteins interacting in many different ways with the polysaccharide matrix require different procedures to elute them from the cell wall. Three categories of CWP are distinguished: labile proteins that have little or no interactions with cell wall components, weakly bound proteins extractable with salts, and strongly bound proteins. Two alternative protocols are decribed for cell wall proteomics: (1) nondestructive techniques allowing the extraction of labile or weakly bound CWP without damaging the plasma membrane; (2) destructive techniques to isolate cell walls from which weakly or strongly bound CWP can be extracted. These protocols give very low levels of contamination by intracellular proteins. Their application should lead to a realistic view of the cell wall proteome at least for labile and weakly bound CWP extractable by salts.

  3. The Lamportian cell wall

    Energy Technology Data Exchange (ETDEWEB)

    Keiliszewski, M.; Lamport, D. (Michigan State Univ. Plant Research Lab., East Lansing (United States))

    1991-05-01

    The Lamportian Warp-Weft hypothesis suggests a cellulose-extensin interpenetrating network where extensin mechanically couples the load-bearing cellulose microfibrils in a wall matrix that is best described as a microcomposite. This model is based on data gathered from the extensin-rich walls of tomato and sycamore cell suspension culture, wherein extensin precursors are insolubilized into the wall by undefined crosslinks. The authors recent work with cell walls isolated from intact tissue as well as walls from suspension cultured cells of the graminaceous monocots maize and rice, the non-graminaceous monocot asparagus, the primitive herbaceous dicot sugar beet, and the gymnosperm Douglas Fir indicate that although extensins are ubiquitous to all plant species examined, they are not the major structural protein component of most walls examined. Amino acid analyses of intact and HF-treated walls shows a major component neither an HRGP, nor directly comparable to the glycine-rich wall proteins such as those associated with seed coat walls or the 67 mole% glycine-rich proteins cloned from petunia and soybean. Clearly, structural wall protein alternatives to extensin exist and any cell wall model must take that into account. If we assume that extracellular matrices are a priori network structures, then new Hypless' structural proteins in the maize cell wall raise questions about the sort of network these proteins create: the kinds of crosslinks involved; how they are formed; and the roles played by the small amounts of HRGPs.

  4. Proteomic Analysis of Chinese Hamster Ovary Cells

    DEFF Research Database (Denmark)

    Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama;

    2012-01-01

    To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimens......To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis...

  5. Proteomics Study of Cotton Fiber Cells

    Institute of Scientific and Technical Information of China (English)

    LIU Jin-yuan

    2008-01-01

    @@ A comparative proteomic analysis was applied to explore the mechanism of fiber cell development in cotton.Initially,an efficient protein preparation method was established for proteomic analysis of developing cotton fibers by two-dimensional gel electrophoresis,and a microwave enhanced ink staining technique also was created for fast and sensitive protein quantification in proteomic studies.

  6. Plant Cell Wall Proteins: A Large Body of Data, but What about Runaways?

    Science.gov (United States)

    Albenne, Cécile; Canut, Hervé; Hoffmann, Laurent; Jamet, Elisabeth

    2014-04-17

    Plant cell wall proteomics has been a very dynamic field of research for about fifteen years. A full range of strategies has been proposed to increase the number of identified proteins and to characterize their post-translational modifications. The protocols are still improving to enlarge the coverage of cell wall proteomes. Comparisons between these proteomes have been done based on various working strategies or different physiological stages. In this review, two points are highlighted. The first point is related to data analysis with an overview of the cell wall proteomes already described. A large body of data is now available with the description of cell wall proteomes of seventeen plant species. CWP contents exhibit particularities in relation to the major differences in cell wall composition and structure between these plants and between plant organs. The second point is related to methodology and concerns the present limitations of the coverage of cell wall proteomes. Because of the variety of cell wall structures and of the diversity of protein/polysaccharide and protein/protein interactions in cell walls, some CWPs can be missing either because they are washed out during the purification of cell walls or because they are covalently linked to cell wall components.

  7. Plant Cell Wall Proteins: A Large Body of Data, but What about Runaways?

    Directory of Open Access Journals (Sweden)

    Cécile Albenne

    2014-04-01

    Full Text Available Plant cell wall proteomics has been a very dynamic field of research for about fifteen years. A full range of strategies has been proposed to increase the number of identified proteins and to characterize their post-translational modifications. The protocols are still improving to enlarge the coverage of cell wall proteomes. Comparisons between these proteomes have been done based on various working strategies or different physiological stages. In this review, two points are highlighted. The first point is related to data analysis with an overview of the cell wall proteomes already described. A large body of data is now available with the description of cell wall proteomes of seventeen plant species. CWP contents exhibit particularities in relation to the major differences in cell wall composition and structure between these plants and between plant organs. The second point is related to methodology and concerns the present limitations of the coverage of cell wall proteomes. Because of the variety of cell wall structures and of the diversity of protein/polysaccharide and protein/protein interactions in cell walls, some CWPs can be missing either because they are washed out during the purification of cell walls or because they are covalently linked to cell wall components.

  8. 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.

  9. Cell Wall Biology: Perspectives from Cell Wall Imaging

    Institute of Scientific and Technical Information of China (English)

    Kieran J.D.Lee; Susan E.Marcus; J.Paul Knox

    2011-01-01

    Polysaccharide-rich plant cell walls are important biomaterials that underpin plant growth,are major repositories for photosynthetically accumulated carbon,and,in addition,impact greatly on the human use of plants. Land plant cell walls contain in the region of a dozen major polysaccharide structures that are mostly encompassed by cellulose,hemicelluloses,and pectic polysaccharides. During the evolution of land plants,polysaccharide diversification appears to have largely involved structural elaboration and diversification within these polysaccharide groups. Cell wall chemistry is well advanced and a current phase of cell wall science is aimed at placing the complex polysaccharide chemistry in cellular contexts and developing a detailed understanding of cell wall biology. Imaging cell wall glycomes is a challenging area but recent developments in the establishment of cell wall molecular probe panels and their use in high throughput procedures are leading to rapid advances in the molecular understanding of the spatial heterogeneity of individual cell walls and also cell wall differences at taxonomic levels. The challenge now is to integrate this knowledge of cell wall heterogeneity with an understanding of the molecular and physiological mechanisms that underpin cell wall properties and functions.

  10. Proteomic Analysis of Chinese Hamster Ovary Cells

    Science.gov (United States)

    Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama; Chen, Lily; Lewis, Nathan E.; Nagarajan, Harish; Sarkaria, Vishaldeep; Kumar, Amit; Wolozny, Daniel; Colao, Joe; Jacobson, Elena; Tian, Yuan; O'Meally, Robert N.; Krag, Sharon S.; Cole, Robert N.; Palsson, Bernhard O.; Zhang, Hui; Betenbaugh, Michael

    2013-01-01

    In order to complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multi-dimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most a 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using CHO genome exclusively which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. 504 of the detected proteins included N-acetylation modifications and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions. PMID:22971049

  11. Proteomic analysis of bovine blastocoel fluid and blastocyst cells

    DEFF Research Database (Denmark)

    Jensen, Pernille Linnert; Grøndahl, Marie Louise; Beck, Hans Christian

    2014-01-01

    development, little information is available about the protein complement of early embryos. Modern, sensitive proteomic technology (nano HPLC tandem mass spectrometry) allowed us to describe the proteome of the scarce blastocoel fluid and cell material of expanded bovine blastocysts isolated...

  12. Proteomic cornerstones of hematopoietic stem cell differentiation

    DEFF Research Database (Denmark)

    Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon

    2012-01-01

    Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors...... which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem....../progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5,000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical...

  13. Isolation of the Cell Wall.

    Science.gov (United States)

    Canut, Hervé; Albenne, Cécile; Jamet, Elisabeth

    2017-01-01

    This chapter describes a method allowing the purification of the cell wall for studying both polysaccharides and proteins. The plant primary cell wall is mainly composed of polysaccharides (90-95 % in mass) and of proteins (5-10 %). At the end of growth, specialized cells may synthesize a lignified secondary wall composed of polysaccharides (about 65 %) and lignin (about 35 %). Due to its composition, the cell wall is the cellular compartment having the highest density and this property is used for its purification. It plays critical roles during plant development and in response to environmental constraints. It is largely used in the food and textile industries as well as for the production of bioenergy. All these characteristics and uses explain why its study as a true cell compartment is of high interest. The proposed method of purification can be used for large amount of material but can also be downscaled to 500 mg of fresh material. Tools for checking the quality of the cell wall preparation, such as protein analysis and microscopy observation, are also provided.

  14. Cell Wall Metabolism in Response to Abiotic Stress

    Directory of Open Access Journals (Sweden)

    Hyacinthe Le Gall

    2015-02-01

    Full Text Available This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic, transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i an increased level in xyloglucan endotransglucosylase/hydrolase (XTH and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions.

  15. Catalysts of plant cell wall loosening

    OpenAIRE

    Cosgrove, Daniel J.

    2016-01-01

    The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyl...

  16. Advancing cell biology through proteomics in space and time (PROSPECTS)

    DEFF Research Database (Denmark)

    Lamond, A.I.; Uhlen, M.; Horning, S.

    2012-01-01

    a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU...... the proteomics field is moving beyond simply identifying proteins with high sensitivity toward providing a powerful and versatile set of assay systems for characterizing proteome dynamics and thereby creating a new "third generation" proteomics strategy that offers an indispensible tool for cell biology...... and molecular medicine. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc....

  17. Suspension-cultured plant cells as a tool to analyze the extracellular proteome.

    Science.gov (United States)

    Sabater-Jara, Ana B; Almagro, Lorena; Belchí-Navarro, Sarai; Martínez-Esteso, María J; Youssef, Sabry M; Casado-Vela, Juan; Vera-Urbina, Juan C; Sellés-Marchart, Susana; Bru-Martínez, Roque; Pedreño, María A

    2014-01-01

    Suspension-cultured cells (SCC) are generally considered the most suitable cell systems to carry out scientific studies, including the extracellular proteome (secretome). SCC are initiated by transferring friable callus fragments into flasks containing liquid culture medium for cell biomass growth, and they are maintained in an orbital shaker to supply the sufficient oxygen that allows cell growth. SCC increase rapidly during the exponential phase and after 10-20 days (depending on the cell culture nature), the growth rate starts to decrease due to limitation of nutrients, and to maintain for decades these kinds of cell cultures is needed to transfer a portion of these SCC into a fresh culture medium. Despite the central role played by extracellular proteins in most processes that control growth and development, the secretome has been less well characterized than other subcellular compartments, meaning that our understanding of the cell wall physiology is still very limited. Useful proteomic tools have emerged in recent years to unravel metabolic network that occurs in cell walls. With the recent progress made in mass spectrometry technology, it has become feasible to identify proteins from a given organ, tissue, cells, or even a subcellular compartment. Compared with other methods used to isolate cell wall proteins, the spent medium of SCC provides a convenient, continuous, and reliable and unique source of extracellular proteins. Therefore, this biological system could be used as a large-scale cell culture from which these proteins can be secreted, easily separated from cells without cell disruption, and so, without any cytosolic contamination, easily recovered from the extracellular medium. This nondestructive cell wall proteome approach discloses a set of proteins that are specifically expressed in the remodelling of the cell wall architecture and stress defense.

  18. Proteomics

    DEFF Research Database (Denmark)

    Tølbøll, Trine Højgaard; Danscher, Anne Mette; Andersen, Pia Haubro;

    2012-01-01

    different proteins were identified, with 146 proteins available for identification in C, 279 proteins in D and 269 proteins in L. A functional annotation of the identified proteins was obtained using the on-line Blast2GO tool. Three hundred and sixteen of the identified proteins could be subsequently...... grouped manually to one or more of five major functional groups related to metabolism, cell structure, immunity, apoptosis and angiogenesis. These were chosen to represent basic cell functions and biological processes potentially involved in the pathogenesis of CHD. The LC–MS/MS-based proteomic analysis...

  19. Identification of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics and Yield in Rice (Oryza Sativa)

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Zhaohua PEng [Mississippi State University; Ronald, Palmela [UC-Davis; Wang, Guo-Liang [The Ohio State University

    2013-04-26

    This project aims to identify the regulatory genes of rice cell wall synthesis pathways using a cell wall removal and regeneration system. We completed the gene expression profiling studies following the time course from cell wall removal to cell wall regeneration in rice suspension cells. We also completed, total proteome, nuclear subproteome and histone modification studies following the course from cell wall removal and cell wall regeneration process. A large number of differentially expressed regulatory genes and proteins were identified. Meanwhile, we generated RNAi and over-expression transgenic rice for 45 genes with at least 10 independent transgenic lines for each gene. In addition, we ordered T-DNA and transposon insertion mutants for 60 genes from Korea, Japan, and France and characterized the mutants. Overall, we have mutants and transgenic lines for over 90 genes, exceeded our proposed goal of generating mutants for 50 genes. Interesting Discoveries a) Cell wall re-synthesis in protoplasts may involve a novel cell wall synthesis mechanism. The synthesis of the primary cell wall is initiated in late cytokinesis with further modification during cell expansion. Phragmoplast plays an essential role in cell wall synthesis. It services as a scaffold for building the cell plate and formation of a new cell wall. Only one phragmoplast and one new cell wall is produced for each dividing cell. When the cell wall was removed enzymatically, we found that cell wall re-synthesis started from multiple locations simultaneously, suggesting that a novel mechanism is involved in cell wall re-synthesis. This observation raised many interesting questions, such as how the starting sites of cell wall synthesis are determined, whether phragmoplast and cell plate like structures are involved in cell wall re-synthesis, and more importantly whether the same set of enzymes and apparatus are used in cell wall re-synthesis as during cytokinesis. Given that many known cell wall

  20. Cell-specific proteomic analysis in Caenorhabditis elegans.

    Science.gov (United States)

    Yuet, Kai P; Doma, Meenakshi K; Ngo, John T; Sweredoski, Michael J; Graham, Robert L J; Moradian, Annie; Hess, Sonja; Schuman, Erin M; Sternberg, Paul W; Tirrell, David A

    2015-03-03

    Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.

  1. Shape dynamics of growing cell walls

    CERN Document Server

    Banerjee, Shiladitya; Dinner, Aaron R

    2015-01-01

    We introduce a general theoretical framework to study the shape dynamics of actively growing and remodeling surfaces. Using this framework we develop a physical model for growing bacterial cell walls and study the interplay of cell shape with the dynamics of growth and constriction. The model allows us to derive constraints on cell wall mechanical energy based on the observed dynamics of cell shape. We predict that exponential growth in cell size requires a constant amount of cell wall energy to be dissipated per unit volume. We use the model to understand and contrast growth in bacteria with different shapes such as spherical, ellipsoidal, cylindrical and toroidal morphologies. Coupling growth to cell wall constriction, we predict a discontinuous shape transformation, from partial constriction to cell division, as a function of the chemical potential driving cell-wall synthesis. Our model for cell wall energy and shape dynamics relates growth kinetics with cell geometry, and provides a unified framework to d...

  2. Proteome adaptation in cell reprogramming proceeds via distinct transcriptional networks.

    Science.gov (United States)

    Benevento, Marco; Tonge, Peter D; Puri, Mira C; Hussein, Samer M I; Cloonan, Nicole; Wood, David L; Grimmond, Sean M; Nagy, Andras; Munoz, Javier; Heck, Albert J R

    2014-12-10

    The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quantitative mass spectrometry-based analysis to probe in-depth dynamic proteome changes during somatic cell reprogramming. Our data reveal defined waves of proteome resetting, with the first wave occurring 48 h after the activation of the reprogramming transgenes and involving specific biological processes linked to the c-Myc transcriptional network. A second wave of proteome reorganization occurs in a later stage of reprogramming, where we characterize the proteome of two distinct pluripotent cellular populations. In addition, the overlay of our proteome resource with parallel generated -omics data is explored to identify post-transcriptionally regulated proteins involved in key steps during reprogramming.

  3. Microanalysis of Plant Cell Wall Polysaccharides

    Institute of Scientific and Technical Information of China (English)

    Nicolai Obel; Veronika Erben; Tatjana Schwarz; Stefan Kühne; Andrea Fodor; Markus Pauly

    2009-01-01

    Oligosaccharide Mass Profiling (OLIMP) allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry (MALDI-TOF MS). The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first iso-lating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apo-plastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.

  4. Exosome Proteome of U-87MG Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Sohyun Chun

    2016-12-01

    Full Text Available Exosomes are small membrane vesicles between 30 and 100 nm in diameter secreted by many cell types, and are associated with a wide range of physiological and/or pathological processes. Exosomes containing proteins, lipids, mRNA, and microRNA contribute to cell-to-cell communication and cell-to-environment regulation, however, their biological functions are not yet fully understood. In this report, exosomes in the glioblastoma cell line, U-87MG, were isolated and the proteome was investigated. In addition, exosome proteome changes in U-87MG cells exposed to a low temperature were investigated to elucidate whether the exosome proteome could respond to an external stimulus. Cell culture medium was collected, and exosomes were isolated by continuous centrifugation eliminating cell debris, nucleic acids, and other particles. The morphology of exosomes was observed by cryo-tunneling electron microscopy. According to 2-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, certain proteins including collagen type VI alpha 1, putative RNA-binding protein 15B chain A, substrate induced remodeling of the active site regulates HTRA1, coatomer protein complex-subunit beta 2, myosin-heavy chain 1, and keratin-type I cytoskeletal 9 showed differences between the control proteome and the low temperature-exposed proteome.

  5. Advancing cell biology through proteomics in space and time (PROSPECTS).

    Science.gov (United States)

    Lamond, Angus I; Uhlen, Mathias; Horning, Stevan; Makarov, Alexander; Robinson, Carol V; Serrano, Luis; Hartl, F Ulrich; Baumeister, Wolfgang; Werenskiold, Anne Katrin; Andersen, Jens S; Vorm, Ole; Linial, Michal; Aebersold, Ruedi; Mann, Matthias

    2012-03-01

    The term "proteomics" encompasses the large-scale detection and analysis of proteins and their post-translational modifications. Driven by major improvements in mass spectrometric instrumentation, methodology, and data analysis, the proteomics field has burgeoned in recent years. It now provides a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU-funded project that brings together leading European research groups, spanning from instrumentation to biomedicine, in a collaborative five year initiative to develop new methods and applications for the functional analysis of cellular proteins. This special issue of Molecular and Cellular Proteomics presents 16 research papers reporting major recent progress by the PROSPECTS groups, including improvements to the resolution and sensitivity of the Orbitrap family of mass spectrometers, systematic detection of proteins using highly characterized antibody collections, and new methods for absolute as well as relative quantification of protein levels. Manuscripts in this issue exemplify approaches for performing quantitative measurements of cell proteomes and for studying their dynamic responses to perturbation, both during normal cellular responses and in disease mechanisms. Here we present a perspective on how the proteomics field is moving beyond simply identifying proteins with high sensitivity toward providing a powerful and versatile set of assay systems for characterizing proteome dynamics and thereby creating a new "third generation" proteomics strategy that offers an indispensible tool for cell biology and molecular medicine.

  6. The cell surface proteome of Entamoeba histolytica.

    Science.gov (United States)

    Biller, Laura; Matthiesen, Jenny; Kühne, Vera; Lotter, Hannelore; Handal, Ghassan; Nozaki, Tomoyoshi; Saito-Nakano, Yumiko; Schümann, Michael; Roeder, Thomas; Tannich, Egbert; Krause, Eberhard; Bruchhaus, Iris

    2014-01-01

    Surface molecules are of major importance for host-parasite interactions. During Entamoeba histolytica infections, these interactions are predicted to be of prime importance for tissue invasion, induction of colitis and liver abscess formation. To date, however, little is known about the molecules involved in these processes, with only about 20 proteins or protein families found exposed on the E. histolytica surface. We have therefore analyzed the complete surface proteome of E. histolytica. Using cell surface biotinylation and mass spectrometry, 693 putative surface-associated proteins were identified. In silico analysis predicted that ∼26% of these proteins are membrane-associated, as they contain transmembrane domains and/or signal sequences, as well as sites of palmitoylation, myristoylation, or prenylation. An additional 25% of the identified proteins likely represent nonclassical secreted proteins. Surprisingly, no membrane-association sites could be predicted for the remaining 49% of the identified proteins. To verify surface localization, 23 proteins were randomly selected and analyzed by immunofluorescence microscopy. Of these 23 proteins, 20 (87%) showed definite surface localization. These findings indicate that a far greater number of E. histolytica proteins than previously supposed are surface-associated, a phenomenon that may be based on the high membrane turnover of E. histolytica.

  7. [The cell wall of Coelastrum (Chlorophycees)].

    Science.gov (United States)

    Reymond, O

    1975-01-01

    The cell wall of Coelastrum is usually composed of three layers. The outermost layer was studied most extensively. It consists of erect tubules which often bear long bristles whose function may be to stabilize the algae in its enviroment. The cell wall can modify its morphology according to the enviroment.

  8. Red blood cell (RBC) membrane proteomics--Part I: Proteomics and RBC physiology.

    Science.gov (United States)

    Pasini, Erica M; Lutz, Hans U; Mann, Matthias; Thomas, Alan W

    2010-01-03

    Membrane proteomics is concerned with accurately and sensitively identifying molecules involved in cell compartmentalisation, including those controlling the interface between the cell and the outside world. The high lipid content of the environment in which these proteins are found often causes a particular set of problems that must be overcome when isolating the required material before effective HPLC-MS approaches can be performed. The membrane is an unusually dynamic cellular structure since it interacts with an ever changing environment. A full understanding of this critical cell component will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. Devoid of nucleus and organelles in mammalian species other than camelids, and constantly in motion in the blood stream, red blood cells (RBCs) are the sole mammalian oxygen transporter. The fact that mature mammalian RBCs have no internal membrane-bound organelles, somewhat simplifies proteomics analysis of the plasma membrane and the fact that it has no nucleus disqualifies microarray based methods. Proteomics has the potential to provide a better understanding of this critical interface, and thereby assist in identifying new approaches to diseases.

  9. Proteins associated with the size and expansion rate of the abdominal aortic aneurysm wall as identified by proteomic analysis

    DEFF Research Database (Denmark)

    Urbonavicius, Sigitas; Lindholt, Jes S.; Delbosc, Sandrine

    2010-01-01

    Identification of biomarkers for the natural history of abdominal aortic aneurysms (AAA) holds the key to non-surgical intervention and improved selection for AAA repair. We aimed to associate the basic proteomic composition of AAA wall tissue with the expansion rate and size in patients with AAA....

  10. Mitotic spindle proteomics in Chinese hamster ovary cells.

    Directory of Open Access Journals (Sweden)

    Mary Kate Bonner

    Full Text Available Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.

  11. Proteomics

    DEFF Research Database (Denmark)

    Dam, Svend; Stougaard, Jens

    2014-01-01

    proteomics data. Two characteristics of legumes are the high seed protein level and the nitrogen fixing symbiosis. Thus, the majority of the proteomics studies in Lotus have been performed on seed/pod and nodule/root tissues in order to create proteome reference maps and to enable comparative analyses within...... Lotus tissues or toward similar tissues from other legume species. More recently, N-glycan structures and compositions have been determined from mature Lotus seeds using glycomics and glycoproteomics, and finally, phosphoproteomics has been employed...... and annotated Lotus japonicus (Lotus) genome has been essential for obtaining high-quality protein identifications from proteomics studies. Furthermore, additional genomics and transcriptomics studies from several Lotus species/ecotypes support putative gene structures and these can be further supported using...

  12. Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200.

    Science.gov (United States)

    De Angelis, Maria; Siragusa, Sonya; Campanella, Daniela; Di Cagno, Raffaella; Gobbetti, Marco

    2015-07-01

    This study investigated the relative abundance of extracellular and cell wall associated proteins (exoproteome), cytoplasmic proteins (proteome), and related phenotypic traits of Lactobacillus plantarum grown under planktonic and biofilm conditions. Lactobacillus plantarum DB200 was preliminarily selected due to its ability to form biofilms and to adhere to Caco2 cells. As shown by fluorescence microscope analysis, biofilm cells became longer and autoaggregated at higher levels than planktonic cells. The molar ratio between glucose consumed and lactate synthesised was markedly decreased under biofilm compared to planktonic conditions. DIGE analysis showed a differential exoproteome (115 protein spots) and proteome (44) between planktonic and biofilm L. plantarum DB200 cells. Proteins up- or downregulated by at least twofold (p < 0.05) were found to belong mainly to the following functional categories: cell wall and catabolic process, cell cycle and adhesion, transport, glycolysis and carbohydrate metabolism, exopolysaccharide metabolism, amino acid and protein metabolisms, fatty acid and lipid biosynthesis, purine and nucleotide metabolism, stress response, oxidation/reduction process, and energy metabolism. Many of the above proteins showed moonlighting behavior. In accordance with the high expression levels of stress proteins (e.g., DnaK, GroEL, ClpP, GroES, and catalase), biofilm cells demonstrated enhanced survival under conditions of environmental stress.

  13. Accelerating forward genetics for cell wall deconstruction

    Directory of Open Access Journals (Sweden)

    Danielle eVidaurre

    2012-06-01

    Full Text Available One of the biggest challenges of cell wall biology is the elucidation of the genes involved the cell wall and their function due to the recalcitrance of the cell wall. Through traditional genetic approaches, many simple yet elegant screens have been able to identify components of the cell wall and their networks. Despite progress in the identification of several genes of the cell wall, there remain many unknown players whose function has yet to be determined. Exhausting the genetic toolbox by performing secondary screens on a genetically mutated background, chemical genetics using small molecules and improved cell wall imaging hold promise for new gene discovery and function. With the recent introduction of next-generation sequencing technologies, it is now possible to quickly and efficiently map and clone genes of interest in Arabidopsis and any model organism with a completed genome sequence. The combination of a classical genetics approach and cutting edge technology will propel cell wall biology of Arabidopsis and other useful crops forward into the future.

  14. Molecular regulation of plant cell wall extensibility

    Science.gov (United States)

    Cosgrove, D. J.

    1998-01-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized.

  15. 2003 Plant Cell Walls Gordon Conference

    Energy Technology Data Exchange (ETDEWEB)

    Daniel J. Cosgrove

    2004-09-21

    This conference will address recent progress in many aspects of cell wall biology. Molecular, genetic, and genomic approaches are yielding major advances in our understanding of the composition, synthesis, and architecture of plant cell walls and their dynamics during growth, and are identifying the genes that encode the machinery needed to make their biogenesis possible. This meeting will bring together international scientists from academia, industry and government labs to share the latest breakthroughs and perspectives on polysaccharide biosynthesis, wood formation, wall modification, expansion and interaction with other organisms, and genomic & evolutionary analyses of wall-related genes, as well as to discuss recent ''nanotechnological'' advances that take wall analysis to the level of a single cell.

  16. Unique proteomic signatures distinguish macrophages and dendritic cells.

    Directory of Open Access Journals (Sweden)

    Lev Becker

    Full Text Available Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow-derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo.

  17. Function of laccases in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Larsen, Anders; Holm, Preben Bach; Andersen, Jeppe Reitan

    2011-01-01

    substrate specificities and expression patterns. As part of the strategic research centre Bio4Bio, the present project deals with laccase functions in relation to cell wall formation in grasses based on a study of the model species Brachypodium distachyon. Thirty-one isozymes have been retrieved from......Laccases are multicopper oxidases capable of polymerizing monolignols. Histochemical assays have shown temporal and spatial correlation with secondary cell wall formation in both herbs and woody perennials. However, in plants laccases constitutes a relatively large group of isoenzymes with unique...... hybridization. Specific isozymes that show high correlation with the process of secondary cell wall formation will be further studied in a reverse genetic study in which candidates will be knocked out using RNA interference. Phenotypes of knock-out mutants are to be described in relation to cell wall...

  18. Trans-Golgi Network-An Intersection of Trafficking Cell Wall Components

    Institute of Scientific and Technical Information of China (English)

    Natasha Worden; Eunsook Park; Georgia Drakakaki

    2012-01-01

    The cell wall,a crucial cell compartment,is composed of a network of polysaccharides and proteins,providing structural support and protection from external stimuli.While the cell wall structure and biosynthesis have been extensively studied,very little is known about the transport of polysaccharides and other components into the developing cell wall.This review focuses on endomembrane trafficking pathways involved in cell wall deposition.Cellulose synthase complexes are assembled in the Golgi,and are transported in vesicles to the plasma membrane.Non-cellulosic polysaccharides are synthesized in the Golgi apparatus,whereas cellulose is produced by enzyme complexes at the plasma membrane.Polvsaccharides and enzymes that are involved in cell wall modification and assembly are transported by distinct vesicle types to their destinations; however,the precise mechanisms involved in selection,sorting and delivery remain to be identified.The endomembrane system orchestrates the delivery of Golgi-derived and possibly endocytic vesicles carrying cell wall and cell membrane components to the newly-formed cell plate.However,the nature of these vesicles,their membrane compositions,and the timing of their delivery are largely unknown.Emerging technologies such as chemical genomics and proteomics are promising avenues to gain insight into the trafficking of cell wall components.

  19. Biomaterial surface proteomic signature determines interaction with epithelial cells.

    Science.gov (United States)

    Abdallah, Mohamed-Nur; Tran, Simon D; Abughanam, Ghada; Laurenti, Marco; Zuanazzi, David; Mezour, Mohamed A; Xiao, Yizhi; Cerruti, Marta; Siqueira, Walter L; Tamimi, Faleh

    2017-03-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices.

  20. Proteomic profiling of the human T-cell nucleolus.

    Science.gov (United States)

    Jarboui, Mohamed Ali; Wynne, Kieran; Elia, Giuliano; Hall, William W; Gautier, Virginie W

    2011-12-01

    The nucleolus, site of ribosome biogenesis, is a dynamic subnuclear organelle involved in diverse cellular functions. The size, number and organisation of nucleoli are cell-specific and while it remains to be established, the nucleolar protein composition would be expected to reflect lineage-specific transcriptional regulation of rDNA genes and have cell-type functional components. Here, we describe the first characterisation of the human T-cell nucleolar proteome. Using the Jurkat T-cell line and a reproducible organellar proteomic approach, we identified 872 nucleolar proteins. In addition to ribosome biogenesis and RNA processing networks, network modeling and topological analysis of nucleolar proteome revealed distinct macromolecular complexes known to orchestrate chromatin structure and to contribute to the regulation of gene expression, replication, recombination and repair, and chromosome segregation. Furthermore, among our dataset, we identified proteins known to functionally participate in T-cell biology, including RUNX1, ILF3, ILF2, STAT3, LSH, TCF-1, SATB1, CTCF, HMGB3, BCLAF1, FX4L1, ZAP70, TIAM1, RAC2, THEMIS, LCP1, RPL22, TOPK, RETN, IFI-16, MCT-1, ISG15, and 14-3-3τ, which support cell-specific composition of the Jurkat nucleolus. Subsequently, the nucleolar localisation of RUNX1, ILF3, STAT3, ZAP70 and RAC2 was further validated by Western Blot analysis and immunofluorescence microscopy. Overall, our T-cell nucleolar proteome dataset not only further expands the existing repertoire of the human nucleolar proteome but support a cell type-specific composition of the nucleolus in T cell and highlights the potential roles of the nucleoli in lymphocyte biology.

  1. Plant organelle proteomics: collaborating for optimal cell function.

    Science.gov (United States)

    Agrawal, Ganesh Kumar; Bourguignon, Jacques; Rolland, Norbert; Ephritikhine, Geneviève; Ferro, Myriam; Jaquinod, Michel; Alexiou, Konstantinos G; Chardot, Thierry; Chakraborty, Niranjan; Jolivet, Pascale; Doonan, John H; Rakwal, Randeep

    2011-01-01

    Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function. For example, one of the striking differences between plant and animal cells is the plastids in plants. Organelles have their own proteins, and few organelles like mitochondria and chloroplast have their own genome to synthesize proteins for specific function and also require nuclear-encoded proteins. Enormous work has been performed on animal organelle proteomics. However, plant organelle proteomics has seen limited work mainly due to: (i) inter-plant and inter-tissue complexity, (ii) difficulties in isolation of subcellular compartments, and (iii) their enrichment and purity. Despite these concerns, the field of organelle proteomics is growing in plants, such as Arabidopsis, rice and maize. The available data are beginning to help better understand organelles and their distinct and/or overlapping functions in different plant tissues, organs or cell types, and more importantly, how protein components of organelles behave during development and with surrounding environments. Studies on organelles have provided a few good reviews, but none of them are comprehensive. Here, we present a comprehensive review on plant organelle proteomics starting from the significance of organelle in cells, to organelle isolation, to protein identification and to biology and beyond. To put together such a systematic, in-depth review and to translate acquired knowledge in a proper and adequate form, we join minds to provide discussion and viewpoints on the collaborative nature of organelles in

  2. Modes of deformation of walled cells.

    Science.gov (United States)

    Dumais, Jacques

    2013-11-01

    The bewildering morphological diversity found in cells is one of the starkest illustrations of life's ability to self-organize. Yet the morphogenetic mechanisms that produce the multifarious shapes of cells are still poorly understood. The shared similarities between the walled cells of prokaryotes, many protists, fungi, and plants make these groups particularly appealing to begin investigating how morphological diversity is generated at the cell level. In this review, I attempt a first classification of the different modes of surface deformation used by walled cells. Five modes of deformation were identified: inextensional bending, equi-area shear, elastic stretching, processive intussusception, and chemorheological growth. The two most restrictive modes-inextensional and equi-area deformations-are embodied in the exine of pollen grains and the wall-like pellicle of euglenoids, respectively. For these modes, it is possible to express the deformed geometry of the cell explicitly in terms of the undeformed geometry and other easily observable geometrical parameters. The greatest morphogenetic power is reached with the processive intussusception and chemorheological growth mechanisms that underlie the expansive growth of walled cells. A comparison of these two growth mechanisms suggests a possible way to tackle the complexity behind wall growth.

  3. Identification of Novel Cell Wall Components

    Energy Technology Data Exchange (ETDEWEB)

    Michelle Momany

    2009-10-26

    Our DOE Biosciences-funded work focused on the fungal cell wall and morphogenesis. We are especially interested in how new cell wall material is targeted to appropriate areas for polar (asymmetric) growth. Polar growth is the only way that filamentous fungi explore the environment to find suitable substrates to degrade. Work funded by this grant has resulted in a total of twenty peer-reviewed publications. In work funded by this grant, we identified nine Aspergillus nidulans temperature-sensitive (ts) mutants that fail to send out a germ tube and show a swollen cell phenotype at restrictive temperature, the swo mutants. In other organisms, a swollen cell phenotype is often associated with misdirected growth or weakened cell walls. Our work shows that several of the A. nidulans swo mutants have defects in the establishment and maintenance of polarity. Cloning of several swo genes by complementation also showed that secondary modification of proteins seems is important in polarity. We also investigated cell wall biosynthesis and branching based on leads in literature from other organisms and found that branching and nuclear division are tied and that the cell wall reorganizes during development. In our most recent work we have focused on gene expression during the shift from isotropic to polar growth. Surprisingly we found that genes previously thought to be involved only in spore formation are important in early vegetative growth as well.

  4. The proteome of neural stem cells from adult rat hippocampus

    Directory of Open Access Journals (Sweden)

    Fütterer Carsten D

    2003-06-01

    Full Text Available Abstract Background Hippocampal neural stem cells (HNSC play an important role in cerebral plasticity in the adult brain and may contribute to tissue repair in neurological disease. To describe their biological potential with regard to plasticity, proliferation, or differentiation, it is important to know the cellular composition of their proteins, subsumed by the term proteome. Results Here, we present for the first time a proteomic database for HNSC isolated from the brains of adult rats and cultured for 10 weeks. Cytosolic proteins were extracted and subjected to two-dimensional gel electrophoresis followed by protein identification through mass spectrometry, database search, and gel matching. We could map about 1141 ± 209 (N = 5 protein spots for each gel, of which 266 could be identified. We could group the identified proteins into several functional categories including metabolism, protein folding, energy metabolism and cellular respiration, as well as cytoskeleton, Ca2+ signaling pathways, cell cycle regulation, proteasome and protein degradation. We also found proteins belonging to detoxification, neurotransmitter metabolism, intracellular signaling pathways, and regulation of DNA transcription and RNA processing. Conclusions The HNSC proteome database is a useful inventory which will allow to specify changes in the cellular protein expression pattern due to specific activated or suppressed pathways during differentiation or proliferation of neural stem cells. Several proteins could be identified in the HNSC proteome which are related to differentiation and plasticity, indicating activated functional pathways. Moreover, we found a protein for which no expression has been described in brain cells before.

  5. The time is right: proteome biology of stem cells.

    NARCIS (Netherlands)

    Whetton, A.D.; Williamson, A.J.K.; Krijgsveld, J.; Lee, B.H.; Lemischka, I.; Oh, S.; Pera, M.; Mummery, C.L.; Heck, A.J.R.

    2008-01-01

    In stem cell biology, there is a growing need for advanced technologies that may help to unravel the molecular mechanisms of self-renewal and differentiation. Proteomics, the comprehensive analysis of proteins, is such an emerging technique. To facilitate interactions between specialists in proteomi

  6. A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana: Confirmed actors and newcomers

    Directory of Open Access Journals (Sweden)

    Jamet Elisabeth

    2008-09-01

    Full Text Available Abstract Background Cell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time. Results Two developmental stages (active growth and after growth arrest were compared. A new strategy consisting of high performance cation exchange chromatography and mono-dimensional electrophoresis was established for separation of cell wall proteins. This work allowed identification of 137 predicted secreted proteins, among which 51 had not been identified previously. Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function. Conclusion This work highlights the CWP dynamics that takes place between the two developmental stages. The presence of proteins known to be related to cell wall extension after growth arrest showed that these proteins may play other roles in cell walls. Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.

  7. "Steiner trees" between cell walls of sisal

    Institute of Scientific and Technical Information of China (English)

    LI GuanShi; YIN YaJun; LI Yan; ZHONG Zheng

    2009-01-01

    Through careful analysis on the cross-section of sisal fibers,it is found that the middle lamellae between the cell walls have clear geometric characteristics:between the cell walls of three neighboring cells,the middle lamellae form a three-way junction with 120°symmetry. If the neighboring three-way junctions are connected,a network of Steiner tree with angular symmetry and topological invariability is formed. If more and more Steiner trees are connected,a network of Steiner rings is generated. In another word,idealized cell walls and the middle lamellae are dominated by the Steiner geometry. This geometry not only depicts the geometric symmetry,the topological invariability and minimal property of the middle lamellae,but also controls the mechanics of sisal fibers.

  8. Proteomic Applications in the Study of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Jesús Mateos

    2014-02-01

    Full Text Available Mesenchymal stem cells (MSCs are undifferentiated cells with an unlimited capacity for self-renewal and able to differentiate towards specific lineages under appropriate conditions. MSCs are, a priori, a good target for cell therapy and clinical trials as an alternative to embryonic stem cells, avoiding ethical problems and the chance for malignant transformation in the host. However, regarding MSCs, several biological implications must be solved before their application in cell therapy, such as safe ex vivo expansion and manipulation to obtain an extensive cell quantity amplification number for use in the host without risk accumulation of genetic and epigenetic abnormalities. Cell surface markers for direct characterization of MSCs remain unknown, and the precise molecular mechanisms whereby growth factors stimulate their differentiation are still missing. In the last decade, quantitative proteomics has emerged as a promising set of techniques to address these questions, the answers to which will determine whether MSCs retain their potential for use in cell therapy. Proteomics provides tools to globally analyze cellular activity at the protein level. This proteomic profiling allows the elucidation of connections between broad cellular pathways and molecules that were previously impossible to determine using only traditional biochemical analysis. However; thus far, the results obtained must be orthogonally validated with other approaches. This review will focus on how these techniques have been applied in the evaluation of MSCs for their future applications in safe therapies.

  9. Proteomic Applications in the Study of Human Mesenchymal Stem Cells

    Science.gov (United States)

    Mateos, Jesús; Fernández Pernas, Pablo; Fafián Labora, Juan; Blanco, Francisco; Arufe, María del Carmen

    2014-01-01

    Mesenchymal stem cells (MSCs) are undifferentiated cells with an unlimited capacity for self-renewal and able to differentiate towards specific lineages under appropriate conditions. MSCs are, a priori, a good target for cell therapy and clinical trials as an alternative to embryonic stem cells, avoiding ethical problems and the chance for malignant transformation in the host. However, regarding MSCs, several biological implications must be solved before their application in cell therapy, such as safe ex vivo expansion and manipulation to obtain an extensive cell quantity amplification number for use in the host without risk accumulation of genetic and epigenetic abnormalities. Cell surface markers for direct characterization of MSCs remain unknown, and the precise molecular mechanisms whereby growth factors stimulate their differentiation are still missing. In the last decade, quantitative proteomics has emerged as a promising set of techniques to address these questions, the answers to which will determine whether MSCs retain their potential for use in cell therapy. Proteomics provides tools to globally analyze cellular activity at the protein level. This proteomic profiling allows the elucidation of connections between broad cellular pathways and molecules that were previously impossible to determine using only traditional biochemical analysis. However; thus far, the results obtained must be orthogonally validated with other approaches. This review will focus on how these techniques have been applied in the evaluation of MSCs for their future applications in safe therapies.

  10. Proteomics research on muscle-invasive bladder transitional cell carcinoma

    Directory of Open Access Journals (Sweden)

    Cao Yan

    2011-06-01

    Full Text Available Abstract Background Aimed to facilitate candidate biomarkers selection and improve network-based multi-target therapy, we perform comparative proteomics research on muscle-invasive bladder transitional cell carcinoma. Laser capture microdissection was used to harvest purified muscle-invasive bladder cancer cells and normal urothelial cells from 4 paired samples. Two-dimensional liquid chromatography tandem mass spectrometry was used to identify the proteome expression profile. The differential proteins were further analyzed using bioinformatics tools and compared with the published literature. Results A total of 885/890 proteins commonly appeared in 4 paired samples. 295/337 of the 488/493 proteins that specific expressed in tumor/normal cells own gene ontology (GO cellular component annotation. Compared with the entire list of the international protein index (IPI, there are 42/45 GO terms exhibited as enriched and 9/5 exhibited as depleted, respectively. Several pathways exhibit significantly changes between cancer and normal cells, mainly including spliceosome, endocytosis, oxidative phosphorylation, etc. Finally, descriptive statistics show that the PI Distribution of candidate biomarkers have certain regularity. Conclusions The present study identified the proteome expression profile of muscle-invasive bladder cancer cells and normal urothelial cells, providing information for subcellular pattern research of cancer and offer candidate proteins for biomarker panel and network-based multi-target therapy.

  11. Cell wall oxalate oxidase modifies the ferulate metabolism in cell walls of wheat shoots.

    Science.gov (United States)

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki

    2011-11-01

    Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.

  12. Enzymatic Modification of Plant Cell Wall Polysaccharides

    DEFF Research Database (Denmark)

    Øbro, Jens; Hayashi, Takahisa; Mikkelsen, Jørn Dalgaard

    2011-01-01

    fibres, hydrocolloids, paper,textile, animal feeds or biofuels. Classical microbial-based fermentation systems could in the future face serious competition from plant-based expression systems for enzyme production. Plant expressed enzymes can either be targeted to specific cellular compartments......Plant cell walls are intricate structures with remarkable properties, widely used in almost every aspect of our life. Cell walls consist largely of complex polysaccharides and there is often a need for chemical and biochemical processing before industrial use. There is an increasing demand...... for sustainable processes that replace chemical treatments with white biotechnology. Plants can contribute significantly to this sustainable process by producing plant or microbialenzymes in planta that are necessary for plant cell wall modification or total degradation. This will give rise to superior food...

  13. Differential proteome analysis of chikungunya virus infection on host cells.

    Directory of Open Access Journals (Sweden)

    Christina Li-Ping Thio

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach. METHODOLOGY AND PRINCIPAL FINDINGS: The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE. Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP and cell cycle regulation. CONCLUSION/SIGNIFICANCE: This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1 regulation (in favour of virus survival, replication and transmission. While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.

  14. Xyloglucan endotransglucosylase and cell wall extensibility.

    Science.gov (United States)

    Miedes, E; Zarra, I; Hoson, T; Herbers, K; Sonnewald, U; Lorences, E P

    2011-02-15

    Transgenic tomato hypocotyls with altered levels of an XTH gene were used to study how XET activity could affect the hypocotyl growth and cell wall extensibility. Transgenic hypocotyls showed significant over-expression (line 13) or co-suppression (line 33) of the SlXTH1 in comparison with the wild type, with these results being correlated with the results on specific soluble XET activity, suggesting that SlXTH1 translates mainly for a soluble XET isoenzyme. A relationship between XET activity and cell wall extensibility was found, and the highest total extensibility was located in the apical hypocotyl segment of the over-expressing SlXTH1 line, where the XET-specific activity and hypocotyl growth were also highest compared with the wild line. Also, in the co-suppression SlXTH1 line, total extensibility values were lower than in the wild type line. The study of linkages between cell wall polysaccharides by FTIR showed that hypocotyls over-expressing SlXTH1 and having a higher XET-specific activity, were grouped away from the wild line, indicating that the linkages between pectins and between cellulose and xyloglucans might differ. These results suggested that the action of the increased XET activity in the transgenic line could be responsible for the cell wall structural changes, and therefore, alter the cell wall extensibility. On the other hand, results on xyloglucan oligosaccharides composition of the xyloglucan by MALDI TOF-MS showed no differences between lines, indicating that the xyloglucan structure was not affected by the XET action. These results provide evidences that XTHs from group I are involved mainly in the restructuring of the cell wall during growth and development, but they are not the limiting factor for plant growth.

  15. Cell Wall Diversity in Forage Maize

    NARCIS (Netherlands)

    Torres, A.F.; Noordam-Boot, C.M.M.; Dolstra, Oene; Weijde, van der Tim; Combes, Eliette; Dufour, Philippe; Vlaswinkel, Louis; Visser, R.G.F.; Trindade, L.M.

    2015-01-01

    Genetic studies are ideal platforms for assessing the extent of genetic diversity, inferring the genetic architecture, and evaluating complex trait interrelations for cell wall compositional and bioconversion traits relevant to bioenergy applications. Through the characterization of a forage maiz

  16. Microanalysis of Plant Cell Wall Polysaccharides

    NARCIS (Netherlands)

    Obel, N.; Erben, V.; Schwarz, T.; Kühnel, S.; Fodor, A.; Pauly, M.

    2009-01-01

    Oligosaccharide Mass Profiling (OLIMP) allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry (MALDI-TOF MS). The short time required for sample preparation and analysis makes possible the s

  17. A proteome map of primary cultured rat Schwann cells

    Directory of Open Access Journals (Sweden)

    Shen Mi

    2012-03-01

    Full Text Available Abstract Background Schwann cells (SCs are the principal glial cells of the peripheral nervous system with a wide range of biological functions. SCs play a key role in peripheral nerve regeneration and are involved in several hereditary peripheral neuropathies. The objective of this study was to gain new insight into the whole protein composition of SCs. Results Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC-MS/MS was performed to identify the protein expressions in primary cultured SCs of rats. We identified a total of 1,232 proteins, which were categorized into 20 functional classes. We also used quantitative real time RT-PCR and Western blot analysis to validate some of proteomics-identified proteins. Conclusion We showed for the first time the proteome map of SCs. Our data could serve as a reference library to provide basic information for understanding SC biology.

  18. Comprehensive proteomic characterization of stem cell-derived extracellular matrices.

    Science.gov (United States)

    Ragelle, Héloïse; Naba, Alexandra; Larson, Benjamin L; Zhou, Fangheng; Prijić, Miralem; Whittaker, Charles A; Del Rosario, Amanda; Langer, Robert; Hynes, Richard O; Anderson, Daniel G

    2017-06-01

    In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as in vitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering.

  19. Cell Wall Heterogeneity in Root Development of Arabidopsis

    Science.gov (United States)

    Somssich, Marc; Khan, Ghazanfar Abbas; Persson, Staffan

    2016-01-01

    Plant cell walls provide stability and protection to plant cells. During growth and development the composition of cell walls changes, but provides enough strength to withstand the turgor of the cells. Hence, cell walls are highly flexible and diverse in nature. These characteristics are important during root growth, as plant roots consist of radial patterns of cells that have diverse functions and that are at different developmental stages along the growth axis. Young stem cell daughters undergo a series of rapid cell divisions, during which new cell walls are formed that are highly dynamic, and that support rapid anisotropic cell expansion. Once the cells have differentiated, the walls of specific cell types need to comply with and support different cell functions. For example, a newly formed root hair needs to be able to break through the surrounding soil, while endodermal cells modify their walls at distinct positions to form Casparian strips between them. Hence, the cell walls are modified and rebuilt while cells transit through different developmental stages. In addition, the cell walls of roots readjust to their environment to support growth and to maximize nutrient uptake. Many of these modifications are likely driven by different developmental and stress signaling pathways. However, our understanding of how such pathways affect cell wall modifications and what enzymes are involved remain largely unknown. In this review we aim to compile data linking cell wall content and re-modeling to developmental stages of root cells, and dissect how root cell walls respond to certain environmental changes. PMID:27582757

  20. Arrangement of peptidoglycan in the cell wall of Staphylococcus spp.

    OpenAIRE

    Amako, K.; Umeda, A; Murata, K

    1982-01-01

    The arrangement of peptidoglycan in the cell wall of Staphylococcus was observed with the newly developed freeze-fracture technique, using n-octanol instead of water as the freezing medium. The replica of the trichloroacetic acid-extracted cell wall (TCA-wall) showed two areas. One of them has a concentric circular structure, a characteristic surface structure of the staphylococcal cell wall, and the other showed an irregular and rough surface. The chemical analysis of the wall revealed that ...

  1. Interconnections between cell wall polymers, wall mechanics, and cortical microtubules: Teasing out causes and consequences.

    Science.gov (United States)

    Xiao, Chaowen; Anderson, Charles T

    2016-09-01

    In plants, cell wall components including cellulose, hemicelluloses, and pectins interact with each other to form complex extracellular network structures that control cell growth and maintain cell shape. However, it is still not clear exactly how different wall polymers interact, how the conformations and interactions of cell wall polymers relate to wall mechanics, and how these factors impinge on intracellular structures such as the cortical microtubule cytoskeleton. Here, based on studies of Arabidopsis thaliana xxt1 xxt2 mutants, which lack detectable xyloglucan in their walls and display aberrant wall mechanics, altered cellulose patterning and biosynthesis, and reduced cortical microtubule stability, we discuss the potential relationships between cell wall biosynthesis, wall mechanics, and cytoskeletal dynamics in an effort to better understand their roles in controlling plant growth and morphogenesis.

  2. Proteomic analysis of HIV-T cell interaction: an update.

    Directory of Open Access Journals (Sweden)

    Dave eSpeijer

    2012-07-01

    Full Text Available This mini-review summarizes techniques applied in, and results obtained with, proteomic studies of HIV-1 T cell interaction. Our group previously reported on the use of two-dimensional differential gel electrophoresis (2D-DIGE coupled to MALDI-TOF peptide mass fingerprint analysis, to study T cell responses upon HIV-1 infection. Only one in three differentially expressed proteins could be identified using this experimental setup. Here we report on our latest efforts to test models generated by this data set and extend its analysis by using novel bioinformatic algorithms. The 2D-DIGE results are compared with other studies including a pilot study using one-dimensional peptide separation coupled to MSE, a novel mass spectrometric approach. It can be concluded that although the latter method detects fewer proteins, it is much faster and less labor intensive. Last but not least, recent developments and remaining challenges in the field of proteomic studies of HIV-1 infection and proteomics in general are discussed.

  3. Plant Cell Wall Matrix Polysaccharide Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Ajay Pal S. Sandhu; Gursharn S. Randhawa; Kanwarpal S. Dhugga

    2009-01-01

    The wall of an expanding plant cell consists primarily of cellulose microfibrils embedded in a matrix of hemi-cellulosic and pectic polysaccharides along with small amounts of structural and enzymatic proteins. Matrix polysacchar-ides are synthesized in the Golgi and exported to the cell wall by exocytosis, where they intercalate among cellulose microfibrUs, which are made at the plasma membrane and directly deposited into the cell wall. Involvement of Golgi glucan synthesis in auxin-induced cell expansion has long been recognized; however, only recently have the genes corresponding to glucan synthases been identified. Biochemical purification was unsuccessful because of the labile nature and very low abundance of these enzymes. Mutational genetics also proved fruitless. Expression of candidate genes identified through gene expression profiling or comparative genomics in heterologous systems followed by functional characterization has been relatively successful. Several genes from the cellulose synthase-like (Cs/) family have been found to be involved in the synthesis of various hemicellulosic glycans. The usefulness of this approach, however, is limited to those enzymes that probably do not form complexes consisting of unrelated proteins. Nonconventional approaches will continue to incre-mentally unravel the mechanisms of Golgi polysaccharide biosynthesis.

  4. Alfalfa stem tissues: Cell wall deposition, composition, and degradability

    NARCIS (Netherlands)

    Jung, H.G.; Engels, F.M.

    2002-01-01

    Declining cell wall degradability of alfalfa (Medicago sativa L.) stems with maturation limits the nutritional value of alfalfa for ruminants. This study characterized changes in cell wall concentration, composition, and degradability by rumen microbes resulting from alfalfa stem tissue proliferatio

  5. [Structure and function of fungal cell wall].

    Science.gov (United States)

    Ohno, Naohito

    2008-12-01

    Cell wall glycans of fungi/yeasts are reviewed. Fungi/yeasts produce various kinds of polysaccharides. As part of the cell wall they are interlinked with other components forming a huge network. The insolubility and complex with multiple components makes the research very tough. Studies on beta-glucan have been performed from various views, such as chemistry, conformation, solubility, tissue distribution and metabolism, biological activity, clinical application, receptor, biosynthesis, and antibody. Studies on mannan focus on immunotoxicity, such as anaphylactoid reaction and coronary arteritis induction. alpha-glucan, chitin, and capsular polysaccharide were also mentioned in relation to structure and genes. Compared with human and animal polysaccharides, fungi/yeasts polysaccharides have very characteristic properties.

  6. Proteomics and glycoproteomics of pluripotent stem-cell surface proteins.

    Science.gov (United States)

    Sun, Bingyun

    2015-03-01

    Pluripotent stem cells are a unique cell type with promising potential in regenerative and personalized medicine. Yet the difficulty to understand and coax their seemingly stochastic differentiation and spontaneous self-renewal have largely limited their clinical applications. A call has been made by numerous researchers for a better characterization of surface proteins on these cells, in search of biomarkers that can dictate developmental stages and lineage specifications, and can help formulate mechanistic insight of stem-cell fate choices. In the past two decades, proteomics has gained significant recognition in profiling surface proteins at high throughput. This review will summarize the impact of these studies on stem-cell biology, and discuss the used proteomic techniques. A systematic comparison of all the techniques and their results is also attempted here to help reveal pros, cons, and the complementarity of the existing methods. This awareness should assist in selecting suitable strategies for stem-cell related research, and shed light on technical improvements that can be explored in the future.

  7. Association Mapping of Cell Wall Synthesis Regulatory Genes and Cell Wall Quality in Switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Bartley, Laura [Univ. of Oklahoma, Norman, OK (United States). Dept. of Microbiology and Plant Biology; Wu, Y. [Oklahoma State Univ., Stillwater, OK (United States); Zhu, L. [Oklahoma State Univ., Stillwater, OK (United States); Brummer, E. C. [Noble Foundation, Ardmore, OK (United States); Saha, M. [Noble Foundation, Ardmore, OK (United States)

    2016-05-31

    Inefficient conversion of biomass to biofuels is one of the main barriers for biofuel production from such materials. Approximately half of polysaccharides in biomass remain unused by typical biochemical conversion methods. Conversion efficiency is influenced by the composition and structure of cell walls of biomass. Grasses such as wheat, maize, and rice, as well as dedicated perennial bioenergy crops, like switchgrass, make up ~55% of biomass that can be produced in the United States. Grass cell walls have a different composition and patterning compared with dicotyledonous plants, including the well-studied model plant, Arabidopsis. This project identified genetic determinants of cell wall composition in grasses using both naturally occurring genetic variation of switchgrass and gene network reconstruction and functional assays in rice. In addition, the project linked functional data in rice and other species to switchgrass improvement efforts through curation of the most abundant class of regulators in the switchgrass genome. Characterizing natural diversity of switchgrass for variation in cell wall composition and properties, also known as quality, provides an unbiased avenue for identifying biologically viable diversity in switchgrass cell walls. To characterizing natural diversity, this project generated cell wall composition and enzymatic deconstruction data for ~450 genotypes of the Switchgrass Southern Association Collection (SSAC), a diverse collection composed of 36 switchgrass accessions from the southern U.S. distribution of switchgrass. Comparing these data with other measures of cell wall quality for the same samples demonstrated the complementary nature of the diverse characterization platforms now being used for biomass characterization. Association of the composition data with ~3.2K single nucleotide variant markers identified six significant single nucleotide variant markers co-associated with digestibility and another compositional trait. These

  8. Association Mapping of Cell Wall Synthesis Regulatory Genes and Cell Wall Quality in Switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Bartley, Laura [Univ. of Oklahoma, Norman, OK (United States). Dept. of Microbiology and Plant Biology; Wu, Y. [Oklahoma State Univ., Stillwater, OK (United States); Zhu, L. [Oklahoma State Univ., Stillwater, OK (United States); Brummer, E. C. [Noble Foundation, Ardmore, OK (United States); Saha, M. [Noble Foundation, Ardmore, OK (United States)

    2016-05-31

    Inefficient conversion of biomass to biofuels is one of the main barriers for biofuel production from such materials. Approximately half of polysaccharides in biomass remain unused by typical biochemical conversion methods. Conversion efficiency is influenced by the composition and structure of cell walls of biomass. Grasses such as wheat, maize, and rice, as well as dedicated perennial bioenergy crops, like switchgrass, make up ~55% of biomass that can be produced in the United States. Grass cell walls have a different composition and patterning compared with dicotyledonous plants, including the well-studied model plant, Arabidopsis. This project identified genetic determinants of cell wall composition in grasses using both naturally occurring genetic variation of switchgrass and gene network reconstruction and functional assays in rice. In addition, the project linked functional data in rice and other species to switchgrass improvement efforts through curation of the most abundant class of regulators in the switchgrass genome. Characterizing natural diversity of switchgrass for variation in cell wall composition and properties, also known as quality, provides an unbiased avenue for identifying biologically viable diversity in switchgrass cell walls. To characterizing natural diversity, this project generated cell wall composition and enzymatic deconstruction data for ~450 genotypes of the Switchgrass Southern Association Collection (SSAC), a diverse collection composed of 36 switchgrass accessions from the southern U.S. distribution of switchgrass. Comparing these data with other measures of cell wall quality for the same samples demonstrated the complementary nature of the diverse characterization platforms now being used for biomass characterization. Association of the composition data with ~3.2K single nucleotide variant markers identified six significant single nucleotide variant markers co-associated with digestibility and another compositional trait. These

  9. Beyond growth: novel functions for bacterial cell wall hydrolases.

    Science.gov (United States)

    Wyckoff, Timna J; Taylor, Jennifer A; Salama, Nina R

    2012-11-01

    The peptidoglycan cell wall maintains turgor pressure and cell shape of most bacteria. Cell wall hydrolases are essential, together with synthases, for growth and daughter cell separation. Recent work in diverse organisms has uncovered new cell wall hydrolases that act autonomously or on neighboring cells to modulate invasion of prey cells, cell shape, innate immune detection, intercellular communication, and competitor lysis. The hydrolases involved in these processes catalyze the cleavage of bonds throughout the sugar and peptide moities of peptidoglycan. Phenotypes associated with these diverse hydrolases reveal new functions of the bacterial cell wall beyond growth and division.

  10. Proteomic analysis of proton beam irradiated human melanoma cells.

    Directory of Open Access Journals (Sweden)

    Sylwia Kedracka-Krok

    Full Text Available Proton beam irradiation is a form of advanced radiotherapy providing superior distributions of a low LET radiation dose relative to that of photon therapy for the treatment of cancer. Even though this clinical treatment has been developing for several decades, the proton radiobiology critical to the optimization of proton radiotherapy is far from being understood. Proteomic changes were analyzed in human melanoma cells treated with a sublethal dose (3 Gy of proton beam irradiation. The results were compared with untreated cells. Two-dimensional electrophoresis was performed with mass spectrometry to identify the proteins. At the dose of 3 Gy a minimal slowdown in proliferation rate was seen, as well as some DNA damage. After allowing time for damage repair, the proteomic analysis was performed. In total 17 protein levels were found to significantly (more than 1.5 times change: 4 downregulated and 13 upregulated. Functionally, they represent four categories: (i DNA repair and RNA regulation (VCP, MVP, STRAP, FAB-2, Lamine A/C, GAPDH, (ii cell survival and stress response (STRAP, MCM7, Annexin 7, MVP, Caprin-1, PDCD6, VCP, HSP70, (iii cell metabolism (TIM, GAPDH, VCP, and (iv cytoskeleton and motility (Moesin, Actinin 4, FAB-2, Vimentin, Annexin 7, Lamine A/C, Lamine B. A substantial decrease (2.3 x was seen in the level of vimentin, a marker of epithelial to mesenchymal transition and the metastatic properties of melanoma.

  11. Proteome of human colon cancer stem cells: A comparative analysis

    Institute of Scientific and Technical Information of China (English)

    Jian Zou; Xiao-Feng Yu; Zhi-Jun Bao; Jie Dong

    2011-01-01

    AIM: To isolate and identify the biological characteristics of human colon cancer stem cells (SW1116 cells) and further study their proteome. METHODS: SW1116 cells were isolated and cultured with a serum-free medium (SFM). Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SW1116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction (PCR)-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis (2-DE) and differentially expressed proteins were identified by tandem mass spectrometry (MALDI-TOF/TOF). RESULTS: The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusiondegradation 1-like protein, nuclear chloride channel protein, tubulin b, Raichu404X, stratifin, F-actin capping protein a-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein b polypeptide 2-like 1, respectively. CONCLUSION: SW1116 cells are biologically

  12. Integration of Proteomics and Transcriptomics Data Sets for the Analysis of a Lymphoma B-Cell Line in the Context of the Chromosome-Centric Human Proteome Project.

    Science.gov (United States)

    Díez, Paula; Droste, Conrad; Dégano, Rosa M; González-Muñoz, María; Ibarrola, Nieves; Pérez-Andrés, Martín; Garin-Muga, Alba; Segura, Víctor; Marko-Varga, Gyorgy; LaBaer, Joshua; Orfao, Alberto; Corrales, Fernando J; De Las Rivas, Javier; Fuentes, Manuel

    2015-09-04

    A comprehensive study of the molecular active landscape of human cells can be undertaken to integrate two different but complementary perspectives: transcriptomics, and proteomics. After the genome era, proteomics has emerged as a powerful tool to simultaneously identify and characterize the compendium of thousands of different proteins active in a cell. Thus, the Chromosome-centric Human Proteome Project (C-HPP) is promoting a full characterization of the human proteome combining high-throughput proteomics with the data derived from genome-wide expression profiling of protein-coding genes. Here we present a full proteomic profiling of a human lymphoma B-cell line (Ramos) performed using a nanoUPLC-LTQ-Orbitrap Velos proteomic platform, combined to an in-depth transcriptomic profiling of the same cell type. Data are available via ProteomeXchange with identifier PXD001933. Integration of the proteomic and transcriptomic data sets revealed a 94% overlap in the proteins identified by both -omics approaches. Moreover, functional enrichment analysis of the proteomic profiles showed an enrichment of several functions directly related to the biological and morphological characteristics of B-cells. In turn, about 30% of all protein-coding genes present in the whole human genome were identified as being expressed by the Ramos cells (stable average of 30% genes along all the chromosomes), revealing the size of the protein expression-set present in one specific human cell type. Additionally, the identification of missing proteins in our data sets has been reported, highlighting the power of the approach. Also, a comparison between neXtProt and UniProt database searches has been performed. In summary, our transcriptomic and proteomic experimental profiling provided a high coverage report of the expressed proteome from a human lymphoma B-cell type with a clear insight into the biological processes that characterized these cells. In this way, we demonstrated the usefulness of

  13. Fermentation of the endosperm cell walls of monocotyledon and dicotyledon plant species: The relationship between cell wall characteristics and fermentability

    NARCIS (Netherlands)

    Laar, van H.; Tamminga, S.; Williams, B.A.; Verstegen, M.W.A.

    2000-01-01

    Cell walls from the endosperm of four monocotyledons (maize, wheat, rye, and rice) and four dicotyledons (soya bean, lupin, faba bean, and pea) seeds were studied to relate cell wall composition and structure with fermentation characteristics. Cell wall material was isolated from the endosperm of th

  14. Cell Shape and Cardiosphere Differentiation: A Revelation by Proteomic Profiling

    Directory of Open Access Journals (Sweden)

    Nanako Kawaguchi

    2013-01-01

    Full Text Available Stem cells (embryonic stem cells, somatic stem cells such as neural stem cells, and cardiac stem cells and cancer cells are known to aggregate and form spheroid structures. This behavior is common in undifferentiated cells and may be necessary for adapting to certain conditions such as low-oxygen levels or to maintain undifferentiated status in microenvironments including stem cell niches. In order to decipher the meaning of this spheroid structure, we established a cardiosphere clone (CSC-21E derived from the rat heart which can switch its morphology between spheroid and nonspheroid. Two forms, floating cardiospheres and dish-attached flat cells, could be switched reversibly by changing the cell culture condition. We performed differential proteome analysis studies and obtained protein profiles distinct between spherical forms and flat cells. From protein profiling analysis, we found upregulation of glycolytic enzymes in spheroids with some stress proteins switched in expression levels between these two forms. Evidence has been accumulating that certain chaperone/stress proteins are upregulated in concert with cellular changes including proliferation and differentiation. We would like to discuss the possible mechanism of how these aggregates affect cell differentiation and/or other cellular functions.

  15. Proteomic profiling of endorepellin angiostatic activity on human endothelial cells

    Directory of Open Access Journals (Sweden)

    Iozzo Renato V

    2008-02-01

    Full Text Available Abstract Background Endorepellin, the C-terminal domain V of the heparan sulfate proteoglycan perlecan, exhibits powerful and targeted anti-angiogenic activity on endothelial cells. To identify proteins involved with endorepellin anti-angiogenic action, we performed an extensive comparative proteomic analysis between vehicle- and endorepellin-treated human endothelial cells. Results Proteomic analysis of endorepellin influence on human umbilical vein endothelial cells identified five differentially expressed proteins, three of which (β-actin, calreticulin, and chaperonin/Hsp60 were down-regulated and two of which (vimentin and the β subunit of prolyl 4-hydroxylase also known as protein disulfide isomerase were up-regulated in response to endorepellin treatment—and associated with a fold change (endorepellin/control ≤ 0.75 and ≥ 2.00, and a statistically significant p-value as determined by Student's t test. Conclusion The proteins identified represent potential target areas involved with endorepellin anti-angiogenic mechanism of action. Further elucidation as such will ultimately provide useful in utilizing endorepellin as an anti-angiogenic therapy in humans.

  16. Grass Cell Walls: A Story of Cross-Linking

    Science.gov (United States)

    Hatfield, Ronald D.; Rancour, David M.; Marita, Jane M.

    2017-01-01

    Cell wall matrices are complex composites mainly of polysaccharides, phenolics (monomers and polymers), and protein. We are beginning to understand the synthesis of these major wall components individually, but still have a poor understanding of how cell walls are assembled into complex matrices. Valuable insight has been gained by examining intact components to understand the individual elements that make up plant cell walls. Grasses are a prominent group within the plant kingdom, not only for their important roles in global agriculture, but also for the complexity of their cell walls. Ferulate incorporation into grass cell wall matrices (C3 and C4 types) leads to a cross-linked matrix that plays a prominent role in the structure and utilization of grass biomass compared to dicot species. Incorporation of p-coumarates as part of the lignin structure also adds to the complexity of grass cell walls. Feruoylation results in a wall with individual hemicellulosic polysaccharides (arabinoxylans) covalently linked to each other and to lignin. Evidence strongly suggests that ferulates not only cross-link arabinoxylans, but may be important factors in lignification of the cell wall. Therefore, the distribution of ferulates on arabinoxylans could provide a means of structuring regions of the matrix with the incorporation of lignin and have a significant impact upon localized cell wall organization. The role of other phenolics in cell wall formation such as p-coumarates (which can have concentrations higher than ferulates) remains unknown. It is possible that p-coumarates assist in the formation of lignin, especially syringyl rich lignin. The uniqueness of the grass cell wall compared to dicot sepcies may not be so much in the gross composition of the wall, but how the distinctive individual components are organized into a functional wall matrix. These features are discussed and working models are provided to illustrate how changing the organization of feruoylation and p

  17. Enzymes and other agents that enhance cell wall extensibility

    Science.gov (United States)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

  18. Proteomic assessment of a cell model of spinal muscular atrophy

    Directory of Open Access Journals (Sweden)

    Lee Kelvin H

    2011-03-01

    Full Text Available Abstract Background Deletion or mutation(s of the survival motor neuron 1 (SMN1 gene causes spinal muscular atrophy (SMA, a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN protein is embryonically lethal, yet reduced levels of this protein result in selective death of motor neurons. Why motor neurons are specifically targeted by SMN deficiency remains to be determined. In this study, embryonic stem (ES cells derived from a severe SMA mouse model were differentiated into motor neurons in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic and western blot analyses were used to probe protein expression alterations in this cell-culture model of SMA that could be relevant to the disease. Results When ES cells were primed with Noggin/fibroblast growth factors (bFGF and FGF-8 in a more robust neural differentiation medium for 2 days before differentiation induction, the efficiency of in vitro motor neuron differentiation was improved from ~25% to ~50%. The differentiated ES cells expressed a pan-neuronal marker (neurofilament and motor neuron markers (Hb9, Islet-1, and ChAT. Even though SMN-deficient ES cells had marked reduced levels of SMN (~20% of that in control ES cells, the morphology and differentiation efficiency for these cells are comparable to those for control samples. However, proteomics in conjunction with western blot analyses revealed 6 down-regulated and 14 up-regulated proteins with most of them involved in energy metabolism, cell stress-response, protein degradation, and cytoskeleton stability. Some of these activated cellular pathways showed specificity for either undifferentiated or differentiated cells. Increased p21 protein expression indicated that SMA ES cells were responding to cellular stress. Up-regulation of p21 was confirmed in spinal cord tissues from the same SMA mouse model from which the ES cells were derived. Conclusion SMN

  19. Disruption of cell walls for enhanced lipid recovery

    Science.gov (United States)

    Knoshaug, Eric P; Donohoe, Bryon S; Gerken, Henri; Laurens, Lieve; Van Wychen, Stefanie Rose

    2015-03-24

    Presented herein are methods of using cell wall degrading enzymes for recovery of internal lipid bodies from biomass sources such as algae. Also provided are algal cells that express at least one exogenous gene encoding a cell wall degrading enzyme and methods for recovering lipids from the cells.

  20. Two endogenous proteins that induce cell wall extension in plants

    Science.gov (United States)

    McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

    1992-01-01

    Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

  1. Red blood cell (RBC) membrane proteomics--Part II: Comparative proteomics and RBC patho-physiology.

    Science.gov (United States)

    Pasini, Erica M; Lutz, Hans U; Mann, Matthias; Thomas, Alan W

    2010-01-03

    Membrane proteomics offers unprecedented possibilities to compare protein expression in health and disease leading potentially to the identification of markers, of targets for therapeutics and to a better understanding of disease mechanisms. From transfusion medicine to infectious diseases, from cardiovascular affections to diabetes, comparative proteomics has made a contribution to the identification of proteins unique to RBCs of patients with specific illnesses shedding light on possible RBC markers for systemic diseases. In this review we will provide a short overview of some of the main achievements obtained by comparative proteomics in the field of RBC-related local and systemic diseases and suggest some additional areas of RBCs research to which comparative proteomics approaches could be fruitfully applied or extended in combination with biochemical techniques.

  2. Multidimensional solid-state NMR spectroscopy of plant cell walls.

    Science.gov (United States)

    Wang, Tuo; Phyo, Pyae; Hong, Mei

    2016-09-01

    Plant biomass has become an important source of bio-renewable energy in modern society. The molecular structure of plant cell walls is difficult to characterize by most atomic-resolution techniques due to the insoluble and disordered nature of the cell wall. Solid-state NMR (SSNMR) spectroscopy is uniquely suited for studying native hydrated plant cell walls at the molecular level with chemical resolution. Significant progress has been made in the last five years to elucidate the molecular structures and interactions of cellulose and matrix polysaccharides in plant cell walls. These studies have focused on primary cell walls of growing plants in both the dicotyledonous and grass families, as represented by the model plants Arabidopsis thaliana, Brachypodium distachyon, and Zea mays. To date, these SSNMR results have shown that 1) cellulose, hemicellulose, and pectins form a single network in the primary cell wall; 2) in dicot cell walls, the protein expansin targets the hemicellulose-enriched region of the cellulose microfibril for its wall-loosening function; and 3) primary wall cellulose has polymorphic structures that are distinct from the microbial cellulose structures. This article summarizes these key findings, and points out future directions of investigation to advance our fundamental understanding of plant cell wall structure and function.

  3. Advanced technologies for plant cell wall evolution and diversity

    DEFF Research Database (Denmark)

    Fangel, Jonatan Ulrik

    Plant cell walls consist of polysaccharides, glycoproteins and phenolic polymers interlinked together in a highly complex network. The detailed analysis of cell walls is challenging because of their inherent complexity and heterogeneity. Also, complex carbohydrates, unlike proteins and nucleotides...... probes (monoclonal antibodies mAbs and carbohydrate binding modules, CBMs) to rapidly profile polysaccharides across a sample set. During my PhD I have further developed the CoMPP technique and used it for cell wall analysis within the context of a variety of applied and fundamental projects. The data...... produced has provided new insight into cell wall evolution and biosynthesis and has contributed to the commercial development of cell wall materials. A major focus of the work has been the wide scale sampling of cell wall diversity across the plant kingdom, from unicellular algae to highly evolved...

  4. Proteomic Studies of Cholangiocarcinoma and Hepatocellular Carcinoma Cell Secretomes

    Directory of Open Access Journals (Sweden)

    Chantragan Srisomsap

    2010-01-01

    Full Text Available Cholangiocarcinoma (CCA and hepatocellular carcinoma (HCC occur with relatively high incidence in Thailand. The secretome, proteins secreted from cancer cells, are potentially useful as biomarkers of the diseases. Proteomic analysis was performed on the secreted proteins of cholangiocarcinoma (HuCCA-1 and hepatocellular carcinoma (HCC-S102, HepG2, SK-Hep-1, and Alexander cell lines. The secretomes of the five cancer cell lines were analyzed by SDS-PAGE combined with LC/MS/MS. Sixty-eight proteins were found to be expressed only in HuCCA-1. Examples include neutrophil gelatinase-associated lipocalin (lipocalin 2, laminin 5 beta 3, cathepsin D precursor, desmoplakin, annexin IV variant, and annexin A5. Immunoblotting was used to confirm the presence of lipocalin 2 in conditioned media and cell lysate of 5 cell lines. The results showed that lipocalin 2 was a secreted protein which is expressed only in the conditioned media of the cholangiocarcinoma cell line. Study of lipocalin 2 expression in different types of cancer and normal tissues from cholangiocarcinoma patients showed that lipocalin 2 was expressed only in the cancer tissues. We suggest that lipocalin 2 may be a potential biomarker for cholangiocarcinoma.

  5. Cell wall degradation in the autolysis of filamentous fungi.

    Science.gov (United States)

    Perez-Leblic, M I; Reyes, F; Martinez, M J; Lahoz, R

    1982-12-27

    A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes. Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present. In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.

  6. Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells

    OpenAIRE

    Gho, Yong Song; Choi, Dong-Sic; Choi, Do-Young; Hong, Bok Sil; Jang, Su Chul; Kim, Dae-Kyum; Lee, Jaewook; Kim, Yoon-Keun; Kim, Kwang Pyo

    2012-01-01

    Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastat...

  7. Proteomic profile of human monocytic cells infected with dengue virus

    Institute of Scientific and Technical Information of China (English)

    Viviana Martnez-Betancur; Marlen Martnez-Gutierrez

    2016-01-01

    Objective: To identify the changes in the proteome of U937 cells infected with dengue virus (DENV). Methods: In this study, differentiated U937 cultures were infected with two DENV-2 strains, one of which was associated with dengue (DENV-2/NG) and the other one with severe dengue (DENV-2/16681), with the aim of determining the cellular proteomic profiles under different infection conditions. Cellular proteins were extracted and sepa-rated by two-dimensional electrophoresis, and those proteins with differential expression profiles were identified by mass spectrometry. The obtained results were correlated with cellular viability, the number of infectious viral particles, and the viral DNA/protein quantity. Results: In comparison with non-infected cultures, in the cells infected with the DENV-2/NG strain, nine proteins were expressed differentially (five were upregulated and four were downregulated); in those cultures infected with the DENV-2/16681 strain, six proteins were differentially expressed (two were downregulated and four were upregu-lated). The downregulated proteins included fatty acid-binding protein, heterogeneous nuclear ribonucleoprotein 1, protein disulfide isomerase, enolase 1, heat shock 70 kDa protein 9, phosphotyrosyl phosphatase, and annexin IV. The upregulated proteins included heat shock 90 kDa protein AA1, tubulin beta, enolase 1, pyruvate kinase, transaldolase and phospholipase C-alpha. Conclusions: Because the monocyte/macrophage lineage is critical for disease patho-genicity, additional studies on these proteins could provide a better understanding of the cellular response to DENV infection and could help identify new therapeutic targets against infection.

  8. Small molecule probes for plant cell wall polysaccharide imaging

    Directory of Open Access Journals (Sweden)

    Ian eWallace

    2012-05-01

    Full Text Available Plant cell walls are composed of interlinked polymer networks consisting of cellulose, hemicelluloses, pectins, proteins, and lignin. The ordered deposition of these components is a dynamic process that critically affects the development and differentiation of plant cells. However, our understanding of cell wall synthesis and remodeling, as well as the diverse cell wall architectures that result from these processes, has been limited by a lack of suitable chemical probes that are compatible with live-cell imaging. In this review, we summarize the currently available molecular toolbox of probes for cell wall polysaccharide imaging in plants, with particular emphasis on recent advances in small molecule-based fluorescent probes. We also discuss the potential for further development of small molecule probes for the analysis of cell wall architecture and dynamics.

  9. Evaluation of Three Protein-Extraction Methods for Proteome Analysis of Maize Leaf Midrib, a Compound Tissue Rich in Sclerenchyma Cells

    OpenAIRE

    2016-01-01

    Leaf morphology is closely related to the growth and development of maize (Zea mays L.) plants and final kernel production. As an important part of the maize leaf, the midrib holds leaf blades in the aerial position for maximum sunlight capture. Leaf midribs of adult plants contain substantial sclerenchyma cells with heavily thickened and lignified secondary walls and have a high amount of phenolics, making protein extraction and proteome analysis difficult in leaf midrib tissue. In the prese...

  10. Mechanical Properties of Plant Cell Walls Probed by Relaxation Spectra

    DEFF Research Database (Denmark)

    Hansen, Steen Laugesen; Ray, Peter Martin; Karlsson, Anders Ola

    2011-01-01

    Transformants and mutants with altered cell wall composition are expected to display a biomechanical phenotype due to the structural role of the cell wall. It is often quite difficult, however, to distinguish the mechanical behavior of a mutant's or transformant's cell walls from that of the wild...... type. This may be due to the plant’s ability to compensate for the wall modification or because the biophysical method that is often employed, determination of simple elastic modulus and breakstrength, lacks the resolving power necessary for detecting subtle mechanical phenotypes. Here, we apply...... a method, determination of relaxation spectra, which probes, and can separate, the viscoelastic properties of different cell wall components (i.e. those properties that depend on the elastic behavior of load-bearing wall polymers combined with viscous interactions between them). A computer program, Bayes...

  11. Proteomics in Cell Culture: From Genomics to Combined ‘Omics for Cell Line Engineering and Bioprocess Development

    DEFF Research Database (Denmark)

    Heffner, Kelley; Kaas, Christian Schrøder; Kumar, Amit;

    2015-01-01

    The genetic sequencing of Chinese hamster ovary cells has initiated a systems biology era for biotechnology applications. In addition to genomics, critical omics data sets also include proteomics, transcriptomics and metabolomics. Recently, the use of proteomics in cell lines for recombinant...... in media development and cell line engineering to improve growth or productivity, delay the onset of apoptosis, or utilize nutrients efficiently. Mass-spectrometry based and other proteomics methods can provide for the detection of thousands of proteins from cell culture and bioinformatics analysis serves...

  12. Proteomic inventory of "anchorless" proteins on the colon adenocarcinoma cell surface.

    NARCIS (Netherlands)

    Tjalsma, H.; Pluk, W.J.G.; Heuvel, L.P.W.J. van den; Peters, W.H.M.; Roelofs, R.H.W.M.; Swinkels, D.W.

    2006-01-01

    Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several "non-membrane" proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets

  13. Structural Studies of Complex Carbohydrates of Plant Cell Walls

    Energy Technology Data Exchange (ETDEWEB)

    Darvill, Alan [Univ. of Georgia, Athens, GA (United States); Hahn, Michael G. [Univ. of Georgia, Athens, GA (United States); O' Neill, Malcolm A. [Univ. of Georgia, Athens, GA (United States); York, William S. [Univ. of Georgia, Athens, GA (United States)

    2015-02-17

    Most of the solar energy captured by land plants is converted into the polysaccharides (cellulose, hemicellulose, and pectin) that are the predominant components of the cell wall. These walls, which account for the bulk of plant biomass, have numerous roles in the growth and development of plants. Moreover, these walls have a major impact on human life as they are a renewable source of biomass, a source of diverse commercially useful polymers, a major component of wood, and a source of nutrition for humans and livestock. Thus, understanding the molecular mechanisms that lead to wall assembly and how cell walls and their component polysaccharides contribute to plant growth and development is essential to improve and extend the productivity and value of plant materials. The proposed research will develop and apply advanced analytical and immunological techniques to study specific changes in the structures and interactions of the hemicellulosic and pectic polysaccharides that occur during differentiation and in response to genetic modification and chemical treatments that affect wall biosynthesis. These new techniques will make it possible to accurately characterize minute amounts of cell wall polysaccharides so that subtle changes in structure that occur in individual cell types can be identified and correlated to the physiological or developmental state of the plant. Successful implementation of this research will reveal fundamental relationships between polysaccharide structure, cell wall architecture, and cell wall functions.

  14. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the c

  15. Hemicellulose biosynthesis and degradation in tobacco cell walls

    NARCIS (Netherlands)

    Compier, M.G.M.

    2005-01-01

    Natural fibres have a wide range of technological applications, such as in paper and textile industries. The basic properties and the quality of plant fibres are determined by the composition of the plant cell wall. Characteristic for fibres are thick secondary cell walls, which consist of cellulose

  16. Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation

    Directory of Open Access Journals (Sweden)

    Kouki eYoshida

    2013-10-01

    Full Text Available Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs can regulate secondary wall formation in rice (Oryza sativa and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S has very low transcriptional activation ability, but the longer protein (OsSWN2L and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.

  17. Recovery of Chinese hamster ovary host cell proteins for proteomic analysis.

    Science.gov (United States)

    Valente, Kristin N; Schaefer, Amy K; Kempton, Hannah R; Lenhoff, Abraham M; Lee, Kelvin H

    2014-01-01

    Identification and characterization of Chinese hamster ovary (CHO) host cell protein (HCP) impurities by proteomic techniques can aid bioprocess design and lead to more efficient development and improved biopharmaceutical manufacturing operations. Recovery of extracellular CHO HCP for proteomic analysis is particularly challenging due to the relatively low protein concentration and complex composition of media. In this article, we report the development of optimized protocols that improve proteome capture for CHO HCP. Eleven precipitation protocols were screened for protein recovery and optimized for a subset of precipitants by a design of experiments (DOE) approach. Because total protein recovery does not fully replicate a proteomics experiment, or detect non-protein agents that may interfere with proteomic methods, a subset of precipitation conditions were compared by two-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry, with optimized recovery shown to differ between the two proteomic methods. This work demonstrates broadly applicable methods that can be applied as initial steps to optimize sample preparation of any sample type for proteomic analysis, and presents optimized precipitation protocols for extracellular CHO HCP recovery, which can vary appreciably between gel-based and shotgun proteomic methods.

  18. On-Off Switches for Secondary Cell Wall Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Huan-Zhong Wang; Richard A.Dixon

    2012-01-01

    Secondary cell walls provide plants with rigidity and strength to support their body weight and ensure water and nutrient transport.They also provide textiles,timber,and potentially second-generation biofuels for human use.Genes responsible for synthesis of the different cell wall components,namely cellulose,hemicelluloses,and lignin,are coordinately expressed and under transcriptional regulation.In the past several years,cell wall-related NAC and MYB transcription factors have been intensively investigated in different species and shown to be master switches of secondary cell wall biosynthesis.Positive and negative regulators,which function upstream of NAC master switches,have also been identified in different plant tissues.Further elucidation of the regulatory mechanisms of cell wall synthesis will facilitate the engineering of plant feedstocks suitable for biofuel production.

  19. Iron restriction-induced adaptations in the wall proteome of Candida albicans

    NARCIS (Netherlands)

    A.G. Sorgo; S. Brul; C.G. de Koster; L.J. de Koning; F.M. Klis

    2013-01-01

    The opportunistic fungal pathogen Candida albicans has developed various ways to overcome iron restriction in a mammalian host. Using different surface proteins, among them membrane- and wall-localized GPI-proteins, it can exploit iron from host hemoglobin, ferritin, and transferrin. Culturing C. al

  20. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xia [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Fifth People' s Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240 (China); Zhao, Libo [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Third People' s Hospital of Chongqing, 400014 (China); Yang, Yongtao [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Bode, Liv [Bornavirus Research Group affiliated to the Free University of Berlin, Berlin (Germany); Huang, Hua [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Liu, Chengyu [Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Huang, Rongzhong [Department of Rehabilitative Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010 (China); Zhang, Liang [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); and others

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

  1. The Penicillium chrysogenum extracellular proteome. Conversion from a food-rotting strain to a versatile cell factory for white biotechnology.

    Science.gov (United States)

    Jami, Mohammad-Saeid; García-Estrada, Carlos; Barreiro, Carlos; Cuadrado, Abel-Alberto; Salehi-Najafabadi, Zahra; Martín, Juan-Francisco

    2010-12-01

    The filamentous fungus Penicillium chrysogenum is well-known by its ability to synthesize β-lactam antibiotics as well as other secondary metabolites. Like other filamentous fungi, this microorganism is an excellent host for secretion of extracellular proteins because of the high capacity of its protein secretion machinery. In this work, we have characterized the extracellular proteome reference map of P. chrysogenum Wisconsin 54-1255 by two-dimensional gel electrophoresis. This method allowed the correct identification of 279 spots by peptide mass fingerprinting and tandem MS. These 279 spots included 328 correctly identified proteins, which corresponded to 131 different proteins and their isoforms. One hundred and two proteins out of 131 were predicted to contain either classical or nonclassical secretion signal peptide sequences, providing evidence of the authentic extracellular location of these proteins. Proteins with higher representation in the extracellular proteome were those involved in plant cell wall degradation (polygalacturonase, pectate lyase, and glucan 1,3-β-glucosidase), utilization of nutrients (extracellular acid phosphatases and 6-hydroxy-d-nicotine oxidase), and stress response (catalase R). This filamentous fungus also secretes enzymes specially relevant for food industry, such as sulfydryl oxidase, dihydroxy-acid dehydratase, or glucoamylase. The identification of several antigens in the extracellular proteome also highlights the importance of this microorganism as one of the main indoor allergens. Comparison of the extracellular proteome among three strains of P. chrysogenum, the wild-type NRRL 1951, the Wis 54-1255 (an improved, moderate penicillin producer), and the AS-P-78 (a penicillin high-producer), provided important insights to consider improved strains of this filamentous fungus as versatile cell-factories of interest, beyond antibiotic production, for other aspects of white biotechnology.

  2. Dynamic metabolic flux analysis of plant cell wall synthesis.

    Science.gov (United States)

    Chen, Xuewen; Alonso, Ana P; Shachar-Hill, Yair

    2013-07-01

    The regulation of plant cell wall synthesis pathways remains poorly understood. This has become a bottleneck in designing bioenergy crops. The goal of this study was to analyze the regulation of plant cell wall precursor metabolism using metabolic flux analysis based on dynamic labeling experiments. Arabidopsis T87 cells were cultured heterotrophically with (13)C labeled sucrose. The time course of ¹³C labeling patterns in cell wall precursors and related sugar phosphates was monitored using liquid chromatography tandem mass spectrometry until steady state labeling was reached. A kinetic model based on mass action reaction mechanisms was developed to simulate the carbon flow in the cell wall synthesis network. The kinetic parameters of the model were determined by fitting the model to the labeling time course data, cell wall composition, and synthesis rates. A metabolic control analysis was performed to predict metabolic regulations that may improve plant biomass composition for biofuel production. Our results describe the routes and rates of carbon flow from sucrose to cell wall precursors. We found that sucrose invertase is responsible for the entry of sucrose into metabolism and UDP-glucose-4-epimerase plays a dominant role in UDP-Gal synthesis in heterotrophic Aradidopsis cells under aerobic conditions. We also predicted reactions that exert strong regulatory influence over carbon flow to cell wall synthesis and its composition.

  3. Maize development: cell wall changes in leaves and sheaths

    Science.gov (United States)

    Developmental changes occur in maize (Zea mays L.) as it transitions from juvenile stages to the mature plant. Changes also occur as newly formed cells mature into adult cells. Maize leaf blades, including the midribs and sheaths, undergo cell wall changes as cells transition to fully mature cell ty...

  4. Isolation of cell surface proteins for mass spectrometry-based proteomics.

    Science.gov (United States)

    Elschenbroich, Sarah; Kim, Yunee; Medin, Jeffrey A; Kislinger, Thomas

    2010-02-01

    Defining the cell surface proteome has profound importance for understanding cell differentiation and cell-cell interactions, as well as numerous pathogenic abnormalities. Owing to their hydrophobic nature, plasma membrane proteins that reside on the cell surface pose analytical challenges and, despite efforts to overcome difficulties, remain under-represented in proteomic studies. Limitations in the classically employed ultracentrifugation-based approaches have led to the invention of more elaborate techniques for the purification of cell surface proteins. Three of these methods--cell surface coating with cationic colloidal silica beads, biotinylation and chemical capture of surface glycoproteins--allow for marked enrichment of this subcellular proteome, with each approach offering unique advantages and characteristics for different experiments. In this article, we introduce the principles of each purification method and discuss applications from the recent literature.

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

    Directory of Open Access Journals (Sweden)

    Jimin Xiong

    2016-01-01

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

  6. Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell.

    Science.gov (United States)

    Pasini, Erica M; Kirkegaard, Morten; Salerno, Doris; Mortensen, Peter; Mann, Matthias; Thomas, Alan W

    2008-07-01

    Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much research has therefore focused on RBC and cardiovascular disorders of mouse and humans. RBCs also host malaria parasites. Recently we presented an in-depth proteome for the human RBC. Here we present directly comparable data for the mouse RBC as membrane-only, soluble-only, and combined membrane-bound/soluble proteomes (comprising, respectively, 247, 232, and 165 proteins). All proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function, and in comparison with the human RBC, were classified as orthologs, family-related, or unique. Splice isoforms were identified, and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinated or partially degraded complexes. Overall there was close concordance between mouse and human proteomes, confirming the unexpected RBC complexity. Several novel findings in the human proteome have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function.

  7. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging

    Directory of Open Access Journals (Sweden)

    Samuel T. Mindaye

    2015-12-01

    Full Text Available Bone-marrow derived mesenchymal stromal cells (BMSCs have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5,6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results.

  8. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  9. Trypsin-induced proteome alteration during cell subculture in mammalian cells

    Directory of Open Access Journals (Sweden)

    Lin Cheng-Wen

    2010-05-01

    Full Text Available Abstract Background It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions. Methods In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting. Results 36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization. Conclusions In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.

  10. 2D-immunoblotting analysis of Sporothrix schenckii cell wall

    Directory of Open Access Journals (Sweden)

    Estela Ruiz-Baca

    2011-03-01

    Full Text Available We utilized two-dimensional gel electrophoresis and immunoblotting (2D-immunoblotting with anti-Sporothrix schenckii antibodies to identify antigenic proteins in cell wall preparations obtained from the mycelial and yeast-like morphologies of the fungus. Results showed that a 70-kDa glycoprotein (Gp70 was the major antigen detected in the cell wall of both morphologies and that a 60-kDa glycoprotein was present only in yeast-like cells. In addition to the Gp70, the wall from filament cells showed four proteins with molecular weights of 48, 55, 66 and 67 kDa, some of which exhibited several isoforms. To our knowledge, this is the first 2D-immunoblotting analysis of the S. schenckii cell wall.

  11. Up against the wall: is yeast cell wall integrity ensured by mechanosensing in plasma membrane microdomains?

    Science.gov (United States)

    Kock, Christian; Dufrêne, Yves F; Heinisch, Jürgen J

    2015-02-01

    Yeast cell wall integrity (CWI) signaling serves as a model of the regulation of fungal cell wall synthesis and provides the basis for the development of antifungal drugs. A set of five membrane-spanning sensors (Wsc1 to Wsc3, Mid2, and Mtl1) detect cell surface stress and commence the signaling pathway upon perturbations of either the cell wall structure or the plasma membrane. We here summarize the latest advances in the structure/function relationship primarily of the Wsc1 sensor and critically review the evidence that it acts as a mechanosensor. The relevance and physiological significance of the information obtained for the function of the other CWI sensors, as well as expected future developments, are discussed.

  12. Cell wall-associated malate dehydrogenase activity from maize roots.

    Science.gov (United States)

    Hadži-Tašković Šukalović, Vesna; Vuletić, Mirjana; Marković, Ksenija; Vučinić, Zeljko

    2011-10-01

    Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn(2+) and Cu(2+) in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn(2+) and Cu(2+) in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.

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

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik J

    2009-01-01

    characterization of the identified proteins. The strategy is based on subtractive immunization of mice, and we used the two isogenic cell lines, NM-2C5 and M-4A4, derived from the MDA-MB-435 cancer cell line, as a model system. Although the two cell lines are equally tumorigenic, only M-4A4 has metastatic...... of the immune system itself to scan the cell surface proteome for differentially expressed proteins. The subtractive immunization strategy should be broadly applicable as a quantitative and comparative proteomic approach for screening the cell surface and also allow generation of mAbs to study these cell...

  14. Patterns of expression of cell wall related genes in sugarcane

    Directory of Open Access Journals (Sweden)

    Lima D.U.

    2001-01-01

    Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

  15. Pectin, a versatile polysaccharide present in plant cell walls

    NARCIS (Netherlands)

    Voragen, A.G.J.; Coenen, G.J.; Verhoef, R.P.; Schols, H.A.

    2009-01-01

    Pectin or pectic substances are collective names for a group of closely associated polysaccharides present in plant cell walls where they contribute to complex physiological processes like cell growth and cell differentiation and so determine the integrity and rigidity of plant tissue. They also pla

  16. How the deposition of cellulose microfibrils builds cell wall architecture

    NARCIS (Netherlands)

    Emons, A.M.C.; Mulder, B.M.

    2000-01-01

    Cell walls, the extracytoplasmic matrices of plant cells, consist of an ordered array of cellulose microfibrils embedded in a matrix of polysaccharides and glycoproteins. This construction is reminiscent of steel rods in reinforced concrete. How a cell organizes these ordered textures around itself,

  17. Cell wall deposition during morphogenesis in fucoid algae.

    Science.gov (United States)

    Bisgrove, S R; Kropf, D L

    2001-04-01

    Cell was deposition was investigated during morphogenesis in zygotes of Pelvetia compressa (J. Agardh) De Toni. Young zygotes are spherical and wall is deposited uniformly, but at germination (about 10 h after fertilization) wall deposition becomes localized to the apex of the tip-growing rhizoid. Wall deposition was investigated before and after the initiation of tip growth by disrupting cytoskeleton, secretion or cellulose deposition; effects on wall strength and structure were examined. All three were involved in generating wall strength in both spherical and tip-growing zygotes, but their relative importance were different at the two developmental stages. Much of the wall strength in young zygotes was dependent on F-actin, whereas cellulose and a sulfated component, probably a fucan (F2), were most important in tip growing zygotes. Some treatments had contrasting effects at the two developmental stages; for example, disruption of F-actin or inhibition of secretion weakened walls in spherical zygotes but strengthened those in tip-growing zygotes. Transmission electron microscopic analysis showed that most treatments that altered wall strength induced modifications of internal wall structure.

  18. Role of the plant cell wall in gravity resistance.

    Science.gov (United States)

    Hoson, Takayuki; Wakabayashi, Kazuyuki

    2015-04-01

    Gravity resistance, mechanical resistance to the gravitational force, is a principal graviresponse in plants, comparable to gravitropism. The cell wall is responsible for the final step of gravity resistance. The gravity signal increases the rigidity of the cell wall via the accumulation of its constituents, polymerization of certain matrix polysaccharides due to the suppression of breakdown, stimulation of cross-link formation, and modifications to the wall environment, in a wide range of situations from microgravity in space to hypergravity. Plants thus develop a tough body to resist the gravitational force via an increase in cell wall rigidity and the modification of growth anisotropy. The development of gravity resistance mechanisms has played an important role in the acquisition of responses to various mechanical stresses and the evolution of land plants.

  19. Functional role of autophagy-mediated proteome remodeling in cell survival signaling and innate immunity.

    Science.gov (United States)

    Mathew, Robin; Khor, Sinan; Hackett, Sean R; Rabinowitz, Joshua D; Perlman, David H; White, Eileen

    2014-09-18

    Ras-driven cancer cells upregulate basal autophagy that degrades and recycles intracellular proteins and organelles. Autophagy-mediated proteome degradation provides free amino acids to support metabolism and macromolecular synthesis, which confers a survival advantage in starvation and promotes tumorigenesis. While the degradation of isolated protein substrates by autophagy has been implicated in controlling cellular function, the extent and specificity by which autophagy remodels the cellular proteome and the underlying functional consequences were unknown. Here we compared the global proteome of autophagy-functional and -deficient Ras-driven cancer cells, finding that autophagy affects the majority of the proteome yet is highly selective. While levels of vesicle trafficking proteins important for autophagy are preserved during starvation-induced autophagy, deleterious inflammatory response pathway components are eliminated even under basal conditions, preventing cytokine-induced paracrine cell death. This reveals the global, functional impact of autophagy-mediated proteome remodeling on cell survival and identifies critical autophagy substrates that mediate this process.

  20. Medullospheres from DAOY, UW228 and ONS-76 cells: increased stem cell population and proteomic modifications.

    Directory of Open Access Journals (Sweden)

    Cristina Zanini

    Full Text Available BACKGROUND: Medulloblastoma (MB is an aggressive pediatric tumor of the Central Nervous System (CNS usually treated according to a refined risk stratification. The study of cancer stem cells (CSC in MB is a promising approach aimed at finding new treatment strategies. METHODOLOGY/PRINCIPAL FINDINGS: The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76 grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM. In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. CONCLUSIONS/SIGNIFICANCE: Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB.

  1. Sorption of volatile phenols by yeast cell walls

    Directory of Open Access Journals (Sweden)

    Nerea Jiménez-Moreno

    2009-01-01

    Full Text Available Nerea Jiménez-Moreno, Carmen Ancín-AzpilicuetaDepartment of Applied Chemistry, Universidad Pública de Navarra, Pamplona, SpainAbstract: Yeast walls can retain different wine compounds and so its use is interesting in order to eliminate harmful substances from the must which affect alcoholic fermentation (medium chain fatty acids or which affect wine quality in a negative way (ethyl phenols, ochratoxin A. The aim of this study was to examine the capacity of commercial yeast cell walls in eliminating volatile phenols (4-ethylphenol and 4-ethylguaiacol from a synthetic wine that contained 1 mg/L of each one of these compounds. The binding of these compounds to the wall was quite fast which would seem to indicate that the yeast wall-volatile compound union is produced in the outer surface layers of this enological additive. The cell walls used reduced the concentration of 4-ethylphenol and 4-ethylguaiacol, although it would seem that on modifying the matrix of the wine the number of free binding sites on the walls is also modified.Keywords: volatile phenols, yeast cell walls, wine, sorption

  2. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    Directory of Open Access Journals (Sweden)

    Lori B Huberman

    Full Text Available Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

  3. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    Science.gov (United States)

    Huberman, Lori B; Murray, Andrew W

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

  4. Another brick in the cell wall: biosynthesis dependent growth model.

    Science.gov (United States)

    Barbacci, Adelin; Lahaye, Marc; Magnenet, Vincent

    2013-01-01

    Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is possible if (i) a tensile stress is developed in the cell wall due to the coupling effect between turgor pressure and the modulation of its mechanical properties through enzymatic and physicochemical reactions and if (ii) new cell wall elements can be synthesized and assembled to the existing wall. In other words, expansive growth is the result of coupling effects between mechanical, thermal and chemical energy. To have a better understanding of this process, models must describe the interplay between physical or mechanical variable with biological events. In this paper we propose a general unified and theoretical framework to model growth in function of energy forms and their coupling. This framework is based on irreversible thermodynamics. It is then applied to model growth of the internodal cell of Chara corallina modulated by changes in pressure and temperature. The results describe accurately cell growth in term of length increment but also in term of cell pectate biosynthesis and incorporation to the expanding wall. Moreover, the classical growth model based on Lockhart's equation such as the one proposed by Ortega, appears as a particular and restrictive case of the more general growth equation developed in this paper.

  5. Another brick in the cell wall: biosynthesis dependent growth model.

    Directory of Open Access Journals (Sweden)

    Adelin Barbacci

    Full Text Available Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is possible if (i a tensile stress is developed in the cell wall due to the coupling effect between turgor pressure and the modulation of its mechanical properties through enzymatic and physicochemical reactions and if (ii new cell wall elements can be synthesized and assembled to the existing wall. In other words, expansive growth is the result of coupling effects between mechanical, thermal and chemical energy. To have a better understanding of this process, models must describe the interplay between physical or mechanical variable with biological events. In this paper we propose a general unified and theoretical framework to model growth in function of energy forms and their coupling. This framework is based on irreversible thermodynamics. It is then applied to model growth of the internodal cell of Chara corallina modulated by changes in pressure and temperature. The results describe accurately cell growth in term of length increment but also in term of cell pectate biosynthesis and incorporation to the expanding wall. Moreover, the classical growth model based on Lockhart's equation such as the one proposed by Ortega, appears as a particular and restrictive case of the more general growth equation developed in this paper.

  6. Biochemistry, proteomics, and phosphoproteomics of plant mitochondria from non-photosynthetic cells

    DEFF Research Database (Denmark)

    Havelund, Jesper; Thelen, Jay J.; Møller, Ian Max

    2013-01-01

    functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification...

  7. Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood

    Directory of Open Access Journals (Sweden)

    Heiner Falkenberg

    2013-01-01

    Full Text Available Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF, thrombopoietin (THPO, interleukin 6 (IL-6, and fms-related tyrosine kinase 3 ligand (FLT3lg. At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

  8. Altered cell wall disassembly during ripening of Cnr tomato fruit : implications for cell wall adhesion and fruit softening

    NARCIS (Netherlands)

    Orfila, C.; Huisman, M.M.H.; Willats, W.G.T.; Alebeek, van G.J.W.M.; Schols, H.A.; Seymour, G.B.; Knox, J.P.

    2002-01-01

    The Cnr (Colourless non-ripening) tomato (Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic

  9. Primary Cell Wall Structure in the Evolution of Land Plants

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Investigation of the primary cell walls of lower plants improves our understanding of the cell biology of these organisms but also has the potential to improve our understanding of cell wall structure and function in angiosperms that evolved from lower plants. Cell walls were prepared from eight species, ranging from a moss to advanced gymnosperms, and subjected to sequential chemical extraction to separate the main polysaccharide fractions. The glycosyl compositions of these fractions were then determined by gas chromatography. The results were compared among the eight plants and among data from related studies reported in the existing published reports to identify structural features that have been either highly conserved or clearly modified during evolution. Among the highly conserved features are the presence of a cellulose framework, the presence of certain hemicelluloses such as xyloglucan, and the presence of rhamnogalacturonan Ⅱ, a domain in pectic polysaccharides. Among the modified features are the abundance of mannosyl-containing hemicelluloses and the presence of methylated sugars.

  10. Magnetic domain wall conduits for single cell applications

    DEFF Research Database (Denmark)

    Donolato, Marco; Torti, A.; Kostesha, Natalie;

    2011-01-01

    The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls...... generated in micro- and nano-structures fabricated on a chip surface can be used to handle single yeast cells labeled with magnetic beads. In detail, first we show that the proposed approach maintains the microorganism viable, as proven by monitoring the division of labeled yeast cells trapped by domain...... walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain...

  11. Simulated microgravity inhibits cell wall regeneration of Penicillium decumbens protoplasts

    Science.gov (United States)

    Zhao, C.; Sun, Y.; Yi, Z. C.; Rong, L.; Zhuang, F. Y.; Fan, Y. B.

    2010-09-01

    This work compares cell wall regeneration from protoplasts of the fungus Penicillium decumbens under rotary culture (simulated microgravity) and stationary cultures. Using an optimized lytic enzyme mixture, protoplasts were successfully released with a yield of 5.3 × 10 5 cells/mL. Under simulated microgravity conditions, the protoplast regeneration efficiency was 33.8%, lower than 44.9% under stationary conditions. Laser scanning confocal microscopy gave direct evidence for reduced formation of polysaccharides under simulated conditions. Scanning electron microscopy showed the delayed process of cell wall regeneration by simulated microgravity. The delayed regeneration of P. decumbens cell wall under simulated microgravity was likely caused by the inhibition of polysaccharide synthesis. This research contributes to the understanding of how gravitational loads affect morphological and physiological processes of fungi.

  12. Analyzing Cell Wall Elasticity After Hormone Treatment: An Example Using Tobacco BY-2 Cells and Auxin.

    Science.gov (United States)

    Braybrook, Siobhan A

    2017-01-01

    Atomic force microscopy, and related nano-indentation techniques, is a valuable tool for analyzing the elastic properties of plant cell walls as they relate to changes in cell wall chemistry, changes in development, and response to hormones. Within this chapter I will describe a method for analyzing the effect of the phytohormone auxin on the cell wall elasticity of tobacco BY-2 cells. This general method may be easily altered for different experimental systems and hormones of interest.

  13. Diffusion of an organic cation into root cell walls.

    Science.gov (United States)

    Meychik, N R; Yermakov, I P; Prokoptseva, O S

    2003-07-01

    Uptake of a cationic dye (methylene blue) by isolated root cell walls, roots of whole transpiring seedlings, and excised roots was investigated using 7-day-old seedlings of cucumber, maize, and wheat. The number of ionogenic groups per 1 g dry and wet weight of the root cell walls, their swelling capacity (K(cw)), time-dependence of methylene blue (M(cw)) ion exchange capacity, and diffusion coefficients of the cation diffusion in the polymer matrix of the cell walls (D(cw)) were determined. The M(cw) value depended on pH (or carboxyl group dissociation); it changed in accordance with the number of carboxyl groups per 1 g cell wall dry weight. This parameter decreased in the order: cucumber > wheat > maize. For description of experimental kinetic curves and calculation of cation diffusion coefficients, the equation for ion diffusion into a cylinder of infinite length was used. The chosen model adequately described cation diffusion in cell walls and roots. Diffusion coefficient values for cucumber, wheat, and maize were 3.1*10(-8), 1.3*10(-8), and 8.4*10(-8) cm(2)/sec, respectively. There was a statistically significant linear dependence between K(cw) and D(cw) values, which characterize the same property of the polymer matrix, rigidity of its polymer structure or the degree of cross-linkage or permeability. This also confirms the right choice of the model selected for calculation of methylene blue diffusion coefficients, because K(cw) and D(cw) values were obtained in independent experiments. The coefficients determined for methylene blue diffusion in transpiring seedling roots (D(ts)) and excised roots (D(er)) depended on the plant species. The rate of methylene blue diffusion into the excised roots was either 1.5-fold lower (cucumber) or 3-4-times lower (maize, wheat) than in cell walls. The values of diffusion coefficients in roots of whole seedlings were comparable which those for the cell walls. On the basis of the experimental data and results of calculations

  14. Tandem duplication of KIT exon 11 influences the proteome of canine mast cell tumours.

    Science.gov (United States)

    Schlieben, P; Meyer, A; Weise, C; Bondzio, A; Gruber, A D; Klopfleisch, R

    2013-05-01

    Mutations with permanent activation of the stem cell factor receptor KIT have been identified as one potential cause for canine cutaneous mast cell tumours (MCTs). The exact changes in global gene expression patterns associated with permanent activation of KIT in these tumours are unknown. The present study compares, by the use of two dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the proteomes of canine MCTs, with and without KIT exon 11 tandem duplication. Fifteen differentially expressed proteins were identified in mutated MCTs. These are mainly involved in cytoskeleton structure and cell motility (ACTR2, ACTB and CAPPA1), cell signalling (ARHGDIA) and lipid metabolism (ALOX15 and ACSBG4), or are serum proteins. The results therefore support the notion that KIT mutation is associated with changes in the proteome of affected cells with a major effect on the composition of the cytoskeletal proteome and cell motility proteins. No overlaps were identified when the results were compared with a recent study on the proteomic differences between low- and high-grade tumours, suggesting that KIT-mutated tumours may be regarded as a separate entity of high-grade tumours with potential relevance to therapeutic strategies.

  15. Growth and cell wall changes in rice roots during spaceflight.

    Science.gov (United States)

    Hoson, Takayuki; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Tanimoto, Eiichi

    2003-08-01

    We analyzed the changes in growth and cell wall properties of roots of rice (Oryza sativa L. cv. Koshihikari) grown for 68.5, 91.5, and 136 h during the Space Shuttle STS-95 mission. In space, most of rice roots elongated in a direction forming a constant mean angle of about 55 degrees with the perpendicular base line away from the caryopsis in the early phase of growth, but later the roots grew in various directions, including away from the agar medium. In space, elongation growth of roots was stimulated. On the other hand, some of elasticity moduli and viscosity coefficients were higher in roots grown in space than on the ground, suggesting that the cell wall of space-grown roots has a lower capacity to expand than the controls. The levels of both cellulose and the matrix polysaccharides per unit length of roots decreased greatly, whereas the ratio of the high molecular mass polysaccharides in the hemicellulose fraction increased in space-grown roots. The prominent thinning of the cell wall could overwhelm the disadvantageous changes in the cell wall mechanical properties, leading to the stimulation of elongation growth in rice roots in space. Thus, growth and the cell wall properties of rice roots were strongly modified under microgravity conditions during spaceflight.

  16. The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells.

    Science.gov (United States)

    Bauer, W D; Talmadge, K W; Keegstra, K; Albersheim, P

    1973-01-01

    The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 m urea, endoglucanase, or 0.5 n NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the sycamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The structure of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of beta-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues.

  17. Transcriptional Wiring of Cell Wall-Related Genes in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Marek Mutwil; Colin Ruprecht; Federico M. Giorgi; Martin Bringmann; Bj(o)rn Usadel; Staffan Persson

    2009-01-01

    Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the correspond-ing proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of anal-yses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes (CAZy)-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface (http://aranet.mpimp-golm.mpg.de/corecarb) containing downloadable files for all the transcriptional associations.

  18. Fluorescent Probes for Exploring Plant Cell Wall Deconstruction: A Review

    Directory of Open Access Journals (Sweden)

    Gabriel Paës

    2014-07-01

    Full Text Available Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction.

  19. The role of the cell wall in fungal pathogenesis.

    Science.gov (United States)

    Arana, David M; Prieto, Daniel; Román, Elvira; Nombela, César; Alonso-Monge, Rebeca; Pla, Jesús

    2009-05-01

    Fungal infections are a serious health problem. In recent years, basic research is focusing on the identification of fungal virulence factors as promising targets for the development of novel antifungals. The wall, as the most external cellular component, plays a crucial role in the interaction with host cells mediating processes such as adhesion or phagocytosis that are essential during infection. Specific components of the cell wall (called PAMPs) interact with specific receptors in the immune cell (called PRRs), triggering responses whose molecular mechanisms are being elucidated. We review here the main structural carbohydrate components of the fungal wall (glucan, mannan and chitin), how their biogenesis takes place in fungi and the specific receptors that they interact with. Different model fungal pathogens are chosen to illustrate the functional consequences of this interaction. Finally, the identification of the key components will have important consequences in the future and will allow better approaches to treat fungal infections.

  20. Proteomics of the Peroxisome

    OpenAIRE

    2006-01-01

    Genomes provide us with a blue print for the potential of a cell. However, the activity of a cell is expressed in its proteome. Full understanding of the complexity of cells demands a comprehensive view of the proteome; its interactions, activity states and organization. Comprehensive proteomic approaches applied to peroxisomes have yielded new insights into the organelle and its dynamic interplay with other cellular structures. As technologies and methodologies improve proteomics hold the pr...

  1. Characters of Fractal Ultrastructure in Wood Cell Wall

    Institute of Scientific and Technical Information of China (English)

    LI Beimei; ZHAO Guangjie

    2006-01-01

    Fractal theory was introduced in order to describe the ultrastructure of wood cell wall in this paper.The cellulose chain clusters around nano-scale were viewed as a fractal object that consists of many fibrillar structural units with different scales including microfibrils.On the basis of the morphological data of wood cell wall.fractal dimensions of multi-level fibrillar structural units were calculated by fractal-geometry approach,and then the morphological and structural characteristics of fibers as well as the influences on wood properties were investigated according to the dimensions.Besides,the fractal self-nesting character of the ultrastruture was also analyzed.

  2. Hematopoietic Stem Cells Expansionin Rotating Wall Vessel

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 IntroductionClinical trials have demonstrated that ex vivo expanded hematopoietic stem cells (HSCs) and progenitors offer great promise in reconstituting in vivo hematopoiesis in patients who have undergone intensive chemotherapy. It is therefore necessary to develop a clinical-scale culture system to provide the expanded HSCs and progenitors. Static culture systems such as T-flasks and gas-permeable blood bags are the most widely used culture devices for expanding hematopoietic cells. But they reveal sev...

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  4. Development of Advanced Technologies for Complete Genomic and Proteomic Characterization of Quantized Human Tumor Cells

    Science.gov (United States)

    2014-07-01

    four parental cell lines and eleven subclones derived (Figure 11). We have begun deep proteome analysis of the secretomes by performing off- gel ...1 Antihelminthic 33 7 7 Antiarrhythmic 24 0 1 Antibacterial 227 11 11 Antifungal 55 5 5 Antineoplastic 115 29 28 Antihyperlipidemic 12 3 4

  5. Phagocytic properties of lung alveolar wall cells

    Directory of Open Access Journals (Sweden)

    Tanaka,Akisuke

    1974-04-01

    Full Text Available For the purpose to define the mechanism of heavy metal intoxication by inhalation, morphologic observations were made on rat lungs after nasal instillation of iron colloid particles of positive and negative electric charges. Histochemical observation was also made on the liver and spleen of these animals. The instilled iron colloid particles reach the alveolar cavity easily, as can be seen in the tissue sections stained by Prussian blue reaction. Alveolar macrophages do take up them avidly both of positive and negative charges, though much less the positive particles than negative ones. In contrast, the alveolar epithelial cells take up solely positive particles by phagocytosis but not negative ones. Electron microscope observation revealed that the positive particles are ingested by Type I epithelial cells by pinocytosis and by Type II cells by phagocytosis as well. Then the iron colloid particles are transferred into the basement membrane by exocytosis. Travelling through the basement membrane they are again taken up by capillary endothelial cells by phagocytosis. Some particles were found in the intercellular clefts of capillary endothelial cells but not any iron colloid particles in the intercellular spaces of epithelial cells and in the capillary lumen. However, the liver and spleen tissues of the animals given iron colloid showed a strong positive iron reaction. On the basis of these observations, the mechanism of acute intoxication by inhaling heavy metal dusts like lead fume is discussed from the view point of selective uptake of alveolar epithelial and capillary endothelial cells for the particles of the positive electric cha'rge.

  6. Quantitative Membrane Proteomics in a Human Mesenchymal Stem Cell Line Undergoing Osteogenic Differentiation

    DEFF Research Database (Denmark)

    Christiansen, Helle

    -transform mass spectrometry was used due to the high resolution and sensitivity provided by this strategy enabling very high sensitivity data and thus increased possibility of finding specific marker candidates for these cells. We have chosen to employ quantitative mass spectrometry on SILAC-labeled (Stable...... of proteins during several timepoints in osteogenesis and find that nearly all proteins quantified increase in level as osteogenesis progresses. This study expands beyond qualitative identification of markers candidates and into the quantitative profiling of membrane proteome changes over time in osteogenic....... The objectives of this project were to identify and quantify such surface protein markers of human MSCs using quantitative mass spectrometry-based proteomics and to monitor the quantitative changes of these surface markers during timepoints in osteogenic differentiation. Quantitative proteomics by fourier...

  7. Bacterial Cell Wall Growth, Shape and Division

    NARCIS (Netherlands)

    Derouaux, A.; Terrak, M.; den Blaauwen, T.; Vollmer, W.; Remaut, H.; Fronzes, R.

    2014-01-01

    The shape of a bacterial cell is maintained by its peptidoglycan sacculus that completely surrounds the cytoplasmic membrane. During growth the sacculus is enlarged by peptidoglycan synthesis complexes that are controlled by components linked to the cytoskeleton and, in Gram-negative bacteria, by ou

  8. Cell wall modification in grapevine cells in response to UV stress investigated by atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lesniewska, E.; Adrian, M.; Klinguer, A.; Pugin, A

    2004-08-15

    Despite cell wall reinforcement being a well-known defence mechanism of plants, it remains poorly characterized from a physical point of view. The objective of this work was to further describe this mechanism. Vitis vinifera cv Gamay cells were treated with UV-light (254 nm), a well-known elicitor of defence mechanisms in grapevines, and physical cell wall modifications were observed using the atomic force microscopy (AFM) under native conditions. The grapevine cell suspensions were continuously observed in their culture medium from 30 min to 24 h after elicitation. In the beginning, cellulose fibrils covered by a matrix surrounded the control and treated cells. After 3 h, the elicited cells displayed sprouted expansions around the cell wall that correspond to pectin chains. These expansions were not observed on untreated grapevine cells. The AFM tip was used to determine the average surface elastic modulus of cell wall that account for cell wall mechanical properties. The elasticity is diminished in UV-treated cells. In a comparative study, grapevine cells showed the same decrease in cell wall elasticity when treated with a fungal biotic elicitor of defence response. These results demonstrate cell wall strengthening by UV stress.

  9. An emerging role of pectic rhamnogalacturonanII for cell wall integrity.

    Science.gov (United States)

    Reboul, Rebecca; Tenhaken, Raimund

    2012-02-01

    The plant cell wall is a complex network of different polysaccharides and glycoproteins, showing high diversity in nature. The essential components, tethering cell wall are under debate, as novel mutants challenge established models. The mutant ugd2,3 with a reduced supply of the important wall precursor UDP-glucuronic acid reveals the critical role of the pectic compound rhamnogalacturonanII for cell wall stability. This polymer seems to be more important for cell wall integrity than the previously favored xyloglucan.

  10. Elucidation of the avian nucleolar proteome by quantitative proteomics using SILAC and changes in cells infected with the coronavirus infectious bronchitis virus.

    Science.gov (United States)

    Emmott, Edward; Smith, Catriona; Emmett, Stevan R; Dove, Brian K; Hiscox, Julian A

    2010-10-01

    The nucleolus is a dynamic subnuclear compartment involved in ribosome subunit biogenesis, regulation of cell stress and modulation of cellular growth and the cell cycle, among other functions. The nucleolus is composed of complex protein/protein and protein/RNA interactions. It is a target of virus infection with many viral proteins being shown to localize to the nucleolus during infection. Perturbations to the structure of the nucleolus and its proteome have been predicted to play a role in both cellular and infectious disease. Stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS with bioinformatic analysis using Ingenuity Pathway Analysis was used to investigate whether the nucleolar proteome altered in virus-infected cells. In this study, the avian nucleolar proteome was defined in the absence and presence of virus, in this case the positive strand RNA virus, avian coronavirus infectious bronchitis virus. Data sets, potential protein changes and the functional consequences of virus infection were validated using independent assays. These demonstrated that specific rather than generic changes occurred in the nucleolar proteome in infectious bronchitis virus-infected cells.

  11. The Basal Level Ethylene Response is Important to the Wall and Endomembrane Structure in the Hypocotyl Cells of Etiolated Arabidopsis Seedlings

    Institute of Scientific and Technical Information of China (English)

    Chan Xu; Xiaoyan Gao; Xiaobin Sun; Chi-Kuang Wen

    2012-01-01

    The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells.Preventing the basal level ethylene response facilitated cell elongation,and the cells exhibited wall loosening and separation phenotype.Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum (rER) membranes,the presence of paramural bodies,and the circular Golgi formation.The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide.The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses.Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 (ein2) seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking,remodeling,and wall modifications were differentially expressed.FM4-64 staining supported the proteomic changes,which indicated reduced endocytosis activity with alleviation of the ethylene response.The basal level ethylene response has an important role in endomembrane trafficking,biological materials transport and maintenance of the endomembrane organization.It is possible that endomembrane alterations may partly associate with the wall modifications,though the biological significance of the alterations should be addressed in future studies.

  12. Cell wall structure and function in lactic acid bacteria.

    Science.gov (United States)

    Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

    2014-08-29

    The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.

  13. In planta modification of the potato tuber cell wall

    NARCIS (Netherlands)

    Oomen, R.J.F.J.

    2003-01-01

    Apart from its well known uses in the human diet a large amount of the grown potatoes (about one third in the Netherlands) is used for the isolation of starch which is used in several food and non-food applications. The cell wall fibres comprise a large portion of the waste material remaining after

  14. Characterisation of cell wall polysaccharides in bilberries and black currants

    NARCIS (Netherlands)

    Hilz, H.

    2007-01-01

    During berry juice production, polysaccharides are released from the cell walls and cause thickening and high viscosity when the berries are mashed. Consequences are a low juice yield and a poor colour. This can be prevented by the use of enzymes that degrade these polysaccharides. To use these enzy

  15. Analyzing the complex machinery of cell wall biosynthesis

    NARCIS (Netherlands)

    Timmers, J.F.P.

    2009-01-01

    The plant cell wall polymers make up most of the plant biomass and provide the raw material for many economically important products including food, feed, bio-materials, chemicals, textiles, and biofuel. This broad range of functions and applications make the biosynthesis of these polysaccharides a

  16. Evidence for a Melanin Cell Wall Component in Pneumocystis carinii

    OpenAIRE

    Icenhour, Crystal R.; Kottom, Theodore J.; Limper, Andrew H

    2003-01-01

    Fluorescein isothiocyanate-labeled monoclonal antibodies specific for fungal melanin were used in this study to visualize melanin-like components of the Pneumocystis carinii cell wall. A colorimetric enzyme assay confirmed these findings. This is the first report of melanin-like pigments in Pneumocystis.

  17. The role of the cell wall in plant immunity

    DEFF Research Database (Denmark)

    Malinovsky, Frederikke Gro; Fangel, Jonatan Ulrik; Willats, William George Tycho

    2014-01-01

    The battle between plants and microbes is evolutionarily ancient, highly complex, and often co-dependent. A primary challenge for microbes is to breach the physical barrier of host cell walls whilst avoiding detection by the plant's immune receptors. While some receptors sense conserved microbial...

  18. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides

    NARCIS (Netherlands)

    Vries, de R.P.; Visser, J.

    2001-01-01

    Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a m

  19. Characterisation of cell-wall polysaccharides from mandarin segment membranes

    NARCIS (Netherlands)

    Coll-Almela, L.; Saura-Lopez, D.; Laencina-Sanchez, J.; Schols, H.A.; Voragen, A.G.J.; Ros-García, J.M.

    2015-01-01

    In an attempt to develop a process of enzymatic peeling of mandarin segments suitable for use on an industrial scale, the cell wall fraction of the segment membrane of Satsuma mandarin fruits was extracted to obtain a chelating agent-soluble pectin fraction (ChSS), a dilute sodium hydroxide-soluble

  20. Polymer mobility in cell walls of cucumber hypocotyls

    Science.gov (United States)

    Fenwick, K. M.; Apperley, D. C.; Cosgrove, D. J.; Jarvis, M. C.

    1999-01-01

    Cell walls were prepared from the growing region of cucumber (Cucumis sativus) hypocotyls and examined by solid-state 13C NMR spectroscopy, in both enzymically active and inactivated states. The rigidity of individual polymer segments within the hydrated cell walls was assessed from the proton magnetic relaxation parameter, T2, and from the kinetics of cross-polarisation from 1H to 13C. The microfibrils, including most of the xyloglucan in the cell wall, as well as cellulose, behaved as very rigid solids. A minor xyloglucan fraction, which may correspond to cross-links between microfibrils, shared a lower level of rigidity with some of the pectic galacturonan. Other pectins, including most of the galactan side-chain residues of rhamnogalacturonan I, were much more mobile and behaved in a manner intermediate between the solid and liquid states. The only difference observed between the enzymically active and inactive cell walls, was the loss of a highly mobile, methyl-esterified galacturonan fraction, as the result of pectinesterase activity.

  1. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    DEFF Research Database (Denmark)

    Thygesen, Lisbeth Garbrecht; E. Thybring, Emil; Johansen, Katja Salomon;

    2014-01-01

    . Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry......, particularly when it comes to up-scaling of processes based on insoluble feed stocks....

  2. Roles of tRNA in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Dare, Kiley; Ibba, Michael

    2012-01-01

    Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families...

  3. The digestion of yeast cell wall polysaccharides in veal calves

    NARCIS (Netherlands)

    Gaillard, B.D.E.; Weerden, van E.J.

    1976-01-01

    1. The digestibility of the cell wall polysaccharides of an alkane-grown yeast in different parts of the digestive tract of two veal calves fitted with re-entrant cannulas at the end of the ileum was studied by replacing part of the skim-milk powder of their ‘normal’, milk-substitute (all-milk-prote

  4. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

    Science.gov (United States)

    Carinhas, Nuno; Robitaille, Aaron Mark; Moes, Suzette; Carrondo, Manuel José Teixeira; Jenoe, Paul; Oliveira, Rui; Alves, Paula Marques

    2011-01-01

    Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC) to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

  5. Prion Protein Deficiency Causes Diverse Proteome Shifts in Cell Models That Escape Detection in Brain Tissue.

    Science.gov (United States)

    Mehrabian, Mohadeseh; Brethour, Dylan; Williams, Declan; Wang, Hansen; Arnould, Hélène; Schneider, Benoit; Schmitt-Ulms, Gerold

    2016-01-01

    A popular method for studying the function of a given protein is to generate and characterize a suitable model deficient for its expression. For the prion protein (PrP), best known for its role in several invariably fatal neurodegenerative diseases, a natural choice, therefore, would be to undertake such studies with brain samples. We recently documented the surprising observation that PrP deficiency caused a loss or enhancement of NCAM1 polysialylation, dependent on the cell model used. To identify possible causes for this disparity, we set out to systematically investigate the consequence of PrP deficiency on the global proteome in brain tissue and in four distinct cell models. Here we report that PrP deficiency causes robust but surprisingly divergent changes to the global proteomes of cell models but has no discernible impact on the global brain proteome. Amongst >1,500 proteins whose levels were compared in wild-type and PrP-deficient models, members of the MARCKS protein family exhibited pronounced, yet cell model-dependent changes to their steady-state levels. Follow-up experiments revealed that PrP collaborates with members of the MARCKS protein family in its control of NCAM1 polysialylation. We conclude that the physiological function of PrP may be masked in analyses of complex brain samples but its cell-type specific influence on a lipid raft-based NCAM1-related cell biology comes to the fore in investigations of specific cell types.

  6. Vesicular Trans-Cell Wall Transport in Fungi: A Mechanism for the Delivery of Virulence-Associated Macromolecules?

    Directory of Open Access Journals (Sweden)

    Marcio L. Rodrigues

    2008-01-01

    Full Text Available Fungal cells are encaged in rigid, complex cell walls. Until recently, there was remarkably little information regarding the trans-fungal cell wall transfer of intracellular macromolecules to the extracellular space. Recently, several studies have begun to elucidate the mechanisms that fungal cells utilize to secrete a wide variety of macromolecules through the cell wall. The combined use of transmission electron microscopy, serology, biochemistry, proteomics and lipidomics have revealed that the fungal pathogens Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida parapsilosis and Sporothrix schenckii, as well as the model yeast Saccharomyces cerevisiae, each produces extracellular vesicles that carry lipids, proteins, polysaccharides and pigment-like structures of unquestionable biological significance. Compositional analysis of the C. neoformans and H. capsulatum extracellular vesicles suggests that they may function as ‘virulence bags’, with the potential to modulate the host-pathogen interaction in favor of the fungus. The cellular origin of the extracellular vesicles remains unknown, but morphological and biochemical features indicate that they are similar to the well-described mammalian exosomes.

  7. Action of xyloglucan hydrolase within the native cell wall architecture and its effect on cell wall extensibility in azuki bean epicotyls.

    Science.gov (United States)

    Kaku, Tomomi; Tabuchi, Akira; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Hoson, Takayuki

    2002-01-01

    Xyloglucan hydrolase (XGH) has recently been purified from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls as a new type of xyloglucan-degrading enzyme [Tabuchi et al. (2001) Plant Cell Physiol. 42: 154]. In the present study, the effects of XGH on the mechanical properties of the cell wall and on the level and the molecular size of xyloglucans within the native wall architecture were examined in azuki bean epicotyls. When the epidermal tissue strips from the growing regions of azuki bean epicotyls were incubated with XGH, the mechanical extensibility of the cell wall dramatically increased. XGH exogenously applied to cell wall materials (homogenates) or epidermal tissue strips decreased the amount of xyloglucans via the solubilization of the polysaccharides. Also, XGH substantially decreased the molecular mass of xyloglucans in both materials. These results indicate that XGH is capable of hydrolyzing xyloglucans within the native cell wall architecture and thereby increasing the cell wall extensibility in azuki bean epicotyls.

  8. Molecular deformation mechanisms of the wood cell wall material.

    Science.gov (United States)

    Jin, Kai; Qin, Zhao; Buehler, Markus J

    2015-02-01

    Wood is a biological material with outstanding mechanical properties resulting from its hierarchical structure across different scales. Although earlier work has shown that the cellular structure of wood is a key factor that renders it excellent mechanical properties at light weight, the mechanical properties of the wood cell wall material itself still needs to be understood comprehensively. The wood cell wall material features a fiber reinforced composite structure, where cellulose fibrils act as stiff fibers, and hemicellulose and lignin molecules act as soft matrix. The angle between the fiber direction and the loading direction has been found to be the key factor controlling the mechanical properties. However, how the interactions between theses constitutive molecules contribute to the overall properties is still unclear, although the shearing between fibers has been proposed as a primary deformation mechanism. Here we report a molecular model of the wood cell wall material with atomistic resolution, used to assess the mechanical behavior under shear loading in order to understand the deformation mechanisms at the molecular level. The model includes an explicit description of cellulose crystals, hemicellulose, as well as lignin molecules arranged in a layered nanocomposite. The results obtained using this model show that the wood cell wall material under shear loading deforms in an elastic and then plastic manner. The plastic regime can be divided into two parts according to the different deformation mechanisms: yielding of the matrix and sliding of matrix along the cellulose surface. Our molecular dynamics study provides insights of the mechanical behavior of wood cell wall material at the molecular level, and paves a way for the multi-scale understanding of the mechanical properties of wood.

  9. Analysis of membrane proteome and secretome in cells over-expressing ADAM17 using quantitative proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, R.; Simabuco, F.M. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Yokoo, S.; Paes Leme, A.F. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Sherman, N. [University of Virginia, Charlottesville, VA (United States)

    2012-07-01

    Full text: A disintegrin and metalloproteinase (ADAM) protease is involved in proteolytic ectodomain shedding of several membrane-associated proteins and modulation of key cell signaling pathways in the tumor microenvironment. In this study, we examined the effect of over-expressing the full length human ADAM17 in membrane and secreted proteins. To this end, we constructed a stable Flp-In T-RExHEK293 cells expressing ADAM17 by tetracycline induction. These cells were grown in Dulbeccos modified Eagles medium containing light lysine, arginine or heavy, L-Arg-13C615N4 and L-Lys -13C615N2 (SILAC: stable isotope labeling with amino acid in cell culture) media and they were treated with an ADAM17 activator, phorbolester (PMA). Controls such as Flp-In T-RExHEK293 cell without PMA treatment and without ADAM17 cloned were cultivated in light medium. The ADAM17 overexpression was induced with tetracycline 500 ng/ml for 24 hours. Cells in a heavy condition were treated with PMA 50 ng/ml for 1 hour and vehicle DMSO was used as control in a light cell condition. The extracellular media were collected, concentrated and used to evaluate the secretome and a cell surface biotinylation-based approach was used to capture cell surface-associated proteins. The biotinylated proteins were eluted with dithiothreitol, alkylated with iodoacetamide and then digested with trypsin. The resulting peptides were subjected to LC-MS/MS analysis on an ETD enabled Orbitrap Velos instrument. The results showed different proteins up or down regulated in membrane and secretome analysis which might represent potential molecules involved in signaling or ADAM17 regulation events. (author)

  10. Structure of Plant Cell Walls: XI. GLUCURONOARABINOXYLAN, A SECOND HEMICELLULOSE IN THE PRIMARY CELL WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS.

    Science.gov (United States)

    Darvill, J E; McNeil, M; Darvill, A G; Albersheim, P

    1980-12-01

    The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues.

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

    Directory of Open Access Journals (Sweden)

    Euler Kerstin N

    2013-01-01

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

  12. Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells.

    Science.gov (United States)

    Meganathan, Kesavan; Jagtap, Smita; Wagh, Vilas; Winkler, Johannes; Gaspar, John Antonydas; Hildebrand, Diana; Trusch, Maria; Lehmann, Karola; Hescheler, Jürgen; Schlüter, Hartmut; Sachinidis, Agapios

    2012-01-01

    Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.

  13. Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kesavan Meganathan

    Full Text Available Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs. Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE coupled with Tandem Mass spectrometry to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s. Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3 after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2, that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.

  14. Phenotypic screening of Arabidopsis T-DNA insertion lines for cell wall mechanical properties revealed ANTHOCYANINLESS2, a cell wall-related gene.

    Science.gov (United States)

    Mabuchi, Atsushi; Soga, Kouichi; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2016-02-01

    We performed a phenotypic screening of confirmed homozygous T-DNA insertion lines in Arabidopsis for cell wall extensibility, in an attempt to identify genes involved in the regulation of cell wall mechanical properties. Seedlings of each line were cultivated and the cell wall extensibility of their hypocotyls was measured with a tensile tester. Hypocotyls of lines with known cell wall-related genes showed higher or lower extensibility than those of the wild-type at high frequency, indicating that the protocol used was effective. In the first round of screening of randomly selected T-DNA insertion lines, we identified ANTHOCYANINLESS2 (ANL2), a gene involved in the regulation of cell wall mechanical properties. In the anl2 mutant, the cell wall extensibility of hypocotyls was significantly lower than that of the wild-type. Levels of cell wall polysaccharides per hypocotyl, particularly cellulose, increased in anl2. Microarray analysis showed that in anl2, expression levels of the major peroxidase genes also increased. Moreover, the activity of ionically wall-bound peroxidases clearly increased in anl2. The activation of peroxidases as well as the accumulation of cell wall polysaccharides may be involved in decreased cell wall extensibility. The approach employed in the present study could contribute to our understanding of the mechanisms underlying the regulation of cell wall mechanical properties.

  15. Studying biomolecule localization by engineering bacterial cell wall curvature.

    Directory of Open Access Journals (Sweden)

    Lars D Renner

    Full Text Available In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i the cell-shape related protein MreB of Escherichia coli, which is positioned along the long axis of the rod-shaped cell; and ii the negative curvature-sensing cell division protein DivIVA of Bacillus subtilis, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of E. coli and B. subtilis demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.

  16. Cell wall staining with Trypan Blue enables quantitative analysis of morphological changes in yeast cells

    Directory of Open Access Journals (Sweden)

    Johannes eLiesche

    2015-02-01

    Full Text Available Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

  17. Resistance to antibiotics targeted to the bacterial cell wall.

    Science.gov (United States)

    Nikolaidis, I; Favini-Stabile, S; Dessen, A

    2014-03-01

    Peptidoglycan is the main component of the bacterial cell wall. It is a complex, three-dimensional mesh that surrounds the entire cell and is composed of strands of alternating glycan units crosslinked by short peptides. Its biosynthetic machinery has been, for the past five decades, a preferred target for the discovery of antibacterials. Synthesis of the peptidoglycan occurs sequentially within three cellular compartments (cytoplasm, membrane, and periplasm), and inhibitors of proteins that catalyze each stage have been identified, although not all are applicable for clinical use. A number of these antimicrobials, however, have been rendered inactive by resistance mechanisms. The employment of structural biology techniques has been instrumental in the understanding of such processes, as well as the development of strategies to overcome them. This review provides an overview of resistance mechanisms developed toward antibiotics that target bacterial cell wall precursors and its biosynthetic machinery. Strategies toward the development of novel inhibitors that could overcome resistance are also discussed.

  18. Dislocation-mediated growth of bacterial cell walls

    CERN Document Server

    Amir, Ariel

    2012-01-01

    Recent experiments have illuminated a remarkable growth mechanism of rod-shaped bacteria: proteins associated with cell wall extension move at constant velocity in circles oriented approximately along the cell circumference (Garner et al., Science (2011), Dominguez-Escobar et al. Science (2011), van Teeffelen et al. PNAS (2011). We view these as dislocations in the partially ordered peptidoglycan structure, activated by glycan strand extension machinery, and study theoretically the dynamics of these interacting defects on the surface of a cylinder. Generation and motion of these interacting defects lead to surprising effects arising from the cylindrical geometry, with important implications for growth. We also discuss how long range elastic interactions and turgor pressure affect the dynamics of the fraction of actively moving dislocations in the bacterial cell wall.

  19. Targeted and non-targeted effects in cell wall polysaccharides from transgenetically modified potato tubers

    NARCIS (Netherlands)

    Huang, J.H.

    2016-01-01

    The plant cell wall is a chemically complex network composed mainly of polysaccharides. Cell wall polysaccharides surround and protect plant cells and are responsible for the stability and rigidity of plant tissue. Pectin is a major component of primary cell wall and the middle lamella of plants. Ho

  20. Pectic arabinan side chains are essential for pollen cell wall integrity during pollen development

    NARCIS (Netherlands)

    Cankar, K.; Kortstee, A.J.; Toonen, M.A.J.; Wolters-Arts, M.; Houbein, R.; Mariani, C.; Ulvskov, P.; Jorgensen, B.; Schols, H.A.; Visser, R.G.F.; Trindade, L.M.

    2014-01-01

    Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure–function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pec

  1. Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.; Ari, Krakowski; Luo, Kunxin; Chen, David J.; Li, Song

    2004-08-08

    Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference map of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.

  2. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    Hugo Melida; Antonio Encina; Asier Largo-Gosens; Esther Novo-Uzal; Rogelio Santiago; Federico Pomar; Pedro Garca; Penelope Garca-Angulo; Jose Luis Acebes; Jesus Alvarez

    2015-01-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

  3. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells.

    Science.gov (United States)

    Villar, Margarita; Ayllón, Nieves; Alberdi, Pilar; Moreno, Andrés; Moreno, María; Tobes, Raquel; Mateos-Hernández, Lourdes; Weisheit, Sabine; Bell-Sakyi, Lesley; de la Fuente, José

    2015-12-01

    Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host-pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick-Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These results

  4. Technological advances for deciphering the complexity of psychiatric disorders: merging proteomics with cell biology.

    Science.gov (United States)

    Wesseling, Hendrik; Guest, Paul C; Lago, Santiago G; Bahn, Sabine

    2014-08-01

    Proteomic studies have increased our understanding of the molecular pathways affected in psychiatric disorders. Mass spectrometry and two-dimensional gel electrophoresis analyses of post-mortem brain samples from psychiatric patients have revealed effects on synaptic, cytoskeletal, antioxidant and mitochondrial protein networks. Multiplex immunoassay profiling studies have found alterations in hormones, growth factors, transport and inflammation-related proteins in serum and plasma from living first-onset patients. Despite these advances, there are still difficulties in translating these findings into platforms for improved treatment of patients and for discovery of new drugs with better efficacy and side effect profiles. This review describes how the next phase of proteomic investigations in psychiatry should include stringent replication studies for validation of biomarker candidates and functional follow-up studies which can be used to test the impact on physiological function. All biomarker candidates should now be tested in series with traditional and emerging cell biological approaches. This should include investigations of the effects of post-translational modifications, protein dynamics and network analyses using targeted proteomic approaches. Most importantly, there is still an urgent need for development of disease-relevant cellular models for improved translation of proteomic findings into a means of developing novel drug treatments for patients with these life-altering disorders.

  5. Proteomic analysis of cellular response induced by boron neutron capture reaction in human squamous cell carcinoma SAS cells.

    Science.gov (United States)

    Sato, Akira; Itoh, Tasuku; Imamichi, Shoji; Kikuhara, Sota; Fujimori, Hiroaki; Hirai, Takahisa; Saito, Soichiro; Sakurai, Yoshinori; Tanaka, Hiroki; Nakamura, Hiroyuki; Suzuki, Minoru; Murakami, Yasufumi; Baiseitov, Diaz; Berikkhanova, Kulzhan; Zhumadilov, Zhaxybay; Imahori, Yoshio; Itami, Jun; Ono, Koji; Masunaga, Shinichiro; Masutani, Mitsuko

    2015-12-01

    To understand the mechanism of cell death induced by boron neutron capture reaction (BNCR), we performed proteome analyses of human squamous tumor SAS cells after BNCR. Cells were irradiated with thermal neutron beam at KUR after incubation under boronophenylalanine (BPA)(+) and BPA(-) conditions. BNCR mainly induced typical apoptosis in SAS cells 24h post-irradiation. Proteomic analysis in SAS cells suggested that proteins functioning in endoplasmic reticulum, DNA repair, and RNA processing showed dynamic changes at early phase after BNCR and could be involved in the regulation of cellular response to BNCR. We found that the BNCR induces fragments of endoplasmic reticulum-localized lymphoid-restricted protein (LRMP). The fragmentation of LRMP was also observed in the rat tumor graft model 20 hours after BNCT treatment carried out at the National Nuclear Center of the Republic of Kazakhstan. These data suggest that dynamic changes of LRMP could be involved during cellular response to BNCR.

  6. Tomato Fruit Cell Wall Synthesis during Development and Senescence : In Vivo Radiolabeling of Wall Fractions Using [C]Sucrose.

    Science.gov (United States)

    Mitcham, E J; Gross, K C; Ng, T J

    1989-02-01

    The pedicel of tomato fruit (Lycopersicon esculentum Mill., cv ;Rutgers') of different developmental stages from immature-green (IG) to red was injected on the vine with 7 microcuries [(14)C(U)]sucrose and harvested after 18 hours. Cell walls were isolated from outer pericarp and further fractionated yielding ionically associated pectin, covalently bound pectin, hemicellulosic fraction I, hemicellulosic fraction II, and cellulosic fraction II. The dry weight of the total cell wall and of each cell wall fraction per gram fresh weight of pericarp tissue decreased after the mature-green (MG) stage of development. Incorporation of radiolabeled sugars into each fraction decreased from the IG to MG3 (locules jellied but still green) stage. Incorporation in all fractions increased from MG3 to breaker and turning (T) and then decreased from T to red. Data indicate that cell wall synthesis continues throughout ripening and increases transiently from MG4 (locules jellied and yellow to pink in color) to T, corresponding to the peak in respiration and ethylene synthesis during the climacteric. Synthesis continued at a time when total cell wall fraction dry weight decreased indicating the occurrence of cell wall turnover. Synthesis and insertion of a modified polymer with removal of other polymers may produce a less rigid cell wall and allow softening of the tissue integrity during ripening.

  7. IDENTIFYING GENES CONTROLLING FERULATE CROSS-LINKING FORMATION IN GRASS CELL WALLS

    Energy Technology Data Exchange (ETDEWEB)

    de O Buanafina, Marcia Maria

    2013-10-16

    improve their degradability as it might be more effective than the fungal enzymes at degrading maize lignocellulose if overexpressed in the cell wall. Publications supported by this grant: Buanafina, M.M. de O. (2009). Feruloylation in Grasses: Current and Future Perspectives. Molecular Plant, 2:861-872. Buanafina, M. M. de O., Langdon, T., Hauck, B., Dalton, S., Timms-Taravella, E. and Morris, P. (2010). Targeting expression of a fungal ferulic acid esterase to the apoplast, endoplasmic reticulum or golgi can disrupt feruloylation of the growing cell wall and increase the biodegradability of tall fescue (Festuca arundinacea): Plant Biot. J. 8:316-33. Michael L. Robbins, Ansuman Roy, Po-Hao Wang, Iffa Gaffoor, Rajandeep S. Sekhon, Marcia M. de O. Buanafina, Jai S. Rohila, Surinder Chopra (2013). Comparative Proteomics Analysis by DIGE and iTRAQ Provides Insight into the Regulation of Phenylpropanoids in Maize. Journal of Proteomics (In Press http://dx.doi.org/10.1016/j.jprot.2013.06.018)) Publications in Preparation Marcia M. de O. Buanafina, Sue Dalton, Tim Langdon, Emma-Timms-Tavarella, Erica A. Shearer, and Phillip Morris. Improved post harvest cell wall deconstruction of tall fescue by co-expression of a fungal ferulic acid esterase with a 1-4-endoxylanase. Marcia M. de O. Buanafina, Mandeep Sharma, Howard W. Fescemyer and Erica A. Shearer. Functional testing of two PF02458 homologues of putative rice arabinoxylan feruloyl transferase genes in Brachypodium distachyon. Marcia M. de O. Buanafina and Erica A. Shearer. Novel protein identified in maize and other grasses. Presentations/ Posters presented at major scientific meetings: Identifying genes controlling feruloylation in grass cell walls. Genomics: GTL Annual Contractor-Grantee Workshop held jointly with the USDA-DOE Plant Feedstock Genomics for Bioenergy awardees meeting. February 8-11/2009, Bethesda, Maryland. Identifying genes controlling feruloylation in grass cell walls. The American Society of Plant

  8. Orbital wall infarction in child with sickle cell disease.

    Science.gov (United States)

    Janssens, C; Claeys, L; Maes, P; Boiy, T; Wojciechowski, M

    2015-12-01

    We present the case of a 17-year-old boy, known with homozygous sickle cell disease, who was admitted because of generalised pain. He developed bilateral periorbital oedema and proptosis, without pain or visual disturbances. In addition to hyperhydration, oxygen and analgesia IV antibiotics were started, to cover a possible osteomyelitis. Patients with sickle cell disease are at risk for vaso-occlusive crises, when the abnormally shaped red blood cells aggregate and block the capillaries. Such a crisis typically presents at a location with high bone marrow activity, as the vertebrae and long bones. At an early age, the bone marrow is still active at other sites, for example the orbital wall, and thus infarction can also occur there. Thus, in young persons with sickle cell disease, it is important to consider orbital wall infarction in the differential diagnosis, since the approach is different from osteomyelitis. If the disease is complicated by an orbital compression syndrome, corticosteroids or surgical intervention may be necessary to preserve the vision. In our patient, an MRI of the orbitae demonstrated periorbital oedema with bone anomalies in the orbital and frontal bones, confirming orbital wall infarction. Ophthalmological examination revealed no signs of pressure on the nervus opticus. The patient recovered gradually with conservative treatment.

  9. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain...... Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals...

  10. Temporal proteomic profiling of Chlamydia trachomatis-infected HeLa-229 human cervical epithelial cells.

    Science.gov (United States)

    Tan, Grace Min Yi; Lim, Hui Jing; Yeow, Tee Cian; Movahed, Elaheh; Looi, Chung Yeng; Gupta, Rishein; Arulanandam, Bernard P; Abu Bakar, Sazaly; Sabet, Negar Shafiei; Chang, Li-Yen; Wong, Won Fen

    2016-05-01

    Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography-tandem mass spectrometry (LC-MS(3) ) analysis. C. trachomatis (serovar D, MOI 1)-infected HeLa-229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis-infected HeLa-229 cells indicate complex host-pathogen interactions at early phase of chlamydial infection.

  11. Cytoplasmic streaming in plant cells: the role of wall slip.

    Science.gov (United States)

    Wolff, K; Marenduzzo, D; Cates, M E

    2012-06-01

    We present a computer simulation study, via lattice Boltzmann simulations, of a microscopic model for cytoplasmic streaming in algal cells such as those of Chara corallina. We modelled myosin motors tracking along actin lanes as spheres undergoing directed motion along fixed lines. The sphere dimension takes into account the fact that motors drag vesicles or other organelles, and, unlike previous work, we model the boundary close to which the motors move as walls with a finite slip layer. By using realistic parameter values for actin lane and myosin density, as well as for endoplasmic and vacuole viscosity and the slip layer close to the wall, we find that this simplified view, which does not rely on any coupling between motors, cytoplasm and vacuole other than that provided by viscous Stokes flow, is enough to account for the observed magnitude of streaming velocities in intracellular fluid in living plant cells.

  12. Cell wall bound anionic peroxidases from asparagus byproducts.

    Science.gov (United States)

    Jaramillo-Carmona, Sara; López, Sergio; Vazquez-Castilla, Sara; Jimenez-Araujo, Ana; Rodriguez-Arcos, Rocio; Guillen-Bejarano, Rafael

    2014-10-08

    Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters.

  13. Plant cell walls: New insights from ancient species

    DEFF Research Database (Denmark)

    Sørensen, Iben; Willats, William George Tycho

    2008-01-01

    Cell walls are a defining feature of plants and have numerous crucial roles in growth and development. They are also the largest source of terrestrial biomass and have many important industrial applications - ranging from bulk products to functional food ingredients. There is considerable interest......¿4)-linked ß-D-Glcp are joined by occasional (1¿3)-linkages. This mixed linkage glucan (MLG) has been the subject of extensive research because of the economic importance of several Poales species including rice, barley and wheat and because MLG has proven health benefits. The recent discovery of MLG......-D-glucan is not unique to the Poales and is an abundant component of Equisetum arvense cell walls. Plant J 2008; 54:510-21....

  14. Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

    Science.gov (United States)

    Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

    2011-04-01

    Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

  15. Life behind cell walls: paradigm lost, paradigm regained.

    Science.gov (United States)

    Lamport, D T

    2001-09-01

    This review of the living cell wall and its protein components is in two parts. The first is anecdotal. A personal account spanning over 40 years research may perhaps be an antidote to one stereotypical view of scientists as detached and humorless. The second part deals with the meaning of function, particularly as it applies to hydroxyproline-rich glycoproteins. Function is a difficult word to define objectively. However, with help from such luminaries as Humpty Dumpty: "A word means what I want it to mean, neither more nor less," and Wittgenstein: "Giving examples of usage ... is the only way to talk about meaning," it is possible to construct a ziggurat representing increasingly complex levels of organization from molecular structure to ecology. Forty years ago I suggested that hydroxyproline-rich structural proteins played a key role in cell wall functioning. But because the bulk of the wall is carbohydrate, there has been an understandable resistance to paradigm change. Expansins, paradoxically, contribute greatly to this resistance because their modus operandi as cell-wall-loosening proteins is based on the idea that they break hydrogen bonds between polysaccharide chains allowing slippage. However, this view is not consistent with the recent discovery [Grobe et al. (1999) Eur. J. Biochem 263: 33-40] that beta-expansins may be proteases, as it implies that the extensin network is not a straightjacket but a substrate for expansin in muro. Such a direct role for extensins in both negative and positive regulation of cell expansion and elongation may constitute a major morphogenetic mechanism operating at all levels of plant growth and development.

  16. Regulation of plant cells, cell walls and development by mechanical signals

    Energy Technology Data Exchange (ETDEWEB)

    Meyerowitz, Elliot M. [California Inst. of Technology (CalTech), Pasadena, CA (United States)

    2016-06-14

    The overall goal of the revised scope of work for the final year of funding was to characterize cell wall biosynthesis in developing cotyledons and in the shoot apical meristem of Arabidopsis thaliana, as a way of learning about developmental control of cell wall biosynthesis in plants, and interactions between cell wall biosynthesis and the microtubule cytoskeleton. The proposed work had two parts – to look at the effect of mutation in the SPIRAL2 gene on microtubule organization and reorganization, and to thoroughly characterize the glycosyltransferase genes expressed in shoot apical meristems by RNA-seq experiments, by in situ hybridization of the RNAs expressed in the meristem, and by antibody staining of the products of the glycosyltransferases in meristems. Both parts were completed; the spiral2 mutant was found to speed microtubule reorientation after ablation of adjacent cells, supporting our hypothesis that reorganization correlates with microtubule severing, the rate of which is increased by the mutation. The glycosyltransferase characterization was completed and published as Yang et al. (2016). Among the new things learned was that primary cell wall biosynthesis is strongly controlled both by cell type, and by stage of cell cycle, implying not only that different, even adjacent, cells can have different sugar linkages in their (nonshared) walls, but also that a surprisingly large proportion of glycosyltransferases is regulated in the cell cycle, and therefore that the cell cycle regulates wall maturation to a degree previously unrecognized.

  17. Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.METHODS: A proteomic strategy combining two-dimensional gel electrophoresis and ultraperform ance liquid chromatographyelectrospray ionizationtandem mass spectrometry (UPLCESIMS/MS) was used to study the differential expression of proteins and Western blotting was used to validate the re...

  18. Toluene Dose-Response and Preliminary Study of Proteomics for Neuronal Cell Lines

    Science.gov (United States)

    2015-07-01

    Index, 1989). It is widely used in commercial products for paints and paints thinner, nail polish, lacquers, and rust inhibitors. It is also a...fluorocarbon film bottom; this film is vapor permeable and allows toluene vapor to pass, thereby exposing the cells. For control exposures, the...use of vapor permeable Lumox® microplates as suitable culture vessels for toluene in the glass chamber. The quantitative proteomics identified

  19. Quantitative Proteomics Identifies Vasopressin-Responsive Nuclear Proteins in Collecting Duct Cells

    OpenAIRE

    Schenk, Laura K.; Bolger, Steven J.; Luginbuhl, Kelli; Gonzales, Patricia A.; Rinschen, Markus M.; Yu, Ming-Jiun; Hoffert, Jason D.; Pisitkun, Trairak; Knepper, Mark A.

    2012-01-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nucl...

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel...... of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics....

  1. Cell wall proteins in seedling cotyledons of Prosopis chilensis.

    Science.gov (United States)

    Rodríguez, J G; Cardemil, L

    1994-01-01

    Four cell wall proteins of cotyledons of Prosopis chilensis seedlings were characterized by PAGE and Western analyses using a polyclonal antibody, generated against soybean seed coat extensin. These proteins had M(r)s of 180,000, 126,000, 107,000 and 63,000, as determined by SDS-PAGE. The proteins exhibited a fluorescent positive reaction with dansylhydrazine suggesting that they are glycoproteins; they did not show peroxidase activity. The cell wall proteins were also characterized by their amino acid composition and by their amino-terminal sequence. These analyses revealed that there are two groups of related cell wall proteins in the cotyledons. The first group comprises the proteins of M(r)s 180,000, 126,000, 107,000 which are rich in glutamic acid/glutamine and aspartic acid/asparagine and they have almost identical NH2-terminal sequences. The second group comprises the M(r) 63,000 protein which is rich in proline, glycine, valine and tyrosine, with an NH2-terminal sequence which was very similar to that of soybean proline-rich proteins.

  2. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis.

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.

  3. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  4. Adsorption of polycyclic aromatic hydrocarbons (PAHs) on Rhizopus oryzae cell walls: application of cosolvent models for validating the cell wall-water partition coefficient.

    Science.gov (United States)

    Ma, Bin; Xu, Minmin; Wang, Jiaojiao; Chen, Huaihai; He, Yan; Wu, Laosheng; Wang, Haizhen; Xu, Jianming

    2011-11-01

    The cell wall-cosolvent partition coefficients (Km) of polycyclic aromatic hydrocarbons (PAHs) were determined for Rhizopus oryzae cell walls by controlling the volume fraction of methanol (f) ranging from 0.1 to 0.5. Five cosolvent models were employed for extrapolating the cell wall-water partition coefficients (Kw) in pure water. The extrapolated Kw values of four PAHs on R. oryzae cell walls were ranged from 2.9 to 5.1. Comparison of various Kw values of pyrene generated from extrapolation and the QSPR model, together with predicted different (PD), mean percentage deviations (MPD), and root mean square errors (RSE), revealed that the performance of the LL and Bayesian models were the best among all five tested cosolvent models. This study suggests that R. oryzae cell walls play an important role in the partitioning of PAHs during bioremediation because of the high Kw of fungal cell walls.

  5. Proteomic analysis of male 4C germ cell proteins involved in mouse meiosis.

    Science.gov (United States)

    Guo, Xuejiang; Zhang, Ping; Qi, Yujuan; Chen, Wen; Chen, Xiangxiang; Zhou, Zuomin; Sha, Jiahao

    2011-01-01

    Male meiosis is a specialized type of cell division that gives rise to sperm. Errors in this process can result in the generation of aneuploid gametes, which are associated with birth defects and infertility in humans. Until now, there has been a lack of a large-scale identification of proteins involved in male meiosis in mammals. In this study, we report the high-confidence identification of 3625 proteins in mouse male germ cells with 4C DNA content undergoing meiosis I. Of these, 397 were found to be testis specific. Bioinformatics analysis of the proteome led to the identification of 28 proteins known to be essential for male meiosis in mice. We also found 172 proteins that had yeast orthologs known to be essential for meiosis. Chromosome distribution analysis of the proteome showed underrepresentation of the identified proteins on the X chromosome, which may be due to meiotic sex chromosome inactivation. Characterization of the proteome of 4C germ cells from mouse testis provides an inventory of proteins, which is useful for understanding meiosis and the mechanisms of male infertility.

  6. High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources

    Directory of Open Access Journals (Sweden)

    Reka A. Haraszti

    2016-11-01

    Full Text Available Extracellular vesicles (EVs, including exosomes and microvesicles (MVs, are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs. We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types.

  7. Cell wall composition and candidate biosynthesis gene expression during rice development

    DEFF Research Database (Denmark)

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra

    2016-01-01

    Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall compone...

  8. Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II

    DEFF Research Database (Denmark)

    Schneider, Tanja; Kruse, Thomas; Wimmer, Reinhard

    2010-01-01

    that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular...

  9. Proteomic and protein interaction network analysis of human T lymphocytes during cell-cycle entry

    Science.gov (United States)

    Orr, Stephen J; Boutz, Daniel R; Wang, Rong; Chronis, Constantinos; Lea, Nicholas C; Thayaparan, Thivyan; Hamilton, Emma; Milewicz, Hanna; Blanc, Eric; Mufti, Ghulam J; Marcotte, Edward M; Thomas, N Shaun B

    2012-01-01

    Regulating the transition of cells such as T lymphocytes from quiescence (G0) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G0. We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G0 into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle. PMID:22415777

  10. Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method.

    Directory of Open Access Journals (Sweden)

    Ganglong Yang

    Full Text Available The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia, KK47 (low grade nonmuscle invasive bladder cancer, NMIBC, and YTS1 (metastatic bladder cancer have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer.

  11. Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

    Directory of Open Access Journals (Sweden)

    Yoichiro Fukao

    2016-01-01

    Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

  12. Effect of Wall Charge on Striation in Plasma Display Cells

    Institute of Scientific and Technical Information of China (English)

    HE Feng; OUYANG Jiting; CAO Jing; FENG Shuo; MIAO Jinsong; WANG Jianqi

    2007-01-01

    Different configurations and driving voltages have been employed to investigate the effect of the wall charge on the striations in macroscopic plasma display panel (PDP) cells.The experimental results show that a discharge channel near the dielectric layer is indispensable to striation occurring in the anode area during a discharge,while the pre-accumulated charge on the dielectric layer and the surface state are not important.The origin of the striation is related only to the physical process in the cell.The dielectric layer acts as a charge collector during a PDP discharge.

  13. Stress analysis for wall structure in mobile hot cell design

    Energy Technology Data Exchange (ETDEWEB)

    Bahrin, Muhammad Hannan, E-mail: hannan@nuclearmalaysia.gov.my; Rahman, Anwar Abdul, E-mail: anwar@nuclearmalaysia.gov.my; Hamzah, Mohd Arif, E-mail: arif@nuclearmalaysia.gov.my; Mamat, Mohd Rizal; Azman, Azraf; Hasan, Hasni [Prototype and Plant Development Centre, Technical Services Division, Malaysian Nuclear Agency (Malaysia)

    2016-01-22

    Malaysian Nuclear Agency is developing a Mobile Hot Cell (MHC) in order to handle and manage Spent High Activity Radioactive Sources (SHARS) such as teletherapy heads and irradiators. At present, there are only two units of MHC in the world, in South Africa and China. Malaysian Mobile Hot cell is developed by Malaysian Nuclear Agency with the assistance of IAEA expert, based on the design of South Africa and China, but with improved features. Stress analysis has been performed on the design in order to fulfil the safety requirement in operation of MHC. This paper discusses the loading analysis effect from the sand to the MHC wall structure.

  14. Evidence for 'silicon' within the cell walls of suspension-cultured rice cells.

    Science.gov (United States)

    He, Congwu; Wang, Lijun; Liu, Jian; Liu, Xin; Li, Xiuli; Ma, Jie; Lin, Yongjun; Xu, Fangsen

    2013-11-01

    Despite the ubiquity and beneficial role of silicon (Si) in plant biology, structural and chemical mechanisms operating at the single-cell level have not been extensively studied. To obtain insights regarding the effect of Si on individual cells, we cultivated suspended rice (Oryza sativa) cells in the absence and presence of Si and analyzed single cells using a combination of physical techniques including atomic force microscopy (AFM). Si is naturally present as a constituent of the cell walls, where it is firmly bound to the cell wall matrix rather than occurring within intra- or extracellular silica deposition, as determined by using inductively coupled plasma mass spectrometry (ICP-MS) and X-ray photoelectron spectroscopy (XPS). This species of Si, linked with the cell wall matrix, improves the structural stability of cell walls during their expansion and subsequent cell division. Maintaining cell shape is thereby enhanced, which may be crucial for the function and survival of cells. This study provides further evidence that organosilicon is present in plant cell walls, which broadens our understanding of the chemical nature of 'anomalous Si' in plant biology.

  15. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in a...

  16. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in...

  17. Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

    Energy Technology Data Exchange (ETDEWEB)

    Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Lee, Yeon-Mi [Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Park, Bong-Wook; Byun, June-Ho [Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju 660-702 (Korea, Republic of); Ahn, Chun-Seob; Kim, Jae-Won [Department of Microbiology, Division of Life Sciences, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Rho, Gyu-Jin, E-mail: jinrho@gnu.ac.kr [Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701 (Korea, Republic of)

    2014-01-01

    Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs.

  18. In situ analysis of cell wall polymers associated with phloem fibre cells in stems of hemp, Cannabis sativa L.

    Science.gov (United States)

    Blake, Anthony W; Marcus, Susan E; Copeland, James E; Blackburn, Richard S; Knox, J Paul

    2008-06-01

    A study of stem anatomy and the sclerenchyma fibre cells associated with the phloem tissues of hemp (Cannabis sativa L.) plants is of interest for both understanding the formation of secondary cell walls and for the enhancement of fibre utility as industrial fibres and textiles. Using a range of molecular probes for cell wall polysaccharides we have surveyed the presence of cell wall components in stems of hemp in conjunction with an anatomical survey of stem and phloem fibre development. The only polysaccharide detected to occur abundantly throughout the secondary cell walls of phloem fibres was cellulose. Pectic homogalacturonan epitopes were detected in the primary cell walls/intercellular matrices between the phloem fibres although these epitopes were present at a lower level than in the surrounding parenchyma cell walls. Arabinogalactan-protein glycan epitopes displayed a diversity of occurrence in relation to fibre development and the JIM14 epitope was specific to fibre cells, binding to the inner surface of secondary cell walls, throughout development. Xylan epitopes were found to be present in the fibre cells (and xylem secondary cell walls) and absent from adjacent parenchyma cell walls. Analysis of xylan occurrence in the phloem fibre cells of hemp and flax indicated that xylan epitopes were restricted to the primary cell walls of fibre cells and were not present in the secondary cell walls of these cells.

  19. Proteomic analysis of human blastocoel fluid and blastocyst cells

    DEFF Research Database (Denmark)

    Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen

    2013-01-01

    Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst and can differentiate into any cell type in the human body. These cells hold a great potential for regenerative medicine, but to obtain enough cells needed for medical treatment, culture is required......, the blastocoel fluid, which is in contact with all the cells in the blastocyst, including hESCs. Fifty-three surplus human blastocysts were donated after informed consent, and blastocoel fluid was isolated by micromanipulation. Using highly sensitive nano-high-pressure liquid chromatography-tandem mass...

  20. Proteomic analysis of pancreatic endocrine tumor cell lines treated with the histone deacetylase inhibitor trichostatin A.

    Science.gov (United States)

    Cecconi, Daniela; Donadelli, Massimo; Rinalducci, Sara; Zolla, Lello; Scupoli, Maria Teresa; Scarpa, Aldo; Palmieri, Marta; Righetti, Pier Giorgio

    2007-05-01

    Effects of the histone-deacetylases inhibitor trichostatin A (TSA) on the growth of three different human pancreatic endocrine carcinoma cell lines (CM, BON, and QGP-1) have been assessed via dosage-dependent growth inhibition curves. TSA determined strong inhibition of cell growth with similar IC(50) values for the different cell lines: 80.5 nM (CM), 61.6 nM (BON), and 86 nM (QGP-1), by arresting the cell cycle in G2/M phase and inducing apoptosis. 2DE and nano-RP-HPLC-ESI-MS/MS analysis revealed 34, 33, and 38 unique proteins differentially expressed after TSA treatment in the CM, BON, and QGP-1 cell lines, respectively. The most important groups of modulated proteins belong to cell proliferation, cell cycle, and apoptosis classes (such as peroxiredoxins 1 and 2, the diablo protein, and HSP27). Other proteins pertain to processes such as regulation of gene expression (nucleophosmin, oncoprotein dek), signal transduction (calcium-calmodulin), chromatin, and cytoskeleton organization (calgizzarin, dynein, and lamin), RNA splicing (nucleolin, HNRPC), and protein folding (HSP70). The present data are in agreement with previous proteomic analyses performed on pancreatic ductal carcinoma cell lines (Cecconi, D. et al.., Electrophoresis 2003; Cecconi, D. et al., J. Proteome Res. 2005) and place histone-deacetylases inhibitors among the potentially most powerful drugs for the treatment of pancreatic tumors.

  1. Effect of long-term exposure of SH-SY5Y cells to morphine: a whole cell proteomic analysis

    Directory of Open Access Journals (Sweden)

    Moulédous Lionel

    2006-12-01

    Full Text Available Abstract Background Opiate addiction reflects plastic changes that endurably alter synaptic transmission within relevant neuronal circuits. The biochemical mechanisms of these adaptations remain largely unknown and proteomics-based approaches could lead to a broad characterization of the molecular events underlying adaptations to chronic drug exposure. Results Thus, we have started proteomic analyses of the effects of chronic morphine exposure in a recombinant human neuroblastoma SH-SY5Y clone that stably overexpresses the μ-opioid receptor. Cells were treated with morphine for 6, 24 and 72 hours, the proteins were separated by 2-D gel electrophoresis and stained with Coomassie blue, and the protein map was compared with that obtained from untreated cells. Spots showing a statistically significant variation were selected for identification using mass spectrometric analyses. Conclusion A total of 45 proteins were identified, including proteins involved in cellular metabolism, cytoskeleton organization, vesicular trafficking, transcriptional and translational regulation, and cell signaling.

  2. Prion Protein Deficiency Causes Diverse Proteome Shifts in Cell Models That Escape Detection in Brain Tissue.

    Directory of Open Access Journals (Sweden)

    Mohadeseh Mehrabian

    Full Text Available A popular method for studying the function of a given protein is to generate and characterize a suitable model deficient for its expression. For the prion protein (PrP, best known for its role in several invariably fatal neurodegenerative diseases, a natural choice, therefore, would be to undertake such studies with brain samples. We recently documented the surprising observation that PrP deficiency caused a loss or enhancement of NCAM1 polysialylation, dependent on the cell model used. To identify possible causes for this disparity, we set out to systematically investigate the consequence of PrP deficiency on the global proteome in brain tissue and in four distinct cell models. Here we report that PrP deficiency causes robust but surprisingly divergent changes to the global proteomes of cell models but has no discernible impact on the global brain proteome. Amongst >1,500 proteins whose levels were compared in wild-type and PrP-deficient models, members of the MARCKS protein family exhibited pronounced, yet cell model-dependent changes to their steady-state levels. Follow-up experiments revealed that PrP collaborates with members of the MARCKS protein family in its control of NCAM1 polysialylation. We conclude that the physiological function of PrP may be masked in analyses of complex brain samples but its cell-type specific influence on a lipid raft-based NCAM1-related cell biology comes to the fore in investigations of specific cell types.

  3. Clear Cell Adenocarcinoma Arising from Abdominal Wall Endometriosis

    Directory of Open Access Journals (Sweden)

    Thouraya Achach

    2008-01-01

    Full Text Available Endometriosis is a frequent benign disorder. Malignancy arising in extraovarian endometriosis is a rare event. A 49-year-old woman is presented with a large painful abdominal wall mass. She underwent a myomectomy, 20 years before, for uterus leiomyoma. Computed tomography suggested that this was a desmoid tumor and she underwent surgery. Histological examination showed a clear cell adenocarcinoma associated with endometriosis foci. Pelvic ultrasound, computed tomography, and endometrial curettage did not show any malignancy or endometriosis in the uterus and ovaries. Adjuvant chemotherapy was recommended, but the patient was lost to follow up. Six months later, she returned with a recurrence of the abdominal wall mass. She was given chemotherapy and then she was reoperated.

  4. Human lung epithelial cell A549 proteome data after treatment with titanium dioxide and carbon black.

    Science.gov (United States)

    Vuong, Ngoc Q; Goegan, Patrick; Mohottalage, Susantha; Breznan, Dalibor; Ariganello, Marianne; Williams, Andrew; Elisma, Fred; Karthikeyan, Subramanian; Vincent, Renaud; Kumarathasan, Premkumari

    2016-09-01

    Here, we have described the dataset relevant to the A549 cellular proteome changes after exposure to either titanium dioxide or carbon black particles as compared to the non-exposed controls, "Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures" (Vuong et al., 2016) [1]. Detailed methodologies on the separation of cellular proteins by 2D-GE and the subsequent mass spectrometry analyses using MALDI-TOF-TOF-MS are documented. Particle exposure-specific protein expression changes were measured via 2D-GE spot volume analysis. Protein identification was done by querying mass spectrometry data against SwissProt and RefSeq protein databases using Mascot search engine. Two-way ANOVA analysis data provided information on statistically significant A549 protein expression changes associated with particle exposures.

  5. Human lung epithelial cell A549 proteome data after treatment with titanium dioxide and carbon black

    Directory of Open Access Journals (Sweden)

    Ngoc Q. Vuong

    2016-09-01

    Full Text Available Here, we have described the dataset relevant to the A549 cellular proteome changes after exposure to either titanium dioxide or carbon black particles as compared to the non-exposed controls, “Proteomic changes in human lung epithelial cells (A549 in response to carbon black and titanium dioxide exposures” (Vuong et al., 2016 [1]. Detailed methodologies on the separation of cellular proteins by 2D-GE and the subsequent mass spectrometry analyses using MALDI-TOF-TOF-MS are documented. Particle exposure-specific protein expression changes were measured via 2D-GE spot volume analysis. Protein identification was done by querying mass spectrometry data against SwissProt and RefSeq protein databases using Mascot search engine. Two-way ANOVA analysis data provided information on statistically significant A549 protein expression changes associated with particle exposures.

  6. Pressure Dependent Wall Relaxation in Polarized $^3$He Gaseous Cells

    CERN Document Server

    Peng, C; Chu, P -H; Gao, H; Zhang, Y

    2013-01-01

    Pressure dependence of longitudinal relaxation time (T$_1$) due to the cell wall was observed previously at both room temperature and low temperature in valved Rb-coated refillable $^3$He gaseous cells in \\cite{Zheng2}. The diffusion of $^3$He from measurement cell through a capillary tube to the valve and the subsequent depolarization on the surface of the valve was proposed to possibly explain such a pressure dependence at room temperature \\cite{Saam}. In this paper, we investigate this diffusion effect through measurements of T$_1$ with newly designed Rb-coated Pyrex glass cells at 295 K as well as finite element analysis (FEA) studies. Both the experimental results and FEA studies show that the diffusion effect is insufficient to explain the observed linear pressure-dependent behavior of T$_1$.

  7. Change in wall composition of transfer and aleurone cells during wheat grain development.

    Science.gov (United States)

    Robert, P; Jamme, F; Barron, C; Bouchet, B; Saulnier, L; Dumas, P; Guillon, F

    2011-02-01

    In addition to the starchy endosperm, a specialized tissue accumulating storage material, the endosperm of wheat grain, comprises the aleurone layer and the transfer cells next to the crease. The transfer cells, located at the ventral region of the grain, are involved in nutrient transfer from the maternal tissues to the developing endosperm. Immunolabeling techniques, Raman spectroscopy, and synchrotron infrared micro-spectroscopy were used to study the chemistry of the transfer cell walls during wheat grain development. The kinetic depositions of the main cell wall polysaccharides of wheat grain endosperm, arabinoxylan, and (1-3)(1-4)-β-glucan in transfer cell walls were different from kinetics previously observed in the aleurone cell walls. While (1-3)(1-4)-β-glucan appeared first in the aleurone cell walls at 90°D, arabinoxylan predominated in the transfer cell walls from 90 to 445°D. Both aleurone and transfer cell walls were enriched in (1-3)(1-4)-β-glucan at the mature stage of wheat grain development. Arabinoxylan was more substituted in the transfer cell walls than in the aleurone walls. However, arabinoxylan was more feruloylated in the aleurone than in the transfer cell walls, whatever the stage of grain development. In the transfer cells, the ferulic acid was less abundant in the outer periclinal walls while para-coumarate was absent. Possible implications of such differences are discussed.

  8. Species-Related Differences in the Proteome of Rat and Human Pancreatic Beta Cells

    Directory of Open Access Journals (Sweden)

    G. A. Martens

    2015-01-01

    Full Text Available The core proteomes of human and rat pancreatic beta cells were compared by label-free LC-MS/MS: this resulted in quantification of relative molar abundances of 707 proteins belonging to functional pathways of intermediary metabolism, protein synthesis, and cytoskeleton. Relative molar abundances were conserved both within and between pathways enabling the selection of a housekeeping network for geometric normalization and the analysis of potentially relevant differential expressions. Human beta cells differed from rat beta cells in their lower level of enzymes involved in glucose sensing (MDH1, PC, and ACLY and upregulation of lysosomal enzymes. Human cells also expressed more heat shock proteins and radical scavenging systems: apart from SOD2, they expressed high levels of H2O2-scavenger peroxiredoxin 3 (PRDX3, confirmed by microarray, Western blotting, and microscopy. Besides conferring lower susceptibility to oxidative stress to human cells PRDX3 might also play a role in physiological redox regulation as, in rat, its expression was restricted to a beta cell subset with higher metabolic glucose responsiveness. In conclusion, although their core proteomic architecture is conserved, human and rat beta cells differ in their molar expression of key enzymes involved in glucose sensing and redox control.

  9. Proteomic Analysis of Human Blastocoel Fluid and Blastocyst Cells

    DEFF Research Database (Denmark)

    Linnert Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen;

    the cells of the blastocyst are exposed. The ICM is the starting point for the development of undifferentiated human embryonic stem cells (hESCs), which posses the potential to develop into any cell type present in the adult human body [1,2]. This ability makes hESCs a potential source of cells...... for regenerative medicine, such as in the treatment of diabetes, Parkinson’s disease, blindness, and spinal cord injury. In the context of developing regenerative medicine based on hESCs, it remains a challenge to employ safe, xenofree and defined culture conditions. The blastocoel fluid is per se the in vivo......The human blastocyst consists of 100-200 cells that are organized in an outer layer of differentiated trophectoderm (TE) cells lining the blastocyst cavity into which the undifferentiated inner cell mass (ICM) protrudes. The cavity of the blastocyst is filled with blastocoel fluid to which all...

  10. Stimulation of elongation growth and cell wall loosening in rice coleoptiles under microgravity conditions in space.

    Science.gov (United States)

    Hoson, Takayuki; Soga, Kouichi; Mori, Ryuji; Saiki, Mizue; Nakamura, Yukiko; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro

    2002-09-01

    We analyzed the growth rate and the cell wall properties of coleoptiles of rice seedlings grown at 23.6 degrees C for 68.5, 91.5 and 136 h during the Space Shuttle STS-95 mission. In space, elongation growth of coleoptiles was stimulated and the cell wall extensibility increased. Also, the levels of the cell wall polysaccharides per unit length of coleoptiles and the relative content of the high molecular mass matrix polysaccharides decreased in space. These differences in the cell wall polysaccharides could be involved in increasing the cell wall extensibility, leading to growth stimulation of rice coleoptiles in space.

  11. Principles of Bacterial Cell-Size Determination Revealed by Cell-Wall Synthesis Perturbations

    Directory of Open Access Journals (Sweden)

    Carolina Tropini

    2014-11-01

    Full Text Available Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

  12. The nuclear proteome and DNA-binding fraction of human Raji lymphoma cells.

    Science.gov (United States)

    Henrich, Silke; Cordwell, Stuart J; Crossett, Ben; Baker, Mark S; Christopherson, Richard I

    2007-04-01

    Purification of organelles and analysis of their proteins is an important initial step for biological proteomics, simplifying the proteome prior to analysis by established techniques such as two-dimensional liquid chromatography (2-DLC) or two-dimensional gel electrophoresis (2-DE). Nuclear proteins play a central role in regulating gene expression, but are often under-represented in proteomic studies due to their lower abundance in comparison to cellular 'housekeeping' metabolic enzymes and structural proteins. A reliable procedure for separation and proteomic analysis of nuclear proteins would be useful for investigations of cell proliferation and differentiation during disease processes (e.g., human cancer). In this study, we have purified nuclei from the human Burkitt's lymphoma B-cell line, Raji, using sucrose density gradient centrifugation. The integrity and purity of the nuclei were assessed by light microscopy and proteins from the nuclear fractions were separated by 2-DE and identified using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). A total of 124 unique proteins were identified, of which 91% (n=110) were predicted to be nuclear using PSORT. Proteins from the nuclear fraction were subjected to affinity chromatography on DNA-agarose to isolate DNA-binding proteins. From this purified fraction, 131 unique proteins were identified, of which 69% (n=90) were known or predicted DNA-binding proteins. Purification of nuclei and subsequent enrichment of DNA-binding proteins allowed identification of a total of 209 unique proteins, many involved in transcription and/or correlated with lymphoma, leukemia or cancer in general. The data obtained should be valuable for identification of biomarkers and targets for cancer therapy, and for furthering our understanding of the molecular mechanisms underlying lymphoma development and progression.

  13. Measuring the Mechanical Properties of Plant Cell Walls

    Directory of Open Access Journals (Sweden)

    Hannes Vogler

    2015-03-01

    Full Text Available The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM, and its automated successor, real-time CFM (RT-CFM.

  14. [Hydroxyproline: Rich glycoproteins of the plant and cell wall

    Energy Technology Data Exchange (ETDEWEB)

    Varner, J.E.

    1993-01-01

    Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a number of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H[sub 2]O[sub 2] production reinforce the earlier ideas of others that H[sub 2]O[sub 2] is involved in normal lignification.

  15. Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo.

    Science.gov (United States)

    Lombard-Banek, Camille; Moody, Sally A; Nemes, Peter

    2016-02-12

    We advance mass spectrometry from a cell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-μESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng, or frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.

  16. T-cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome

    DEFF Research Database (Denmark)

    Bresciani, Anne Gøther; Paul, Sinu; Schommer, Nina

    2016-01-01

    or allergen with the conservation of its sequence in the human proteome or the healthy human microbiome. Indeed, performing such comparisons on large sets of validated T-cell epitopes, we found that epitopes that are similar with self-antigens above a certain threshold showed lower immunogenicity, presumably...... as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. These findings indicate that the existence (and potentially...

  17. System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T.G.; Prokhorova, Tatyana; Akimov, Vyacheslav;

    2011-01-01

    To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned...... by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those...

  18. Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.

    Science.gov (United States)

    Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A

    2015-09-23

    The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations.

  19. Direct measurement of cell wall stress-stiffening and turgor pressure in live bacterial cells

    CERN Document Server

    Deng, Yi; Shaevitz, Joshua W

    2011-01-01

    The mechanical properties of gram-negative bacteria are governed by a rigid peptidoglycan (PG) cell wall and the turgor pressure generated by the large concentration of solutes in the cytoplasm. The elasticity of the PG has been measured in bulk and in isolated sacculi and shown to be compliant compared to the overall stiffness of the cell itself. However, the stiffness of the cell wall in live cells has not been measured. In particular, the effects that pressure-induced stress might have on the stiffness of the mesh-like PG network have not been addressed even though polymeric materials often exhibit large amounts of stress-stiffening. We study bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress-stiffening in the E. coli cell wall, with an exponent of $1.07 \\pm 0.25$, such that the wall is significantly stiffer in live cells ($E\\sim32\\pm10$ MPa) than in unpres...

  20. Biochemistry, proteomics and phosphoproteomics of plant mitochondria from non-photosynthetic cells

    Directory of Open Access Journals (Sweden)

    Jesper Foged Havelund

    2013-03-01

    Full Text Available Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents (NAD(PH and they provide the cell with intermediates for a range of biosynthetic pathways. In addition to this, mitochondria contribute to a number of specialized functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of mitochondria and general biochemical properties such as oxidative phosphorylation. We will then mention a few adaptive properties in response to water stress, seed maturation and germination and the ability to function under hypoxic conditions. The discussion will mainly focus on Arabidopsis cell cultures, etiolated germinating rice seedlings and potato tubers as model plants. It will cover the general proteome as well as the posttranslational modification protein phosphorylation. To date 64 phosphorylated mitochondrial proteins with a total of 103 phosphorylation sites have been identified.

  1. A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations.

    Science.gov (United States)

    Nie, Song; McDermott, Sean P; Deol, Yadwinder; Tan, Zhijing; Wicha, Max S; Lubman, David M

    2015-11-01

    Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem-like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24- cell populations) and the mature luminal cells (CD49f-EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label-free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed potential molecular regulatory networks that may regulate CSCs. Selected differential proteins were validated by Western blot assay and immunohistochemical staining. The use of proteomics analysis may increase our understanding of the underlying molecular mechanisms of breast CSCs. This may be of importance in the future development of anti-CSC therapeutics.

  2. Multi-Walled Carbon Nanotubes Inhibit Breast Cancer Cell Migration.

    Science.gov (United States)

    Graham, Elizabeth G; Wailes, Elizabeth M; Levi-Polyachenko, Nicole H

    2016-02-01

    According to the American Cancer Society, breast cancer is the second leading cause of cancer death in the US. Cancerous cells may have inadequate adhesions to the extracellular matrix and adjacent cells. Previous work has suggested that restoring these contacts may negate the cancer phenotype. This work aims to restore those contacts using multi-walled carbon nanotubes (MWNTs). Varying concentrations of carboxylated MWNTs in water, with or without type I collagen, were dried to create a thin film upon which one of three breast cell lines were seeded: cancerous and metastatic MDA- MB-231 cells, cancerous but non-metastatic MCF7 cells, or non-cancerous MCF10A cells. Proliferation, adhesion, scratch and autophagy assays, western blots, and immunochemical staining were used to assess adhesion and E-cadherin expression. Breast cancer cells grown on a MWNT-collagen coated surface displayed increased adhesion and decreased migration which correlated with an increase in E-cadherin. This work suggests an alternative approach to cancer treatment by physically mediating the cells' microenvironment.

  3. Proteome adaptation in cell reprogramming proceeds via distinct transcriptional networks

    NARCIS (Netherlands)

    Benevento, Marco; Tonge, Peter D; Puri, Mira C; Hussein, Samer M I; Cloonan, Nicole; Wood, David L; Grimmond, Sean M; Nagy, Andras; Munoz, Javier; Heck, Albert J R

    2014-01-01

    The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quan

  4. Challenges for red blood cell biomarker discovery through proteomics

    NARCIS (Netherlands)

    Barasa, B.A.; Slijper, M.

    2014-01-01

    Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. T

  5. Transient sedimentation in a cell with top and bottom walls

    Science.gov (United States)

    Dance, Sarah; Maxey, Martin

    2002-11-01

    Wall boundary conditions may play a role in the screening of particle velocity fluctuations in Stokes suspensions. Using a Force-Coupling Method (Maxey and Patel, Int. J. Multiphase Flow 27 (2001)) we simulate transient sedimentation. The numerical scheme is a mixed Fourier-spectral element method, based on the Uzawa algorithm for Stokes flows. The sedimentation cell has top and bottom wall boundaries and periodic boundaries in the horizontal. These boundaries are chosen both for computational convenience, and to determine the relative importance of bottom and side walls in screening the velocity fluctuations. We consider several different box sizes, in an attempt to elucidate the connection between particle velocity fluctuation levels and box width. We quantify the evolution of particle mean velocities and fluctuations as well as the particle microstructure. In each case we observe an initial growth, followed by a decay in both the mean particle velocity and fluctuations. We also observe that a stable stratification develops. We suggest that the stratification is important in the evolution of the bulk mean velocity. We propose a mechanism involving particle cluster dynamics to explain the behaviour of the velocity fluctuations.

  6. Proteomic and phosphoproteomic comparison of human ES and iPS cells.

    Science.gov (United States)

    Phanstiel, Douglas H; Brumbaugh, Justin; Wenger, Craig D; Tian, Shulan; Probasco, Mitchell D; Bailey, Derek J; Swaney, Danielle L; Tervo, Mark A; Bolin, Jennifer M; Ruotti, Victor; Stewart, Ron; Thomson, James A; Coon, Joshua J

    2011-01-01

    Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.

  7. Cell-based proteome profiling of potential dasatinib targets by use of affinity-based probes.

    Science.gov (United States)

    Shi, Haibin; Zhang, Chong-Jing; Chen, Grace Y J; Yao, Shao Q

    2012-02-15

    Protein kinases (PKs) play an important role in the development and progression of cancer by regulating cell growth, survival, invasion, metastasis, and angiogenesis. Dasatinib (BMS-354825), a dual Src/Abl inhibitor, is a promising therapeutic agent with oral bioavailability. It has been used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). Most kinase inhibitors, including Dasatinib, inhibit multiple cellular targets and do not possess exquisite cellular specificity. Recent efforts in kinase research thus focus on the development of large-scale, proteome-wide chemical profiling methods capable of rapid identification of potential cellular (on- and off-) targets of kinase inhibitors. Most existing approaches, however, are still problematic and in many cases not compatible with live-cell studies. In this work, we have successfully developed a cell-permeable kinase probe (DA-2) capable of proteome-wide profiling of potential cellular targets of Dasatinib. In this way, highly regulated, compartmentalized kinase-drug interactions were maintained. By comparing results obtained from different proteomic setups (live cells, cell lysates, and immobilized affinity matrix), we found DA-2 was able to identify significantly more putative kinase targets. In addition to Abl and Src family tyrosine kinases, a number of previously unknown Dasatinib targets have been identified, including several serine/threonine kinases (PCTK3, STK25, eIF-2A, PIM-3, PKA C-α, and PKN2). They were further validated by pull-down/immunoblotting experiments as well as kinase inhibition assays. Further studies are needed to better understand the exact relevance of Dasatinib and its pharmacological effects in relation to these newly identified cellular targets. The approach developed herein should be amenable to the study of many of the existing reversible drugs/drug candidates.

  8. Cellulose-hemicellulose interaction in wood secondary cell-wall

    Science.gov (United States)

    Zhang, Ning; Li, Shi; Xiong, Liming; Hong, Yu; Chen, Youping

    2015-12-01

    The wood cell wall features a tough and relatively rigid fiber reinforced composite structure. It acts as a pressure vessel, offering protection against mechanical stress. Cellulose microfibrils, hemicellulose and amorphous lignin are the three major components of wood. The structure of secondary cell wall could be imagined as the same as reinforced concrete, in which cellulose microfibrils acts as reinforcing steel bar and hemicellulose-lignin matrices act as the concrete. Therefore, the interface between cellulose and hemicellulose/lignin plays a significant role in determine the mechanical behavior of wood secondary cell wall. To this end, we present a molecular dynamics (MD) simulation study attempting to quantify the strength of the interface between cellulose microfibrils and hemicellulose. Since hemicellulose binds with adjacent cellulose microfibrils in various patterns, the atomistic models of hemicellulose-cellulose composites with three typical binding modes, i.e. bridge, loop and random binding modes are constructed. The effect of the shape of hemicellulose chain on the strength of hemicellulose-cellulose composites under shear loadings is investigated. The contact area as well as hydrogen bonds between cellulose and hemicellulose, together with the covalent bonds in backbone of hemicellulose chain are found to be the controlling parameters which determine the strength of the interfaces in the composite system. For the bridge binding model, the effect of shear loading direction on the strength of the cellulose material is also studied. The obtained results suggest that the shear strength of wood-inspired engineering composites can be optimized through maximizing the formations of the contributing hydrogen bonds between cellulose and hemicellulose.

  9. Effect of Turkish propolis extracts on proteome of prostate cancer cell line

    Directory of Open Access Journals (Sweden)

    Barlak Yaşam

    2011-12-01

    Full Text Available Abstract Background Propolis is a natural, resinous hive product that has several pharmacological activities. Its composition varies depending on the vegetation, climate, season and environmental conditions of the area from where it was collected. Surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS is a proteomic approach which has been used in cancer proteomics studies. Prostate cancer is one of the most commonly diagnosed cancers in men. It has shown that nutritional supplements rich in polyphenolic compounds such as propolis play a significant role in prostate cancer chemoprevention. The aim of this study is to evaluate if protein expression profile in PC-3 prostate cancer cell lines could be differentiated when incubated with dimethyl sulfoxide and water extracts of Turkish propolis. Results The antioxidant potentials of dimethyl sulfoxide and water extracts of propolis were found in correlation with the amount of total phenolic compounds of them. Dimethyl sulfoxide and water extracts of propolis of 20 μg/mL reduced the cell viability to 24.5% and 17.7%, respectively. Statistically significant discriminatory peaks between control PC-3 cells and dimethyl sulfoxide extract of propolis-treated PC-3 cells were found to be the proteomic features at m/z 5143, 8703, 12661, 20184 and 32794, detected by CM10 ProteinChip, and the peak at m/z 3772, detected by Q10 ProteinChip. Between control PC-3 cells and water extract of propolis-treated PC-3 cells, statistically significant discriminatory peaks were found to be the proteomic features at m/z 15846, 16052 and 24658, detected by CM10 ProteinChip and the peaks at m/z 10348, 10899 and 11603, detected by Q10 ProteinChip. Conclusions It was concluded that dimethyl sulfoxide and water extracts of Turkish propolis may have anti-proliferative activity through differentiating protein expression profile in PC-3 prostate cancer cell lines along with their antioxidant

  10. Modification of chemical properties of cell walls by silicon and its role in regulation of the cell wall extensibility in oat leaves.

    Science.gov (United States)

    Hossain, Mohammad Talim; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Fujii, Shuhei; Yamamoto, Ryoichi; Hoson, Takayuki

    2007-04-01

    Effects of silicon on the mechanical and chemical properties of cell walls in the second leaf of oat (Avena sativa L.) seedlings were investigated. The cell wall extensibility in the basal region of the second leaf was considerably higher than that in the middle and subapical regions. Externally applied silicon increased the cell wall extensibility in the basal region, but it did not affect the extensibility in the middle and subapical regions. The amounts of cell wall polysaccharides and phenolic compounds, such as diferulic acid (DFA) and ferulic acid (FA), per unit length were lower in the basal region than in the middle and subapical regions of the leaf, and silicon altered these amounts in the basal region. In this region, silicon decreased the amounts of matrix polymers and cellulose per unit length and of DFA and FA, both per unit length and unit matrix polymer content. Silicon treatment also lowered the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) in the basal region. In contrast, the amount of silicon in cell walls increased in response to silicon treatment in three regions. These results suggest that in the basal region, silicon reduces the net wall mass and the formation of phenolic acid-mediated cross-linkages between wall polysaccharides. Such modifications of wall architecture may be responsible for the silicon-induced increase in the cell wall extensibility in oat leaves.

  11. A radioimmunoassay for lignin in plant cell walls

    Energy Technology Data Exchange (ETDEWEB)

    Dawley, R.M.

    1989-01-01

    Lignin detection and determination in herbaceous tissue requires selective, specific assays which are not currently available. A radioimmunoassay (RIA) was developed to study lignin metabolism in these tissues. A {beta}-aryl ether lignin model compound was synthesized, linked to keyhole limpet hemocyanin using a water-soluble carbodiimide, and injected into rabbits. The highest titer of the antiserum obtained was 34 {eta}g/mL of model derivatized BSA. An in vitro system was developed to characterize the RIA. The model compound was linked to amino activated polyacrylamide beads to mimic lignin in the cell walls. {sup 125}I Radiolabelled protein A was used to detect IgG antibody binding. The RIA was shown in the in vitro system to exhibit saturable binding. The amount of antibody bound decreased when the serum was diluted. Immunoelectrophoresis and competitive binding experiments confirmed that both aromatic rings of the lignin model compound had been antigenic. Chlorogenic acid, a phenolic known to be present in plant cells, did not compete for antibody binding. The RIA was used to measure lignin in milled plant samples and barley seedlings. Antiserum binding to wheat cell walls and stressed barley segments was higher than preimmune serum binding. Antibody binding to stressed barley tissue decreased following NaClO{sub 2} delignification. The RIA was found to be less sensitive than expected, so several avenues for improving the method are discussed.

  12. Plant fluid proteomics: Delving into the xylem sap, phloem sap and apoplastic fluid proteomes.

    Science.gov (United States)

    Rodríguez-Celma, Jorge; Ceballos-Laita, Laura; Grusak, Michael A; Abadía, Javier; López-Millán, Ana-Flor

    2016-08-01

    The phloem sap, xylem sap and apoplastic fluid play key roles in long and short distance transport of signals and nutrients, and act as a barrier against local and systemic pathogen infection. Among other components, these plant fluids contain proteins which are likely to be important players in their functionalities. However, detailed information about their proteomes is only starting to arise due to the difficulties inherent to the collection methods. This review compiles the proteomic information available to date in these three plant fluids, and compares the proteomes obtained in different plant species in order to shed light into conserved functions in each plant fluid. Inter-species comparisons indicate that all these fluids contain the protein machinery for self-maintenance and defense, including proteins related to cell wall metabolism, pathogen defense, proteolysis, and redox response. These analyses also revealed that proteins may play more relevant roles in signaling in the phloem sap and apoplastic fluid than in the xylem sap. A comparison of the proteomes of the three fluids indicates that although functional categories are somewhat similar, proteins involved are likely to be fluid-specific, except for a small group of proteins present in the three fluids, which may have a universal role, especially in cell wall maintenance and defense. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

  13. A novel method for sample preparation of fresh lung cancer tissue for proteomics analysis by tumor cell enrichment and removal of blood contaminants

    OpenAIRE

    Orre Lotta; Bergman Per; Elmberger Göran; Pernemalm Maria; De Petris Luigi; Lewensohn Rolf; Lehtiö Janne

    2010-01-01

    Abstract Background In-depth proteomics analyses of tumors are frequently biased by the presence of blood components and stromal contamination, which leads to large experimental variation and decreases the proteome coverage. We have established a reproducible method to prepare freshly collected lung tumors for proteomics analysis, aiming at tumor cell enrichment and reduction of plasma protein contamination. We obtained enriched tumor-cell suspensions (ETS) from six lung cancer cases (two ade...

  14. Proteomic changes resulting from gene copy number variations in cancer cells.

    Directory of Open Access Journals (Sweden)

    Tamar Geiger

    2010-09-01

    Full Text Available Along the transformation process, cells accumulate DNA aberrations, including mutations, translocations, amplifications, and deletions. Despite numerous studies, the overall effects of amplifications and deletions on the end point of gene expression--the level of proteins--is generally unknown. Here we use large-scale and high-resolution proteomics combined with gene copy number analysis to investigate in a global manner to what extent these genomic changes have a proteomic output and therefore the ability to affect cellular transformation. We accurately measure expression levels of 6,735 proteins and directly compare them to the gene copy number. We find that the average effect of these alterations on the protein expression is only a few percent. Nevertheless, by using a novel algorithm, we find the combined impact that many of these regional chromosomal aberrations have at the protein level. We show that proteins encoded by amplified oncogenes are often overexpressed, while adjacent amplified genes, which presumably do not promote growth and survival, are attenuated. Furthermore, regulation of biological processes and molecular complexes is independent of general copy number changes. By connecting the primary genome alteration to their proteomic consequences, this approach helps to interpret the data from large-scale cancer genomics efforts.

  15. Inference Method for Developing Mathematical Models of Cell Signaling Pathways Using Proteomic Datasets.

    Science.gov (United States)

    Tian, Tianhai; Song, Jiangning

    2017-01-01

    The progress in proteomics technologies has led to a rapid accumulation of large-scale proteomic datasets in recent years, which provides an unprecedented opportunity and valuable resources to understand how living organisms perform necessary functions at systems levels. This work presents a computational method for designing mathematical models based on proteomic datasets. Using the mitogen-activated protein (MAP) kinase pathway as the test system, we first develop a mathematical model including the cytosolic and nuclear subsystems. A key step of modeling is to apply a genetic algorithm to infer unknown model parameters. Then the robustness property of mathematical models is used as a criterion to select appropriate rate constants from the estimated candidates. Moreover, quantitative information such as the absolute protein concentrations is used to further refine the mathematical model. The successful application of this inference method to the MAP kinase pathway suggests that it is a useful and powerful approach for developing accurate mathematical models to gain important insights into the regulatory mechanisms of cell signaling pathways.

  16. Proteomic analysis of blood cells in fish exposed to chemotherapeutics: evidence for long term effects.

    Science.gov (United States)

    Pierrard, Marie-Aline; Kestemont, Patrick; Phuong, Nguyen Thanh; Tran, Minh Phu; Delaive, Edouard; Thezenas, Marie-Laëtitia; Dieu, Marc; Raes, Martine; Silvestre, Frédéric

    2012-04-18

    Proteomics technology are increasingly used in ecotoxicological studies to characterize and monitor biomarkers of exposure. The present study aims at identifying long term effects of malachite green (MG) exposure on the proteome of peripheral blood mononuclear cells (PBMC) from the Asian catfish, Pangasianodon hypophthalmus. A common (0.1 ppm) concentration for therapeutic treatment was applied twice with a 72 h interval. PBMC were collected directly at the end of the second bath of MG (T1) and after 1 month of decontamination (T2). Analytical 2D-DIGE gels were run and a total of 2551±364 spots were matched. Among them, MG induced significant changes in abundance of 116 spots with no recovery after one month of decontamination. Using LC-MS/MS and considering single identification per spot, we could identify 25 different proteins. Additionally, MG residues were measured in muscle and in blood indicating that leuco-MG has almost totally disappeared after one month of decontamination. This work highlights long term effects of MG treatment on the PBMC proteome from fish intended for human consumption.

  17. Chitosan Obtained from Cell Wall of Aspergillus Niger Mycelium

    Institute of Scientific and Technical Information of China (English)

    HUANG Hui-li; LIN Wen-luan; LIN Jian-ming

    2004-01-01

    Chitin from cell walls of Aspergillus Niger mycelium was prepared. A new method for the preparation of high deacetylation degree chitosan was studied in a dilute sodium hydroxide solution at a high pressure. The experimental results indicate that the deacetylation degree of the chitosan can reach 80% under the condition of a 5.00 mol/L sodium hydroxide solution at 0.1 MPa of pressure for 1 h. This method shows the advantages of the applications in the industry production and environment protection.

  18. Mass spectrometry for characterizing plant cell wall polysaccharides

    Directory of Open Access Journals (Sweden)

    Stefan eBauer

    2012-03-01

    Full Text Available Mass spectrometry is a selective and powerful technique to obtain identification and structural information on compounds present in complex mixtures. Since it requires only small sample amount it is an excellent tool for researchers interested in detecting changes in composition of complex carbohydrates of plants. This mini-review gives an overview of common mass spectrometry techniques applied to the analysis of plant cell wall carbohydrates. It presents examples in which mass spectrometry has been used to elucidate the structure of oligosaccharides derived from hemicelluloses and pectins and illustrates how information on sequence, linkages, branching and modifications are obtained from characteristic fragmentation patterns.

  19. Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells.

    Science.gov (United States)

    de Roos, Baukje; Duthie, Susan J; Polley, Abigael C J; Mulholland, Francis; Bouwman, Freek G; Heim, Carolin; Rucklidge, Garry J; Johnson, Ian T; Mariman, Edwin C; Daniel, Hannelore; Elliott, Ruan M

    2008-06-01

    This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.

  20. Transchromosomic cell model of Down syndrome shows aberrant migration, adhesion and proteome response to extracellular matrix

    Directory of Open Access Journals (Sweden)

    Cotter Finbarr E

    2009-08-01

    Full Text Available Abstract Background Down syndrome (DS, caused by trisomy of human chromosome 21 (HSA21, is the most common genetic birth defect. Congenital heart defects (CHD are seen in 40% of DS children, and >50% of all atrioventricular canal defects in infancy are caused by trisomy 21, but the causative genes remain unknown. Results Here we show that aberrant adhesion and proliferation of DS cells can be reproduced using a transchromosomic model of DS (mouse fibroblasts bearing supernumerary HSA21. We also demonstrate a deacrease of cell migration in transchromosomic cells independently of their adhesion properties. We show that cell-autonomous proteome response to the presence of Collagen VI in extracellular matrix is strongly affected by trisomy 21. Conclusion This set of experiments establishes a new model system for genetic dissection of the specific HSA21 gene-overdose contributions to aberrant cell migration, adhesion, proliferation and specific proteome response to collagen VI, cellular phenotypes linked to the pathogenesis of CHD.

  1. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-jun; WANG Bin; YAN Zhi-yong; QIAN Dong-meng; SONG Xu-xia; Ding Shou-yi; BAI Zhi-qiang

    2007-01-01

    Objective: To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods: Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 (Immobilized Mental Affinity Capture) and WCX2 (Weak Cation Exchange) chips on the SELDI-TOF-MS ProteinChip reader. Results: Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion: Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

  2. Proteome data from a host-pathogen interaction study with Staphylococcus aureus and human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Kristin Surmann

    2016-06-01

    Full Text Available To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP encoding a continuously expressed green fluorescent protein (GFP. Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed. Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC–MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]. They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset.

  3. Proteome data from a host-pathogen interaction study with Staphylococcus aureus and human lung epithelial cells.

    Science.gov (United States)

    Surmann, Kristin; Simon, Marjolaine; Hildebrandt, Petra; Pförtner, Henrike; Michalik, Stephan; Dhople, Vishnu M; Bröker, Barbara M; Schmidt, Frank; Völker, Uwe

    2016-06-01

    To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP) encoding a continuously expressed green fluorescent protein (GFP). Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed). Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC) standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC-MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]). They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset.

  4. Binding of /sup 18/F by cell membranes and cell walls of Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Yotis, W.W.; Zeb, M.; McNulty, J.; Kirchner, F.; Reilly, C.; Glendenin, L.

    1983-07-01

    The binding of /sup 18/F to isolated cell membranes and cell walls of Streptococcus mutans GS-5 or other bacteria was assayed. The attachment of /sup 18/F to these cell envelopes proceeded slowly and reached equilibrium within 60 min. /sup 18/F binding was stimulated by Ca/sup 2 +/ (1 mM). The binding of /sup 18/F to cellular components was dependent upon the pH, as well as the amount of /sup 18/F and dose of the binder employed. The binding of /sup 18/F by cell walls prepared from fluoride-sensitive and fluoride-resistant cells of S. salivarius and S. mutans did not differ significantly. The pretreatment of cell walls or cell membranes for 60 min at 30 degrees C with 1 mg of RNase, DNase, or trypsin per ml did not influence the binding of /sup 18/F by the walls and membranes of S. mutans GS-5. However, prior exposure of cell membranes to sodium dodecyl sulfate caused a significant reduction in the number of /sup 18/F atoms bound by the membranes. In saturated assay systems, cell membranes of S. mutans GS-5 bound 10(15) to 10(16) atoms of /sup 18/F per mg (dry weight), whereas cell walls from S. mutans GS-5, FA-1, and HS-6 or Actinomyces viscosus T14V and T14AV bound 10(12) to 10(13) atoms of /sup 18/F per mg (dry weight). /sup 18/F in this quantity (10(12) to 10(13) atoms) cannot be detected with the fluoride electrode. The data provide, for the first time, a demonstration of /sup 18/F binding by cell membranes and walls of oral flora.

  5. SILAC-based quantitative proteomic analysis of human lung cell response to copper oxide nanoparticles.

    Science.gov (United States)

    Edelmann, Mariola J; Shack, Leslie A; Naske, Caitlin D; Walters, Keisha B; Nanduri, Bindu

    2014-01-01

    Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level.

  6. SILAC-based quantitative proteomic analysis of human lung cell response to copper oxide nanoparticles.

    Directory of Open Access Journals (Sweden)

    Mariola J Edelmann

    Full Text Available Copper (II oxide (CuO nanoparticles (NP are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level.

  7. Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study

    OpenAIRE

    Wang, Hualin; ZHANG Jing; Sit, Wai-Hung; Lee, Chung-Yung Jetty; Wan, Jennifer Man-Fan

    2014-01-01

    Background Cordyceps cicadae is a medicinal fungus that is often used for treating cancer. However, the anticancer mechanisms of C. cicadae are largely unknown. This study aims to investigate the anticancer mechanisms of C. cicadae against hepatocellular carcinoma cells in vitro using a proteomic approach. Methods Human hepatocellular carcinoma MHCC97H cells were treated with a water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) for 48 h and harvested for cell viability assays. The...

  8. Properties of lead deposits in cell walls of radish (Raphanus sativus) roots.

    Science.gov (United States)

    Inoue, Hiroshi; Fukuoka, Daisuke; Tatai, Yuri; Kamachi, Hiroyuki; Hayatsu, Manabu; Ono, Manami; Suzuki, Suechika

    2013-01-01

    Various mechanisms are involved in detoxification of heavy metals such as lead (Pb) in plant cells. Most of the Pb taken up by plants accumulates in their roots. However, the detailed properties of Pb complexes in roots remain unclear. We have investigated the properties of Pb deposits in root cell walls of radish (Raphanus sativus L.) seedlings grown on glass beads bed containing Pb pellets, which are the source of Pb-contamination in shooting range soils. Pb deposits were tightly bound to cell walls. Cell wall fragments containing about 50,000 ppm Pb were prepared from the roots. After extracting Pb from the cell wall fragments using HCl, Pb ions were recombined with the Pb-extracted cell wall fragments in a solution containing Pb acetate. When the cell wall fragments were treated with pectinase (E.C. 3.2.1.15) and were chemically modified with 1-ethyl-3-dimethylamino-propylcarboimide, the Pb-rebinding ability of the treated cell wall fragments decreased. When acid-treated cell wall fragments were incubated in a solution containing Pb(2+) and excess amounts of a chelating agent, Pb recombined with the cell wall fragments were measured to estimate the affinity between Pb(2+) and the cell wall fragments. Our data show that Pb(2+) binds to carboxyl groups of cell walls. The source of the carboxyl groups is suggested to be pectic compounds. A stability constant of the Pb-cell wall complex was estimated to be about 10(8). The role of root cell walls in the mechanism underlying heavy metal tolerance was discussed.

  9. A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells.

    Science.gov (United States)

    Liu, Zhenke; Dai, Shujia; Bones, Jonathan; Ray, Somak; Cha, Sangwon; Karger, Barry L; Li, Jingyi Jessica; Wilson, Lee; Hinckle, Greg; Rossomando, Anthony

    2015-01-01

    A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.

  10. Root exudate-induced alterations in Bacillus cereus cell wall contribute to root colonization and plant growth promotion.

    Directory of Open Access Journals (Sweden)

    Swarnalee Dutta

    Full Text Available The outcome of an interaction between plant growth promoting rhizobacteria and plants may depend on the chemical composition of root exudates (REs. We report the colonization of tobacco, and not groundnut, roots by a non-rhizospheric Bacillus cereus (MTCC 430. There was a differential alteration in the cell wall components of B. cereus in response to the REs from tobacco and groundnut. Attenuated total reflectance infrared spectroscopy revealed a split in amide I region of B. cereus cells exposed to tobacco-root exudates (TRE, compared to those exposed to groundnut-root exudates (GRE. In addition, changes in exopolysaccharides and lipid-packing were observed in B. cereus grown in TRE-amended minimal media that were not detectable in GRE-amended media. Cell-wall proteome analyses revealed upregulation of oxidative stress-related alkyl hydroperoxide reductase, and DNA-protecting protein chain (Dlp-2, in response to GRE and TRE, respectively. Metabolism-related enzymes like 2-amino-3-ketobutyrate coenzyme A ligase and 2-methylcitrate dehydratase and a 60 kDa chaperonin were up-regulated in response to TRE and GRE. In response to B. cereus, the plant roots altered their exudate-chemodiversity with respect to carbohydrates, organic acids, alkanes, and polyols. TRE-induced changes in surface components of B. cereus may contribute to successful root colonization and subsequent plant growth promotion.

  11. Dental pulp response to bacterial cell wall material.

    Science.gov (United States)

    Warfvinge, J; Dahlén, G; Bergenholtz, G

    1985-08-01

    Lipopolysaccharides (LPS) from Bacteroides oralis and Veillonella parvula and cell wall material from Lactobacillus casei were studied for their capacity to induce leukocyte migration in the dental pulp and in an implanted wound chamber. Three adult monkeys were challenged using lyophilized material sealed into buccal Class V cavities prepared in dentin. Pulp tissue responses were observed histologically eight and 72 hours after initiation of the experiment. Subjacent to cut dentinal tubules, bacterial materials induced polymorphonuclear leukocyte (PMN's) infiltration in the pulp tissue of the majority of test teeth examined. Responses were similar for the three bacterial test materials at both time periods. Topical applications of bovine serum albumin (BSA), used as a control, induced significantly less accumulation of PMN's. Assessments of induced exudate volumes and leukocyte densities in chambers implanted in rats showed comparable rankings with pulpal experiment between test (i.e., bacterial) and control (BSA) materials. Analysis of the data indicates that high-molecular-weight complexes of bacterial cell walls may adversely affect pulpal tissue across freshly exposed dentin.

  12. Chemical Profiling of the Plant Cell Wall through Raman Microspectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Singh, Seema; Sun, Lan; Simmons, Blake; Auer, Manfred; Parvin, Bahram

    2010-03-02

    This paper presents a computational framework for chemical pro.ling of the plant cell wall through the Raman spectroscopy. The system enables query of known spectral signatures and clustering of spectral data based on intrinsic properties. As a result, presence and relative concentration of speci.c chemical bonds can be quanti.ed. The primary contribution of this paper is in representation of raman pro.le in terms of .uorescence background and multiscale peak detection at each grid point (voxel). Such a representation allows ef.cient spatial segmentation based on the coupling between high-level salient properties and low-level symbolic representation at each voxel. The high-level salient properties refer to preferred peaks and their attributes for the entire image. The low-level symbolic representations are based on .uorescence background, spectral peak locations, and their attributes. We present results on a corn stover tissue section that is imaged through Raman microscopy, and the results are consistent with the literature. In addition, automatic clustering indicates several distinct layers of the cell walls with different spectral signatures.

  13. Proteomic characterization of Her2/neu-overexpressing breast cancer cells.

    Science.gov (United States)

    Chen, Hexin; Pimienta, Genaro; Gu, Yiben; Sun, Xu; Hu, Jianjun; Kim, Min-Sik; Chaerkady, Raghothama; Gucek, Marjan; Cole, Robert N; Sukumar, Saraswati; Pandey, Akhilesh

    2010-11-01

    The receptor tyrosine kinase HER2 is an oncogene amplified in invasive breast cancer and its overexpression in mammary epithelial cell lines is a strong determinant of a tumorigenic phenotype. Accordingly, HER2-overexpressing mammary tumors are commonly indicative of a poor prognosis in patients. Several quantitative proteomic studies have employed two-dimensional gel electrophoresis in combination with MS/MS, which provides only limited information about the molecular mechanisms underlying HER2/neu signaling. In the present study, we used a SILAC-based approach to compare the proteomic profile of normal breast epithelial cells with that of Her2/neu-overexpressing mammary epithelial cells, isolated from primary mammary tumors arising in mouse mammary tumor virus-Her2/neu transgenic mice. We identified 23 proteins with relevant annotated functions in breast cancer, showing a substantial differential expression. This included overexpression of creatine kinase, retinol-binding protein 1, thymosin 4 and tumor protein D52, which correlated with the tumorigenic phenotype of Her2-overexpressing cells. The differential expression pattern of two genes, gelsolin and retinol binding protein 1, was further validated in normal and tumor tissues. Finally, an in silico analysis of published cancer microarray data sets revealed a 23-gene signature, which can be used to predict the probability of metastasis-free survival in breast cancer patients.

  14. Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

    Indian Academy of Sciences (India)

    Jianxiong He; Canhua Huang; Aiping Tong; Bin Chen; Zhi Zeng; Peng Zhang; Chunting Wang; Yuquan Wei

    2008-12-01

    Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.

  15. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

    Directory of Open Access Journals (Sweden)

    Nuno Carinhas

    Full Text Available Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

  16. Murein and pseudomurein cell wall binding domains of bacteria and archaea-a comparative view

    NARCIS (Netherlands)

    Visweswaran, Ganesh Ram R.; Dijkstra, Bauke W.; Kok, Jan

    2011-01-01

    The cell wall, a major barrier protecting cells from their environment, is an essential compartment of both bacteria and archaea. It protects the organism from internal turgor pressure and gives a defined shape to the cell. The cell wall serves also as an anchoring surface for various proteins and a

  17. Molecular Mechanisms for Vascular Development and Secondary Cell Wall Formation

    Science.gov (United States)

    Yang, Jung Hyun; Wang, Huanzhong

    2016-01-01

    Vascular tissues are important for transporting water and nutrients throughout the plant and as physical support of upright growth. The primary constituents of vascular tissues, xylem, and phloem, are derived from the meristematic vascular procambium and cambium. Xylem cells develop secondary cell walls (SCWs) that form the largest part of plant lignocellulosic biomass that serve as a renewable feedstock for biofuel production. For the last decade, research on vascular development and SCW biosynthesis has seen rapid progress due to the importance of these processes to plant biology and to the biofuel industry. Plant hormones, transcriptional regulators and peptide signaling regulate procambium/cambium proliferation, vascular patterning, and xylem differentiation. Transcriptional regulatory pathways play a pivot role in SCW biosynthesis. Although most of these discoveries are derived from research in Arabidopsis, many genes have shown conserved functions in biofuel feedstock species. Here, we review the recent advances in our understanding of vascular development and SCW formation and discuss potential biotechnological uses. PMID:27047525

  18. MreB: pilot or passenger of cell wall synthesis?

    Science.gov (United States)

    White, Courtney L; Gober, James W

    2012-02-01

    The discovery that the bacterial cell shape determinant MreB is related to actin spurred new insights into bacterial morphogenesis and development. The trafficking and mechanical roles of the eukaryotic cytoskeleton were hypothesized to have a functional ancestor in MreB based on evidence implicating MreB as an organizer of cell wall synthesis. Genetic, biochemical and cytological studies implicate MreB as a coordinator of a large multi-protein peptidoglycan (PG) synthesizing holoenzyme. Recent advances in microscopy and new biochemical evidence, however, suggest that MreB may function differently than previously envisioned. This review summarizes our evolving knowledge of MreB and attempts to refine the generalized model of the proteins organizing PG synthesis in bacteria. This is generally thought to be conserved among eubacteria and the majority of the discussion will focus on studies from a few well-studied model organisms.

  19. Cell wall proteins of Sporothrix schenckii as immunoprotective agents.

    Science.gov (United States)

    Alba-Fierro, Carlos A; Pérez-Torres, Armando; López-Romero, Everardo; Cuéllar-Cruz, Mayra; Ruiz-Baca, Estela

    2014-01-01

    Sporothrix schenckii is the etiological agent of sporotrichosis, an endemic subcutaneous mycosis in Latin America. Cell wall (CW) proteins located on the cell surface are inducers of cellular and humoral immune responses, potential candidates for diagnosis purposes and to generate vaccines to prevent fungal infections. This mini-review emphasizes the potential use of S. schenckii CW proteins as protective and therapeutic immune response inducers against sporotrichosis. A number of pathogenic fungi display CW components that have been characterized as inducers of protective cellular and humoral immune responses against the whole pathogen from which they were originally purified. The isolation and characterization of immunodominant protein components of the CW of S. schenckii have become relevant because of their potential in the development of protective and therapeutic immune responses against sporotrichosis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

  20. Nuclear proteome analysis of benzo(a)pyrene-treated HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Chen Zhaojun; Li Huanrong; Zhang Guanglin [The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003 (China); Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058 (China); Li Feng [The First Renmin Hospital, Houma, Shanxi 043000 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003 (China); Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang 310036 (China)

    2012-03-01

    Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to 10 {mu}M benzo(a)pyrene (BaP) during a 24 h frame can lead to changes in nuclear protein expression and alternative splicing. To further expand our knowledge about the DNA damage response (DDR) induced by BaP, we investigated the nuclear protein expression profiles in HeLa cells treated with different concentrations of BaP (0.1, 1, and 10 {mu}M) using this proteomics-based 2-D gel electrophoresis assay. We found 125 differentially expressed proteins in BaP-treated cells compared to control cells. Among them, 79 (63.2%) were down-regulated, 46 (36.8%) were up-regulated; 8 showed changes in the 1 {mu}M and 10 {mu}M BaP-treated groups, 2 in the 0.1 {mu}M and 10 {mu}M BaP-treated groups, 4 in the 0.1 {mu}M and 1 {mu}M BaP-treated groups, and only one showed changes in all three groups. Fifty protein spots were chosen for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and of these, 39 were identified, including subunits of the 26S proteasome and Annexin A1. The functions of some identified proteins were further examined and the results showed that they might be involved in BaP-induced DDR. Taken together, these data indicate that proteomics is a valuable approach in the study of environmental chemical-host interactions, and the identified proteins could provide new leads for better understanding BaP-induced mutagenesis and carcinogenesis.

  1. Proteomic responses of human intestinal Caco-2 cells exposed to silver nanoparticles and ionic silver.

    Science.gov (United States)

    Oberemm, Axel; Hansen, Ulf; Böhmert, Linda; Meckert, Christine; Braeuning, Albert; Thünemann, Andreas F; Lampen, Alfonso

    2016-03-01

    Even although quite a number of studies have been performed so far to demonstrate nanoparticle-specific effects of substances in living systems, clear evidence of these effects is still under debate. The present study was designed as a comparative proteomic analysis of human intestinal cells exposed to a commercial silver nanoparticle reference material and ions from AgNO3. A two-dimensional gel electrophoresis/MALDI mass spectrometry (MS)-based proteomic analysis was conducted after 24-h incubation of differentiated Caco-2 cells with non-cytotoxic and low cytotoxic silver concentrations (2.5 and 25 µg ml(-1) nanosilver, 0.5 and 5 µg ml(-1) AgNO3). Out of an overall number of 316 protein spots differentially expressed at a fold change of ≥ 1.4 or ≤ -1.4 in all treatments, 169 proteins could be identified. In total, 231 spots were specifically deregulated in particle-treated groups compared with 41 spots, which were limited to AgNO3-treatments. Forty-four spots (14 %) were commonly deregulated by both types of treatment. A considerable fraction of the proteins differentially expressed after treatment with nanoparticles is related to protein folding, synthesis or modification of proteins as well as cellular assembly and organization. Overlays of networks obtained for particulate and ionic treatments showed matches, indicating common mechanisms of combined particle and ionic silver exposure and exclusive ionic silver treatment. However, proteomic responses of Caco-2 cells treated with higher concentrations of silver species also showed some differences, for example regarding proteins related to fatty acid and energy metabolism, suggesting an induction of also some different molecular mechanisms for particle exposure and ionic treatment.

  2. Soya beans and Maize : The effect of chemical and physical structure of cell wall polysaccharides on fermentation kinetics

    OpenAIRE

    Laar, van de, P.

    2000-01-01

    The analysis of the relationship between cell wall composition and fermentation of endosperm cell walls of soya beans and maize was approached from three different angles. Firstly, the fermentation (rate and extent of fermentation, the sugar degradation pattern, and volatile fatty acid production) of soya bean and maize cell walls was analysed, both in situ and in vitro. This analysis revealed that the physical structure of the cell wall (particle size and cell wall thickness) influences cell...

  3. A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

    Science.gov (United States)

    Herget, Meike; Scheibinger, Mirko; Guo, Zhaohua; Jan, Taha A; Adams, Christopher M; Cheng, Alan G; Heller, Stefan

    2013-01-01

    Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

  4. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian

    2009-01-01

    proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins...... is associated with actin-bundling and defence against oxidative cellular stress, whereas down-regulated proteins were associated with collagen biosynthesis. Bioinformatic analyses of the entire data set confirmed these findings that represent significant steps towards the understanding of DFPC differentiation....... The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic...

  5. The connection of cytoskeletal network with plasma membrane and the cell wall

    Institute of Scientific and Technical Information of China (English)

    Zengyu Liu; Staffan Persson; Yi Zhang

    2015-01-01

    The cell wall provides external support of the plant cells, while the cytoskeletons including the microtubules and the actin filaments constitute an internal framework. The cytoskeletons contribute to the cell wall biosynthesis by spatially and temporarily regulating the transportation and deposition of cell wall components. This tight control is achieved by the dynamic behavior of the cytoskeletons, but also through the tethering of these structures to the plasma membrane. This tethering may also extend beyond the plasma membrane and impact on the cell wall, possibly in the form of a feedback loop. In this review, we discuss the linking components between the cytoskeletons and the plasma membrane, and/or the cell wall. We also discuss the prospective roles of these components in cell wall biosyn-thesis and modifications, and aim to provide a platform for further studies in this field.

  6. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Horn, Heiko; Jungmichel, Stephanie;

    2014-01-01

    The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein......, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers...

  7. Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells

    Directory of Open Access Journals (Sweden)

    Kuster Niels

    2010-10-01

    Full Text Available Abstract Background Use of mobile phones has widely increased over the past decade. However, in spite of the extensive research, the question of potential health effects of the mobile phone radiation remains unanswered. We have earlier proposed, and applied, proteomics as a tool to study biological effects of the mobile phone radiation, using as a model human endothelial cell line EA.hy926. Exposure of EA.hy926 cells to 900 MHz GSM radiation has caused statistically significant changes in expression of numerous proteins. However, exposure of EA.hy926 cells to 1800 MHz GSM signal had only very small effect on cell proteome, as compared with 900 MHz GSM exposure. In the present study, using as model human primary endothelial cells, we have examined whether exposure to 1800 MHz GSM mobile phone radiation can affect cell proteome. Results Primary human umbilical vein endothelial cells and primary human brain microvascular endothelial cells were exposed for 1 hour to 1800 MHz GSM mobile phone radiation at an average specific absorption rate of 2.0 W/kg. The cells were harvested immediately after the exposure and the protein expression patterns of the sham-exposed and radiation-exposed cells were examined using two dimensional difference gel electrophoresis-based proteomics (2DE-DIGE. There were observed numerous differences between the proteomes of human umbilical vein endothelial cells and human brain microvascular endothelial cells (both sham-exposed. These differences are most likely representing physiological differences between endothelia in different vascular beds. However, the exposure of both types of primary endothelial cells to mobile phone radiation did not cause any statistically significant changes in protein expression. Conclusions Exposure of primary human endothelial cells to the mobile phone radiation, 1800 MHz GSM signal for 1 hour at an average specific absorption rate of 2.0 W/kg, does not affect protein expression, when the

  8. Proteomic analysis of mouse thymoma EL4 cells treated with bis (tri-n-butyltin)oxide (TBTO)

    NARCIS (Netherlands)

    Osman, A.M.; Kol, S.; Peijnenburg, A.A.C.M.; Blokland, M.H.; Pennings, J.L.A.; Kleinjans, J.C.S.; Loveren, van H.

    2009-01-01

    Here, we report the results of proteomic analysis of the mouse thymoma EL4 cell line exposed to bis(tri-n-butylin)oxide (TBTO), an immunotoxic organotin compound. The objective of the work was to examine whether TBTO affects the expression of proteins in this cell line and to compare the differentia

  9. Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2

    NARCIS (Netherlands)

    Stierum, R.; Gaspari, M.; Dommels, Y.; Ouatas, T.; Pluk, H.; Jespersen, S.; Vogels, J.; Verhoeckx, K.; Groten, J.; Ommen, B. van

    2003-01-01

    Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvi

  10. Hematopoietic Stem Cells Expansion in Rotating Wall Vessel

    Institute of Scientific and Technical Information of China (English)

    Yang LIU; Tian-Qing LIU; Xiu-Bo FAN; Dan GE; Zhan-Feng CUI; Xue-Hu MA

    2005-01-01

    @@ 1 Introduction Clinical trials have demonstrated that ex vivo expanded hematopoietic stem cells (HSCs) and progenitors offer great promise in reconstituting in vivo hematopoiesis in patients who have undergone intensive chemotherapy.It is therefore necessary to develop a clinical-scale culture system to provide the expanded HSCs and progenitors.Static culture systems such as T-flasks and gas-permeable blood bags are the most widely used culture devices for expanding hematopoietic cells. But they reveal several inherent limitations: ineffective mixing, lack of control options for dissolved oxygen and pH and difficulty in continuous feeding, which restricts the usefulness of static systems. Several advanced bioreactors have been used in the field of HSCs expansion. But hematopoietic cells are extremely sensitive to shear, so cells in bioreactors such as stirred and perfusion culture systems may suffer physical damage. This problem will be improved by applying the rotating wall vessel (RWV) bioreactor in clinic because of its low shear and unique structure. In this research, cord blood (CB) HSCs were expanded by means of a cell-dilution feeding protocol in RWV.

  11. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells

    DEFF Research Database (Denmark)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas

    2016-01-01

    as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human...... kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified...... to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially...

  12. Evaluation of Three Protein-Extraction Methods for Proteome Analysis of Maize Leaf Midrib, a Compound Tissue Rich in Sclerenchyma Cells.

    Science.gov (United States)

    Wang, Ning; Wu, Xiaolin; Ku, Lixia; Chen, Yanhui; Wang, Wei

    2016-01-01

    Leaf morphology is closely related to the growth and development of maize (Zea mays L.) plants and final kernel production. As an important part of the maize leaf, the midrib holds leaf blades in the aerial position for maximum sunlight capture. Leaf midribs of adult plants contain substantial sclerenchyma cells with heavily thickened and lignified secondary walls and have a high amount of phenolics, making protein extraction and proteome analysis difficult in leaf midrib tissue. In the present study, three protein-extraction methods that are commonly used in plant proteomics, i.e., phenol extraction, TCA/acetone extraction, and TCA/acetone/phenol extraction, were qualitatively and quantitatively evaluated based on 2DE maps and MS/MS analysis using the midribs of the 10th newly expanded leaves of maize plants. Microscopy revealed the existence of substantial amounts of sclerenchyma underneath maize midrib epidermises (particularly abaxial epidermises). The spot-number order obtained via 2DE mapping was as follows: phenol extraction (655) > TCA/acetone extraction (589) > TCA/acetone/phenol extraction (545). MS/MS analysis identified a total of 17 spots that exhibited 2-fold changes in abundance among the three methods (using phenol extraction as a control). Sixteen of the proteins identified were hydrophilic, with GRAVY values ranging from -0.026 to -0.487. For all three methods, we were able to obtain high-quality protein samples and good 2DE maps for the maize leaf midrib. However, phenol extraction produced a better 2DE map with greater resolution between spots, and TCA/acetone extraction produced higher protein yields. Thus, this paper includes a discussion regarding the possible reasons for differential protein extraction among the three methods. This study provides useful information that can be used to select suitable protein extraction methods for the proteome analysis of recalcitrant plant tissues that are rich in sclerenchyma cells.

  13. SILAC Proteomics of Planarians Identifies Ncoa5 as a Conserved Component of Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Alexander Böser

    2013-11-01

    Full Text Available Planarian regeneration depends on the presence of pluripotent stem cells in the adult. We developed an in vivo stable isotope labeling by amino acids in cell culture (SILAC protocol in planarians to identify proteins that are enriched in planarian stem cells. Through a comparison of SILAC proteomes of normal and stem cell-depleted planarians and of a stem cell-enriched population of sorted cells, we identified hundreds of stem cell proteins. One of these is an ortholog of nuclear receptor coactivator-5 (Ncoa5/CIA, which is known to regulate estrogen-receptor-mediated transcription in human cells. We show that Ncoa5 is essential for the maintenance of the pluripotent stem cell population in planarians and that a putative mouse ortholog is expressed in pluripotent cells of the embryo. Our study thus identifies a conserved component of pluripotent stem cells, demonstrating that planarians, in particular, when combined with in vivo SILAC, are a powerful model in stem cell research.

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

    Science.gov (United States)

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

    2015-01-13

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

  15. Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated Proteins

    Science.gov (United States)

    CRUZ*, ISA N.; COLEY*, HELEN M.; KRAMER, HOLGER B.; MADHURI, THUMULURU KAVITAH; SAFUWAN, NUR A.M.; ANGELINO, ANA RITA; YANG, MIN

    2016-01-01

    Background: Carboplatin and paclitaxel form the cornerstone of chemotherapy for epithelial ovarian cancer, however, drug resistance to these agents continues to present challenges. Despite extensive research, the mechanisms underlying this resistance remain unclear. Materials and Methods: A 2D-gel proteomics method was used to analyze protein expression levels of three human ovarian cancer cell lines and five biopsy samples. Representative proteins identified were validated via western immunoblotting. Ingenuity pathway analysis revealed metabolomic pathway changes. Results: A total of 189 proteins were identified with restricted criteria. Combined treatment targeting the proteasome-ubiquitin pathway resulted in re-sensitisation of drug-resistant cells. In addition, examination of five surgical biopsies of ovarian tissues revealed α-enolase (ENOA), elongation factor Tu, mitochondrial (EFTU), glyceraldehyde-3-phosphate dehydrogenase (G3P), stress-70 protein, mitochondrial (GRP75), apolipoprotein A-1 (APOA1), peroxiredoxin (PRDX2) and annexin A (ANXA) as candidate biomarkers of drug-resistant disease. Conclusion: Proteomics combined with pathway analysis provided information for an effective combined treatment approach overcoming drug resistance. Analysis of cell lines and tissues revealed potential prognostic biomarkers for ovarian cancer. *These Authors contributed equally to this study. PMID:28031236

  16. Proteome responses of Citrobacter werkmanii BF-6 planktonic cells and biofilms to calcium chloride.

    Science.gov (United States)

    Zhou, Gang; Shi, Qing-shan; Huang, Xiao-mo; Xie, Xiao-bao

    2016-02-05

    Calcium ions are well-known as intracellular second messengers that also have an important extracellular structural role for bacteria. Recently, we found that denser biofilms were formed by Citrobacter werkmanii BF-6 in the presence of 400 mM Ca(2+) than that of 12.5mM Ca(2+). Therefore, we employed two-dimensional (2-D) electrophoresis methods to investigate the proteome profiles of planktonic cells and biofilms in BF-6 under different concentrations of Ca(2+). Meanwhile, BF-6 biofilm architecture was also visualized with confocal laser scanning microscopy (CLSM). The results demonstrated that BF-6 biofilms formed at the bottom of microtiter plates when grown in the presence of 400 mM Ca(2+). A total of 151 proteins from planktonic cells and biofilms after exposure of BF-6 cells to 12.5 and 400 mM Ca(2+) were successfully identified. Different gene ontology (GO) and KEGG pathways were categorized and enriched for the above proteins. Growth in the presence of 400 mM Ca(2+) induced more complex signal pathways in BF-6 than 12.5mM Ca(2+). In addition, the biofilm architectures were also affected by Ca(2+). Our results show two different modes of biofilm enhancement for C. werkmanii in the presence of excess Ca(2+) and provide a preliminary expression of these differences based on proteomic assays.

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

    Science.gov (United States)

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

    2015-11-01

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

  18. Proteomics profile changes in cisplatin-treated human ovarian cancer cell strain

    Institute of Scientific and Technical Information of China (English)

    LI Zhengyu; ZHAO Xia; YANG Jinliang; WEI Yuquan

    2005-01-01

    To compare the alterations in proteomes between cisplatin-treated and -untreated human ovarian cancer SKOV3 cells, and to explore the feasibility of proteomics in research about antitumor mechanisms of agents, SKOV3 cells were exposed to cisplatin (6 μg/mL) for 6 h. Then, the cells were collected and solubilized and global proteins were extracted by lysis buffer; two-dimensional electrophoresis was conducted with the IPG readystrips as carriers; the gels were stained with Coomassie blue and alterations between gels were compared by PDQuest. Eventually, 11 spots with significant differences were selected and excised and the proteins were identified by PMF and MS/MS analysis. The results revealed that exposure to cisplatin could notably increase expressions of some proteins, such as tropomyosin family, actin family, triosephosphate isomerase family, and HSP60, etc.; while expressions of some other proteins decreased, such as enolase family, etc. Those proteins were involved in cellular energy metabolism, transformation, apoptosis and morphologic maintenance, which suggested that alterations of those physiological processes might be involved in anti-tumor mechanism of cisplatin.

  19. Role of the cell wall integrity and filamentous growth mitogen-activated protein kinase pathways in cell wall remodeling during filamentous growth.

    Science.gov (United States)

    Birkaya, Barbara; Maddi, Abhiram; Joshi, Jyoti; Free, Stephen J; Cullen, Paul J

    2009-08-01

    Many fungal species including pathogens exhibit filamentous growth (FG) as a means of foraging for nutrients. Genetic screens were performed to identify genes required for FG in the budding yeast Saccharomyces cerevisiae. Genes encoding proteins with established functions in transcriptional activation (MCM1, MATalpha2, PHD1, MSN2, SIR4, and HMS2), cell wall integrity (MPT5, WSC2, and MID2), and cell polarity (BUD5) were identified as potential regulators of FG. The transcription factors MCM1 and MATalpha2 induced invasive growth by promoting diploid-specific bipolar budding in haploid cells. Components of the cell wall integrity pathway including the cell surface proteins Slg1p/Wsc1p, Wsc2p, Mid2p, and the mitogen-activated protein kinase (MAPK) Slt2p/Mpk1p contributed to multiple aspects of the FG response including cell elongation, cell-cell adherence, and agar invasion. Mid2p and Wsc2p stimulated the FG MAPK pathway through the signaling mucin Msb2p and components of the MAPK cascade. The FG pathway contributed to cell wall integrity in parallel with the cell wall integrity pathway and in opposition with the high osmolarity glycerol response pathway. Mass spectrometry approaches identified components of the filamentous cell wall including the mucin-like proteins Msb2p, Flo11p, and subtelomeric (silenced) mucin Flo10p. Secretion of Msb2p, which occurs as part of the maturation of the protein, was inhibited by the ss-1,3-glucan layer of the cell wall, which highlights a new regulatory aspect to cell wall remodeling in this organism. Disruption of ss-1,3-glucan linkages induced mucin shedding and resulted in defects in cell-cell adhesion and invasion of cells into the agar matrix.

  20. Subcellular Proteomics of Soybean under Flooding Stress

    Institute of Scientific and Technical Information of China (English)

    Setsuko Komatsu

    2012-01-01

    Flooding is an environmental stress found worldwide and may increase in frequency due to changes in global climate,and causes significant reductions in the growth and yield of several crops.The application of proteomics techniques to clarify the molecular mechanisms underlying crop responses to flooding stress may facilitate the development of flood tolerant crops.To understand the response mechanism of soybean under flooding stress,proteomics analysis was carried out.Especially,subcellular proteomics studies have led to a better understanding of the mechanism of flooding stress tolerance in soybean.The effects of flooding stress on root plasma membrane were analyzed using an aqueous two-phase partitioning method in combination with gel-based and gel-free proteomics techniques.The results led to the following conclusions:proteins located in the cell wall were increased in the plasma membrane of flooded plants,indicating the contribution of plasma membrane to modification of the cell wall; superoxide dismutase was increased,indicating that the antioxidative system may play a crucial role in protecting cells from oxidative damage following exposure to flooding stress; heat shock cognate 70 kDa protein likely plays a significant role in protecting other proteins from denaturation and degradation during flooding stress; and signaling proteins might work cooperatively to regulate plasma membrane H +-ATPase and maintain ion homeostasis.Cell wall proteins were isolated from root of flooding stressed plants via sucrose gradient centrifugation and analyzed using gel-based proteomics technique.Cell wall proteins identified were related to lignification,and these results indicated that a decrease of lignification-related proteins is related to flooding decreased ROS and jasmonate biosynthesis.And also,lignin staining confirmed that lignification was suppressed in the roots of flooding stressed soybeans.Mitochondrial fractions were purified from root of flooding stressed

  1. Differential proteome analysis of conditioned medium of BPH-1 and LNCaP cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Wen-zheng; PANG Bo; YANG Bo; ZHOU Jian-guang; SUN Ying-hao

    2011-01-01

    Background Although the introduction of serum prostate-specific antigen (PSA) measurements into clinical practice has revolutionized the care of patients with prostate cancer,there are well-recognized limitations of PSA,and there is a critical need to identify additional prostate cancer biomarkers to assist in early detection and prognosis.In this regard,high resolution proteomic technology has the unexceptionable superiority to find those high abundance biomarkers.The purpose of this study was to search new tumor markers by proteomic technology.Methods The proteins in conditioned medium (CM) of BPH-1 and LNCaP cells were profiled by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS).The corresponding mRNA levels of some identified proteins were analyzed by RT-PCR.Results Totally 11 differentially expressed proteins (6 up-regulated including creatine kinase,brain (CKB),triosephosphate isomerase 1 (TPI1),isocitrate dehydrogenase 2 (IDH2) and 5 down-regulated including glutathione S-transferase pi (GST-pi)) in the CM were identified using MALDI-TOF-MS and database search.The expression pattern between mRNA and CM protein levels of CKB,IDH2,TPI1 and GST-pi in BPH-1 and LNCaP was similar.Conclusion We proved a feasible and effective way to search new tumor markers by a proteomics-based strategy and identified 11 potentially useful proteins in CM of BPH-1 and LNCaP cells to distinguish prostate cancer from benign prostatic hypertrophy.

  2. Antiandrogens act as selective androgen receptor modulators at the proteome level in prostate cancer cells.

    Science.gov (United States)

    Brooke, Greg N; Gamble, Simon C; Hough, Michael A; Begum, Shajna; Dart, D Alwyn; Odontiadis, Michael; Powell, Sue M; Fioretti, Flavia M; Bryan, Rosie A; Waxman, Jonathan; Wait, Robin; Bevan, Charlotte L

    2015-05-01

    Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic

  3. Penium margaritaceum as a model organism for cell wall analysis of expanding plant cells

    DEFF Research Database (Denmark)

    Rydahl, Maja Gro; Fangel, Jonatan Ulrik; Mikkelsen, Maria Dalgaard

    2015-01-01

    organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion...... have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying...

  4. Transcriptomic and proteomic analysis of human hepatic stellate cells treated with natural taurine.

    Science.gov (United States)

    Liang, Jian; Deng, Xin; Wu, Fa-Sheng; Tang, Yan-Fang

    2013-05-01

    The aim of this study was to investigate the differential expression of genes and proteins between natural taurine (NTau)‑treated hepatic stellate cells (HSCs) and control cells as well as the underlying mechanism of NTau in inhibiting hepatic fibrosis. A microculture tetrazolium (MTT) assay was used to analyze the proliferation of NTau‑treated HSCs. Flow cytometry was performed to compare the apoptosis rate between NTau-treated and non‑treated HSCs. Proteomic analysis using a combination of 2-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) was conducted to identify the differentially expressed proteins. Microarray analysis was performed to investigate the differential expression of genes and real-time polymerase chain reaction (PCR) was used to validate the results. The experimental findings obtained demonstrated that NTau decreased HSC proliferation, resulting in an increased number of cells in the G0/G1 phase and a reduced number of cells in the S phase. Flow cytometric analysis showed that NTau-treated HSCs had a significantly increased rate of apoptosis when compared with the non‑treated control group. A total of 15 differentially expressed proteins and 658 differentially expressed genes were identified by 2DE and MS, and microarray analysis, respectively. Gene ontology (GO) functional analysis indicated that these genes and proteins were enriched in the function clusters and pathways related to cell proliferation, cellular apoptosis and oxidation. The transcriptome and proteome analyses of NTau-treated HSCs demonstrated that NTau is able to significantly inhibit cell proliferation and promote cell apoptosis, highlighting its potential therapeutic benefits in the treatment of hepatic fibrosis.

  5. Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

    Science.gov (United States)

    Xu, Tao; Pang, Qiuying; Zhou, Dong; Zhang, Aiqin; Luo, Shaman; Wang, Yang; Yan, Xiufeng

    2014-01-01

    Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

  6. Area Expansivity Moduli of Regenerating Plant Protoplast Cell Walls Exposed to Shear Flows

    Science.gov (United States)

    Fujimura, Yuu; Iino, Masaaki; Watanabe, Ugai

    2005-05-01

    To control the elasticity of the plant cell wall, protoplasts isolated from cultured Catharanthus roseus cells were regenerated in shear flows of 115 s-1 (high shear) and 19.2 s-1 (low shear, as a control). The surface area expansivity modulus and the surface breaking strength of these regenerating protoplasts were measured by a micropipette aspiration technique. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye. High shear exposure for 3 h doubled both the surface area modulus and breaking strength observed under low shear, significantly decreased cell wall synthesis, and roughly quadrupled the moduli of the cell wall. Based on the cell wall synthesis data, we estimated the three-dimensional modulus of the cell wall to be 4.1± 1.2 GPa for the high shear, and 0.35± 0.2 GPa for the low shear condition, using the surface area expansivity modulus divided by the cell wall thickness, which is identical with the Young’s modulus divided by 2(1-σ), where σ is Poisson's ratio. We concluded that high shear exposure considerably strengthens the newly synthesized cell wall.

  7. Leaf-cutting ant fungi produce cell wall degrading pectinase complexes reminiscent of phytopathogenic fungi

    Directory of Open Access Journals (Sweden)

    Boomsma Jacobus J

    2010-12-01

    Full Text Available Abstract Background Leaf-cutting (attine ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth. Results We tested this hypothesis by using proteomics methods to determine the gene sequences of fecal proteins in Acromyrmex echinatior leaf-cutting ants. Seven (21% of the 33 identified proteins were pectinolytic enzymes that originated from the fungal symbiont and which were still active in the fecal droplets produced by the ants. We show that these enzymes are found in the fecal material only when the ants had access to fungus garden food, and we used quantitative polymerase chain reaction analysis to show that the expression of six of these enzyme genes was substantially upregulated in the fungal gongylidia. These unique structures serve as food for the ants and are produced only by the evolutionarily advanced garden symbionts of higher attine ants, but not by the fungi reared by the basal lineages of this ant clade. Conclusions Pectinolytic enzymes produced in the gongylidia of the fungal symbiont are ingested but not digested by Acromyrmex leaf-cutting ants so that they end up in the fecal fluid and become mixed with new garden substrate. Substantial quantities of pectinolytic enzymes are typically found in pathogenic fungi that attack live plant tissue, where they are known to breach the cell walls to allow the fungal mycelium access to the cell contents. As the leaf-cutting ant symbionts are derived from fungal clades that decompose dead plant material, our results suggest that their pectinolytic enzymes represent secondarily evolved adaptations that are convergent to

  8. Proteome Profiling in Lung Injury after Hematopoietic Stem Cell Transplantation.

    Science.gov (United States)

    Bhargava, Maneesh; Viken, Kevin J; Dey, Sanjoy; Steinbach, Michael S; Wu, Baolin; Jagtap, Pratik D; Higgins, LeeAnn; Panoskaltsis-Mortari, Angela; Weisdorf, Daniel J; Kumar, Vipin; Arora, Mukta; Bitterman, Peter B; Ingbar, David H; Wendt, Chris H

    2016-08-01

    Pulmonary complications due to infection and idiopathic pneumonia syndrome (IPS), a noninfectious lung injury in hematopoietic stem cell transplant (HSCT) recipients, are frequent causes of transplantation-related mortality and morbidity. Our objective was to characterize the global bronchoalveolar lavage fluid (BALF) protein expression of IPS to identify proteins and pathways that differentiate IPS from infectious lung injury after HSCT. We studied 30 BALF samples from patients who developed lung injury within 180 days of HSCT or cellular therapy transfusion (natural killer cell transfusion). Adult subjects were classified as having IPS or infectious lung injury by the criteria outlined in the 2011 American Thoracic Society statement. BALF was depleted of hemoglobin and 14 high-abundance proteins, treated with trypsin, and labeled with isobaric tagging for relative and absolute quantification (iTRAQ) 8-plex reagent for two-dimensional capillary liquid chromatography (LC) and data dependent peptide tandem mass spectrometry (MS) on an Orbitrap Velos system in higher-energy collision-induced dissociation activation mode. Protein identification employed a target-decoy strategy using ProteinPilot within Galaxy P. The relative protein abundance was determined with reference to a global internal standard consisting of pooled BALF from patients with respiratory failure and no history of HSCT. A variance weighted t-test controlling for a false discovery rate of ≤5% was used to identify proteins that showed differential expression between IPS and infectious lung injury. The biological relevance of these proteins was determined by using gene ontology enrichment analysis and Ingenuity Pathway Analysis. We characterized 12 IPS and 18 infectious lung injury BALF samples. In the 5 iTRAQ LC-MS/MS experiments 845, 735, 532, 615, and 594 proteins were identified for a total of 1125 unique proteins and 368 common proteins across all 5 LC-MS/MS experiments. When comparing IPS to

  9. Comparison of Normal and Breast Cancer Cell lines using Proteome, Genome and Interactome data

    Energy Technology Data Exchange (ETDEWEB)

    Patwardhan, Anil J.; Strittmatter, Eric F.; Camp, David G.; Smith, Richard D.; Pallavicini, Maria

    2005-12-01

    Normal and cancer cell line proteomes were profiled using high throughput mass spectrometry techniques. Application of both protein-level and peptide-level sample fractionation combined with LC-MS/MS analysis enabled the confident identification of 2,235 unmodified proteins representing a broad range of functional and compartmental classes. An iterative multi-step search strategy was used to identify post-translational modifications and detected several proteins that are preferentially modified in cancer cells. Information regarding both unmodified and modified protein forms was combined with publicly available gene expression and protein-protein interaction data. The resulting integrated dataset revealed several functionally related proteins that are differentially regulated between normal and cancer cell lines.

  10. H Ferritin Gene Silencing in a Human Metastatic Melanoma Cell Line: A Proteomic Analysis

    DEFF Research Database (Denmark)

    Di Sanzo, Maddalena; Gaspari, Marco; Misaggi, Roberta

    2011-01-01

    and pathologic states (i.e., neurodegeneration and cancer). This study is aimed at investigating the whole-cell proteome of FHC-expressing and sh-RNA-silenced human metastatic melanoma cells (MM07(m)) in the attempt to identify and classify the highest number of proteins directly or indirectly controlled...... by the FHC. We identified about 200 differentially expressed proteins and classified them in clusters on the basis of their functions, as proteins involved in metabolic processes, cell adhesion, migration, and proliferation processes. Some of them have captured our attention because of their involvement...... of H ferritin signaling pathways and lend support to the hypothesis that specific targeting of this gene might be an attractive and potentially effective strategy for the management of metastatic melanoma....

  11. 309 proteomic analysis of the blastocoel fluid and remaining cells of bovine blastocysts

    DEFF Research Database (Denmark)

    Jensen, P L; Groendahl, M L; Beck, Helle;

    2012-01-01

    Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions...... such as Parkinson's disease, diabetes, and spinal cord injury. One of today's challenges in regenerative medicine is to define proper culture conditions for hESC. The natural milieu in the blastocyst may provide clues on how to improve culture conditions, and the aim of the present study was to determine...... the proteome of the blastocoel fluid and the remaining cells of bovine blastocysts. Bovine blastocysts were produced by in vitro fertilization of oocytes retrieved from slaughterhouse ovaries. The blastocoel from 195 blastocysts (1-8nL per blastocyst) were isolated by micromanipulation and analysed by nano...

  12. Proteomic analysis of proteins associated with tt-DDE induced toxicity in BEAS-2B cells.

    Science.gov (United States)

    Lin, Pin-Pin; Yang, Ming-Hui; Liao, Pao-Chi; Wu, Hsin-Yi; Chang, Louis W; Tsai, Hui-Ti; Tyan, Yu-Chang

    2008-11-21

    Trans, trans-2,4-decadienal (tt-DDE), a specific type of dienaldehyde, is abundant in heated oils or cooking oil fumes. Ingestion of heated oils and exposure to cooking oil fumes has been suggested to have a great health impact in a variety of organs, including the lungs. Previous studies have demonstrated that acute exposures to high doses of tt-DDE have induced oxidative stress, genotoxicity, and cytotoxicity in human lung cells. The objective in utilizing proteomic techniques of this study was to identify protein biomarkers associated with tt-DDE-induced oxidative stress and cytotoxicity in human bronchial epithelial cells BEAS-2B. Experimental results suggested that DJ-1 and cofilin proteins were protein biomarkers for tt-DDE-induced cytotoxicity and oxidative stress in lung cells. DJ-1 was especially an early biomarker for tt-DDE exposure.

  13. Composition and architecture of the cell walls of grasses and the mechanisms of synthesis of cell wall polysaccharides. Final report for period September 1, 1988 - April 30, 2001

    Energy Technology Data Exchange (ETDEWEB)

    Carpita, Nicholas C.

    2001-10-18

    This program was devoted toward complete understanding of the polysaccharide structure and architecture of the primary cell walls grasses and cereals, and the biosynthesis of the mixed-linkage beta-glucane, a cellulose interacting polymer that is synthesized uniquely by grass species and close relatives. With these studies as focal point, the support from DOE was instrumental in the development of new analytical means that enabled us to characterize carbohydrate structure, to reveal new features of cell wall dynamics during cell growth, and to apply these techniques in other model organisms. The support by DOE in these basic studies was acknowledged on numerous occasions in review articles covering current knowledge of cell wall structure, architecture, dynamics, biosynthesis, and in all genes related to cell wall biogenesis.

  14. Integrated proteomic analysis of human cancer cells and plasma from tumor bearing mice for ovarian cancer biomarker discovery.

    Directory of Open Access Journals (Sweden)

    Sharon J Pitteri

    Full Text Available The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery.We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease.Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.

  15. Pectinous cell wall thickenings formation - A common defense strategy of plants to cope with Pb.

    Science.gov (United States)

    Krzesłowska, Magdalena; Rabęda, Irena; Basińska, Aneta; Lewandowski, Michał; Mellerowicz, Ewa J; Napieralska, Anna; Samardakiewicz, Sławomir; Woźny, Adam

    2016-07-01

    Lead, one of the most abundant and hazardous trace metals affecting living organisms, has been commonly detected in plant cell walls including some tolerant plants, mining ecotypes and hyperaccumulators. We have previously shown that in tip growing Funaria sp. protonemata cell wall is remodeled in response to lead by formation of thickenings rich in low-methylesterified pectins (pectin epitope JIM5 - JIM5-P) able to bind metal ions, which accumulate large amounts of Pb. Hence, it leads to the increase of cell wall capacity for Pb compartmentalization. Here we show that diverse plant species belonging to different phyla (Arabidopsis, hybrid aspen, star duckweed), form similar cell wall thickenings in response to Pb. These thickenings are formed in tip growing cells such as the root hairs, and in diffuse growing cells such as meristematic and root cap columella cells of root apices in hybrid aspen and Arabidopsis and in mesophyll cells in star duckweed fronds. Notably, all analyzed cell wall thickenings were abundant in JIM5-P and accumulated high amounts of Pb. In addition, the co-localization of JIM5-P and Pb commonly occurred in these cells. Hence, cell wall thickenings formed the extra compartment for Pb accumulation. In this way plant cells increased cell wall capacity for compartmentalization of this toxic metal, protecting protoplast from its toxicity. As cell wall thickenings occurred in diverse plant species and cell types differing in the type of growth we may conclude that pectinous cell wall thickenings formation is a widespread defense strategy of plants to cope with Pb. Moreover, detection of natural defense strategy, increasing plant cell walls capacity for metal accumulation, reveals a promising direction for enhancing plant efficiency in phytoremediation.

  16. The Karyote physico-chemical genomic, proteomic, metabolic cell modeling system.

    Science.gov (United States)

    Ortoleva, P; Berry, E; Brun, Y; Fan, J; Fontus, M; Hubbard, K; Jaqaman, K; Jarymowycz, L; Navid, A; Sayyed-Ahmad, A; Shreif, Z; Stanley, F; Tuncay, K; Weitzke, E; Wu, L-C

    2003-01-01

    Modeling approaches to the dynamics of a living cell are presented that are strongly based on its underlying physical and chemical processes and its hierarchical spatio-temporal organization. Through the inclusion of a broad spectrum of processes and a rigorous analysis of the multiple scale nature of cellular dynamics, we are attempting to advance cell modeling and its applications. The presentation focuses on our cell modeling system, which integrates data archiving and quantitative physico-chemical modeling and information theory to provide a seamless approach to the modeling/data analysis endeavor. Thereby the rapidly growing mess of genomic, proteomic, metabolic, and cell physiological data can be automatically used to develop and calibrate a predictive cell model. The discussion focuses on the Karyote cell modeling system and an introduction to the CellX and VirusX models. The Karyote software system integrates three elements: (1) a model-building and data archiving module that allows one to define a cell type to be modeled through its reaction network, structure, and transport processes as well as to choose the surrounding medium and other parameters of the phenomenon to be modeled; (2) a genomic, proteomic, metabolic cell simulator that solves the equations of metabolic reaction, transcription/translation polymerization and the exchange of molecules between parts of the cell and with the surrounding medium; and (3) an information theory module (ITM) that automates model calibration and development, and integrates a variety of data types with the cell dynamic computations. In Karyote, reactions may be fast (equilibrated) or slow (finite rate), and the special effects of enzymes and other minority species yielding steady-state cycles of arbitrary complexities are accounted for. These features of the dynamics are handled via rigorous multiple scale analysis. A user interface allows for an automated generation and solution of the equations of multiple timescale

  17. Platelet proteomics.

    Science.gov (United States)

    Zufferey, Anne; Fontana, Pierre; Reny, Jean-Luc; Nolli, Severine; Sanchez, Jean-Charles

    2012-01-01

    Platelets are small cell fragments, produced by megakaryocytes, in the bone marrow. They play an important role in hemostasis and diverse thrombotic disorders. They are therefore primary targets of antithrombotic therapies. They are implicated in several pathophysiological pathways, such as inflammation or wound repair. In blood circulation, platelets are activated by several pathways including subendothelial matrix and thrombin, triggering the formation of the platelet plug. Studying their proteome is a powerful approach to understand their biology and function. However, particular attention must be paid to different experimental parameters, such as platelet quality and purity. Several technologies are involved during the platelet proteome processing, yielding information on protein identification, characterization, localization, and quantification. Recent technical improvements in proteomics combined with inter-disciplinary strategies, such as metabolomic, transcriptomics, and bioinformatics, will help to understand platelets biological mechanisms. Therefore, a comprehensive analysis of the platelet proteome under different environmental conditions may contribute to elucidate complex processes relevant to platelet function regarding bleeding disorders or platelet hyperreactivity and identify new targets for antiplatelet therapy.

  18. A comparative proteome analysis of Escherichia coli DeltarelA mutant cells

    Directory of Open Access Journals (Sweden)

    Sónia Carneiro

    2016-10-01

    Full Text Available The bacterial RelA-dependent stringent response exerts a strong influence over a wide variety of processes. In this work, the impact of the relA gene mutation in E. coli cells was evaluated by a quantitative proteomics analysis, employing stable-isotope labelling and high resolution mass spectrometry. Chemostat cultures of E. coli W3110 and ∆relA mutant strains were performed at two dilution rates (0.1 and 0.2 h-1 to assess the influence of the relA gene mutation in steady-state protein levels. A total of 121 proteins showed significant alterations in their abundance when comparing the proteome of mutant to wild-type cells. The relA gene mutation induced changes on key cellular processes, including the amino acids and nucleotide biosynthesis, the lipid metabolism, transport activities, transcription and translation processes and responses to stress. Furthermore, some of those changes were more pronounced under specific growth conditions, as the most significant differences in protein ratios were observed at one of the dilution rates. An effect of the relA gene mutation in the acetate overflow was also observed, which confers interesting characteristics to this mutant strain that could be useful in the production of recombinant proteins. Overall, these results provide a valuable insight into the E. coli stringent response under defined steady-state conditions, suggesting that this stress response might influence multiple metabolic processes like the acetate overflow or the catabolite repression.

  19. Proteome Alterations of Hippocampal Cells Caused by Clostridium botulinum C3 Exoenzyme.

    Science.gov (United States)

    Schröder, Anke; Rohrbeck, Astrid; Just, Ingo; Pich, Andreas

    2015-11-06

    C3bot from Clostridium botulinum is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells.

  20. Cell Wall Microstructure Analysis Implicates Hemicellulose Polysaccharides in Cell Adhesion in Tomato Fruit Pericarp Parenchyma

    Institute of Scientific and Technical Information of China (English)

    Jose J. Ordaz-Ortiz; Susan E. Marcus; J. Paul Knox

    2009-01-01

    Methods developed to isolate intact cells from both unripe and ripe tomato fruit pericarp parenchyma have allowed the cell biological analysis of polysaccharide epitopes at the surface of separated cells. The LM7 pectic homoga-lacturonan epitope is a marker of the junctions of adhesion planes and intercellular spaces in parenchyma systems. The LM7 epitope persistently marked the former edge of adhesion planes at the surface of cells separated from unripe and ripened tomato fruit and also from fruits with the Cnr mutation. The LM 11 xylan epitope was associated, in sections, with cell walls lining intercellular space but the epitope was not detected at the surface of isolated cells, being lost during cell isolation. The LM15 xyloglucan epitope was present at the surface of cells isolated from unripe fruit in a pattern reflecting the former edge of cell adhesion planes/intercellular space but with gaps and apparent breaks, An equivalent pattern ofLM15 epitope occurrence was revealed at the surface of cells isolated by pectate lyase action but was not present in cells isolated from ripe fruit or from Cnr fruit. In contrast to wild-type cells, the LM5 galactan and LM21 mannan epitopes oc-curred predominantly in positions reflecting intercellular space in Cnr, suggesting a concerted alteration in cell wall mi-crostructure in response to this mutation. Galactanase and mannanase, along with pectic homogalacturonan-degrading enzymes, were capable of releasing cells from unripe fruit parenchyma. These observations indicate that hemicellulose polymers are present in architectural contexts reflecting cell adhesion and that several cell wall polysaccharide classes are likely to contribute to cell adhesion/cell separation in tomato fruit pericarp parenchyma.

  1. Plant cell walls throughout evolution: towards a molecular understanding of their design principles

    Energy Technology Data Exchange (ETDEWEB)

    Sarkar, Purbasha; Bosneaga, Elena; Auer, Manfred

    2009-02-16

    Throughout their life, plants typically remain in one location utilizing sunlight for the synthesis of carbohydrates, which serve as their sole source of energy as well as building blocks of a protective extracellular matrix, called the cell wall. During the course of evolution, plants have repeatedly adapted to their respective niche,which is reflected in the changes of their body plan and the specific design of cell walls. Cell walls not only changed throughout evolution but also are constantly remodelled and reconstructed during the development of an individual plant, and in response to environmental stress or pathogen attacks. Carbohydrate-rich cell walls display complex designs, which together with the presence of phenolic polymers constitutes a barrier for microbes, fungi, and animals. Throughout evolution microbes have co-evolved strategies for efficient breakdown of cell walls. Our current understanding of cell walls and their evolutionary changes are limited as our knowledge is mainly derived from biochemical and genetic studies, complemented by a few targeted yet very informative imaging studies. Comprehensive plant cell wall models will aid in the re-design of plant cell walls for the purpose of commercially viable lignocellulosic biofuel production as well as for the timber, textile, and paper industries. Such knowledge will also be of great interest in the context of agriculture and to plant biologists in general. It is expected that detailed plant cell wall models will require integrated correlative multimodal, multiscale imaging and modelling approaches, which are currently underway.

  2. Proteomic profiling of nuclear fractions from native renal inner medullary collecting duct cells.

    Science.gov (United States)

    Pickering, Christina M; Grady, Cameron; Medvar, Barbara; Emamian, Milad; Sandoval, Pablo C; Zhao, Yue; Yang, Chin-Rang; Jung, Hyun Jun; Chou, Chung-Lin; Knepper, Mark A

    2016-02-01

    The control of renal water excretion occurs in part by regulation of transcription in response to vasopressin in cells of the collecting duct. A systems biology-based approach to understanding transcriptional control in renal collecting duct cells depends on knowledge of what transcription factors and other regulatory proteins are present in the cells' nuclei. The goal of this article is to report comprehensive proteomic profiling of cellular fractions enriched in nuclear proteins from native inner medullary collecting duct (IMCD) cells of the rat. Multidimensional separation procedures and state-of-the art protein mass spectrometry produced 18 GB of spectral data that allowed the high-stringency identification of 5,048 proteins in nuclear pellet (NP) and nuclear extract (NE) fractions of biochemically isolated rat IMCD cells (URL: https://helixweb.nih.gov/ESBL/Database/IMCD_Nucleus/). The analysis identified 369 transcription factor proteins out of the 1,371 transcription factors coded by the rat genome. The analysis added 1,511 proteins to the recognized proteome of rat IMCD cells, now amounting to 8,290 unique proteins. Analysis of samples treated with the vasopressin analog dDAVP (1 nM for 30 min) or its vehicle revealed 99 proteins in the NP fraction and 88 proteins in the NE fraction with significant changes in spectral counts (Fisher exact test, P < 0.005). Among those altered by vasopressin were seven distinct histone proteins, all of which showed decreased abundance in the NP fraction, consistent with a possible effect of vasopressin to induce chromatin remodeling. The results provide a data resource for future studies of vasopressin-mediated transcriptional regulation in the renal collecting duct.

  3. Antioxidant properties of cell wall polysaccharides of Stevia rebaudiana leaves

    Directory of Open Access Journals (Sweden)

    Mediesse Kengne Francine

    2014-12-01

    Full Text Available Objective: To examine the total phenolic and protein contents, and the antioxidant activities of cell wall polysaccharide fractions of Stevia rebaudiana leaves. Methods: Three different polysaccharide-enriched fractions, namely FPE (extract with 50 mmol/ L ethylene diamine tetra acetic acid, FPK (extract with 0.05 mol/L KOH and FH (extract with 4 mol/L KOH were extracted from Stevia rebaudiana leaves. The antioxidant activity of these fractions was evaluated based on their ability to scavenge DPPH (1, 1-diphenyl-2-picryl hydrazyl free radical, to reduce ferric power, to chelate ferrous ion and to protect human DNA. Results: The results indicated that protein content was found to be higher in FPK polysaccharide enriched fraction (47.48 µg per mg of FPK. Furthermore, the phenolic compound analysis according to the Folin-Ciocalteu method was higher in FPK (17.71 µg ferulic acid. The DPPH maximal inhibition percentage of the three polysaccharide-enriched fractions at 400 µg/mL was 27.66%, 59.90% and 23.21% respectively for FPE, FPK and FH. All the polysaccharide fractions exhibited a ferric reducing power except the FH one. The three fractions also exhibited lipid peroxidation inhibition, and they completely reverted the DNA damage induced by H2O2/FeCl2. FPK showed the strongest scavenging activity against the DPPH radical, the best chelating ability and lipid peroxidation inhibition. Conclusions: Stevia cell wall polysaccharide fractions are potent protective agents against oxidative stress. The analysis revealed major differences in the antioxidant activity in the three polysaccharides fractions. However, the 0.05 mol/L KOH pectin fraction (FPK showed better antioxidant activity.

  4. Antioxidant properties of cell wall polysaccharides of Stevia rebaudiana leaves

    Institute of Scientific and Technical Information of China (English)

    Mediesse Kengne Francine; Woguia Alice Louise; Fogue Souopgui Pythagore; Atogho-Tiedeu Barbara; Simo Gustave; Thadde Boudjeko

    2014-01-01

    Objective: To examine the total phenolic and protein contents, and the antioxidant activities of cell wall polysaccharide fractions of Stevia rebaudiana leaves.Methods:L ethylene diamine tetra acetic acid), FPK (extract with 0.05 mol/L KOH) and FH (extract with 4 mol/L KOH) were extracted from Stevia rebaudiana leaves. The antioxidant activity of these fractions was evaluated based on their ability to scavenge DPPH (1, 1-diphenyl-2-picryl hydrazyl) free radical, to reduce ferric power, to chelate ferrous ion and to protect human DNA. Three different polysaccharide-enriched fractions, namely FPE (extract with 50 mmol/Results: The results indicated that protein content was found to be higher in FPK polysaccharide enriched fraction (47.48 µg per mg of FPK). Furthermore, the phenolic compound analysis according to the Folin-Ciocalteu method was higher in FPK (17.71 µg ferulic acid). The DPPH maximal inhibition percentage of the three polysaccharide-enriched fractions at 400 µg/mL was 27.66%, 59.90% and 23.21% respectively for FPE, FPK and FH. All the polysaccharide fractions exhibited a ferric reducing power except the FH one. The three fractions also exhibited lipid peroxidation inhibition, and they completely reverted the DNA damage induced by H2O2/FeCl2. FPK showed the strongest scavenging activity against the DPPH radical, the best chelating ability and lipid peroxidation inhibition.Conclusions: Stevia cell wall polysaccharide fractions are potent protective agents against oxidative stress. The analysis revealed major differences in the antioxidant activity in the three polysaccharides fractions. However, the 0.05 mol/L KOH pectin fraction (FPK) showed better antioxidant activity.

  5. Evidence that pulsed electric field treatment enhances the cell wall porosity of yeast cells.

    Science.gov (United States)

    Ganeva, Valentina; Galutzov, Bojidar; Teissie, Justin

    2014-02-01

    The application of rectangular electric pulses, with 0.1-2 ms duration and field intensity of 2.5-4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2-8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.

  6. CELL-WALL GROWTH AND PROTEIN SECRETION IN FUNGI

    NARCIS (Netherlands)

    SIETSMA, JH; WOSTEN, HAB; WESSELS, JGH

    1995-01-01

    Secretion of proteins is a vital process in fungi. Because hyphal walls form a diffusion barrier for proteins, a mechanism different from diffusion probably exist to transport proteins across the wall. In Schizophyllum commune, evidence has been obtained for synthesis at the hyphal apex of wall comp

  7. Wall extensibility: its nature, measurement and relationship to plant cell growth

    Science.gov (United States)

    Cosgrove, D. J.

    1993-01-01

    Expansive growth of plant cells is controlled principally by processes that loosen the wall and enable it to expand irreversibly. The central role of wall relaxation for cell expansion is reviewed. The most common methods for assessing the extension properties of plant cell walls ( wall extensibility') are described, categorized and assessed critically. What emerges are three fundamentally different approaches which test growing cells for their ability (a) to enlarge at different values of turgor, (b) to induce wall relaxation, and (c) to deform elastically or plastically in response to an applied tensile force. Analogous methods with isolated walls are similarly reviewed. The results of these different assays are related to the nature of plant cell growth and pertinent biophysical theory. I argue that the extensibilities' measured by these assays are fundamentally different from one another and that some are more pertinent to growth than others.

  8. DBIO Best Thesis Award: Mechanics, Dynamics, and Organization of the Bacterial Cytoskeleton and Cell Wall

    Science.gov (United States)

    Wang, Siyuan

    2012-02-01

    Bacteria come in a variety of shapes. While the peptidoglycan (PG) cell wall serves as an exoskeleton that defines the static cell shape, the internal bacterial cytoskeleton mediates cell shape by recruiting PG synthesis machinery and thus defining the pattern of cell-wall synthesis. While much is known about the chemistry and biology of the cytoskeleton and cell wall, much of their biophysics, including essential aspects of the functionality, dynamics, and organization, remain unknown. This dissertation aims to elucidate the detailed biophysical mechanisms of cytoskeleton guided wall synthesis. First, I find that the bacterial cytoskeleton MreB contributes nearly as much to the rigidity of an Escherichia coli cell as the cell wall. This conclusion implies that the cytoskeletal polymer MreB applies meaningful force to the cell wall, an idea favored by theoretical modeling of wall growth, and suggests an evolutionary origin of cytoskeleton-governed cell rigidity. Second, I observe that MreB rotates around the long axis of E. coli, and the motion depends on wall synthesis. This is the first discovery of a cell-wall assembly driven molecular motor in bacteria. Third, I prove that both cell-wall synthesis and the PG network have chiral ordering, which is established by the spatial pattern of MreB. This work links the molecular structure of the cytoskeleton and of the cell wall with organismal-scale behavior. Finally, I develop a mathematical model of cytoskeleton-cell membrane interactions, which explains the preferential orientation of different cytoskeleton components in bacteria.

  9. Structural basis of cell wall cleavage by a staphylococcal autolysin.

    Directory of Open Access Journals (Sweden)

    Sebastian Zoll

    2010-03-01

    Full Text Available The major autolysins (Atl of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several alpha-helices surrounding a central beta-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.

  10. Neural network analyses of infrared spectra for classifying cell wall architectures.

    Science.gov (United States)

    McCann, Maureen C; Defernez, Marianne; Urbanowicz, Breeanna R; Tewari, Jagdish C; Langewisch, Tiffany; Olek, Anna; Wells, Brian; Wilson, Reginald H; Carpita, Nicholas C

    2007-03-01

    About 10% of plant genomes are devoted to cell wall biogenesis. Our goal is to establish methodologies that identify and classify cell wall phenotypes of mutants on a genome-wide scale. Toward this goal, we have used a model system, the elongating maize (Zea mays) coleoptile system, in which cell wall changes are well characterized, to develop a paradigm for classification of a comprehensive range of cell wall architectures altered during development, by environmental perturbation, or by mutation. Dynamic changes in cell walls of etiolated maize coleoptiles, sampled at one-half-d intervals of growth, were analyzed by chemical and enzymatic assays and Fourier transform infrared spectroscopy. The primary walls of grasses are composed of cellulose microfibrils, glucuronoarabinoxylans, and mixed-linkage (1 --> 3),(1 --> 4)-beta-D-glucans, together with smaller amounts of glucomannans, xyloglucans, pectins, and a network of polyphenolic substances. During coleoptile development, changes in cell wall composition included a transient appearance of the (1 --> 3),(1 --> 4)-beta-D-glucans, a gradual loss of arabinose from glucuronoarabinoxylans, and an increase in the relative proportion of cellulose. Infrared spectra reflected these dynamic changes in composition. Although infrared spectra of walls from embryonic, elongating, and senescent coleoptiles were broadly discriminated from each other by exploratory principal components analysis, neural network algorithms (both genetic and Kohonen) could correctly classify infrared spectra from cell walls harvested from individuals differing at one-half-d interval of growth. We tested the predictive capabilities of the model with a maize inbred line, Wisconsin 22, and found it to be accurate in classifying cell walls representing developmental stage. The ability of artificial neural networks to classify infrared spectra from cell walls provides a means to identify many possible classes of cell wall phenotypes. This classification

  11. Mycobacterium tuberculosis CwsA overproduction modulates cell division and cell wall synthesis.

    Science.gov (United States)

    Plocinski, P; Martinez, L; Sarva, K; Plocinska, R; Madiraju, M; Rajagopalan, M

    2013-12-01

    We recently showed that two small membrane proteins of Mycobacterium tuberculosis, CwsA and CrgA, interact with each other, and that loss of CwsA in M. smegmatis is associated with defects in the cell division and cell wall synthesis processes. Here we show that CwsA overproduction also affected growth, cell division and cell shape of M. smegmatis and M. tuberculosis. CwsA overproduction in M. tuberculosis led to increased sensitivity to cefsulodin, a penicillin-binding protein (PBP) 1A/1B targeting beta (β) -lactam, but was unaffected by other β-lactams and vancomycin. A M. smegmatis cwsA overexpressing strain showed bulgy cells, increased fluorescent vancomycin staining and altered localization of Wag31-mCherry fusion protein. However, the levels of phosphorylated Wag31, important for optimal peptidoglycan synthesis and growth in mycobacteria, were not affected. Interestingly, CwsA overproduction in E. coli led to the formation of large rounded cells that eventually lysed whereas the overproduction of FtsZ along with CwsA reversed this phenotype. Together, our results emphasize that optimal levels of CwsA are required for regulated cell wall synthesis, hence maintenance of cell shape, and that CwsA likely interacts with and modulates the activities of other cell wall synthetic components including PBPs.

  12. Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

    Science.gov (United States)

    Radtke, Andrea L; Herbst-Kralovetz, Melissa M

    2012-04-03

    Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties

  13. Comparative secretome analysis suggests low plant cell wall degrading capacity in Frankia symbionts

    Directory of Open Access Journals (Sweden)

    Normand Philippe

    2008-01-01

    genomes, suggesting that plant cell wall polysaccharide degradation may not be crucial to root infection, or that this degradation varies among strains. We hypothesize that the relative lack of secreted polysaccharide-degrading enzymes in Frankia reflects a strategy used by these bacteria to avoid eliciting host defense responses. The esterases, lipases, and proteases found in the core Frankia secretome might facilitate hyphal penetration through the cell wall, release carbon sources, or modify chemical signals. The core secretome also includes extracellular solute-binding proteins and Frankia-specific hypothetical proteins that may enable the actinorhizal symbiosis.

  14. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

    DEFF Research Database (Denmark)

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha;

    2013-01-01

    . The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco) mannan, and xyloglucan as well as overall cell wall acetylation is affected differently...

  15. Cell wall dynamics modulate acetic acid-induced apoptotic cell death of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    António Rego

    2014-08-01

    Full Text Available Acetic acid triggers apoptotic cell death in Saccharomyces cerevisiae, similar to mammalian apoptosis. To uncover novel regulators of this process, we analyzed whether impairing MAPK signaling affected acetic acid-induced apoptosis and found the mating-pheromone response and, especially, the cell wall integrity pathways were the major mediators, especially the latter, which we characterized further. Screening downstream effectors of this pathway, namely targets of the transcription factor Rlm1p, highlighted decreased cell wall remodeling as particularly important for acetic acid resistance. Modulation of cell surface dynamics therefore emerges as a powerful strategy to increase acetic acid resistance, with potential application in industrial fermentations using yeast, and in biomedicine to exploit the higher sensitivity of colorectal carcinoma cells to apoptosis induced by acetate produced by intestinal propionibacteria.

  16. Evaluation of preparation methods for MS-based analysis of intestinal epithelial cell proteomes

    DEFF Research Database (Denmark)

    Hesselager, Marianne Overgaard; Codrea, Marius Cosmin; Bendixen, Emøke

    2015-01-01

    The gut epithelium formed between an organism and the environment plays an essential role in host–microbe interactions, yet remains one of the least characterized mammalian tissues. Especially the membrane proteins, which are critical to bacterial adhesion, are understudied, because these proteins...... are low in abundance, and large amounts of sample is needed for their preparation and for undertaking MS-based analysis. The aim of this study was to evaluate three different methods for isolation and preparation of pig intestinal epithelial cells for MS-based analysis of the proteome. Samples were...... of ease and speed of sample preparation, as well as protein recovery. In comparison, more gentle methods where intestinal epithelial cells are harvested by shaking are more time consuming, result in lower protein yield, and are prone to increased technical variation due to multiple steps involved....

  17. Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study

    Science.gov (United States)

    2014-01-01

    Background Cordyceps cicadae is a medicinal fungus that is often used for treating cancer. However, the anticancer mechanisms of C. cicadae are largely unknown. This study aims to investigate the anticancer mechanisms of C. cicadae against hepatocellular carcinoma cells in vitro using a proteomic approach. Methods Human hepatocellular carcinoma MHCC97H cells were treated with a water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) for 48 h and harvested for cell viability assays. The significant differences in protein expression between control and C. cicadae-treated cells were analyzed by two-dimensional gel-based proteomics coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Flow cytometry analysis was employed to investigate the cell cycle and cell death. The anticancer molecular mechanism was analyzed by whole proteome mapping. Results The water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) inhibited the growth of MHCC97H cells in a dose-dependent manner via G2/M phase cell cycle arrest with no evidence of apoptosis. Among the identified proteins with upregulated expression were dynactin subunit 2, N-myc downstream-regulated gene 1, heat shock protein beta-1, alpha-enolase isoform 1, phosphatidylinositol transfer protein, and WD repeat-containing protein 1. Meanwhile, the proteins with downregulated expression were 14-3-3 gamma, BUB3, microtubule-associated protein RP/EB family member 1, thioredoxin-like protein, chloride intracellular channel protein 1, ectonucleoside triphosphate diphosphohydrolase 5, xaa-Pro dipeptidase, enoyl-CoA delta isomerase 1, protein-disulfide isomerase-related chaperone Erp29, hnRNP 2H9B, peroxiredoxin 1, WD-40 repeat protein, and serine/threonine kinase receptor-associated protein. Conclusion The water extract of C. cicadae reduced the growth of human hepatocellular carcinoma MHCC97H cells via G2/M cell cycle arrest. PMID:24872842

  18. Differential proteome analysis of human embryonic kidney cell line (HEK-293 following mycophenolic acid treatment

    Directory of Open Access Journals (Sweden)

    Rahman Hazir

    2011-09-01

    Full Text Available Abstract Background Mycophenolic acid (MPA is widely used as a post transplantation medicine to prevent acute organ rejection. In the present study we used proteomics approach to identify proteome alterations in human embryonic kidney cells (HEK-293 after treatment with therapeutic dose of MPA. Following 72 hours MPA treatment, total protein lysates were prepared, resolved by two dimensional gel electrophoresis and differentially expressed proteins were identified by QTOF-MS/MS analysis. Expressional regulations of selected proteins were further validated by real time PCR and Western blotting. Results The proliferation assay demonstrated that therapeutic MPA concentration causes a dose dependent inhibition of HEK-293 cell proliferation. A significant apoptosis was observed after MPA treatment, as revealed by caspase 3 activity. Proteome analysis showed a total of 12 protein spots exhibiting differential expression after incubation with MPA, of which 7 proteins (complement component 1 Q subcomponent-binding protein, electron transfer flavoprotein subunit beta, cytochrome b-c1 complex subunit, peroxiredoxin 1, thioredoxin domain-containing protein 12, myosin regulatory light chain 2, and profilin 1 showed significant increase in their expression. The expression of 5 proteins (protein SET, stathmin, 40S ribosomal protein S12, histone H2B type 1 A, and histone H2B type 1-C/E/F/G/I were down-regulated. MPA mainly altered the proteins associated with the cytoskeleton (26%, chromatin structure/dynamics (17% and energy production/conversion (17%. Both real time PCR and Western blotting confirmed the regulation of myosin regulatory light chain 2 and peroxiredoxin 1 by MPA treatment. Furthermore, HT-29 cells treated with MPA and total kidney cell lysate from MMF treated rats showed similar increased expression of myosin regulatory light chain 2. Conclusion The emerging use of MPA in diverse pathophysiological conditions demands in-depth studies to

  19. Osmotic Stress Suppresses Cell Wall Stiffening and the Increase in Cell Wall-Bound Ferulic and Diferulic Acids in Wheat Coleoptiles.

    Science.gov (United States)

    Wakabayashi, K.; Hoson, T.; Kamisaka, S.

    1997-01-01

    The relationship between the mechanical properties of cell walls and the levels of wall-bound ferulic (FA) and diferulic (DFA) acids was investigated in wheat (Triticum aestivum L.) coleoptiles grown under osmotic stress (60 mM polyethylene glycol [PEG] 4000) conditions. The cell walls of stressed coleoptiles remained extensible compared with those of the unstressed ones. The contents of wall-bound FA and DFA increased under unstressed conditions, but the increase was substantially reduced by osmotic stress. In response to PEG removal, these contents increased and reached almost the same levels as those of the unstressed coleoptiles. A close correlation was observed between the contents of FA and DFA and the mechanical properties of cell walls. The activities of phenylalanine ammonia-lyase and tyrosine ammonia-lyase increased rapidly under unstressed conditions. Osmotic stress substantially reduced the increases in enzyme activities. When PEG was removed, however, the enzyme activities increased rapidly. There was a close correlation between the FA levels and enzyme activities. These results suggest that in osmotically stressed wheat coleoptiles, reduced rates of increase in phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities suppress phenylpropanoid biosynthesis, resulting in the reduced level of wall-bound FA that, in turn, probably causes the reduced level of DFA and thereby maintains cell wall extensibility. PMID:12223657

  20. Cell-free layer and wall shear stress variation in microvessels.

    Science.gov (United States)

    Yin, Xuewen; Zhang, Junfeng

    2012-01-01

    In this study, we simulated multiple red blood cells flowing through straight microvessels with the immersed-boundary lattice-Boltzmann model to examine the shear stress variation on the microvessel surface and its relation to the properties of cell-free layer. Significant variation in shear stress has been observed due to the irregular configuration of blood cells flowing near the microvessel wall. A low shear stress is typically found at locations where there is a cell flowing close to the wall, and a large shear stress at locations with a relatively wide gap between cell and wall. This relationship between the shear stress magnitude and the distance between cell and wall has been attributed to the reverse pressure difference developed between the front and rear sides of a cell flowing near the vessel wall. We further studied the effects of several hemodynamic factors on the variation of shear stress, including the cell deformability, the flow rate, and the aggregation among red blood cells. These simulations show that the shear stress variation is less profound in situations with wider cell-free layers, since the reverse pressure difference around the edge cells is less evident, and the influence of this pressure difference on wall shear stress becomes weaker. This study also demonstrates the complexity of the flow field in the gap between cell and wall. More precise experimental techniques are required accurately measure such shear stress variation in microcirculation.

  1. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Applied to Quantitative Proteomics of Bacillus subtilis

    DEFF Research Database (Denmark)

    Soufi, Boumediene; Kumar, C.; Gnad, F.

    2010-01-01

    We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic...... of the most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under...

  2. Primary abdominal wall clear cell carcinoma arising from incisional endometriosis

    Institute of Scientific and Technical Information of China (English)

    Burcu Gundogdu; Isin Ureyen; Gunsu Kimyon; Hakan Turan; Nurettin Boran; Gokhan Tulunay; Dilek Bulbul; Taner Turan; M Faruk Kose

    2013-01-01

    A 49 year-old patient with the complaint of a mass located in the caesarean scar was admitted. There was a fixed mass 30í30 mm in diameter with regular contour located at the right corner of the pfannenstiel incision. Computed tomography revealed a (40í50í50) mm solid mass lesion with margins that cannot be distinguished from the uterus, bladder and small intestines and a heterogeneous mass lesion (50í45í55) mm in diameter, located in the right side of the anterior abdominal wall. Cytoreductive surgery including total abdominal hysterectomy and bilateral salpingo-oophorectomy was performed. Final pathology was clear cell carcinoma. Clear cell carcinoma arising from an extraovarian endometriotic focus was diagnosed and the patient received 6 cycles paclitaxel-carboplatin chemotherapy as adjuvant treatment. The patient who was lost to follow-up applied to our clinic 2 years after surgery with a recurrent mass in the left inguinal region. After 3 cycles of chemotherapy, the patient's tumoral mass in the left inguinal region was excised. The result of the pathology was carcinoma metastasis. It is decided that the following treatment of the patient should be palliative radiation therapy. The patient who underwent palliative radiation therapy died of disease after 4 months of the second operation.

  3. Advances of Salivary Proteomics in Oral Squamous Cell Carcinoma (OSCC) Detection: An Update

    Science.gov (United States)

    Sannam Khan, Rabia; Khurshid, Zohaib; Akhbar, Shazia; Faraz Moin, Syed

    2016-01-01

    Oral cancer refers to malignancies that have higher morbidity and mortality rates due to the late stage diagnosis and no early detection of a reliable diagnostic marker, while oral squamous cell carcinoma (OSCC) is amongst the world’s top ten most common cancers. Diagnosis of cancer requires highly sensitive and specific diagnostic tools which can support untraceable hidden sites of OSCC, yet to be unleashed, for which plenty of biomarkers are identified; the most recommended biomarker detection medium for OSCC includes biological fluids, such as blood and saliva. Saliva holds a promising future in the search for new clinical biomarkers that are easily accessible, less complex, accurate, and cost effective as well as being a non-invasive technique to follow, by analysing the malignant cells’ molecular pathology obtained from saliva through proteomic, genomic and transcriptomic approaches. However, protein biomarkers provide an immense potential for developing novel marker-based assays for oral cancer, hence this current review offers an overall focus on the discovery of a panel of candidates as salivary protein biomarkers, as well as the proteomic tools used for their identification and their significance in early oral cancer detection. PMID:28248250

  4. Uncovering stem cell differentiation factors for salivary gland regeneration by quantitative analysis of differential proteomes

    Science.gov (United States)

    Park, Yun-Jong; Koh, Jin; Kwon, Jin Teak; Park, Yong-Seok; Yang, Lijun; Cha, Seunghee

    2017-01-01

    Severe xerostomia (dry mouth) compromises the quality of life in patients with Sjögren’s syndrome or radiation therapy for head and neck cancer. A clinical management of xerostomia is often unsatisfactory as most interventions are palliative with limited efficacy. Following up our previous study demonstrating that mouse BM-MSCs are capable of differentiating into salivary epithelial cells in a co-culture system, we further explored the molecular basis that governs the MSC reprogramming by utilizing high-throughput iTRAQ-2D-LC-MS/MS-based proteomics. Our data revealed the novel induction of pancreas-specific transcription factor 1a (PTF1α), muscle, intestine and stomach expression-1 (MIST-1), and achaete-scute complex homolog 3 (ASCL3) in 7 day co-cultured MSCs but not in control MSCs. More importantly, a common notion of pancreatic-specific expression of PTF1 α was challenged for the first time by our verification of PTF1 α expression in the mouse salivary glands. Furthermore, a molecular network simulation of our selected putative MSC reprogramming factors demonstrated evidence for their perspective roles in salivary gland development. In conclusion, quantitative proteomics with extensive data analyses narrowed down a set of MSC reprograming factors potentially contributing to salivary gland regeneration. Identification of their differential/synergistic impact on MSC conversion warrants further investigation. PMID:28158262

  5. Advances of Salivary Proteomics in Oral Squamous Cell Carcinoma (OSCC Detection: An Update

    Directory of Open Access Journals (Sweden)

    Rabia Sannam Khan

    2016-12-01

    Full Text Available Oral cancer refers to malignancies that have higher morbidity and mortality rates due to the late stage diagnosis and no early detection of a reliable diagnostic marker, while oral squamous cell carcinoma (OSCC is amongst the world’s top ten most common cancers. Diagnosis of cancer requires highly sensitive and specific diagnostic tools which can support untraceable hidden sites of OSCC, yet to be unleashed, for which plenty of biomarkers are identified; the most recommended biomarker detection medium for OSCC includes biological fluids, such as blood and saliva. Saliva holds a promising future in the search for new clinical biomarkers that are easily accessible, less complex, accurate, and cost effective as well as being a non-invasive technique to follow, by analysing the malignant cells’ molecular pathology obtained from saliva through proteomic, genomic and transcriptomic approaches. However, protein biomarkers provide an immense potential for developing novel marker-based assays for oral cancer, hence this current review offers an overall focus on the discovery of a panel of candidates as salivary protein biomarkers, as well as the proteomic tools used for their identification and their significance in early oral cancer detection.

  6. Two-dimensional gel electrophoresis analysis of the proteomes expressed in the human hepatoma cell line BEL-7404 and normal liver cell line L-02

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Proteome analysis technology has been used extensively in conducting discovery research of biology and has become one of the most essential technologies in functional genomics. The proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 have been separated by high resolution two-dimensional gel electrophoresis (2-DE) with immobilized pH gradient isoelectric focusing (IPG-IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension (IPG-DALT). The resulting images have been analyzed using 2-D analysis software. Quantitative analysis reveals that 7 protein spots are detected only in hepatoma BEL-7404 cells, 14 only in L-02 cells, and 78 protein spots show significant fluctuation in quantity in both cell lines (P<0.01).These protein spots have been displayed on a proteome differential expression map. Analysis for the reproducibility of 2-DE indicates that the positional variability in the IEF dimension is 0.73 mm, while the variability in the SDS-PAGE dimension is 0.44 mm, and the quantitative variability is 17.6%-19.2%. These results suggest that the reproducibility of 2-DE has been suitable for the study of differential expression of proteomes. Proteome differential expression maps can be useful tools for disease diagnosis, drug-target validation analysis and biological process elucidation.

  7. Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

    Directory of Open Access Journals (Sweden)

    Pedersen Henriette L

    2008-05-01

    Full Text Available Abstract Background Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. Results Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15 to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types. Conclusion These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell

  8. Modification of antioxidant systems in cell walls of maize roots by different nitrogen sources

    Directory of Open Access Journals (Sweden)

    Vesna Hadži-Tašković Šukalović

    2016-12-01

    Full Text Available Antioxidant systems of maize root cell walls grown on different nitrogen sources were evaluated. Plants were grown on a medium containing only NO3- or the mixture of NO3-+NH4+, in a 2:1 ratio. Eleven-day old plants, two days after the initiation of lateral roots, were used for the experiments. Cell walls were isolated from lateral roots and primary root segments, 2-7 cm from tip to base, representing zones of intense or decreased growth rates, respectively. Protein content and the activity of enzymes peroxidase, malate dehydrogenase and ascorbate oxidase ionically or covalently bound to the walls, as well as cell wall phenolic content and antioxidant capacity, were determined. Cell walls of plants grown on mixed N possess more developed enzymatic antioxidant systems and lower non-enzymatic antioxidant defenses than cell walls grown on NO3-. Irrespective of N treatment, the activities of all studied enzymes and protein content were higher in cell walls of lateral compared to primary roots. Phenolic content of cell walls isolated from lateral roots was higher in NO3--grown than in mixed N grown plants. No significant differences could be observed in the isozyme patterns of cell wall peroxidases isolated from plants grown on different nutrient solution. Our results indicate that different N treatments modify the antioxidant systems of root cell walls. Treatment with NO3- resulted in an increase of constitutive phenolic content, while the combination of NO3-+NH4+ elevated the redox enzyme activities in root cell walls.

  9. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

    Science.gov (United States)

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J; Zhang, Jianyi; Ge, Ying

    2015-08-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.

  10. Nitrosothiol-Trapping-Based Proteomic Analysis of S-Nitrosylation in Human Lung Carcinoma Cells

    Science.gov (United States)

    Ben-Lulu, Shani; Ziv, Tamar; Weisman-Shomer, Pnina; Benhar, Moran

    2017-01-01

    Nitrosylation of cysteines residues (S-nitrosylation) mediates many of the cellular effects of nitric oxide in normal and diseased cells. Recent research indicates that S-nitrosylation of certain proteins could play a role in tumor progression and responsiveness to therapy. However, the protein targets of S-nitrosylation in cancer cells remain largely unidentified. In this study, we used our recently developed nitrosothiol trapping approach to explore the nitrosoproteome of human A549 lung carcinoma cells treated with S-nitrosocysteine or pro-inflammatory cytokines. Using this approach, we identified about 300 putative nitrosylation targets in S-nitrosocysteine-treated A549 cells and approximately 400 targets in cytokine-stimulated cells. Among the more than 500 proteins identified in the two screens, the majority represent novel targets of S-nitrosylation, as revealed by comparison with publicly available nitrosoproteomic data. By coupling the trapping procedure with differential thiol labeling, we identified nearly 300 potential nitrosylation sites in about 150 proteins. The proteomic results were validated for several proteins by an independent approach. Bioinformatic analysis highlighted important cellular pathways that are targeted by S-nitrosylation, notably, cell cycle and inflammatory signaling. Taken together, our results identify new molecular targets of nitric oxide in lung cancer cells and suggest that S-nitrosylation may regulate signaling pathways that are critically involved in lung cancer progression. PMID:28081246

  11. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Directory of Open Access Journals (Sweden)

    Michael Rosu-Myles

    Full Text Available Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC (CD105+ and non-multipotent (CD105- stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  12. Proteomics Technologies and Challenges

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Proteomics is the study of proteins and their interactions in a cell. With the completion of the Human Genome Project, the emphasis is shifting to the protein compliment of the human organism. Because proteome reflects more accurately on the dynamic state of a cell, tissue, or organism, much is expected from proteomics to yield better disease markers for diagnosis and therapy monitoring. The advent of proteomics technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of diseases. High-throughput proteomics technologies combining with advanced bioinformatics are extensively used to identify molecular signatures of diseases based on protein pathways and signaling cascades. Mass spectrometry plays a vital role in proteomics and has become an indispensable tool for molecular and cellular biology. While the potential is great, many challenges and issues remain to be solved, such as mining low abundant proteins and integration of proteomics with genomics and metabolomics data. Nevertheless, proteomics is the foundation for constructing and extracting useful knowledge to biomedical research. In this review, a snapshot of contemporary issues in proteomics technologies is discussed.

  13. Single‐Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16‐Cell Frog (Xenopus) Embryo

    OpenAIRE

    Lombard‐Banek, Camille; Moody, Sally A.; Nemes, Peter

    2016-01-01

    Abstract We advance mass spectrometry from a cell population‐averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh‐resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE‐μESI‐HRMS enabled the identification of 500–800 nonredundant protein groups by measuring 20 ng, or...

  14. The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements

    Science.gov (United States)

    Procaccini, Claudio; Carbone, Fortunata; Di Silvestre, Dario; Brambilla, Francesca; De Rosa, Veronica; Galgani, Mario; Faicchia, Deriggio; Marone, Gianni; Tramontano, Donatella; Corona, Marco; Alviggi, Carlo; Porcellini, Antonio; La Cava, Antonio; Mauri, Pierluigi; Matarese, Giuseppe

    2016-01-01

    Summary Human CD4+CD25hiFoxp3+CD127− Treg and CD4+CD25−Foxp3− Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells. PMID:26885861

  15. Multipronged functional proteomics approaches for global identification of altered cell signalling pathways in B-cell chronic lymphocytic leukaemia.

    Science.gov (United States)

    Díez, Paula; Lorenzo, Seila; Dégano, Rosa M; Ibarrola, Nieves; González-González, María; Nieto, Wendy; Almeida, Julia; González, Marcos; Orfao, Alberto; Fuentes, Manuel

    2016-04-01

    Chronic lymphocytic leukaemia (CLL) is a malignant B cell disorder characterized by its high heterogeneity. Although genomic alterations have been broadly reported, protein studies are still in their early stages. Herein, a 224-antibody microarray has been employed to study the intracellular signalling pathways in a cohort of 14 newly diagnosed B-CLL patients as a preliminary study for further investigations. Several protein profiles were differentially identified across the cytogenetic and molecular alterations presented in the samples (deletion 13q14 and 17p13.1, trisomy 12, and NOTCH1 mutations) by a combination of affinity and MS/MS proteomics approaches. Among others altered cell signalling pathways, PKC family members were identified as down-regulated in nearly 75% of the samples tested with the antibody arrays. This might explain the rapid progression of the disease when showing p53, Rb1, or NOTCH1 mutations due to PKC-proteins family plays a critical role favouring the slowly progressive indolent behaviour of CLL. Additionally, the antibody microarray results were validated by a LC-MS/MS quantification strategy and compared to a transcriptomic CLL database. In summary, this research displays the usefulness of proteomic strategies to globally evaluate the protein alterations in CLL cells and select the possible biomarkers to be further studied with larger sample sizes.

  16. Immuno and affinity cytochemical analysis of cell wall composition in the moss Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Berry

    2016-03-01

    Full Text Available In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalacturonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogeneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.

  17. CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM BHARUCHAE F. MINOR (OEDOGONIALES, CHLOROPHYTA)(1).

    Science.gov (United States)

    Estevez, José M; Leonardi, Patricia I; Alberghina, Josefina S

    2008-10-01

    Cell wall changes in vegetative and suffultory cells (SCs) and in oogonial structures from Oedogonium bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several carbohydrate epitopes. Vegetative cells and SCs develop only a primary cell wall (PCW), whereas mature oogonial cells secrete a second wall, the oogonium cell wall (OCW). Based on histochemical and immunolabeling results, (1→4)-β-glucans in the form of crystalline cellulose together with a variable degree of Me-esterified homogalacturonans (HGs) and hydroxyproline-rich glycoprotein (HRGP) epitopes were detected in the PCW. The OCW showed arabinosides of the extensin type and low levels of arabinogalactan-protein (AGP) glycans but lacked cellulose, at least in its crystalline form. Surprisingly, strong colabeling in the cytoplasm of mature oogonia cells with three different antibodies (LM-5, LM-6, and CCRC-M2) was found, suggesting the presence of rhamnogalacturonan I (RG-I)-like structures. Our results are discussed relating the possible functions of these cell wall epitopes with polysaccharides and O-glycoproteins during oogonium differentiation. This study represents the first attempt to characterize these two types of cell walls in O. bharuchae, comparing their similarities and differences with those from other green algae and land plants. This work represents a contribution to the understanding of how cell walls have evolved from simple few-celled to complex multicelled organisms.

  18. Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens.

    Science.gov (United States)

    Berry, Elizabeth A; Tran, Mai L; Dimos, Christos S; Budziszek, Michael J; Scavuzzo-Duggan, Tess R; Roberts, Alison W

    2016-01-01

    In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.

  19. The Single Cell Proteome Project - Cell-Cycle Dependent Protein Expression in Breast Cancer Cell Lines

    Science.gov (United States)

    2005-01-01

    sequencing or hybridization array capillary chromatography. After a 6-min-long preliminary technologies.30,31 separation, fractions from the first...characterize single cells. These tools include mass cating cells contain diploid, S-phase and tetraploid frac- spectrometry, electrochemistry and capillary...separation tions; and some advanced tumors contain tetraploid and methods. This review focuses on the use of capillary aneuploid cells [2

  20. Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

    Science.gov (United States)

    Ruiz-Baca, Estela; Hernández-Mendoza, Gustavo; Cuéllar-Cruz, Mayra; Toriello, Conchita; López-Romero, Everardo; Gutiérrez-Sánchez, Gerardo

    2014-07-01

    The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

  1. CONSTITUTIVE MELANIN IN THE CELL WALL OF THE ETIOLOGIC AGENT OF LOBO'S DISEASE

    Directory of Open Access Journals (Sweden)

    TABORDA Valeria B.A.

    1999-01-01

    Full Text Available Lobo's disease is a chronic granulomatous disease caused by the obligate pathogenic fungus, whose cell walls contain constitutive melanin. In contrast, melanin does not occur in the cell walls of Paracoccidioides brasiliensis when stained by the Fontana-Masson stain.

  2. Modification of cell wall architecture of wheat coleoptiles grown under hypergravity conditions.

    Science.gov (United States)

    Wakabayashi, Kazuyuki; Soga, Kouichi; Kamisaka, Seiichiro; Hoson, Takayuki

    2003-10-01

    Cell wall structure of wheat coleoptiles grown under continuous hypergravity (300 g) conditions was investigated. Length of coleoptiles exposed to hypergravity for 2-4 days from germination stage was 60-70% of that of 1 g control. The amounts of cell wall polysaccharides substantially increased during the incubation period both in 1 g control and hypergravity-treated coleoptiles. As a results, the levels of cell wall polysaccharides per unit length of coleoptile, which mean the thickness of cell walls, largely increased under hypergravity conditions. The major sugar components of the hemicellulose fraction, a polymer fraction extracted from cell walls with strong alkali, were arabinose (Ara), xylose (Xyl) and glucose (Glc). The molar ratios of Ara and Xyl to Glc in hypergravity-treated coleoptiles were higher than those in control coleoptiles. Furthermore, the fractionation of hemicellulosic polymers into the neutral and acidic polymers by the anion-exchange column showed that the levels of acidic polymers in cell walls of hypergravity-treated coleoptiles were higher than those of control coleoptiles. These results suggest that hypergravity stimuli bias the synthesis of hemicellulosic polysaccharides and increase the proportion of acidic polymers, such as arabinoxylans, in cell walls of wheat coleoptiles. These structural changes in cell walls may contribute to plant resistance to hypergravity stimuli.

  3. Cell wall composition as a maize defense mechanism against corn borers.

    Science.gov (United States)

    Barros-Rios, Jaime; Malvar, Rosa A; Jung, Hans-Joachim G; Santiago, Rogelio

    2011-04-01

    European and Mediterranean corn borers are two of the most economically important insect pests of maize (Zea mays L.) in North America and southern Europe, respectively. Cell wall structure and composition were evaluated in pith and rind tissues of resistant and susceptible inbred lines as possible corn borer resistance traits. Composition of cell wall polysaccharides, lignin concentration and composition, and cell wall bound forms of hydroxycinnamic acids were measured. As expected, most of the cell wall components were found at higher concentrations in the rind than in the pith tissues, with the exception of galactose and total diferulate esters. Pith of resistant inbred lines had significantly higher concentrations of total cell wall material than susceptible inbred lines, indicating that the thickness of cell walls could be the initial barrier against corn borer larvae attack. Higher concentrations of cell wall xylose and 8-O-4-coupled diferulate were found in resistant inbreds. Stem tunneling by corn borers was negatively correlated with concentrations of total diferulates, 8-5-diferulate and p-coumarate esters. Higher total cell wall, xylose, and 8-coupled diferulates concentrations appear to be possible mechanisms of corn borer resistance.

  4. In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

    NARCIS (Netherlands)

    Muller, K.; Linkies, A.; Vreeburg, R.A.M.; Fry, S.C.; Krieger-Liszkay, A.; Leubner-Metzger, G.

    2009-01-01

    Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (·OH)-mediated cell wall loosening during plant seed germination and se

  5. Cell wall growth during elongation and division : one ring to bind them?

    NARCIS (Netherlands)

    Scheffers, Dirk-Jan

    2007-01-01

    The role of the cell division protein FtsZ in bacterial cell wall (CW) synthesis is believed to be restricted to localizing proteins involved in the synthesis of the septal wall. Elsewhere, compelling evidence is provided that in Caulobacter crescentus, FtsZ plays an additional role in CW synthesis

  6. Proteomic Analysis of Anti-Tumor Effects of 11-Dehydrosinulariolide on CAL-27 Cells

    Directory of Open Access Journals (Sweden)

    Yu-Jen Wu

    2011-07-01

    Full Text Available The anti-tumor effects of 11-dehydrosinulariolide, an active ingredient isolated from soft coral Sinularia leptoclados, on CAL-27 cells were investigated in this study. In the MTT assay for cell proliferation, increasing concentrations of 11-dehydrosinulariolide decreased CAL-27 cell viability. When a concentration of 1.5 µg/mL of 11-dehydrosinulariolide was applied, the CAL-27 cells viability was reduced to a level of 70% of the control sample. The wound healing function decreased as the concentration of 11-dehydrosinulariolide increased. The results in this study indicated that treatment with 11-dehydrosinulariolide for 6 h significantly induced both early and late apoptosis of CAL-27 cells, observed by flow cytometric measurement and microscopic fluorescent observation. A comparative proteomic analysis was conducted to investigate the effects of 11-dehydrosinulariolide on CAL-27 cells at the molecular level by comparison between the protein profiling (revealed on a 2-DE map of CAL-27 cells treated with 11-dehydrosinulariolide and that of CAL-27 cells without the treatment. A total of 28 differential proteins (12 up-regulated and 16 down-regulated in CAL-27 cells treated with 11-dehydrosinulariolide have been identified by LC-MS/MS analysis. Some of the differential proteins are associated with cell proliferation, apoptosis, protein synthesis, protein folding, and energy metabolism. The results of this study provided clues for the investigation of biochemical mechanisms of the anti-tumor effects of 11-dehydrosinulariolide on CAL-27 cells and could be valuable information for drug development and progression monitoring of oral squamous cell carcinoma (OSCC.

  7. Proteomics-based systems biology modeling of bovine germinal vesicle stage oocyte and cumulus cell interaction.

    Directory of Open Access Journals (Sweden)

    Divyaswetha Peddinti

    Full Text Available BACKGROUND: Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at germinal vesicle (GV stage are considered essential for proper maturation or 'programming' of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication. METHODOLOGY/PRINCIPAL FINDINGS: We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation. CONCLUSIONS/SIGNIFICANCE: Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level.

  8. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

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    Stanton Peter G

    2011-05-01

    Full Text Available Abstract Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL 11 regulates human endometrial epithelial cells (hEEC adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2 and flotillin-1 (FLOT1, were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle. Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary h

  9. A targeted quantitative proteomics strategy for global kinome profiling of cancer cells and tissues.

    Science.gov (United States)

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2014-04-01

    Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anti-cancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here, we introduced a novel global kinome profiling method, based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity, reproducibility, and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally, the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines, which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore, this facile and accurate kinome profiling assay, together with the kinome-inhibitor interaction map, could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy.

  10. Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides.

    Science.gov (United States)

    Ishii, T; Thomas, J; Darvill, A; Albersheim, P

    1989-02-01

    Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-alpha-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-alpha-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na(2)CO(3) at 1 and 22 degrees C. These previously uncharacterized polysaccharides accounted for approximately 4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO(3)-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na(2)CO(3) at 1 degrees C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.

  11. Shotgun proteomics and network analysis between plasma membrane and extracellular matrix proteins from rat olfactory ensheathing cells.

    Science.gov (United States)

    Liu, Yisong; Teng, Xiaohua; Yang, Xiaoxu; Song, Qing; Lu, Rong; Xiong, Jixian; Liu, Bo; Zeng, Nianju; Zeng, Yu; Long, Jia; Cao, Rui; Lin, Yong; He, Quanze; Chen, Ping; Lu, Ming; Liang, Songping

    2010-01-01

    Olfactory ensheathing cells (OECs) are a special type of glial cells that have characteristics of both astrocytes and Schwann cells. Evidence suggests that the regenerative capacity of OECs is induced by soluble, secreted factors that influence their microenvironment. These factors may regulate OECs self-renewal and/or induce their capacity to augment spinal cord regeneration. Profiling of plasma membrane and extracellular matrix through a high-throughput expression proteomics approach was undertaken to identify plasma membrane and extracellular matrix proteins of OECs under serum-free conditions. 1D-shotgun proteomics followed with gene ontology (GO) analysis was used to screen proteins from primary culture rat OECs. Four hundred and seventy nonredundant plasma membrane proteins and 168 extracellular matrix proteins were identified, the majority of which were never before reported to be produced by OECs. Furthermore, plasma membrane and extracellular proteins were classified based on their protein-protein interaction predicted by STRING quantitatively integrates interaction data. The proteomic profiling of the OECs plasma membrane proteins and their connection with the secretome in serum-free culture conditions provides new insights into the nature of their in vivo microenvironmental niche. Proteomic analysis for the discovery of clinical biomarkers of OECs mechanism warrants further study.

  12. Proteome Analysis of Human Follicular Thyroid Cancer Cells Exposed to the Random Positioning Machine.

    Science.gov (United States)

    Bauer, Johann; Kopp, Sascha; Schlagberger, Elisabeth Maria; Grosse, Jirka; Sahana, Jayashree; Riwaldt, Stefan; Wehland, Markus; Luetzenberg, Ronald; Infanger, Manfred; Grimm, Daniela

    2017-03-03

    Several years ago, we detected the formation of multicellular spheroids in experiments with human thyroid cancer cells cultured on the Random Positioning Machine (RPM), a ground-based model to simulate microgravity by continuously changing the orientation of samples. Since then, we have studied cellular mechanisms triggering the cells to leave a monolayer and aggregate to spheroids. Our work focused on spheroid-related changes in gene expression patterns, in protein concentrations, and in factors secreted to the culture supernatant during the period when growth is altered. We detected that factors inducing angiogenesis, the composition of integrins, the density of the cell monolayer exposed to microgravity, the enhanced production of caveolin-1, and the nuclear factor kappa B p65 could play a role during spheroid formation in thyroid cancer cells. In this study, we performed a deep proteome analysis on FTC-133 thyroid cancer cells cultured under conditions designed to encourage or discourage spheroid formation. The experiments revealed more than 5900 proteins. Their evaluation confirmed and explained the observations mentioned above. In addition, we learned that FTC-133 cells growing in monolayers or in spheroids after RPM-exposure incorporate vinculin, paxillin, focal adhesion kinase 1, and adenine diphosphate (ADP)-ribosylation factor 6 in different ways into the focal adhesion complex.

  13. Quantitative proteome profiling of respiratory virus-infected lung epithelial cells.

    Science.gov (United States)

    van Diepen, Angela; Brand, H Kim; Sama, Iziah; Lambooy, Lambert H J; van den Heuvel, Lambert P; van der Well, Leontine; Huynen, Martijn; Osterhaus, Albert D M E; Andeweg, Arno C; Hermans, Peter W M

    2010-08-05

    Respiratory virus infections are among the primary causes of morbidity and mortality in humans. Influenza virus, respiratory syncytial virus (RSV), parainfluenza (PIV) and human metapneumovirus (hMPV) are major causes of respiratory illness in humans. Especially young children and the elderly are susceptible to infections with these viruses. In this study we aim to gain detailed insight into the molecular pathogenesis of respiratory virus infections by studying the protein expression profiles of infected lung epithelial cells. A549 cells were exposed to a set of respiratory viruses [RSV, hMPV, PIV and Measles virus (MV)] using both live and UV-inactivated virus preparations. Cells were harvested at different time points after infection and processed for proteomics analysis by 2-dimensional difference gel electrophoresis. Samples derived from infected cells were compared to mock-infected cells to identify proteins that are differentially expressed due to infection. We show that RSV, hMPV, PIV3, and MV induced similar core host responses and that mainly proteins involved in defense against ER stress and apoptosis were affected which points towards an induction of apoptosis upon infection. By 2-D DIGE analyses we have gathered information on the induction of apoptosis by respiratory viruses in A549 cells.

  14. Proteome characterization of sea star coelomocytes--the innate immune effector cells of echinoderms.

    Science.gov (United States)

    Franco, Catarina F; Santos, Romana; Coelho, Ana V

    2011-09-01

    Sea star coelomic fluid is in contact with all internal organs, carrying signaling molecules and a large population of circulating cells, the coelomocytes. These cells, also known as echinoderm blood cells, are responsible for the innate immune responses and are also known to have an important role in the first stage of regeneration, i.e. wound closure, necessary to prevent disruption of the body fluid balance and to limit the invasion of pathogens. This study focuses on the proteome characterization of these multifunctional cells. The identification of 358 proteins was achieved using a combination of two techniques for protein separation (1-D SDS-PAGE followed by nanoLC and 2-D SDS-PAGE) and MALDI-TOF/TOF MS for protein identification. To our knowledge, the present report represents the first comprehensive list of sea star coelomocyte proteins, constituting an important database to validate many echinoderm-predicted proteins. Evidence for new pathways in these particular echinoderm cells are also described, and thus representing a valuable resource to stimulate future studies aiming to unravel the homology with vertebrate immune cells and particularly the origins of the immune system itself.

  15. Proteomic exploration of the impacts of pomegranate fruit juice on the global gene expression of prostate cancer cell.

    Science.gov (United States)

    Lee, Song-Tay; Wu, Yi-Ling; Chien, Lan-Hsiang; Chen, Szu-Ting; Tzeng, Yu-Kai; Wu, Ting-Feng

    2012-11-01

    Prostate cancer has been known to be the second highest cause of death in cancer among men. Pomegranate is rich in polyphenols with the potent antioxidant activity and inhibits cell proliferation, invasion, and promotes apoptosis in various cancer cells. This study demonstrated that pomegranate fruit juice could effectively hinder the proliferation of human prostate cancer DU145 cell. The results of apoptotic analyses implicated that fruit juice might trigger the apoptosis in DU145 cells via death receptor signaling and mitochondrial damage pathway. In this study, we exploited 2DE-based proteomics to compare nine pairs of the proteome maps collected from untreated and treated DU145 cells to identify the differentially expressed proteins. Comparative proteomics indicated that 11 proteins were deregulated in affected DU145 cells with three upregulated and eight downregulated proteins. These dys-regulated proteins participated in cytoskeletal functions, antiapoptosis, proteasome activity, NF-κB signaling, cancer cell proliferation, invasion, and angiogenesis. Western immunoblotting were implemented to confirm the deregulated proteins and the downstream signaling proteins. The analytical results of this study help to provide insight into the molecular mechanism of inducing prostate cancer cell apoptosis by pomegranate fruit juice and to develop a novel mechanism-based chemopreventive strategy for prostate cancer.

  16. Clinostation influence on regeneration of cell wall in Solanum Tuberosum L. protoplasts

    Science.gov (United States)

    Nedukha, Elena M.; Sidorov, V. A.; Samoylov, V. M.

    1994-08-01

    Regeneration of cell walls in protoplasts was investigated using light- and electronmicroscopic methods. The protoplasts were isolated from mesophyll of Solanum tuberosum leaves and were cultivated on the horizontal low rotating clinostat (2 rpm) and in control for 10 days. Using a fluorescent method (with Calcofluor white) it was demonstrated that changes in vector gravity results in an regeneration inhibition of cell wall. With electron-microscopical and electro-cytochemical methods (staining with alcianum blue) dynamics of the regeneration of cell walls in protoplasts was studied; carbohydrate matrix of cell walls is deposited at the earliest stages of this process. The influence of microgravity on the cell wall regeneration is discussed in higher plants.

  17. Interactions between grape skin cell wall material and commercial enological tannins. Practical implications.

    Science.gov (United States)

    Bautista-Ortín, Ana Belén; Cano-Lechuga, Mario; Ruiz-García, Yolanda; Gómez-Plaza, Encarna

    2014-01-01

    Commercial enological tannins were used to investigate the role that cell wall material plays in proanthocyanidin adsorption. Insoluble cell wall material, prepared from the skin of Vitis vinifera L. cv. Monastrell berries, was combined with solutions containing six different commercial enological tannins (proanthocyanidin-type tannins). Analysis of the proanthocyanidins in the solution, after fining with cell wall material, using phloroglucinolysis and size exclusion chromatography, provided quantitative and qualitative information on the non-adsorbed compounds. Cell wall material showed strong affinity for the proanthocyanidins, one of the commercial tannins being bound up to 61% in the experiment. Comparison of the molecular mass distribution of the commercial enological tannins in solution, before and after fining, suggested that cell walls affinity for proanthocyanidins was more related with the proanthocyanidin molecular mass than with their percentage of galloylation. These interactions may have some enological implications, especially as regards the time of commercial tannins addition to the must/wine.

  18. Generation of hydroxyl radical in isolated pea root cell wall, and the role of cell wall-bound peroxidase, Mn-SOD and phenolics in their production.

    Science.gov (United States)

    Kukavica, Biljana; Mojovic, Milos; Vuccinic, Zeljko; Maksimovic, Vuk; Takahama, Umeo; Jovanovic, Sonja Veljovic

    2009-02-01

    The hydroxyl radical produced in the apoplast has been demonstrated to facilitate cell wall loosening during cell elongation. Cell wall-bound peroxidases (PODs) have been implicated in hydroxyl radical formation. For this mechanism, the apoplast or cell walls should contain the electron donors for (i) H(2)O(2) formation from dioxygen; and (ii) the POD-catalyzed reduction of H(2)O(2) to the hydroxyl radical. The aim of the work was to identify the electron donors in these reactions. In this report, hydroxyl radical (.OH) generation in the cell wall isolated from pea roots was detected in the absence of any exogenous reductants, suggesting that the plant cell wall possesses the capacity to generate .OH in situ. Distinct POD and Mn-superoxide dismutase (Mn-SOD) isoforms different from other cellular isoforms were shown by native gel electropho-resis to be preferably bound to the cell walls. Electron paramagnetic resonance (EPR) spectroscopy of cell wall isolates containing the spin-trapping reagent, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used for detection of and differentiation between .OH and the superoxide radical (O(2)(-).). The data obtained using POD inhibitors confirmed that tightly bound cell wall PODs are involved in DEPMPO/OH adduct formation. A decrease in DEPMPO/OH adduct formation in the presence of H(2)O(2) scavengers demonstrated that this hydroxyl radical was derived from H(2)O(2). During the generation of .OH, the concentration of quinhydrone structures (as detected by EPR spectroscopy) increased, suggesting that the H(2)O(2) required for the formation of .OH in isolated cell walls is produced during the reduction of O(2) by hydroxycinnamic acids. Cell wall isolates in which the proteins have been denaturated (including the endogenous POD and SOD) did not produce .OH. Addition of exogenous H(2)O(2) again induced the production of .OH, and these were shown to originate from the Fenton reaction with tightly bound metal ions

  19. The Cell Wall Lipid PDIM Contributes to Phagosomal Escape and Host Cell Exit of Mycobacterium tuberculosis

    Science.gov (United States)

    Quigley, Jeff; Hughitt, V. Keith; Velikovsky, Carlos A.; Mariuzza, Roy A.

    2017-01-01

    ABSTRACT The cell wall of Mycobacterium tuberculosis is composed of unique lipids that are important for pathogenesis. Indeed, the first-ever genetic screen in M. tuberculosis identified genes involved in the biosynthesis and transport of the cell wall lipid PDIM (phthiocerol dimycocerosates) as crucial for the survival of M. tuberculosis in mice. Here we show evidence for a novel molecular mechanism of the PDIM-mediated virulence in M. tuberculosis. We characterized the DNA interaction and the regulon of Rv3167c, a transcriptional repressor that is involved in virulence regulation of M. tuberculosis, and discovered that it controls the PDIM operon. A loss-of-function genetic approach showed that PDIM levels directly correlate with the capacity of M. tuberculosis to escape the phagosome and induce host cell necrosis and macroautophagy. In conclusion, our study attributes a novel role of the cell wall lipid PDIM in intracellular host cell modulation, which is important for host cell exit and dissemination of M. tuberculosis. PMID:28270579

  20. Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens

    Directory of Open Access Journals (Sweden)

    Nathan T Reem

    2016-05-01

    Full Text Available The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity and function remains unclear. Modifications of cell wall composition can induce plant responses known as Cell Wall Integrity control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, increased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant cell wall integrity, which contributes to plant resistance to necrotrophic pathogens.

  1. Comprehensive quantitative comparison of the membrane proteome and PTM-ome of human embryonic stem cells and neural stem cells

    DEFF Research Database (Denmark)

    Braga, Marcella Nunes de Melo; Schulz, Melanie; Jakobsen, Lene

    Introduction: Human embryonic stem cells (hESCs) can differentiate into all three germ layers and self-renew. Due to its ability to differentiate in vitro into human neural stem cells (hNSCs), which can further be differentiated into motor neurons and dopaminergic neurons, these cells are potential...... source for treatment of neurological diseases such as Parkinson´s disease. Membrane proteins are very important in cellular signaling and they are regulated by post-translational modifications such as phosphorylation and glycosylation. In order to obtain more information about important membrane proteins...... and modification sites involved in the differentiation of hESCs to hNSCs and also investigate potential new markers for two stages, we have performed a comprehensive mass-spectrometry-based quantitative proteomics and PTMomics study. Methods: The hESC and hNSC were subject to Na2CO3 and ultracentrifugation...

  2. Immunoprofiling reveals unique cell-specific patterns of wall epitopes in the expanding Arabidopsis stem.

    Science.gov (United States)

    Hall, Hardy C; Cheung, Jingling; Ellis, Brian E

    2013-04-01

    The Arabidopsis inflorescence stem undergoes rapid directional growth, requiring massive axial cell-wall extension in all its tissues, but, at maturity, these tissues are composed of cell types that exhibit markedly different cell-wall structures. It is not clear whether the cell-wall compositions of these cell types diverge rapidly following axial growth cessation, or whether compositional divergence occurs at earlier stages in differentiation, despite the common requirement for cell-wall extensibility. To examine this question, seven cell types were assayed for the abundance and distribution of 18 major cell-wall glycan classes at three developmental stages along the developing inflorescence stem, using a high-throughput immunolabelling strategy. These stages represent a phase of juvenile growth, a phase displaying the maximum rate of stem extension, and a phase in which extension growth is ceasing. The immunolabelling patterns detected demonstrate that the cell-wall composition of most stem tissues undergoes pronounced changes both during and after rapid extension growth. Hierarchical clustering of the immunolabelling signals identified cell-specific binding patterns for some antibodies, including a sub-group of arabinogalactan side chain-directed antibodies whose epitope targets are specifically associated with the inter-fascicular fibre region during the rapid cell expansion phase. The data reveal dynamic, cell type-specific changes in cell-wall chemistry across diverse cell types during cell-wall expansion and maturation in the Arabidopsis inflorescence stem, and highlight the paradox between this structural diversity and the uniform anisotropic cell expansion taking place across all tissues during stem growth.

  3. [Heterocysts with reduced cell walls in populations of cycad cyanobionts].

    Science.gov (United States)

    Baulina, O I; Lobakova, E S

    2003-01-01

    The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycads circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. In addition to heterocysts with the typical ultrastructure, the cyanobiont microcolonies also contained altered heterocysts with reduced cell walls, which might dominate in all regions of the coralloid roots. The altered heterocysts represented a protoplast enclosed in a heterocyst-specific envelope with additional layers. Some heterocysts contained an additional reticular protoplast-enclosing sheath below the heterocyst-specific envelope, whereas the other heterocysts contained an additional electron-opaque outer layer. The substance of the inner sheath of the former heterocysts resembled the polysaccharides of mucilage, which fills the intercellular space of plant tissues, whereas the electron-opaque outer layer of the latter heterocysts probably had a protein nature. The substances that constitute the sheath and the outer layer are likely to be synthesized intracellularly and then released with the aid of membrane-bounded vesicles or by channels in the cytoplasmic membrane.

  4. Modifications of Saccharomyces pastorianus cell wall polysaccharides with brewing process.

    Science.gov (United States)

    Bastos, Rita; Coelho, Elisabete; Coimbra, Manuel A

    2015-06-25

    The cell wall polysaccharides of brewers spent yeast Saccharomyces pastorianus (BSY) and the inoculum yeast (IY) were studied in order to understand the changes induced by the brewing process. The hot water and alkali extractions performed solubilized mainly mannoproteins, more branched for BSY than those of IY. Also, (31)P solid state NMR showed that the BSY mannoproteins were 3 times more phosphorylated. By electron microscopy it was observed that the final residues of alkali sequential extraction until 4M KOH preserved the yeast three-dimensional structure. The final residues, composed mainly by glucans (92%), showed that the BSY, when compared with IY, contained higher amount of (1→4)-linked Glc (43% for BSY and 16% for IY) and lower (1→3)-linked Glc (17% for BSY and 42% for IY). The enzymatic treatment of final residue showed that both BSY and IY had (α1→4)-linked Glc and (β1→4)-linked Glc, in a 2:1 ratio, showing that S. pastorianus increases their cellulose-like linkages with the brewing process.

  5. Proteomic shifts in embryonic stem cells with gene dose modifications suggest the presence of balancer proteins in protein regulatory networks.

    Directory of Open Access Journals (Sweden)

    Lei Mao

    Full Text Available Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of "balancer" proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the "elasticity" of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions.

  6. Proteomic analysis of an Aedes albopictus cell line infected with Dengue serotypes 1 and 3 viruses

    Directory of Open Access Journals (Sweden)

    Thomas Frédéric

    2011-07-01

    Full Text Available Abstract Background Proteomic analysis was performed to identify proteins regulated during infection by Dengue serotypes 1 and 3 in an Aedes albopictus cell line. The potential of these viruses to cause severe disease at primary infection is of interest although few studies have been performed with these two Dengue serotypes. Results The most relevant observation of our study is the significant overexpression of proteins involved in the cellular stress response and the glycolysis pathway after 48 hours of infection. Viral infection activates the translation of some host genes, which may result in stress due to responses involving unfolded proteins. Conclusions Therefore, the oxidation reduction and glycolytic mechanisms could participate in the antiviral response against Dengue virus. The results of our study should help to improve our knowledge of the virus-mosquito interaction at a cellular level with the aim of designing efficient strategies for the control of Dengue virus.

  7. Proteome Changes of Human Bone Marrow Mesenchymal Stem Cells Induced by 1,4-Benzoquinone

    Science.gov (United States)

    2016-01-01

    Benzene is metabolized to hydroquinone in liver and subsequently transported to bone marrow for further oxidization to 1,4-benzoquinone (1,4-BQ), which may be related to the leukemia and other blood disorders. In the present study, we investigated the proteome profiles of human primary bone marrow mesenchymal stem cells (hBM-MSCs) treated by 1,4-BQ. We identified 32 proteins that were differentially expressed. Two of them, HSP27 and Vimentin, were verified at both mRNA and protein levels and their cellular localization was examined by immunofluorescence. We also found increased mRNA level of RAP1GDS1, a critical factor of metabolism that has been identified as a fusion partner in various hematopoietic malignancies. Therefore, these differentially expressed proteins can play important roles in benzene-mediated hematoxicity. PMID:28119923

  8. Proteomics reveals the importance of the dynamic redistribution of the subcellular location of proteins in breast cancer cells.

    Science.gov (United States)

    Pinto, Gabriella; Alhaiek, Abdulrab Ahmed M; Godovac-Zimmermann, Jasminka

    2015-02-01

    At the molecular level, living cells are enormously complicated complex adaptive systems in which intertwined genomic, transcriptomic, proteomic and metabolic networks all play a crucial role. At the same time, cells are spatially heterogeneous systems in which subcellular compartmentalization of different functions is ubiquitous and requires efficient cross-compartmental communication. Dynamic redistribution of multitudinous proteins to different subcellular locations in response to cellular functional state is increasingly recognized as a crucial characteristic of cellular function that seems to be at least as important as overall changes in protein abundance. Characterization of the subcellular spatial dynamics of protein distribution is a major challenge for proteomics and recent results with MCF7 breast cancer cells suggest that this may be of particular importance for cancer cells.

  9. Comparative proteomic analysis of normal and tumor stromal cells by tissue on chip based mass spectrometry (toc-MS

    Directory of Open Access Journals (Sweden)

    Friedrich Karlheinz

    2010-01-01

    Full Text Available Abstract In carcinoma tissues, genetic and metabolic changes not only occur at the tumor cell level, but also in the surrounding stroma. This carcinoma-reactive stromal tissue is heterogeneous and consists e.g. of non-epithelial cells such as fibroblasts or fibrocytes, inflammatory cells and vasculature-related cells, which promote carcinoma growth and progression of carcinomas. Nevertheless, there is just little knowledge about the proteomic changes from normal connective tissue to tumor stroma. In the present study, we acquired and analysed specific protein patterns of small stromal sections surrounding head and neck cell complexes in comparison to normal subepithelial connective tissue. To gain defined stromal areas we used laser-based tissue microdissection. Because these stromal areas are limited in size we established the highly sensitive 'tissue on chip based mass spectrometry' (toc-MS. Therefore, the dissected areas were directly transferred to chromatographic arrays and the proteomic profiles were subsequently analysed with mass spectrometry. At least 100 cells were needed for an adequate spectrum. The locating of differentially expressed proteins enables a precise separation of normal and tumor stroma. The newly described toc-MS technology allows an initial insight into proteomic differences between small numbers of exactly defined cells from normal and tumor stroma.

  10. Serologic response to cell wall mannoproteins and proteins of Candida albicans.

    Science.gov (United States)

    Martínez, J P; Gil, M L; López-Ribot, J L; Chaffin, W L

    1998-01-01

    The cell wall of Candida albicans not only is the structure in which many biological functions essential for the fungal cells reside but also is a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both the carbohydrate and protein moieties are able to trigger immune responses. Although cell-mediated immunity is often considered to be the most important line of defense against candidiasis, cell wall protein and glycoprotein components also elicit a potent humoral response from the host that may include some protective antibodies. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to influence profoundly the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins for host ligands. In this review, the various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo are examined. Although a number of proteins have been shown to stimulate an antibody response, for some of these species the response is not universal. On the other hand, some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidasis, particularly the disseminated form. In addition, recent studies have focused on the potential for antibodies to cell wall protein determinants to protect the host against infection. Hence, a better understanding of the humoral response to cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures

  11. Proteomic analysis of rice endosperm cells in response to expression of hGM-CSF.

    Science.gov (United States)

    Luo, Junling; Ning, Tingting; Sun, Yunfang; Zhu, Jinghua; Zhu, Yingguo; Lin, Qishan; Yang, Daichang

    2009-02-01

    The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm.

  12. Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies.

    Science.gov (United States)

    Cheng, Hsien-Jen; Lin, Chiou-Feng; Lei, Huan-Yao; Liu, Hsiao-Sheng; Yeh, Trai-Ming; Luo, Yueh-Hsia; Lin, Yee-Shin

    2009-01-01

    We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope.

  13. Proteomic analysis of the effect of iptakalim on human pulmonary arterial smooth muscle cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Mingxia YANG; Zhengxia LIU; Shu ZHANG; Yu JING; Shijiang ZHANG; Weiping XIE; Lei MA; Changliang ZHU; Hong WANG

    2009-01-01

    Aim:To investigate the anti-proliferative effect of iptakalim (Ipt),a newly selective KATP channel opener,in endothelin-1 (ET-1)-induced human pulmonary arterial smooth muscle cells (PASMCs) using proteomic analysis.Methods: Human PASMCs were incubated with ET-1 (10-8 mol/L) and ETA (10-8 mol/L) plus iptaklim (10-5 mol/L) for 24 h.Analysis via 2-DE gel electrophoresis and MALDI-TOF-MS was employed to display the different protein profiles of whole-cell protein from cultures of control,ET-1 treatment alone,and treatment with ET-1 and iptaklim combined.Real time RT-PCR and Western blot analysis were used to confirm the proteomic analysis.Results: When iptakalim inhibited the proliferative effect of ET-1 in human PASMCs by opening the KATP channels,the expression of different groups of cellular proteins was changed,including cytoskeleton-associated proteins,plasma mem-brane proteins and receptors,chaperone proteins,ion transport-associated proteins,and glycolytic and metabolism-associ-ated proteins.We found that iptakalim could inhibit the proliferation of human PASMCs partly by affecting the expression of Hsp60,vimentin,nucleoporin P54 (NUP54) and Bcl-XL by opening the KATP channel.Conclusion: The data suggest that a wide range of signaling pathways may be involved in abolishing ET-1-induced prolif-eration of human PASMCs following iptakalim treatment.

  14. Proteomic analysis of human Sonic Hedgehog (SHH) medulloblastoma stem-like cells.

    Science.gov (United States)

    Ronci, Maurizio; Catanzaro, Giuseppina; Pieroni, Luisa; Po, Agnese; Besharat, Zein Mersini; Greco, Viviana; Levi Mortera, Stefano; Screpanti, Isabella; Ferretti, Elisabetta; Urbani, Andrea

    2015-06-01

    Human medulloblastoma (MB) is a malignant brain tumor that comprises four distinct molecular subgroups including the Sonic Hedgehog (SHH)-MB group. A leading cause of the SHH subgroup is an aberrant activation of the SHH pathway, a developmental signaling that regulates postnatal development of the cerebellum by promoting the mitotic expansion of granule neural precursors (GNPs) in the external granule layer (EGL). The abnormal SHH signaling pathway drives not only SHH-MB but also its cancer stem-like cells (SLCs), which represent a fraction of the tumor cell population that maintain cancer growth and have been associated with high grade tumors. Here, we report the first proteomic analysis of human SHH-MB SLCs before and after Retinoic Acid (RA)-induced differentiation. A total of 994 nLC-MS buckets were statistically analysed returning 68 modulated proteins between SLCs and their differentiated counterparts. Heat Shock Protein 70 (Hsp70) was one of the proteins that characterized the protein profile of SLCs. By means of Ingenuity Pathway Analysis (IPA), Genomatix analysis and extending the network obtained using the differentially expressed proteins we found a correlation between Hsp70 and the NF-κB complex. A key driver of the SHH-MB group is cMET whose downstream proliferation/survival signalling is indeed via PI3K/Akt/NF-κB. We confirmed the results of the proteomic analysis by western blot, underlining that a P-p65/NF-κB activatory complex is highly expressed in SLCs. Taking together these results we define a new protein feature of SHH-MB SLCs.

  15. Data for proteomic analysis of murine cardiomyocytic HL-1 cells treated with siRNA against tissue factor

    Directory of Open Access Journals (Sweden)

    Maura Brioschi

    2015-06-01

    Tissue Factor (TF is a key player in the coagulation cascade, but it has additional functions ranging from angiogenesis, tumour invasion and, in the heart, the maintenance of the integrity of cardiac cells. This article reports the nano-LC–MSE analysis of the cardiomyocytic HL-1 cell line proteome and describes the results obtained from a Gene Ontology analysis of those proteins affected by TF-gene silencing.

  16. Combination of hydrogel nanoparticles and proteomics to reveal secreted proteins associated with decidualization of human uterine stromal cells

    Directory of Open Access Journals (Sweden)

    Stephens Andrew N

    2011-09-01

    Full Text Available Abstract Background Identification of secreted proteins of low abundance is often limited by abundant and high molecular weight (MW proteins. We have optimised a procedure to overcome this limitation. Results Low MW proteins in the conditioned media of cultured cells were first captured using dual-size exclusion/affinity hydrogel nanoparticles and their identities were then revealed by proteomics. Conclusions This technique enables the analysis of secreted proteins of cultured cells low MW and low abundance.

  17. Proteomics analysis of human umbilical vein endothelial cells (HUVEC) after treatment with low molecular weight heparin

    Institute of Scientific and Technical Information of China (English)

    YanPAN; Jun-huaWANG; He-mingYU; Xue-junLI

    2004-01-01

    AIM: The endothelium is involved in the generation and the regulation of multiple physiological and pathological processes of blood vessels. Previously we confirmed low molecular weight heparin (LMWH) could inhibit tumor metastasis by protecting human umbilical vein endothelial cells (HUVEC). To understand the effects of LMWH on the protein expression of HUVEC, we performed a comprehensive proteomics to survey global changes in proteins after LMWH treatment in HUVEC cells. METHODS:

  18. Qualitative and quantitative proteomic profiling of cripto(-/-) embryonic stem cells by means of accurate mass LC-MS analysis.

    Science.gov (United States)

    Chambery, Angela; Vissers, Johannes P C; Langridge, James I; Lonardo, Enza; Minchiotti, Gabriella; Ruvo, Menotti; Parente, Augusto

    2009-02-01

    Cripto is one of the key regulators of embryonic stem cells (ESCs) differentiation into cardiomyocites vs neuronal fate. Cripto(-/-) murine ESCs have been utilized to investigate the molecular mechanisms underlying early events of mammalian lineage differentiation. 2D/LC-MS/MS and a label-free LC-MS approaches were used to qualitatively and quantitatively profile the cripto(-/-) ESC proteome, providing an integral view of the alterations induced in stem cell functions by deleting the cripto gene.

  19. Genetic modification of plant cell walls to enhance biomass yield and biofuel production in bioenergy crops.

    Science.gov (United States)

    Wang, Yanting; Fan, Chunfen; Hu, Huizhen; Li, Ying; Sun, Dan; Wang, Youmei; Peng, Liangcai

    2016-01-01

    Plant cell walls represent an enormous biomass resource for the generation of biofuels and chemicals. As lignocellulose property principally determines biomass recalcitrance, the genetic modification of plant cell walls has been posed as a powerful solution. Here, we review recent progress in understanding the effects of distinct cell wall polymers (cellulose, hemicelluloses, lignin, pectin, wall proteins) on the enzymatic digestibility of biomass under various physical and chemical pretreatments in herbaceous grasses, major agronomic crops and fast-growing trees. We also compare the main factors of wall polymer features, including cellulose crystallinity (CrI), hemicellulosic Xyl/Ara ratio, monolignol proportion and uronic acid level. Furthermore, the review presents the main gene candidates, such as CesA, GH9, GH10, GT61, GT43 etc., for potential genetic cell wall modification towards enhancing both biomass yield and enzymatic saccharification in genetic mutants and transgenic plants. Regarding cell wall modification, it proposes a novel groove-like cell wall model that highlights to increase amorphous regions (density and depth) of the native cellulose microfibrils, providing a general strategy for bioenergy crop breeding and biofuel processing technology.

  20. Proteomic analysis as a means to approach limbal stem cell biology in a search for stem cell markers.

    Science.gov (United States)

    Honoré, Bent; Vorum, Henrik

    2014-04-01

    The cornea consists of three main layers: an outer surface epithelium, the stroma, and the endothelium. A clear cornea is necessary for optimal vision and is maintained and repaired from limbal epithelial stem cells located in the limbus between the cornea and the sclera. Diseases and injury may result in deficiency of the stem cells impairing their ability to renew the corneal epithelium. Patients with limbal stem cell deficiency experience chronic pain and ultimately blindness. Attempts to treat the disease are based on replacement of the stem cells by transplantation or by culturing the stem cells. We here review the proteomic techniques that so far have been used to approach characterization of limbal stem cells and markers to identify them. It is apparent that the field is in a rather inchoate state due to the scarcity and relative inaccessibility of the stem cells. However, the importance of revealing limbal stem cell biology and identifying stem cell biomarkers calls for greater use of emerging methodology. Strategies for future studies are discussed.

  1. Changes in Cell Wall Composition during Ripening of Grape Berries1

    Science.gov (United States)

    Nunan, Kylie J.; Sims, Ian M.; Bacic, Antony; Robinson, Simon P.; Fincher, Geoffrey B.

    1998-01-01

    Cell walls were isolated from the mesocarp of grape (Vitis vinifera L.) berries at developmental stages from before veraison through to the final ripe berry. Fluorescence and light microscopy of intact berries revealed no measurable change in cell wall thickness as the mesocarp cells expanded in the ripening fruit. Isolated walls were analyzed for their protein contents and amino acid compositions, and for changes in the composition and solubility of constituent polysaccharides during development. Increases in protein content after veraison were accompanied by an approximate 3-fold increase in hydroxyproline content. The type I arabinogalactan content of the pectic polysaccharides decreased from approximately 20 mol % of total wall polysaccharides to about 4 mol % of wall polysaccharides during berry development. Galacturonan content increased from 26 to 41 mol % of wall polysaccharides, and the galacturonan appeared to become more soluble as ripening progressed. After an initial decrease in the degree of esterification of pectic polysaccharides, no further changes were observed nor were there large variations in cellulose (30–35 mol % of wall polysaccharides) or xyloglucan (approximately 10 mol % of wall polysaccharides) contents. Overall, the results indicate that no major changes in cell wall polysaccharide composition occurred during softening of ripening grape berries, but that significant modification of specific polysaccharide components were observed, together with large changes in protein composition. PMID:9808722

  2. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells

    DEFF Research Database (Denmark)

    Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T;

    2009-01-01

    : Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell......-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human...

  3. SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi.

    Directory of Open Access Journals (Sweden)

    Stéphanie Val

    Full Text Available Chronic Otitis Media with effusion (COME develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi, the most common acute Otitis Media (OM pathogen, is postulated to promote middle ear epithelial remodeling in the progression of OM from acute to chronic. The goals of this study were to examine histopathological and quantitative proteomic epithelial effects of NTHi challenge in a murine middle ear epithelial cell line.NTHi lysates were generated and used to stimulate murine epithelial cells (mMEEC cultured at air-liquid interface over 48 hours- 1 week. Conditional quantitative Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC of cell lysates was performed to interrogate the global protein production in the cells, using the SuperSILAC technique. Histology of the epithelium over time was done to measure bacterial dependent remodeling.Mass spectrometry analysis identified 2,565 proteins across samples, of which 74 exhibited differential enrichment or depletion in cell lysates (+/-2.0 fold-change; p value<0.05. The key molecular functions regulated by NTHi lysates exposure were related to cell proliferation, death, migration, adhesion and inflammation. Finally, chronic exposure induced significant epithelial thickening of cells grown at air liquid interface.NTHi lysates drive pathways responsible of cell remodeling in murine middle ear epithelium which likely contributes to observed epithelial hyperplasia in vitro. Further elucidation of these mediators will be critical in understanding the progression of OM from acute to chronic at the molecular level.

  4. Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells.

    Science.gov (United States)

    Moore, P J; Darvill, A G; Albersheim, P; Staehelin, L A

    1986-11-01

    PLANT CELL WALLS SERVE SEVERAL FUNCTIONS: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.

  5. Lipid Transfer Proteins Enhance Cell Wall Extension in TobaccoW⃞

    Science.gov (United States)

    Nieuwland, Jeroen; Feron, Richard; Huisman, Bastiaan A.H.; Fasolino, Annalisa; Hilbers, Cornelis W.; Derksen, Jan; Mariani, Celestina

    2005-01-01

    Plant cells are enclosed by a rigid cell wall that counteracts the internal osmotic pressure of the vacuole and limits the rate and direction of cell enlargement. When developmental or physiological cues induce cell extension, plant cells increase wall plasticity by a process called loosening. It was demonstrated previously that a class of proteins known as expansins are mediators of wall loosening. Here, we report a type of cell wall–loosening protein that does not share any homology with expansins but is a member of the lipid transfer proteins (LTPs). LTPs are known to bind a large range of lipid molecules to their hydrophobic cavity, and we show here that this cavity is essential for the cell wall–loosening activity of LTP. Furthermore, we show that LTP-enhanced wall extension can be described by a logarithmic time function. We hypothesize that LTP associates with hydrophobic wall compounds, causing nonhydrolytic disruption of the cell wall and subsequently facilitating wall extension. PMID:15937228

  6. Dissecting plasmodesmata molecular composition by mass spectrometry-based proteomics.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Maria Françoise Bayer

    2013-01-01

    Full Text Available In plants, the intercellular communication through the membranous channels called plasmodesmata (PD; singular plasmodesma plays pivotal roles in the orchestration of development, defence responses and viral propagation. PD are dynamic structures embedded in the plant cell wall that are defined by specialised domains of the endoplasmic reticulum and the plasma membrane. PD structure and unique functions are guaranteed by their particular molecular composition. Yet, up to recent years and despite numerous approaches such as mutant screens, immunolocalisation or screening of random cDNAs, only few PD proteins had been conclusively identified and characterised. A clear breakthrough in the search of PD constituents came from mass-spectrometry-based proteomic approaches coupled with subcellular fractionation strategies. Due to their position, firmly anchored in the extracellular matrix, PD are notoriously difficult to isolate for biochemical analysis. Proteomic-based approaches have therefore first relied on the use of cell wall fractions containing embedded PD then on free PD fractions whereby PD membranes were released from the walls by enzymatic degradation. To discriminate between likely contaminants and PD protein candidates, bioinformatics tools have often been used in combination with proteomic approaches. GFP fusion proteins of selected candidates have confirmed the PD association of several protein families. Here we review the accomplishments and limitations of the proteomic based strategies to unravel the functional and structural complexity of PD. We also discuss the role of the identified PD associated proteins.

  7. Proteomic alteration of Marc-145 cells and PAMs after infection by porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Ding, Zhuang; Li, Zhi-jie; Zhang, Xiao-dong; Li, Ya-gang; Liu, Chang-jun; Zhang, Yan-Ping; Li, Yang

    2012-01-15

    Viral infections usually result in alterations in the host cell proteome, which determine the fate of infected cells and the progress of pathogenesis. To uncover cellular protein responses in porcine reproductive and respiratory syndrome virus (PRRSV), infected pulmonary alveolar macrophages (PAMs) and Marc-145 cells were subjected to proteomic analysis involving two-dimensional electrophoresis (2-DE) followed by MALDI-TOF-MS/MS identification. Altered expression of 44 protein spots in infected cells was identified in 2D gels, of which the 29 characterised by MALDI-TOF-MS/MS included 17 up-regulated and 12 down-regulated proteins. Some of these proteins were further confirmed at the mRNA level using real-time RT-PCR. Moreover, Western blot analysis confirmed the up-regulation of HSP27, vimentin and the down-regulation of galectin-1. Our study is the first attempt to analyze the cellular protein profile of PRRSV-infected Marc-145 cells using proteomics to provide valuable information about the effects of PRRSV-induced alterations on Marc-145 cell function. Further study of the affected proteins may facilitate our understanding of the mechanisms of PRRSV infection and pathogenesis.

  8. Comprehensive Proteomic Analysis of Mesenchymal Stem Cell Exosomes Reveals Modulation of Angiogenesis via Nuclear Factor-KappaB Signaling.

    Science.gov (United States)

    Anderson, Johnathon D; Johansson, Henrik J; Graham, Calvin S; Vesterlund, Mattias; Pham, Missy T; Bramlett, Charles S; Montgomery, Elizabeth N; Mellema, Matt S; Bardini, Renee L; Contreras, Zelenia; Hoon, Madeline; Bauer, Gerhard; Fink, Kyle D; Fury, Brian; Hendrix, Kyle J; Chedin, Frederic; El-Andaloussi, Samir; Hwang, Billie; Mulligan, Michael S; Lehtiö, Janne; Nolta, Jan A

    2016-03-01

    Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.

  9. The Effects of Bifidobacterium breve on Immune Mediators and Proteome of HT29 Cells Monolayers

    Directory of Open Access Journals (Sweden)

    Borja Sánchez

    2015-01-01

    Full Text Available The use of beneficial microorganisms, the so-called probiotics, to improve human health is gaining popularity. However, not all of the probiotic strains trigger the same responses and they differ in their interaction with the host. In spite of the limited knowledge on mechanisms of action some of the probiotic effects seem to be exerted through maintenance of the gastrointestinal barrier function and modulation of the immune system. In the present work, we have addressed in vitro the response of the intestinal epithelial cell line HT29 to the strain Bifidobacterium breve IPLA20004. In the array of 84 genes involved in inflammation tested, the expression of 12 was modified by the bifidobacteria. The genes of chemokine CXCL6, the chemokine receptor CCR7, and, specially, the complement component C3 were upregulated. Indeed, HT29 cells cocultivated with B. breve produced significantly higher levels of protein C3a. The proteome of HT29 cells showed increased levels of cytokeratin-8 in the presence of B. breve. Altogether, it seems that B. breve IPLA20004 could favor the recruitment of innate immune cells to the mucosa reinforcing, as well as the physical barrier of the intestinal epithelium.

  10. A large-scale proteomic analysis of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Sherrer Eric

    2007-12-01

    Full Text Available Abstract Background Much of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells. Results Here we describe the use of two large-scale western blot assays to identify over 600 proteins expressed in undifferentiated hESCs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and/or post-translational modifications. Twenty-two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hESCs were also identified. We confirmed the expression of a subset of the identified proteins by immunofluorescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hESCs. These analyses also indicated that hESCs exhibit several features of polarized epithelia, including expression of tight junction proteins. Conclusion Our approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self-renewal.

  11. Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Atay, Safinur [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Gercel-Taylor, Cicek [Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States); Kesimer, Mehmet [Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC (United States); Taylor, Douglas D., E-mail: ddtaylor@louisville.edu [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States)

    2011-05-01

    Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.

  12. Comparative proteomics analysis of lanthanum citrate complex-induced apoptosis in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    In a previous study,the lanthanum citrate complex([LaCit2]3-) has been found to induce apoptosis in the human HeLa cervical cancer cell line.To clarify the mechanism,we carried out comparative proteomics analysis between treated and control cells.Differentially expressed proteins were separated electrophoretically and identified by MALDI-TOF/TOF tandem mass spectrometry.There were profound changes in 14 proteins related to mitochondrial function and oxidative stress,suggesting that mitochondrial dysfunction plays a key role in [LaCit2]3--induced apoptosis.This was confirmed by a decrease in the mitochondrial transmembrane potential,and increases in cytochrome c release and reactive oxygen species generation in [LaCit2]3--treated cells.Western blotting analyses show that [LaCit2]3--induced apoptosis was accompanied by the activation of caspase-9 and the specific proteolytic cleavage of PARP,leading to an increase in the proapoptotic protein Bax and a decrease in the antiapoptotic protein Bcl-2.These results suggest that [LaCit2]3-induced the apoptosis of HeLa cells through oxidative stress mediated pathway involving MT participation.

  13. Profiling of Host Cell Response to Successive Canine Parvovirus Infection Based on Kinetic Proteomic Change Identification

    Science.gov (United States)

    Zhao, Hang; Cheng, Yuening; Wang, Jianke; Lin, Peng; Yi, Li; Sun, Yaru; Ren, Jingqiang; Tong, Mingwei; Cao, Zhigang; Li, Jiawei; Deng, Jinliang; Cheng, Shipeng

    2016-01-01

    Canine parvovirus (CPV) reproduces by co-opting the resources of host cells, inevitably causing cytotoxic effects to the host cells. Feline kidney F81 cells are sensitive to CPV infection and show disparate growing statuses at different time points post-infection. This study analysed the response of F81 cells to CPV infection at successive infection time points by iTRAQ-based quantitative proteomics. Differentially expressed proteins (DEPs) during 60 h of infection and at selected time points post-infection were identified by an analysis of variance test and a two-tailed unpaired t test, respectively. DEPs with similar quantitative changes were clustered by hierarchical clustering and analysed by gene ontology enrichment, revealing that 12 h and 60 h post-infection were the optimal times to analyse the autonomous parvovirus replication and apoptosis processes, respectively. Using the MetacoreTM database, 29 DEPs were enriched in a network involved in p53 regulation. Besides, a significantly enriched pathway suggests that the CPV-induced cytopathic effect was probably due to the deficiency of functional CFTR caused by CPV infection. This study uncovered the systemic changes in key cellular factors involved in CPV infection and help to understand the molecular mechanisms of the anti-cancer activity of CPV and the cytopathic effects induced by CPV infection. PMID:27406444

  14. Pre-fractionation strategies to resolve pea (Pisum sativum) sub-proteomes

    Science.gov (United States)

    Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana; Kukavica, Biljana M.; Lüthje, Sabine

    2015-01-01

    Legumes are important crop plants and pea (Pisum sativum L.) has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula Gaertner) allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins). Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed. PMID:26539198

  15. Pre-fractionation strategies to resolve pea (Pisum sativum sub-proteomes

    Directory of Open Access Journals (Sweden)

    Claudia Nicole Meisrimler

    2015-10-01

    Full Text Available Legumes are important crop plants and pea (Pisum sativum L. has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula G. allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins. Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed.

  16. Phenylalanine ammonia-lyase and cell wall peroxidase are cooperatively involved in the extensive formation of ferulate network in cell walls of developing rice shoots.

    Science.gov (United States)

    Wakabayashi, Kazuyuki; Soga, Kouichi; Hoson, Takayuki

    2012-02-15

    The relationship between the formation of cell wall-bound ferulic acid (FA) and diferulic acid (DFA) and the change in activities of phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) was studied in rice shoots. The length and the fresh mass of shoots increased during the growth period from day 4 to 6, while coleoptiles ceased elongation growth on day 5. The amounts of FA and DFA isomers as well as cell wall polysaccharides continued to increase during the whole period. The activities of PAL and CW-PRX greatly increased in the same manner during the period. There were close correlations between the PAL activity and ferulate content or between the CW-PRX activity and DFA content. The expression levels of investigated genes for PAL and putative CW-PRX showed good accordance with the activities of these enzymes. These results suggest that increases in PAL and CW-PRX activities are cooperatively involved in the formation of ferulate network in cell walls of rice shoots and that investigated genes may be, at least in part, associated with the enzyme activities. The substantial increase in such network probably causes the maturation of cell walls and thus the cessation of elongation growth of coleoptiles.

  17. Proteomic analysis of nuclear matrix proteins during arsenic trioxide induced apoptosis in leukemia K562 cells

    Institute of Scientific and Technical Information of China (English)

    WANG Zi-hui; YU Ding; CHEN Yan; HAO Jian-zhong

    2005-01-01

    Background Arsenic trioxide (As2O3) has been identified as a very potent anti-acute leukemic agent. However its role in apoptosis needs to be elucidated. As2O3 interferes with the proliferation and survival of tumor cells via a variety of mechanisms. Drug-target interactions at the level of nuclear matrix (NM) may be critical events in the induction of cell death by As2O3. This study dealt with As2O3-target interactions at the level of NM in chronic myelogenous leukemia cell line K562 by proteomics. Methods K562 cells were cultured in MEM and treated with different concentrations of As2O3. The nuclear matrix proteins were analyzed by high-resolution two-dimensional gel electrophoresis and computer-assisted image analysis. Results As2O3 significantly inhibited the growth of chronic myelogenous leukemia cell line K562 at low concentrations. While more than 200 protein spots were shared among the nuclear matrices, about 18 distinct spots in the nuclear matrices were found characteristic for As2O3 treated cells. Conclusions: As2O3 induces apoptosis in K562 cells in a dose and time-dependent manner. Our results demonstrated that for the detection of the onset of apoptosis, the alteration in the composition of nuclear matrix proteins was a more sensitive indicator than nucleosomal DNA fragmentation test. These results indicated that As2O3 might be clinically useful in the treatment of chronic myelogenous leukemia. The changes of nuclear matrix proteins in the treated cells can be used as a useful indicator for this treatment.

  18. Growth regulation mechanisms in higher plants under microgravity conditions - changes in cell wall metabolism.

    Science.gov (United States)

    Hoson, T; Kamisaka, S; Wakabayashi, K; Soga, K; Tabuchi, A; Tokumoto, H; Okamura, K; Nakamura, Y; Mori, R; Tanimoto, E; Takeba, G; Nishitani, K; Izumi, R; Ishioka, N; Kamigaichi, S; Aizawa, S; Yoshizaki, I; Shimazu, T; Fukui, K

    2000-06-01

    During Space Shuttle STS-95 mission, we cultivated seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) and Arabidopsis (Arabidopsis thaliana L. cv. Columbia and cv. etr1-1) for 68.5, 91.5, and 136 hr on board, and then analyzed changes in the nature of their cell walls, growth, and morphogenesis under microgravity conditions. In space, elongation growth of both rice coleoptiles and Arabidopsis hypocotyls was stimulated. Also, the increase in the cell wall extensibility, especially that in the irreversible extensibility, was observed for such materials. The analyses of the amounts, the structure, and the physicochemical properties of the cell wall constituents indicated that the decreases in levels and molecular masses of cell wall polysaccharides were induced under microgravity conditions, which appeared to contribute to the increase in the wall extensibility. The activity of certain wall enzymes responsible for the metabolic turnover of the wall polysaccharides was increased in space. By the space flight, we also confirmed the occurrence of automorphogenesis of both seedlings under microgravity conditions; rice coleoptiles showed an adaxial bending, whereas Arabidopsis hypocotyls elongated in random directions. Furthermore, it was shown that spontaneous curvatures of rice coleoptiles in space were brought about uneven modifications of cell wall properties between the convex and the concave sides.

  19. Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

    Science.gov (United States)

    Borovikova, Diana; Teparić, Renata; Mrša, Vladimir; Rapoport, Alexander

    2016-08-01

    The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd.

  20. Critical cell wall hole size for lysis in Gram-positive bacteria

    Science.gov (United States)

    Mitchell, Gabriel; Wiesenfeld, Kurt; Nelson, Daniel; Weitz, Joshua

    2013-03-01

    Gram-positive bacteria transport molecules necessary for their survival through holes in their cell wall. The holes in cell walls need to be large enough to let critical nutrients pass through. However, the cell wall must also function to prevent the bacteria's membrane from protruding through a large hole into the environment and lysing the cell. As such, we hypothesize that there exists a range of cell wall hole sizes that allow for molecule transport but prevent membrane protrusion. Here we develop and analyze a biophysical theory of the response of a Gram-positive cell's membrane to the formation of a hole in the cell wall. We predict a critical hole size in the range 15-24nm beyond which lysis occurs. To test our theory, we measured hole sizes in Streptococcus pyogenes cells undergoing enzymatic lysis via transmission electron microscopy. The measured hole sizes are in strong agreement with our theoretical prediction. Together, the theory and experiments provide a means to quantify the mechanisms of death of Gram-positive cells via enzymatically mediated lysis and provides insight into the range of cell wall hole sizes compatible with bacterial homeostasis.

  1. Combination of metallomics and proteomics to study the effects of the metallodrug RAPTA-T on human cancer cells.

    Science.gov (United States)

    Wolters, Dirk A; Stefanopoulou, Maria; Dyson, Paul J; Groessl, Michael

    2012-11-01

    An approach to characterize the interactions of RAPTA-T, a novel ruthenium-based anticancer drug candidate with intriguing antimetastatic properties, with human ovarian cancer cells in vitro is described. The distribution profile of the metallodrug within the cancer cells was determined by (size exclusion chromatography)-inductively coupled mass spectrometry combined with subcellular fractionation procedures (metallomics). Multidimensional protein identification technology (MudPIT) was then used to obtain insight into the alteration of the cellular proteome upon RAPTA-T treatment. The metallomics approach reveals striking differences in the intracellular behavior of the drug between cisplatin-sensitive and resistant cell lines and provides clues on possible mechanisms of action as well as detoxification, quantitative proteomics based on spectral counting sheds light on cellular response mechanisms to metallodrug treatment.

  2. Superoxide generation in extracts from isolated plant cell walls is regulated by fungal signal molecules.

    Science.gov (United States)

    Kiba, A; Miyake, C; Toyoda, K; Ichinose, Y; Yamada, T; Shiraishi, T

    1997-08-01

    ABSTRACT Fractions solubilized with NaCl from cell walls of pea and cowpea plants catalyzed the formation of blue formazan from nitroblue tetrazolium. Because superoxide dismutase decreased formazan production by over 90%, superoxide anion (O(2) ) may participate in the formation of formazan in the solubilized cell wall fractions. The formazan formation in the fractions solubilized from pea and cowpea cell walls was markedly reduced by exclusion of NAD(P)H, manganese ion, or p-coumaric acid from the reaction mixture. The formazan formation was severely inhibited by salicylhydroxamic acid and catalase, but not by imidazole, pyridine, quinacrine, and diphenyleneiodonium. An elicitor preparation from the pea pathogen Mycosphaerella pinodes enhanced the activities of formazan formation nonspecifically in both pea and cowpea fractions. The suppressor preparation from M. pinodes inhibited the activity in the pea fraction in the presence or absence of the elicitor. In the cowpea fraction, however, the suppressor did not inhibit the elicitor-enhanced activity, and the suppressor alone stimulated formazan formation. These results indicated that O(2) generation in the fractions solubilized from pea and cowpea cell walls seems to be catalyzed by cell wall-bound peroxidase(s) and that the plant cell walls alone are able to respond to the elicitor non-specifically and to the suppressor in a species-specific manner, suggesting the plant cell walls may play an important role in determination of plant-fungal pathogen specificity.

  3. Altered cell wall properties are responsible for ammonium-reduced aluminium accumulation in rice roots.

    Science.gov (United States)

    Wang, Wei; Zhao, Xue Qiang; Chen, Rong Fu; Dong, Xiao Ying; Lan, Ping; Ma, Jian Feng; Shen, Ren Fang

    2015-07-01

    The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH4(+)) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al-binding assay revealed that neither NH4(+) nor NO3(-) altered the Al-binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH4(+)-supplied roots displayed lower Al-binding capacity than those from NO3(-)-supplied roots when grown in non-buffered solutions. Fourier-transform infrared microspectroscopy analysis revealed that, compared with NO3(-)-supplied roots, NH4(+)-supplied roots possessed fewer Al-binding groups (-OH and COO-) and lower contents of pectin and hemicellulose. However, when grown in pH-buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al-binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH4(+)-reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH4(+) uptake rather than direct competition for the cell wall binding sites between Al(3+) and NH4(+).

  4. Plumbagin elicits differential proteomic responses mainly involving cell cycle, apoptosis, autophagy, and epithelial-to-mesenchymal transition pathways in human prostate cancer PC-3 and DU145 cells

    Directory of Open Access Journals (Sweden)

    Qui JX

    2015-01-01

    Full Text Available Jia-Xuan Qiu,1,2 Zhi-Wei Zhou, 3,4 Zhi-Xu He,4 Ruan Jin Zhao,5 Xueji Zhang,6 Lun Yang,7 Shu-Feng Zhou,3,4 Zong-Fu Mao11School of Public Health, Wuhan University, Wuhan, Hubei, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 3Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 4Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China; 5Center for Traditional Chinese Medicine, Sarasota, FL, USA; 6Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 7Bio-X Institutes, Key Laboratory for the Genetics of Development and Neuropsychiatric Disorders (Ministry of Education, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaAbstract: Plumbagin (PLB has exhibited a potent anticancer effect in preclinical studies, but the molecular interactome remains elusive. This study aimed to compare the quantitative proteomic responses to PLB treatment in human prostate cancer PC-3 and DU145 cells using the approach of stable-isotope labeling by amino acids in cell culture (SILAC. The data were finally validated using Western blot assay. First, the bioinformatic analysis predicted that PLB could interact with 78 proteins that were involved in cell proliferation and apoptosis, immunity, and signal transduction. Our quantitative proteomic study using SILAC revealed that there were at least 1,225 and 267 proteins interacting with PLB and there were 341 and 107 signaling pathways and cellular functions potentially regulated by PLB in PC-3 and DU145 cells, respectively. These proteins and pathways played a

  5. Malignant transformation of ectopic pancreatic cells in the duodenal wall

    Institute of Scientific and Technical Information of China (English)

    Roberto; Bini; Paolo; Voghera; Alberto; Tapparo; Raffaele; Nunziata; Andrea; Demarchi; Matteo; Capocefalo; Renzo; Leli

    2010-01-01

    Ectopic pancreas (EP) is the relatively uncommon presence of pancreatic tissue outside the normal location of the pancreas. This condition is usually asymptomatic and rarely complicated by pancreatitis and malignant transformation. A few cases of neoplastic phenomena that developed from EP into the duodenal wall are described in the literature. Herein we report a case of gastric outlet obstruction due to adenocarcinoma arising from EP of the duodenal wall. The patient underwent a Whipple's procedure and had...

  6. Navigating the transcriptional roadmap regulating plant secondary cell wall deposition

    Directory of Open Access Journals (Sweden)

    Steven Grant Hussey

    2013-08-01

    Full Text Available The current status of lignocellulosic biomass as an invaluable resource in industry, agriculture and health has spurred increased interest in understanding the transcriptional regulation of secondary cell wall (SCW biosynthesis. The last decade of research has revealed an extensive network of NAC, MYB and other families of transcription factors regulating Arabidopsis SCW biosynthesis, and numerous studies have explored SCW-related transcription factors in other dicots and monocots. Whilst the general structure of the Arabidopsis network has been a topic of several reviews, they have not comprehensively represented the detailed protein-DNA and protein-protein interactions described in the literature, and an understanding of network dynamics and functionality has not yet been achieved for SCW formation. Furthermore the methodologies employed in studies of SCW transcriptional regulation have not received much attention, especially in the case of non-model organisms. In this review, we have reconstructed the most exhaustive literature-based network representations to date of SCW transcriptional regulation in Arabidopsis. We include a manipulable Cytoscape representation of the Arabidopsis SCW transcriptional network to aid in future studies, along with a list of supporting literature for each documented interaction. Amongst other topics, we discuss the various components of the network, its evolutionary conservation in plants, putative modules and dynamic mechanisms that may influence network function, and the approaches that have been employed in network inference. Future research should aim to better understand network function and its response to dynamic perturbations, whilst the development and application of genome-wide approaches such as ChIP-seq and systems genetics are in progress for the study of SCW transcriptional regulation in non-model organisms.

  7. Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.

    Science.gov (United States)

    Schenk, Laura K; Bolger, Steven J; Luginbuhl, Kelli; Gonzales, Patricia A; Rinschen, Markus M; Yu, Ming-Jiun; Hoffert, Jason D; Pisitkun, Trairak; Knepper, Mark A

    2012-06-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (β-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCβ), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in β-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct.

  8. Integrated Cellular and Plasma Proteomics of Contrasting B-cell Cancers Reveals Common, Unique and Systemic Signatures.

    Science.gov (United States)

    Johnston, Harvey E; Carter, Matthew J; Cox, Kerry L; Dunscombe, Melanie; Manousopoulou, Antigoni; Townsend, Paul A; Garbis, Spiros D; Cragg, Mark S

    2017-03-01

    Approximately 800,000 leukemia and lymphoma cases are diagnosed worldwide each year. Burkitt's lymphoma (BL) and chronic lymphocytic leukemia (CLL) are examples of contrasting B-cell cancers; BL is a highly aggressive lymphoid tumor, frequently affecting children, whereas CLL typically presents as an indolent, slow-progressing leukemia affecting the elderly. The B-cell-specific overexpression of the myc and TCL1 oncogenes in mice induce spontaneous malignancies modeling BL and CLL, respectively. Quantitative mass spectrometry proteomics and isobaric labeling were employed to examine the biology underpinning contrasting Eμ-myc and Eμ-TCL1 B-cell tumors. Additionally, the plasma proteome was evaluated using subproteome enrichment to interrogate biomarker emergence and the systemic effects of tumor burden. Over 10,000 proteins were identified (qIL5) receptor. IL5 treatment promoted Eμ-TCL1 tumor proliferation, suggesting an amplification of IL5-induced AKT signaling by TCL1. Tumor plasma contained a substantial tumor lysis signature, most prominent in Eμ-myc plasma, whereas Eμ-TCL1 plasma contained signatures of immune-response, inflammation and microenvironment interactions, with putative biomarkers in early-stage cancer. These findings provide a detailed characterization of contrasting B-cell tumor models, identifying common and specific tumor mechanisms. Integrated plasma proteomics allowed the dissection of a systemic response and a tumor lysis signature present in early- and late-stage cancers, respectively. Overall, this study suggests common B-cell cancer signatures exist and illustrates the potential of the further evaluation of B-cell cancer subtypes by integrative proteomics.

  9. Altered cell wall disassembly during ripening of Cnr tomato fruit: implications for cell adhesion and fruit softening

    DEFF Research Database (Denmark)

    Orfila, C.; Huisman, M.M.H.; Willats, William George Tycho;

    2002-01-01

    The Cnr (Colourless non-ripening) tomato (Lycopersicon esculentum Mill.) mutant has an aberrant fruit-ripening phenotype in which fruit do not soften and have reduced cell adhesion between pericarp cells. Cell walls from Cnr fruit were analysed in order to assess the possible contribution of pectic...... polysaccharides to the non-softening and altered cell adhesion phenotype. Cell wall material (CWM) and solubilised fractions of mature green and red ripe fruit were analysed by chemical, enzymatic and immunochemical techniques. No major differences in CWM sugar composition were detected although differences were...... that was chelator-soluble was 50% less in Cnr cell walls at both the mature green and red ripe stages. Chelator-soluble material from ripe-stage Cnr was more susceptible to endo-polygalacturonase degradation than the corresponding material from wild-type fruit. In addition, cell walls from Cnr fruit contained...

  10. A proteomic analysis during serial subculture and osteogenic differentiation of human mesenchymal stem cell.

    Science.gov (United States)

    Sun, Hyun Jin; Bahk, Young Yil; Choi, Yon Rak; Shim, Jung Hye; Han, Seung Hwan; Lee, Jin Woo

    2006-11-01

    Although previous studies have reported the effects of extensive subculturing on proliferation rates and osteogenic potential of human mesenchymal stem cells (hMSCs), the results remain controversial. The aim of our study was to characterize the proliferation and osteogenic potential of hMSCs during serial subculture, and also to identify proteins that are differentially regulated in hMSCs during serial subculture and osteogenic differentiation using proteome analysis. Here we show that the proliferation and osteogenic capacity of hMSCs decrease during serial subculturing. Several proteins were shown to be differentially regulated during serial subculture; among these the expression of T-complex protein 1 alpha subunit (TCP-1alpha), a protein known to be associated with cell proliferation, cell cycle, morphological changes, and apoptosis, gradually decreased during serial subculture. Among proteins that were differentially regulated during osteogenic differentiation, chloride intracellular channel 1 (CLIC1) was downregulated only during the early passages eukaryotic translation elongation factor, and acidic ribosomal phosphoprotein P0 was downregulated during the middle passages, while annexin V, LIM, and SH3 domain protein 1 (LASP-1), and 14-3-3 protein gamma (YWHAG) were upregulated during the later passage. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of osteogenic differentiation potential under serial subculturing.

  11. A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture.

    Science.gov (United States)

    Ibarrola, Nieves; Kalume, Dario E; Gronborg, Mads; Iwahori, Akiko; Pandey, Akhilesh

    2003-11-15

    Posttranslational modifications are major mechanisms of regulating protein activity and function in vertebrate cells. It is essential to obtain qualitative information about posttranslational modification patterns of proteins to understand signal transduction mechanisms in greater detail. However, it is equally important to measure the dynamics of posttranslational modifications such as phosphorylation to approach signaling networks from a systems biology perspective. Despite a number of advances, methods to quantitate posttranslational modifications remain difficult to implement due to a number of factors including lack of a generic method, elaborate chemical steps, and requirement for large amounts of sample. We have previously shown that stable isotope-containing amino acids in cell culture (SILAC) can be used to differentially label growing cell populations for quantitation of protein levels. In this report, we extend the use of SILAC as a novel proteomic approach for the relative quantitation of posttranslational modifications such as phosphorylation. We have used SILAC to quantitate the extent of known phosphorylation sites as well as to identify and quantitate novel phosphorylation sites.

  12. Comparative characterization of stromal vascular cells derived from three types of vascular wall and adipose tissue.

    Science.gov (United States)

    Yang, Santsun; Eto, Hitomi; Kato, Harunosuke; Doi, Kentaro; Kuno, Shinichiro; Kinoshita, Kahori; Ma, Hsu; Tsai, Chi-Han; Chou, Wan-Ting; Yoshimura, Kotaro

    2013-12-01

    Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 μm in diameter), venous wall (VN; >500 μm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristics of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34(+)/CD31(-) and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in the osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as arterial wall calcification and possible applications to regenerative therapies

  13. Proteomics and aging : studying the influence of aging on endothelial cells and human plasma

    NARCIS (Netherlands)

    Eman, M.R.

    2007-01-01

    In general, human aging is considered one of the most complex and less-well understood process in biology. In this thesis the possibilities of proteomics technology in the field of aging were explored. The complexity of the aging process was supposed to accompanied by relatively subtle proteome vari

  14. Rice Brittleness Mutants: A Way to Open the 'Black Box' of Monocot Cell Wall Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Baocai Zhang; Yihua Zhou

    2011-01-01

    Rice is a model organism for studying the mechanism of cell wall biosynthesis and remolding in Gramineae.Mechanical strength is an important agronomy trait of rice(Oryza sativa L.)plants that affects crop lodging and grain yield.As a prominent physical property of cell walls,mechanical strength reflects upon the structure of different wall polymers and how they interact.Studies on the mechanisms that regulate the mechanical strength therefore consequently results in uncovering the genes functioning in cell wall biosynthesis and remodeling.Our group focuses on the study of isolation of brittle culm(bc)mutants and characterization of their corresponding genes.To date,several bc mutants have been reported.The identified genes have covered several pathways of cell wall biosynthesis,revealing many secrets of monocot cell wall biosynthesis.Here,we review the progress achieved in this research field and also highlight the perspectives in expectancy.All of those lend new insights into mechanisms of cell wall formation and are helpful for harnessing the waste rice straws for biofuel production.

  15. Evidence for land plant cell wall biosynthetic mechanisms in charophyte green algae

    DEFF Research Database (Denmark)

    Mikkelsen, Maria Dalgaard; Harholt, Jesper; Ulvskov, Peter

    2014-01-01

    characterized in land plants. In addition, gene cloning was employed in two cases to answer important evolutionary questions. KEY RESULTS: Genetic evidence was obtained indicating that many of the most important core cell wall polysaccharides have their evolutionary origins in the CGA, including cellulose...... to colonize land. These cell walls provide support and protection, are a source of signalling molecules, and provide developmental cues for cell differentiation and elongation. The cell wall of land plants is a highly complex fibre composite, characterized by cellulose cross-linked by non......-cellulosic polysaccharides, such as xyloglucan, embedded in a matrix of pectic polysaccharides. How the land plant cell wall evolved is currently unknown: early-divergent chlorophyte and prasinophyte algae genomes contain a low number of glycosyl transferases (GTs), while land plants contain hundreds. The number of GTs...

  16. Effect of commercial enzymes on berry cell wall deconstruction in the context of intravineyard ripeness variation under winemaking conditions

    DEFF Research Database (Denmark)

    Gao, Yu; Fangel, Jonatan Ulrik; Willats, William George Tycho;

    2016-01-01

    at the berry cell wall polymer level and occurred within the experimental vineyard block. Furthemore, all enzyme treatments reduced cell wall variation via depectination. Interestingly, cell wall esterification levels were unaffected by enzyme treatments. This study provides clear evidence that enzymes can...

  17. Expression of S-adenosylmethionine Hydrolase in Tissues Synthesizing Secondary Cell Walls Alters Specific Methylated Cell Wall Fractions and Improves Biomass Digestibility

    Directory of Open Access Journals (Sweden)

    Aymerick Eudes

    2016-07-01

    Full Text Available Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine (AdoMet. In this study, we demonstrate in Arabidopsis stems that targeted expression of S-adenosylmethionine hydrolase (AdoMetase, E.C. 3.3.1.2 in secondary cell-wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl (H units and a reduction of dimethylated syringyl (S units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock.

  18. Combined gene expression and proteomic analysis of EGF induced apoptosis in A431 cells suggests multiple pathways trigger apoptosis.

    Science.gov (United States)

    Alanazi, Ibrahim; Ebrahimie, Esmaeil; Hoffmann, Peter; Adelson, David L

    2013-11-01

    A431 cells, derived from epidermoid carcinoma, overexpress the epidermal growth factor receptor (EGFR) and when treated with a high dose of EGF will undergo apoptosis. We exploited microarray and proteomics techniques and network prediction to study the regulatory mechanisms of EGF-induced apoptosis in A431 cells. We observed significant changes in gene expression in 162 genes, approximately evenly split between pro-apoptotic and anti-apoptotic genes and identified 30 proteins from the proteomic data that had either pro or anti-apoptotic annotation. Our correlation analysis of gene expression and proteome modeled a number of distinct sub-networks that are associated with the onset of apoptosis, allowing us to identify specific pathways and components. These include components of the interferon signalling pathway, and down stream components, including cytokines and suppressors of cytokine signalling. A central component of almost all gene expression sub-networks identified was TP53, which is mutated in A431 cells, and was down regulated. This down regulation of TP53 appeared to be correlated with proteomic sub-networks of cytoskeletal or cell adhesion components that might induce apoptosis by triggering cytochrome C release. Of the only three genes also differentially expressed as proteins, only serpinb1 had a known association with apoptosis. We confirmed that up regulation and cleavage of serpinb1 into L-DNAaseII was correlated with the induction of apoptosis. It is unlikely that a single pathway, but more likely a combination of pathways is needed to trigger EGF induced apoptosis in A431cells.

  19. System-wide survey of proteomic responses of human bone marrow stromal cells (hBMSCs to in vitro cultivation

    Directory of Open Access Journals (Sweden)

    Samuel T. Mindaye

    2015-11-01

    Full Text Available Human bone marrow stromal cells (hBMSCs, also loosely called bone marrow-derived mesenchymal stem cells are the subject of increasing numbers of clinical trials and laboratory research. Our group recently reported on the optimization of a workflow for a sensitive proteomic study of hBMSCs. Here, we couple this workflow with a label-free protein quantitation method to investigate the molecular responses of hBMSCs to long-term in vitro passaging. We explored the proteomic responses of hBMSCs by assessing the expression levels of proteins at early passage (passage 3, P3 and late passage (P7. We used multiple biological as well as technical replicates to ensure that the detected proteomic changes are repeatable between cultures and thus likely to be biologically relevant. Over 1700 proteins were quantified at three passages and a list of differentially expressed proteins was compiled. Bioinformatics-based network analysis and term enrichment revealed that metabolic pathways are largely altered, where many proteins in the glycolytic, pentose phosphate, and TCA pathways were shown to be largely upregulated in late passages. We also observed significant proteomic alterations in functional categories including apoptosis, and ER-based protein processing and sorting following in vitro cell aging. We posit that the comprehensive map outlined in this report of affected phenotypes as well as the underpinning molecular factors tremendously benefit the effort to uncovering targets that are not just used only to monitor cell fitness but can be employed to slowdown the in vitro aging process in hBMSCs and hence ensure manufacturing of cells with known quality, efficacy and stability.

  20. Proteomic Identification of LASP-1 Down-regulation After RNAi Urokinase Silencing in Human Hepatocellular Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Alessandro Salvi

    2009-02-01

    Full Text Available In human hepatocellular carcinoma (HCC, the high expression of urokinase-type plasminogen activator (uPA is an unfavorable prognostic factor and a therapeutic target. To identify the downstream effects of uPA silencing by RNA interference, we studied proteome modifications of uPA-inhibited SKHep1C3 cells, an HCC-derived cell line. The study with two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry showed Lim and SH3 protein 1 (LASP-1, cytokeratin 1 (CK-1, cytokeratin 10 (CK-10, and heterogeneous nuclear ribonucleoprotein H1 down-modulation after uPA inhibition. LASP-1, CK-1, and CK-10 are involved in cytoskeleton dynamics as heterogeneous nuclear ribonucleoprotein H1 takes part in the mRNA processing and stability. We first confirmed the proteomic data by Western blot and immunoflorescence and then explored the link between uPA and LASP-1. The ectopic expression of uPA and LASP-1 supported the proteomic results and showed that uPA up-regulation increased LASP-1 expression and that both were implicated in SKHep1C3 motility. siRNA LASP-1 inhibition showed that LASP-1 was involved in actin microfilaments organization of SKHep1C3 cells. The disruption of the actin microfilaments after LASP-1 depletion increased uPA secretion and SKHep1C3 motility. Our results would suggest the hypothesis that uPA and LASP-1 expression may be coordinated in HCC-derived cells. In summary, the proteomic identification of a set of uPA downstream proteins provides new insight into the function of uPA in HCC cells.

  1. Comparative proteomic analysis of drug sodium iron chlorophyllin addition to Hep 3B cell line.

    Science.gov (United States)

    Zhang, Jun; Wang, Wenhai; Yang, Fengying; Zhou, Xinwen; Jin, Hong; Yang, Peng-yuan

    2012-09-21

    The human hepatoma 3B cell line was chosen as an experimental model for in vitro test of drug screening. The drugs included chlorophyllin and its derivatives such as fluo-chlorophyllin, sodium copper chlorophyllin, and sodium iron chlorophyllin. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method was used in this study to obtain the primary screening results. The results showed that sodium iron chlorophyllin had the best LC(50) value. Proteomic analysis was then performed for further investigation of the effect of sodium iron chlorophyllin addition to the Hep 3B cell line. The proteins identified from a total protein extract of Hep 3B before and after the drug addition were compared by two-dimensional-gel-electrophoresis. Then 32 three-fold differentially expressed proteins were successfully identified by MALDI-TOF-TOF-MS. There are 29 unique proteins among those identified proteins. These proteins include proliferating cell nuclear antigen (PCNA), T-complex protein, heterogeneous nuclear protein, nucleophosmin, heat shock protein A5 (HspA5) and peroxiredoxin. HspA5 is one of the proteins which are involved in protecting cancer cells against stress-induced apoptosis in cultured cells, protecting them against apoptosis through various mechanisms. Peroxiredoxin has anti-oxidant function and is related to cell proliferation, and signal transduction. It can protect the oxidation of other proteins. Peroxiredoxin has a close relationship with cancer and can eventually become a disease biomarker. This might help to develop a novel treatment method for carcinoma cancer.

  2. Growth factor priming differentially modulates components of the extracellular matrix proteome in chondrocytes and synovium-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Elena Alegre-Aguarón

    Full Text Available To make progress in cartilage repair it is essential to optimize protocols for two-dimensional cell expansion. Chondrocytes and SDSCs are promising cell sources for cartilage repair. We previously observed that priming with a specific growth factor cocktail (1 ng/mL transforming growth factor-β1, 5 ng/mL basic fibroblast growth factor, and 10 ng/mL platelet-derived growth factor-BB in two-dimensional culture, led to significant improvement in mechanical and biochemical properties of synovium-derived stem cell (SDSC-seeded constructs. The current study assessed the effect of growth factor priming on the proteome of canine chondrocytes and SDSCs. In particular, growth factor priming modulated the proteins associated with the extracellular matrix in two-dimensional cultures of chondrocytes and SDSCs, inducing a partial dedifferentiation of chondrocytes (most proteins associated with cartilage were down-regulated in primed chondrocytes and a partial differentiation of SDSCs (some collagen-related proteins were up-regulated in primed SDSCs. However, when chondrocytes and SDSCs were grown in pellet culture, growth factor-primed cells maintained their chondrogenic potential with respect to glycosaminoglycan and collagen production. In conclusion, the strength of the label-free proteomics technique is that it allows for the determination of changes in components of the extracellular matrix proteome in chondrocytes and SDSCs in response to growth factor priming, which could help in future tissue engineering strategies.

  3. Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis

    Directory of Open Access Journals (Sweden)

    Jurate Savickiene

    2015-01-01

    Full Text Available Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.

  4. Investigation on Adsorption of Lithospermum erythrorhizon onto Fungal Cell Wall Polysaccharides

    Institute of Scientific and Technical Information of China (English)

    孟琴; 薛莲

    2003-01-01

    A culture of Lithosperrnum erythrorhizon adsorbed on fungal cell wall polysaccharides, a novel bioadsorbent made from fungal cell wall, has been established in this paper. Three steps were involved in this immobilization. The first step was preparation of suspended plant cells from tightly aggregated plant cell clumps. The disassembled ratio of 0.715g·g-1 (the disassembled cells over total cells) was obtained under optimum condition for the enzymatic reaction. Then, the adsorption of plant cells onto fungal cell wall polysaccharides was conducted and the saturated capacity of 12g cell per gram of carrier was obtained in adsorption immobilization. Finally, the culture of cells adsorbed on fungal cell wall polysaccharides was compared with that of cells entrapped in alginate or suspension cell culture. While exposed to in situ liquid paraffin extraction coupled with cell culture, the shikonin productivity of immobilized cells by adsorption was 10.67g·L-1, which was 1.8 times of that in suspension culture and 1.5 times of that entrapped in alginate.

  5. Data from proteomic characterization of the role of Snail1 in murine mesenchymal stem cells and 3T3-L1 fibroblasts differentiation

    Directory of Open Access Journals (Sweden)

    A. Peláez-García

    2015-09-01

    Full Text Available The transcription factor (TF Snail1 is a major inducer of the epithelial–mesenchymal transition (EMT during embryonic development and cancer progression. Ectopic expression of Snail in murine mesenchymal stem cells (mMSC abrogated their differentiation to osteoblasts or adipocytes. We used either stable isotopic metabolic labeling (SILAC for 3T3-L1 cells or isobaric labeling with tandem mass tags (TMT for mMSC stably transfected cells with Snail1 or control. We carried out a proteomic analysis on the nuclear fraction since Snail is a nuclear TF that mediates its effects mainly through the regulation of other TFs. Proteomics data have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifiers PXD001529 and PXD002157 (Vizcaino et al., 2014 [1]. Data are associated with a research article published in Molecular and Cellular Proteomics (Pelaez-Garcia et al., 2015 [2].

  6. Proteomic data analysis of glioma cancer stem-cell lines based on novel nonlinear dimensional data reduction techniques

    Science.gov (United States)

    Lespinats, Sylvain; Pinker-Domenig, Katja; Wengert, Georg; Houben, Ivo; Lobbes, Marc; Stadlbauer, Andreas; Meyer-Bäse, Anke

    2016-05-01

    Glioma-derived cancer stem cells (GSCs) are tumor-initiating cells and may be refractory to radiation and chemotherapy and thus have important implications for tumor biology and therapeutics. The analysis and interpretation of large proteomic data sets requires the development of new data mining and visualization approaches. Traditional techniques are insufficient to interpret and visualize these resulting experimental data. The emphasis of this paper lies in the application of novel approaches for the visualization, clustering and projection representation to unveil hidden data structures relevant for the accurate interpretation of biological experiments. These qualitative and quantitative methods are applied to the proteomic analysis of data sets derived from the GSCs. The achieved clustering and visualization results provide a more detailed insight into the protein-level fold changes and putative upstream regulators for the GSCs. However the extracted molecular information is insufficient in classifying GSCs and paving the pathway to an improved therapeutics of the heterogeneous glioma.

  7. Comprehensive analysis of temporal alterations in cellular proteome of Bacillus subtilis under curcumin treatment.

    Directory of Open Access Journals (Sweden)

    Panga Jaipal Reddy

    Full Text Available Curcumin is a natural dietary compound with antimicrobial activi