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Sample records for cell membrane proteome

  1. Proteomic characterization of golgi membranes enriched from Arabidopsis suspension cell cultures

    DEFF Research Database (Denmark)

    Hansen, Sara Fasmer; Ebert, Berit; Rautengarten, Carsten;

    2016-01-01

    from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization......The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has...... historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes...

  2. Systems analysis of endothelial cell plasma membrane proteome of rat lung microvasculature

    Directory of Open Access Journals (Sweden)

    Witkiewicz Halina

    2011-03-01

    Full Text Available Abstract Background Endothelial cells line all blood vessels to form the blood-tissue interface which is critical for maintaining organ homeostasis and facilitates molecular exchange. We recently used tissue subcellular fractionation combined with several multi-dimensional mass spectrometry-based techniques to enhance identification of lipid-embedded proteins for large-scale proteomic mapping of luminal endothelial cell plasma membranes isolated directly from rat lungs in vivo. The biological processes and functions of the proteins expressed at this important blood-tissue interface remain unexplored at a large scale. Results We performed an unbiased systems analysis of the endothelial cell surface proteome containing over 1800 proteins to unravel the major functions and pathways apparent at this interface. As expected, many key functions of plasma membranes in general (i.e., cell surface signaling pathways, cytoskeletal organization, adhesion, membrane trafficking, metabolism, mechanotransduction, membrane fusion, and vesicle-mediated transport and endothelial cells in particular (i.e., blood vessel development and maturation, angiogenesis, regulation of endothelial cell proliferation, protease activity, and endocytosis were significantly overrepresented in this proteome. We found that endothelial cells express multiple proteins that mediate processes previously reported to be restricted to neuronal cells, such as neuronal survival and plasticity, axon growth and regeneration, synaptic vesicle trafficking and neurotransmitter metabolic process. Surprisingly, molecular machinery for protein synthesis was also detected as overrepresented, suggesting that endothelial cells, like neurons, can synthesize proteins locally at the cell surface. Conclusion Our unbiased systems analysis has led to the potential discovery of unexpected functions in normal endothelium. The discovery of the existence of protein synthesis at the plasma membrane in endothelial

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

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    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)

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

    International Nuclear Information System (INIS)

    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)

  5. Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines

    International Nuclear Information System (INIS)

    Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers

  6. 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

  7. 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.

  8. Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells

    DEFF Research Database (Denmark)

    Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue;

    2014-01-01

    Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important......ESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well...... in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development...

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  10. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

    DEFF Research Database (Denmark)

    Foster, Leonard J; Zeemann, Patricia A; Li, Chen;

    2005-01-01

    One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely...... in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane...... or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another...

  11. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.

    Science.gov (United States)

    Zielke, Ryszard A; Wierzbicki, Igor H; Weber, Jacob V; Gafken, Philip R; Sikora, Aleksandra E

    2014-05-01

    Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC

  12. 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.

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

    DEFF Research Database (Denmark)

    Christiansen, Helle

    Isotope Labelling with Amino acids in Culture) membrane protein fractions of the hMSC under undifferentiated as well as differentiated conditions. Our stem cell lab has developed a human MSC cell line over-expressing the hTERT subunit of the telomerase gene, enabling stable long term in vitro culture...... devoid of cell senescence as encountered in primary cultures. The cell line used in this study has been shown capable of forming bone in vivo and osteogenic differentiation in vitro. We quantified a total number of 972 proteins of which 80 % fall within the categories of: transmembrane, membrane......  Mesenchymal stem cells (MSCs) (or marrow stromal cells) represent a population of cells located in the bone marrow that are capable of differentiation into several mesodermal cell types including osteoblasts. MSCs cannot presently be isolated based on protein markers, as no specific markers exist...

  14. Proteomics and the dynamic plasma membrane

    DEFF Research Database (Denmark)

    Sprenger, Richard R; Jensen, Ole Nørregaard

    2010-01-01

    membrane proteome is crucial for understanding fundamental biological processes, disease mechanisms and for finding drug targets. Protein identification, characterization of dynamic PTMs and protein-ligand interactions, and determination of transient changes in protein expression and composition are among...

  15. Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type

    Directory of Open Access Journals (Sweden)

    Rust Steven

    2013-02-01

    Full Text Available Abstract Background The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. Methods The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. Results All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated

  16. 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

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research...... embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers......: 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...

  17. Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

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    Soderblom Erik J

    2013-01-01

    Full Text Available Abstract Background In sickle cell disease (SCD, the mitogen-activated protein kinase (MAPK ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs. ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown. Results To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2

  18. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann;

    2014-01-01

    Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line...... T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13-16-fold increased exosome yield and facilitated...... quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ- proteomics. We identified several proteins linked...

  19. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane

    DEFF Research Database (Denmark)

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels Henrik Helweg;

    2013-01-01

    granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in...

  20. Organellar proteomics: analysis of pancreatic zymogen granule membranes.

    Science.gov (United States)

    Chen, Xuequn; Walker, Angela K; Strahler, John R; Simon, Eric S; Tomanicek-Volk, Sarah L; Nelson, Bradley B; Hurley, Mary C; Ernst, Stephen A; Williams, John A; Andrews, Philip C

    2006-02-01

    The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.

  1. Evaluation of strategy for analyzing mouse liver plasma membrane proteome

    Institute of Scientific and Technical Information of China (English)

    CHEN; Ping; ZHANG; LiJun; LI; XuanWen; WANG; XiE; CAO; Rui; LIU; Zhen; XIONG; JiXian; PENG; Xia; WEI; YingJuan; YING; XingFeng; WANG; XianChun; LIANG; SongPing

    2007-01-01

    Plasma membrane (PM) proteome is one of the major subproteomes present in the cell,and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.

  2. Cell wall proteins: a new insight through proteomics

    OpenAIRE

    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-translation...

  3. Membrane labeling of coral gastrodermal cells by biotinylation: the proteomic identification of surface proteins involving cnidaria-dinoflagellate endosymbiosis.

    Directory of Open Access Journals (Sweden)

    Hsing-Hui Li

    Full Text Available The cellular and molecular-scale processes underlying the stability of coral-Symbiodinium endosymbioses remain unclear despite decades of investigation. As the coral gastroderm is the only tissue layer characterized by this unique symbiotic association, the membranes of these symbiotic gastrodermal cells (SGCs may play important roles in the initiation and maintenance of the endosymbiosis. In order to elucidate the interactions between the endosymbiotic dinoflagellates and their coral hosts, a thorough characterization of SGC membranes is therefore required. Cell surface proteins of isolated SGCs were biotinylated herein by a cell impermeant agent, biotin-XX sulfosuccinimidyl ester. The in situ distribution of these biotinylated proteins was uncovered by both fluorescence and transmission electron microscopic imaging of proteins bound to Alexa Fluor® 488-conjugated streptavidin. The identity of these proteins was then determined by two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. Nineteen (19 proteins were identified, and they are known to be involved in the molecular chaperone/stress response, cytoskeletal remodeling, and energy metabolism. These results not only reveal the molecular characters of the host SGC membrane, but also provide critical insight into understanding the possible role of host membranes in this ecologically important endosymbiotic association.

  4. Recent advances in yeast organelle and membrane proteomics.

    Science.gov (United States)

    Premsler, Thomas; Zahedi, René Peiman; Lewandrowski, Urs; Sickmann, Albert

    2009-10-01

    Yeast proteome research comprises two different aspects: with respect to systemic fungal infections (fungemias), invasive candidiasis, for instance by Candida albicans, is among the most common causes of morbidity and mortality particularly in the expanding population of immunocompromised patients, which rises a high medical and pharmaceutical interest in this facultative pathogenic organism. Apart from its clinical relevance, yeast research moreover provides an indispensable source of knowledge regarding fundamental biochemical processes of eukaryotic cells. In this context, the budding yeast Saccharomyces cerevisiae is, in addition to its multiple industrial applications, one of the most extensively used microorganisms and serves as the best understood eukaryotic model system so far. Consequently, numerous studies have focused on gaining insight into the yeast proteome, with protein MS providing a very efficient technology to cope with this task since it enables both protein identification and differential quantification of cellular material. In this review we present an overview of recent advances in yeast organelle and membrane proteomics focusing on the cell wall, plasma membrane, mitochondria and vacuole.

  5. Proteomic Substrate Identification for Membrane Proteases in the Brain

    Science.gov (United States)

    Müller, Stephan A.; Scilabra, Simone D.; Lichtenthaler, Stefan F.

    2016-01-01

    Cell-cell communication in the brain is controlled by multiple mechanisms, including proteolysis. Membrane-bound proteases generate signaling molecules from membrane-bound precursor proteins and control the length and function of cell surface membrane proteins. These proteases belong to different families, including members of the “a disintegrin and metalloprotease” (ADAM), the beta-site amyloid precursor protein cleaving enzymes (BACE), membrane-type matrix metalloproteases (MT-MMP) and rhomboids. Some of these proteases, in particular ADAM10 and BACE1 have been shown to be essential not only for the correct development of the mammalian brain, but also for myelination and maintaining neuronal connections in the adult nervous system. Additionally, these proteases are considered as drug targets for brain diseases, including Alzheimer’s disease (AD), schizophrenia and cancer. Despite their biomedical relevance, the molecular functions of these proteases in the brain have not been explored in much detail, as little was known about their substrates. This has changed with the recent development of novel proteomic methods which allow to identify substrates of membrane-bound proteases from cultured cells, primary neurons and other primary brain cells and even in vivo from minute amounts of mouse cerebrospinal fluid (CSF). This review summarizes the recent advances and highlights the strengths of the individual proteomic methods. Finally, using the example of the Alzheimer-related proteases BACE1, ADAM10 and γ-secretase, as well as ADAM17 and signal peptide peptidase like 3 (SPPL3), we illustrate how substrate identification with novel methods is instrumental in elucidating broad physiological functions of these proteases in the brain and other organs.

  6. Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins

    DEFF Research Database (Denmark)

    Elortza, Felix; Nühse, Thomas S; Foster, Leonard J;

    2003-01-01

    Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains...... development. We here present a general mass spectrometry-based proteomic "shave-and-conquer" strategy that specifically targets GPI-APs. Using a combination of biochemical methods, mass spectrometry, and computational sequence analysis we identified six GPI-APs in a Homo sapiens lipid raft-enriched fraction...... and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date....

  7. Proteomics of MUC1-containing lipid rafts from plasma membranes and exosomes of human breast carcinoma cells MCF-7.

    Science.gov (United States)

    Staubach, Simon; Razawi, Hanieh; Hanisch, Franz-Georg

    2009-05-01

    Apically expressed human MUC1 is known to become endocytosed and either to re-enter the secretory pathway for recycling to the plasma membrane or to be exported by the cells via the formation of multi-vesicular bodies and the release of exosomes. By using recombinant fusion-tagged MUC1 as a bait protein we followed an anti-myc affinity-based approach for isolating subpopulations of lipid rafts from the plasma membranes and exosomes of MCF-7 breast cancer cells. MUC1(+) lipid rafts were not only found to contain genuine raft proteins (flotillin-1, prohibitin, G protein, annexin A2), but also raft-associated proteins linking these to the cytoskeleton (ezrin/villin-2, profilin II, HSP27, gamma-actin, beta-actin) or proteins in complexes with raft proteins, including the bait protein (HSP60, HSP70). Major overlaps were revealed for the subproteomes of plasma membranous and exosomal lipid raft preparations, indicating that MUC1 is sorted into subpopulations of rafts for its trafficking via flotillin-dependent pathways and export via exosomes.

  8. Analysis of the membrane proteome of ciprofloxacin-resistant macrophages by stable isotope labeling with amino acids in cell culture (SILAC.

    Directory of Open Access Journals (Sweden)

    Nancy E Caceres

    Full Text Available Overexpression of multidrug transporters is a well-established mechanism of resistance to chemotherapy, but other changes may be co-selected upon exposure to drugs that contribute to resistance. Using a model of J774 macrophages made resistant to the fluoroquinolone antibiotic ciprofloxacin and comparing it with the wild-type parent cell line, we performed a quantitative proteomic analysis using the stable isotope labeling with amino acids in cell culture technology coupled with liquid chromatography electrospray ionization Fourier transform tandem mass spectrometry (LC-ESI-FT-MS/MS on 2 samples enriched in membrane proteins (fractions F1 and F2 collected from discontinuous sucrose gradient. Nine hundred proteins were identified with at least 3 unique peptides in these 2 pooled fractions among which 61 (F1 and 69 (F2 showed a significantly modified abundance among the 2 cell lines. The multidrug resistance associated protein Abcc4, known as the ciprofloxacin efflux transporter in these cells, was the most upregulated, together with Dnajc3, a protein encoded by a gene located downstream of Abcc4. The other modulated proteins are involved in transport functions, cell adhesion and cytoskeleton organization, immune response, signal transduction, and metabolism. This indicates that the antibiotic ciprofloxacin is able to trigger a pleiotropic adaptative response in macrophages that includes the overexpression of its efflux transporter.

  9. 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...... identified phosphorylated and SA glycosylated proteins, respectively. This study allowed us to identify several significantly regulated proteins during the differentiation process, including proteins involved in the early embryonic development as well as in the neural development. In the latter group...... of proteins we could identify a number of proteins associated with synaptic vesicles, which are vesicles that store neurotransmitters in the nerve-terminals. An example of an upregulated protein in hESCs is the gap junction alpha 1 (GJA1), a phosphorylated protein which plays a crucial role in embryonic...

  10. Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics.

    Science.gov (United States)

    Moore, Stephanie M; Hess, Stephanie M; Jorgenson, James W

    2016-04-01

    The importance of membrane proteins in biological systems is indisputable; however, their amphipathic nature makes them difficult to analyze. In this study, the most popular techniques for extraction, enrichment, solubilization, and digestion are compared, resulting in an overall improved workflow for the insoluble portion of Saccharomyces cerevisiae cell lysate. Yeast cells were successfully lysed using a French press pressure cell at 20 000 psi, and resulting proteins were fractionated prior to digestion to reduce sample complexity. The proteins were best solubilized with the addition of ionic detergent sodium deoxycholate (1%) and through the application of high-frequency sonication prior to a tryptic digestion at 37 °C. Overall, the improved membrane proteomic workflow resulted in a 26% increase in membrane protein identifications for baker's yeast. In addition, more membrane protein identifications were unique to the improved protocol. When comparing membrane proteins that were identified in the improved protocol and the standard operating procedure (176 proteins), 93% of these proteins were present in greater abundance (higher intensity) when using the improved method. PMID:26979493

  11. Cell wall proteomics of crops

    OpenAIRE

    Komatsu, Setsuko; Yanagawa, Yuki

    2013-01-01

    Cell wall proteins play key roles in cell structure and metabolism, cell enlargement, signal transduction, responses to environmental stress, and many other physiological events. Agricultural crops are often used for investigating stress tolerance because cultivars with differing degrees of tolerance are available. Abiotic and biotic stress factors markedly influence the geographical distribution and yields of many crop species. Crop cell wall proteomics is of particular importance for improv...

  12. Cassava root membrane proteome reveals activities during storage root maturation.

    Science.gov (United States)

    Naconsie, Maliwan; Lertpanyasampatha, Manassawe; Viboonjun, Unchera; Netrphan, Supatcharee; Kuwano, Masayoshi; Ogasawara, Naotake; Narangajavana, Jarunya

    2016-01-01

    Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. Its storage roots are used as food, feed, starch production, and be the important source for biofuel and biodegradable plastic production. Despite the importance of cassava storage roots, little is known about the mechanisms involved in their formation. This present study has focused on comparison of the expression profiles of cassava root proteome at various developmental stages using two-dimensional gel electrophoresis and LC-MS/MS. Based on an anatomical study using Toluidine Blue, the secondary growth was confirmed to be essential during the development of cassava storage root. To investigate biochemical processes occurring during storage root maturation, soluble and membrane proteins were isolated from storage roots harvested from 3-, 6-, 9-, and 12-month-old cassava plants. The proteins with differential expression pattern were analysed and identified to be associated with 8 functional groups: protein folding and degradation, energy, metabolism, secondary metabolism, stress response, transport facilitation, cytoskeleton, and unclassified function. The expression profiling of membrane proteins revealed the proteins involved in protein folding and degradation, energy, and cell structure were highly expressed during early stages of development. Integration of these data along with the information available in genome and transcriptome databases is critical to expand knowledge obtained solely from the field of proteomics. Possible role of identified proteins were discussed in relation with the activities during storage root maturation in cassava.

  13. 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.

  14. Characterization of membrane-shed micro-vesicles from cytokine-stimulated beta-cells using proteomics strategies

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Jensen, Soren Skov; Le Bihan, Marie Catherine;

    2012-01-01

    amino acids in cell culture (SILAC) combined with mass spectrometry. We identified and quantified a large number of beta-cell specific proteins and proteins previously described in micro-vesicles from other cell types in addition to new proteins located to these vesicles. In addition, we quantified...... specific sites of protein phosphorylation and sialylated glycosylation in proteins associated with micro-vesicles from beta-cells. Using pathway analysis software we were able to map the most distinctive changes between micro-vesicles generated during growth and after cytokine stimulation to several cell...

  15. 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.

  16. Purification and proteomics of pathogen-modified vacuoles and membranes

    Directory of Open Access Journals (Sweden)

    Jo-Ana eHerweg

    2015-06-01

    Full Text Available Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e. the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.

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

    Directory of Open Access Journals (Sweden)

    Christian Niehage

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

  18. A Proteomics Approach to Membrane Trafficking

    NARCIS (Netherlands)

    Groen, A.J.; Vries, de S.C.; Lilley, K.S.

    2008-01-01

    Membrane trafficking, including that of integral membrane proteins as well as peripherally associated proteins, appears to be a vital process common to all eukaryotes. An important element of membrane trafficking is to determine the protein composition of the various endomembrane compartments. A maj

  19. 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...

  20. Proteomic Profiling of Detergent Resistant Membranes (Lipid Rafts) of Prostasomes.

    Science.gov (United States)

    Dubois, Louise; Ronquist, Karl K Göran; Ek, Bo; Ronquist, Gunnar; Larsson, Anders

    2015-11-01

    Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.

  1. 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

    2012-11-01

    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, multidimensional 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 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 the 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. Five-hundred four 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

  2. Neural Stem Cells (NSCs) and Proteomics.

    Science.gov (United States)

    Shoemaker, Lorelei D; Kornblum, Harley I

    2016-02-01

    Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. PMID:26494823

  3. A Shotgun Proteomic Method for the Identification of Membrane-Embedded Proteins and Peptides

    OpenAIRE

    Blackler, Adele R.; Speers, Anna E.; Ladinsky, Mark S.; Wu, Christine C

    2008-01-01

    Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome.

  4. RNAi-mediated downregulation of poplar plasma membrane intrinsic proteins (PIPs) changes plasma membrane proteome composition and affects leaf physiology.

    Science.gov (United States)

    Bi, Zhen; Merl-Pham, Juliane; Uehlein, Norbert; Zimmer, Ina; Mühlhans, Stefanie; Aichler, Michaela; Walch, Axel Karl; Kaldenhoff, Ralf; Palme, Klaus; Schnitzler, Jörg-Peter; Block, Katja

    2015-10-14

    Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. PMID:26248320

  5. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

    Science.gov (United States)

    Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

    2005-07-01

    Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

  6. The pancreatic beta cell surface proteome

    OpenAIRE

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

    2012-01-01

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

  7. Proteomics of cell-cell interactions in health and disease.

    Science.gov (United States)

    Lindoso, Rafael S; Sandim, Vanessa; Collino, Federica; Carvalho, Adriana B; Dias, Juliana; da Costa, Milene R; Zingali, Russolina B; Vieyra, Adalberto

    2016-01-01

    The mechanisms of cell-cell communications are now under intense study by proteomic approaches. Proteomics has unraveled changes in protein profiling as the result of cell interactions mediated by ligand/receptor, hormones, soluble factors, and the content of extracellular vesicles. Besides being a brief overview of the main and profitable methodologies now available (evaluating theory behind the methods, their usefulness, and pitfalls), this review focuses on-from a proteome perspective-some signaling pathways and post-translational modifications (PTMs), which are essential for understanding ischemic lesions and their recovery in two vital organs in mammals, the heart, and the kidney. Knowledge of misdirection of the proteome during tissue recovery, such as represented by the convergence between fibrosis and cancer, emerges as an important tool in prognosis. Proteomics of cell-cell interaction is also especially useful for understanding how stem cells interact in injured tissues, anticipating clues for rational therapeutic interventions. In the effervescent field of induced pluripotency and cell reprogramming, proteomic studies have shown what proteins from specialized cells contribute to the recovery of infarcted tissues. Overall, we conclude that proteomics is at the forefront in helping us to understand the mechanisms that underpin prevalent pathological processes. PMID:26552723

  8. 鼻咽癌5-8F细胞膜蛋白质组初步分析%A Preliminary Analysis of Plasma Membrane Proteome of Nasopharyngeal Carcinoma Cell 5-8F

    Institute of Scientific and Technical Information of China (English)

    张鹏飞; 曾谷清; 易红; 梁宋平; 陈主初; 肖志强

    2012-01-01

    The proteins on the membrane act as"doorbells"and"doorways"playing crucial roles in cell function including intercellular communication, cellular development, cell migration, and immune response. To comprehensively identify metastasis associated proteins of nasopharyngeal carcinoma (NPC) cell 5-8F plasma membrane, we have adopted a proteomics strategy that utilizes differential centrifugation combined with aqueous two-phase partition for plasma membrane purification, followed by SDS-PAGE for protein isolation, mass spectrometry and bioinformatics for proteins identification. After LC-MS/MS analysis, a total of 316 proteins of 5-8F cell plasma membrane preparations were identified. Of the identified proteins 152 were plasma membrane and membrane-associated proteins (accounting for 48.5%). Through differentially expressed proteins in human cancers database (dbDEPD) searching, 49 kinds of membrane proteins in 114 i-dentified membrane proteins were tumor related proteins, of which 21 membrane proteins associated with tumor metastasis. Further analysis found that CD 104, VDAC2, CD298 and SLC25A3 were head and neck cancer (oral cancer) metastasis related proteins, suggesting that these membrane proteins may be potential metastasis associated proteins in NPC. The present experimental results accumulated valuable data for understanding the carcinogenesis mechanism of NPC and constructing the protein database specific for NPC cell.%细胞膜蛋白是细胞的重要组成部分,作为细胞的“门铃”与“门户”,参与细胞内外物质交换、信息转换、细胞生长发育、细胞迁移以及免疫应答等重要生理活动.为鉴定鼻咽癌转移相关膜蛋白,运用差速离心联合双水相方法分离纯化鼻咽癌高转移细胞5-8F的细胞膜,SDS-PAGE分离膜蛋白,液相色谱/电喷雾串联质谱分析(LC-MS/MS)结合生物信息学分析鉴定出316种非冗余蛋白质,其中152种(48.5%)被注释为膜蛋白或膜相关蛋白.通过肿瘤

  9. A workflow for peptide-based proteomics in a poorly sequenced plant: A case study on the plasma membrane proteome of banana

    DEFF Research Database (Denmark)

    Vertommen, A.; Laurell Blom Møller, Anders; Cordewener, J. H. G.;

    2011-01-01

    for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins.In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i......, integral plasma membrane proteins from banana leaves were successfully identified....

  10. Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

    Directory of Open Access Journals (Sweden)

    Campbell Kevin P

    2007-08-01

    Full Text Available Abstract Background Polymorphonuclear neutrophils (PMN constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.

  11. Membrane proteomics of phagosomes suggests a connection to autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Shui, Wenqing; Sheu, Leslie; Liu, Jun; Smart, Brian; Petzold, Christopher J.; Hsieh, Tsung-yen; Pitcher, Austin; Keasling*, Jay D.; Bertozzi*, Carolyn R.

    2008-11-25

    Phagocytosis is the central process by which macrophage cellsinternalize and eliminate infectious microbes as well as apoptoticcells. During maturation, phagosomes containing engulfed particlesfuse with various endosomal compartments through theaction of regulatory molecules on the phagosomal membrane. Inthis study, we performed a proteomic analysis of the membranefraction from latex bead-containing (LBC) phagosomes isolatedfrom macrophages. The profile, which comprised 546 proteins,suggests diverse functions of the phagosome and potential connectionsto secretory processes, toll-like receptor signaling, andautophagy. Many identified proteins were not previously knownto reside in the phagosome. We characterized several proteins inLBC phagosomes that change in abundance on induction of autophagy,a process that has been previously implicated in the hostdefense against microbial pathogens. These observations suggestcrosstalk between autophagy and phagocytosis that may be relevantto the innate immune response of macrophages.

  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. Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (¤Pisum sativum¤) symbiosomes

    DEFF Research Database (Denmark)

    Saalbach, G.; Erik, P.; Wienkoop, S.

    2002-01-01

    The legume Rhizobium symbiosis leads to the formation of a new compartment in the plant cell, the symbiosome. This compartment harbours the bacteroids surrounded by a peribacteroid membrane (PBM) originating from the plant plasma membrane. The PBM and the space between the PBM and the bacteroid...... membrane, called peribacteroid space (PS), mediate the exchange of metabolites between the symbionts. Proteome analysis was used as an approach to characterise the proteins in the PBM and the PS. A standard differential centrifugation procedure including a Percoll gradient was used for symbiosome isolation...

  14. Membrane glucocorticoid receptor activation induces proteomic changes aligning with classical glucocorticoid effects.

    Science.gov (United States)

    Vernocchi, Sara; Battello, Nadia; Schmitz, Stephanie; Revets, Dominique; Billing, Anja M; Turner, Jonathan D; Muller, Claude P

    2013-07-01

    Glucocorticoids exert rapid nongenomic effects by several mechanisms including the activation of a membrane-bound glucocorticoid receptor (mGR). Here, we report the first proteomic study on the effects of mGR activation by BSA-conjugated cortisol (Cort-BSA). A subset of target proteins in the proteomic data set was validated by Western blot and we found them responding to mGR activation by BSA-conjugated cortisol in three additional cell lines, indicating a conserved effect in cells originating from different tissues. Changes in the proteome of BSA-conjugated cortisol treated CCRF-CEM leukemia cells were associated with early and rapid pro-apoptotic, immune-modulatory and metabolic effects aligning with and possibly "priming" classical activities of the cytosolic glucocorticoid receptor (cGR). PCR arrays investigating target genes of the major signaling pathways indicated that the mGR does not exert its effects through the transcriptional activity of any of the most common kinases in these leukemic cells, but RhoA signaling emerged from our pathway analysis. All cell lines tested displayed very low levels of mGR on their surface. Highly sensitive and specific in situ proximity ligation assay visualized low numbers of mGR even in cells previously thought to be mGR negative. We obtained similar results when using three distinct anti-GR monoclonal antibodies directed against the N-terminal half of the cGR. This strongly suggests that the mGR and the cGR have a high sequence homology and most probably originate from the same gene. Furthermore, the mGR appears to reside in caveolae and its association with caveolin-1 (Cav-1) was clearly detected in two of the four cell lines investigated using double recognition proximity ligation assay. Our results indicate however that Cav-1 is not necessary for membrane localization of the GR since CCRF-CEM and Jurkat cells have a functional mGR, but did not express this caveolar protein. However, if expressed, this membrane protein

  15. Quantitative Proteomics Analysis of Leukemia Cells.

    Science.gov (United States)

    Halbach, Sebastian; Dengjel, Jörn; Brummer, Tilman

    2016-01-01

    Chronic myeloid leukemia (CML) is driven by the oncogenic fusion kinase Bcr-Abl, which organizes its own signaling network with various proteins. These proteins, their interactions, and their role in relevant signaling pathways can be analyzed by quantitative mass spectrometry (MS) approaches in various models systems, e.g., in cell culture models. In this chapter, we describe in detail immunoprecipitations and quantitative proteomics analysis using stable isotope labeling by amino acids in cell culture (SILAC) of components of the Bcr-Abl signaling pathway in the human CML cell line K562. PMID:27581145

  16. Rapid label-free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome.

    Science.gov (United States)

    Papanastasiou, Malvina; Orfanoudaki, Georgia; Kountourakis, Nikos; Koukaki, Marina; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Tsolis, Konstantinos C; Karamanou, Spyridoula; Economou, Anastassios

    2016-01-01

    Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.

  17. Differential proteomic analysis of outer membrane enriched extracts of Bacteroides fragilis grown under bile salts stress.

    Science.gov (United States)

    Boente, Renata F; Pauer, Heidi; Silva, Deborah N S; Filho, Joaquim Santos; Sandim, Vanessa; Antunes, Luis Caetano M; Ferreira, Rosana Barreto Rocha; Zingali, Russolina B; Domingues, Regina M C P; Lobo, Leandro A

    2016-06-01

    Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses. PMID:26948242

  18. The Cell Surface Proteome of Human Mesenchymal Stromal Cells

    OpenAIRE

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

    2011-01-01

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

  19. 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...

  20. Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing.

    Science.gov (United States)

    Chick, J M; Haynes, P A; Molloy, M P; Bjellqvist, B; Baker, M S; Len, A C L

    2008-03-01

    Membrane proteins are of particular interest in proteomics because of their potential therapeutic utility. Past proteomic approaches used to investigate membrane proteins have only been partially successful at providing a comprehensive analysis due to the inherently hydrophobic nature and low abundance for some of these proteins. Recently, these difficulties have been improved by analyzing membrane protein enriched samples using shotgun proteomics. In addition, the recent application of methanol-assisted trypsin digestion of membrane proteins has been shown to be a method to improve membrane protein identifications. In this study, a comparison of different concentrations of methanol was assessed for assisting membrane protein digestion with trypsin prior to analysis using a gel-based shotgun proteomics approach called peptide immobilized pH gradient isoelectric focusing (IPG-IEF). We demonstrate the use of peptide IEF on pH 3-10 IPG strips as the first dimension of two-dimensional shotgun proteomics for protein identifications from the membrane fraction of rat liver. Tryptic digestion of proteins was carried out in varying concentrations of methanol in 10 mM ammonium bicarbonate: 0% (v/v), 40% (v/v), and 60% (v/v). A total of 800 proteins were identified from 60% (v/v) methanol, which increased the protein identifications by 17% and 14% compared to 0% (v/v) methanol and 40% (v/v) methanol assisted digestion, respectively. In total, 1549 nonredundant proteins were identified from all three concentrations of methanol including 690 (42%) integral membrane proteins of which 626 of these proteins contained at least one transmembrane domain. Peptide IPG-IEF separation of peptides was successful as the peptides were separated into discrete pI regions with high resolution. The results from this study prove utility of 60% (v/v) methanol assisted digestion in conjunction with peptide IPG-IEF as an optimal shotgun proteomics technique for the separation and identification of

  1. Proteomics of the milk fat globule membrane from Camelus dromedarius.

    Science.gov (United States)

    Saadaoui, Besma; Henry, Céline; Khorchani, Touhami; Mars, Mohamed; Martin, Patrice; Cebo, Christelle

    2013-04-01

    Camel milk has been widely characterized with regards to casein and whey proteins. However, in camelids, almost nothing is known about the milk fat globule membrane (MFGM), the membrane surrounding fat globules in milk. The purpose of this study was thus to identify MFGM proteins from Camelus dromedarius milk. Major MFGM proteins (namely, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already evidenced in cow milk were identified in camel milk using MS. In addition, a 1D-LC-MS/MS approach led us to identify 322 functional groups of proteins associated with the camel MFGM. Dromedary MFGM proteins were then classified into functional categories using DAVID (the Database for Annotation, Visualization, and Integrated Discovery) bioinformatics resources. More than 50% of MFGM proteins from camel milk were found to be integral membrane proteins (mostly belonging to the plasma membrane), or proteins associated to the membrane. Enriched GO terms associated with MFGM proteins from camel milk were protein transport (p-value = 1.73 × 10(-14)), translation (p-value = 1.08 × 10(-11)), lipid biosynthetic process (p-value = 6.72 × 10(-10)), hexose metabolic process (p-value = 1.89 × 10(-04)), and actin cytoskeleton organization (p-value = 2.72 × 10(-04)). These findings will help to contribute to a better characterization of camel milk. Identified MFGM proteins from camel milk may also provide new insight into lipid droplet formation in the mammary epithelial cell.

  2. 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...

  3. Modification-specific proteomics of plasma membrane proteins

    DEFF Research Database (Denmark)

    Elortza, Felix; Mohammed, Shabaz; Bunkenborg, Jakob;

    2006-01-01

    that phospholipase D (PLD) treatment of human and plant plasma membrane fractions leads to the release of GPI-anchored proteins that were identified and characterized by capillary liquid chromatography and tandem mass spectrometry. In contrast to phospholipase C, the PLD enzyme is not affected by structural......-recognized as they are candidate cell surface biomarker molecules with potential diagnostic and therapeutic applications in molecular medicine. GPI-APs have also attracted interest in plant biotechnology because of their role in root development and cell remodeling. Using a shave-and-conquer concept, we demonstrate...

  4. Proton exchange membrane fuel cells

    CERN Document Server

    Qi, Zhigang

    2013-01-01

    Preface Proton Exchange Membrane Fuel CellsFuel CellsTypes of Fuel CellsAdvantages of Fuel CellsProton Exchange Membrane Fuel CellsMembraneCatalystCatalyst LayerGas Diffusion MediumMicroporous LayerMembrane Electrode AssemblyPlateSingle CellStackSystemCell Voltage Monitoring Module (CVM)Fuel Supply Module (FSM)Air Supply Module (ASM)Exhaust Management Module (EMM)Heat Management Module (HMM)Water Management Module (WMM)Internal Power Supply Module (IPM)Power Conditioning Module (PCM)Communications Module (COM)Controls Module (CM)SummaryThermodynamics and KineticsTheoretical EfficiencyVoltagePo

  5. Cell Membrane Softening in Cancer Cells

    Science.gov (United States)

    Schmidt, Sebastian; Händel, Chris; Käs, Josef

    Biomechanical properties are useful characteristics and regulators of the cell's state. Current research connects mechanical properties of the cytoskeleton to many cellular processes but does not investigate the biomechanics of the plasma membrane. We evaluated thermal fluctuations of giant plasma membrane vesicles, directly derived from the plasma membranes of primary breast and cervical cells and observed a lowered rigidity in the plasma membrane of malignant cells compared to non-malignant cells. To investigate the specific role of membrane rigidity changes, we treated two cell lines with the Acetyl-CoA carboxylase inhibitor Soraphen A. It changed the lipidome of cells and drastically increased membrane stiffness by up regulating short chained membrane lipids. These altered cells had a decreased motility in Boyden chamber assays. Our results indicate that the thermal fluctuations of the membrane, which are much smaller than the fluctuations driven by the cytoskeleton, can be modulated by the cell and have an impact on adhesion and motility.

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Enrichment and proteomic analysis of plasma membrane from rat dorsal root ganglions

    Directory of Open Access Journals (Sweden)

    Lin Yong

    2009-11-01

    Full Text Available Abstract Background Dorsal root ganglion (DRG neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. Plasma membrane (PM of DRG cells plays important roles in their functions. PM proteins are main performers of the functions. However, mainly due to the very low amount of DRG that leads to the difficulties in PM sample collection, few proteomic analyses on the PM have been reported and it is a subject that demands further investigation. Results By using aqueous polymer two-phase partition in combination with high salt and high pH washing, PMs were efficiently enriched, demonstrated by western blot analysis. A total of 954 non-redundant proteins were identified from the plasma membrane-enriched preparation with CapLC-MS/MS analysis subsequent to protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE or shotgun digestion. 205 (21.5% of the identified proteins were unambiguously assigned as PM proteins, including a large number of signal proteins, receptors, ion channel and transporters. Conclusion The aqueous polymer two-phase partition is a simple, rapid and relatively inexpensive method. It is well suitable for the purification of PMs from small amount of tissues. Therefore, it is reasonable for the DRG PM to be enriched by using aqueous two-phase partition as a preferred method. Proteomic analysis showed that DRG PM was rich in proteins involved in the fundamental biological processes including material exchange, energy transformation and information transmission, etc. These data would help to our further understanding of the fundamental DRG functions.

  8. Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis

    Directory of Open Access Journals (Sweden)

    Mukhopadhyay Nishit K

    2008-06-01

    Full Text Available Abstract Background Cholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry. Results DRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins

  9. Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach

    KAUST Repository

    Zhang, Huoming

    2010-07-02

    Human monocytes\\' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

  10. Proteomic analysis of liver plasma membrane from hepatitis B surface antigen transgenic mice

    Institute of Scientific and Technical Information of China (English)

    贾小芳

    2012-01-01

    Objective To explore the differential liver plasma membrane( PM) proteins that may be related to the occurrence,development and reversal process of hepatitis and to understand the pathogenesis of hepatitis and the new drug targets by performing a comparative proteomics research of liver PM between

  11. New proteomic approaches to study the organization of yeast mitochondrial membranes

    NARCIS (Netherlands)

    Gubbens, J.

    2008-01-01

    Despite their importance, membrane proteins have traditionally been underrepresented in proteomics studies due to their incompatibility with common methods used in this field. Therefore, new methods have to be developed for studying this class of proteins. In this thesis, new approaches are describe

  12. Fuel cell with ionization membrane

    Science.gov (United States)

    Hartley, Frank T. (Inventor)

    2007-01-01

    A fuel cell is disclosed comprising an ionization membrane having at least one area through which gas is passed, and which ionizes the gas passing therethrough, and a cathode for receiving the ions generated by the ionization membrane. The ionization membrane may include one or more openings in the membrane with electrodes that are located closer than a mean free path of molecules within the gas to be ionized. Methods of manufacture are also provided.

  13. Insights into physiological traits of Bifidobacterium animalis subsp. lactis BB-12 through membrane proteome analysis

    DEFF Research Database (Denmark)

    Gilad, Ofir; Hjernø, Karin; Østerlund, Eva Christina;

    2012-01-01

    Bifidobacterium animalis subsp. lactis BB-12 is a widely used probiotic strain associated with a variety of health-promoting traits. There is, however, only limited knowledge available regarding the membrane proteome and the proteins involved in oligosaccharide transport in BB-12. We applied two...... enrichment strategies to improve the identification of membrane proteins from BB-12 cultures grown on glucose and on xylo-oligosaccharides, the latter being an emerging prebiotic substrate recently reported to be fermented by BB-12. Our approach encompassed consecutive steps of detergent- and carbonate......-treatment in order to generate inside-out membrane vesicles and to interfere with binding of membrane-associated proteins to the membrane, respectively. Proteins in the enriched membrane fraction and membrane-associated fraction were digested by lysyl endopeptidase and trypsin followed by peptide sequencing by LC...

  14. Proteomic Analysis of Rice Plasma Membrane-associated Proteins in Response to Chitooligosaccharide Elicitors

    Institute of Scientific and Technical Information of China (English)

    Fang Chen; Qun Li; Zuhua He

    2007-01-01

    Chitooligomers or chitooligosaccharides (COS) are elicitors that bind to the plasma membrane (PM) and elicit various defense responses. However, the PM-bound proteins involved in elicitor-mediated plant defense responses still remain widely unknown. In order to get more information about PM proteins involved in rice defense responses, we conducted PM proteomic analysis of the rice suspension cells elicited by COS. A total of 14 up- or down-regulated protein spots were observed on 2-D gels of PM fractions at 12 h and 24 h after COS incubation. Of them, eight protein spots were successfully identified by MS (mass spectrography) and predicted to be associated to the PM and function in plant defense, including a putative PKN/PRK1 protein kinase, a putative pyruvate kinase isozyme G, a putative zinc finger protein, a putative MAR-binding protein MFP1, and a putative calcium-dependent protein kinase. Interestingly, a COS-induced pM5-like protein was identified for the first time in plants, which is a trans-membrane nodal modulator in transforming growth factor-β(TGFβ) signaling in vertebrates. We also identified two members of a rice polyprotein family, which were up-regulated by COS. Our study would provide a starting point for functionality of PM proteins in the rice basal defense.

  15. Membrane proteomics characterization of brush border membrane proteins of mice intestinal mucosa : case study: cholesterol absorption

    OpenAIRE

    Tsirogianni, Eirini

    2009-01-01

    The epithelial absorbing cells of the small intestinal villi, the enterocytes, are the main protagonists for the transport of nutrients from the intestinal lumen to the interstitial fluids. The oriented flow of nutrients is carried out by different and complementary transport systems present in the apical and the basolateral domains of the enterocyte’s plasma membrane. One of the distinctive characteristics of those intestinal cells is the presence of numerous structurally distinct protrusion...

  16. Model cell membranes

    DEFF Research Database (Denmark)

    Günther-Pomorski, Thomas; Nylander, Tommy; Cardenas Gomez, Marite

    2014-01-01

    The high complexity of biological membranes has motivated the development and application of a wide range of model membrane systems to study biochemical and biophysical aspects of membranes in situ under well defined conditions. The aim is to provide fundamental understanding of processes control...

  17. Yeast membrane proteomics using leucine metabolic labelling: Bioinformatic data processing and exemplary application to the ER-intramembrane protease Ypf1.

    Science.gov (United States)

    Nilse, Lars; Avci, Dönem; Heisterkamp, Patrick; Serang, Oliver; Lemberg, Marius K; Schilling, Oliver

    2016-10-01

    We describe in detail the usage of leucine metabolic labelling in yeast in order to monitor quantitative proteome alterations, e.g. upon removal of a protease. Since laboratory yeast strains are typically leucine auxotroph, metabolic labelling with trideuterated leucine (d3-leucine) is a straightforward, cost-effective, and ubiquitously applicable strategy for quantitative proteomic studies, similar to the widely used arginine/lysine metabolic labelling method for mammalian cells. We showcase the usage of advanced peptide quantification using the FeatureFinderMultiplex algorithm (part of the OpenMS software package) for robust and reliable quantification. Furthermore, we present an OpenMS bioinformatics data analysis workflow that combines accurate quantification with high proteome coverage. In order to enable visualization, peptide-mapping, and sharing of quantitative proteomic data, especially for membrane-spanning and cell-surface proteins, we further developed the web-application Proteator (http://proteator.appspot.com). Due to its simplicity and robustness, we expect metabolic leucine labelling in yeast to be of great interest to the research community. As an exemplary application, we show the identification of the copper transporter Ctr1 as a putative substrate of the ER-intramembrane protease Ypf1 by yeast membrane proteomics using d3-leucine isotopic labelling. PMID:27426920

  18. Streamlined Membrane Proteome Preparation for Shotgun Proteomics Analysis with Triton X-100 Cloud Point Extraction and Nanodiamond Solid Phase Extraction

    Directory of Open Access Journals (Sweden)

    Minh D. Pham

    2016-05-01

    Full Text Available While mass spectrometry (MS plays a key role in proteomics research, characterization of membrane proteins (MP by MS has been a challenging task because of the presence of a host of interfering chemicals in the hydrophobic protein extraction process, and the low protease digestion efficiency. We report a sample preparation protocol, two-phase separation with Triton X-100, induced by NaCl, with coomassie blue added for visualizing the detergent-rich phase, which streamlines MP preparation for SDS-PAGE analysis of intact MP and shot-gun proteomic analyses. MP solubilized in the detergent-rich milieu were then sequentially extracted and fractionated by surface-oxidized nanodiamond (ND at three pHs. The high MP affinity of ND enabled extensive washes for removal of salts, detergents, lipids, and other impurities to ensure uncompromised ensuing purposes, notably enhanced proteolytic digestion and down-stream mass spectrometric (MS analyses. Starting with a typical membranous cellular lysate fraction harvested with centrifugation/ultracentrifugation, MP purities of 70%, based on number (not weight of proteins identified by MS, was achieved; the weight-based purity can be expected to be much higher.

  19. Detection and identification of stable oligomeric protein complexes in Escherichi coli inner membranes: a proteomics approach.

    Science.gov (United States)

    Spelbrink, Robin E J; Kolkman, Annemieke; Slijper, Monique; Killian, J Antoinette; de Kruijff, Ben

    2005-08-01

    In this study we present a new technology to detect stable oligomeric protein complexes in membranes. The technology is based on the ability of small membrane-active alcohols to dissociate the highly stable homotetrameric potassium channel KcsA. It is shown via a proteomics approach, using diagonal electrophoresis and nano-flow liquid chromatography coupled to tandem mass spectrometry, that a large number of both integral and peripheral Escherichia coli inner membrane proteins are part of stable oligomeric complexes that can be dissociated by small alcohols. This study gives insight into the composition and stability of these complexes. PMID:15919657

  20. Plasma membrane proteomics in the maize primary root growth zone: novel insights into root growth adaptation to water stress.

    Science.gov (United States)

    Voothuluru, Priyamvada; Anderson, Jeffrey C; Sharp, Robert E; Peck, Scott C

    2016-09-01

    Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress.

  1. Plasma membrane proteomics in the maize primary root growth zone: novel insights into root growth adaptation to water stress.

    Science.gov (United States)

    Voothuluru, Priyamvada; Anderson, Jeffrey C; Sharp, Robert E; Peck, Scott C

    2016-09-01

    Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress. PMID:27341663

  2. The plasma membrane proteome of germinating barley embryos

    DEFF Research Database (Denmark)

    Hynek, Radovan; Svensson, Birte; Jensen, O.N.;

    2009-01-01

    with amphiphilicity and low abundance of membrane proteins. A fraction enriched in plasma membranes was prepared from embryos dissected from 18 h germinated barley seeds using aqueous two-phase partitioning. Reversed-phase chromatography on C-4 resin performed in micro-spin columns with stepwise elution by 2-propanol...

  3. Comparison of the Proteome of Adult and Cord Erythroid Cells, and Changes in the Proteome Following Reticulocyte Maturation.

    Science.gov (United States)

    Wilson, Marieangela C; Trakarnsanga, Kongtana; Heesom, Kate J; Cogan, Nicola; Green, Carole; Toye, Ashley M; Parsons, Steve F; Anstee, David J; Frayne, Jan

    2016-06-01

    Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared with adult peripheral blood progenitors and cord blood banks usually being more representative of national populations than blood donors. Consequently, it is important to establish how similar cord RBCs are to adult cells. In this study, we used multiplex tandem mass tag labeling combined with nano-LC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria, 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focused on proteins critical for RBC function. Of these, only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 and 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2- and 100-fold following maturation. However, ∼5% were at a higher level in RBCs, localized almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that, with respect to the proteome, there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal, not adult, hemoglobin. PMID:27006477

  4. 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.

  5. Hydroponics on a chip: analysis of the Fe deficient Arabidopsis thylakoid membrane proteome.

    Science.gov (United States)

    Laganowsky, Arthur; Gómez, Stephen M; Whitelegge, Julian P; Nishio, John N

    2009-04-13

    The model plant Arabidopsis thaliana was used to evaluate the thylakoid membrane proteome under Fe-deficient conditions. Plants were cultivated using a novel hydroponic system, called "hydroponics on a chip", which yields highly reproducible plant tissue samples for physiological analyses, and can be easily used for in vivo stable isotope labeling. The thylakoid membrane proteome, from intact chloroplasts isolated from Fe-sufficient and Fe-deficient plants grown with hydroponics on a chip, was analyzed using liquid chromatography coupled to mass spectrometry. Intact masses of thylakoid membrane proteins were measured, many for the first time, and several proteins were identified with post-translational modifications that were altered by Fe deficiency; for example, the doubly phosphorylated form of the photosystem II oxygen evolving complex, PSBH, increased under Fe-deficiency. Increased levels of photosystem II protein subunit PSBS were detected in the Fe-deficient samples. Antioxidant enzymes, including ascorbate peroxidase and peroxiredoxin Q, were only detected in the Fe-deficient samples. We present the first biochemical evidence that the two major LHC IIb proteins (LHCB1 and LHCB2) may have significantly different functions in the thylakoid membrane. The study illustrates the utility of intact mass proteomics as an indispensable tool for functional genomics. "Hydroponics on a chip" provides the ability to grow A. thaliana under defined conditions that will be useful for systems biology.

  6. Drafting the proteome landscape of myeloid-derived suppressor cells.

    Science.gov (United States)

    Gato, María; Blanco-Luquin, Idoia; Zudaire, Maribel; de Morentin, Xabier Martínez; Perez-Valderrama, Estela; Zabaleta, Aintzane; Kochan, Grazyna; Escors, David; Fernandez-Irigoyen, Joaquín; Santamaría, Enrique

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are defined by their myeloid origin, immature state, and ability to potently suppress T-cell responses. They regulate immune responses and the population significantly increases in the tumor microenvironment of patients with glioma and other malignant tumors. For their study, MDSCs are usually isolated from the spleen or directly of tumors from a large number of tumor-bearing mice although promising ex vivo differentiated MDSC production systems have been recently developed. During the last years, proteomics has emerged as a powerful approach to analyze MDSCs proteomes using shotgun-based mass spectrometry (MS), providing functional information about cellular homeostasis and metabolic state at a global level. Here, we will revise recent proteome profiling studies performed in MDSCs from different origins. Moreover, we will perform an integrative functional analysis of the protein compilation derived from these large-scale proteomic studies in order to obtain a comprehensive view of MDSCs biology. Finally, we will also discuss the potential application of high-throughput proteomic approaches to study global proteome dynamics and post-translational modifications (PTMs) during the differentiation process of MDSCs that will greatly boost the identification of novel MDSC-specific therapeutic targets to apply in cancer immunotherapy. PMID:26403437

  7. The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane

    Directory of Open Access Journals (Sweden)

    Cacciotto Carla

    2010-08-01

    Full Text Available Abstract Background Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition. Results The selective enrichment for M. agalactiae PG2T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events. Conclusions This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization.

  8. Comparison of Milk Fat Globule Membrane (MFGM Proteins of Chianina and Holstein Cattle Breed Milk Samples Through Proteomics Methods

    Directory of Open Access Journals (Sweden)

    Lorraine Pariset

    2009-12-01

    Full Text Available Identification of proteins involved in milk production is important to understand the biology of lactation. Many studies have advanced the understanding of mammary function and milk secretion, but the critical molecular mechanisms implicated in milk fat secretion is still incomplete. Milk Fat Globules are secreted from the apical surface of the mammary cells, surrounded by a thin membrane bilayer, the Milk Fat Globule Membrane (MFGM, formed by proteins which have been suggested to be cholesterolemia-lowering factors, inhibitors of cancer cell growth, vitamin binders, bactericidal, suppressors of multiple sclerosis. Using a proteomic approach, we compared MFGM from milk samples of individuals belonging to two different cattle breeds, Chianina and Holstein, representative of selection for milk and meat traits, respectively. We were able to isolate some of the major MFGM proteins in the examined samples and to identify differences between the protein fractions of the two breeds. We detected differences in the amount of proteins linked to mammary gland development and lipid droplets formation, as well as host defence mechanisms. We have shown that proteomics is a suitable, unbiased method for the study of milk fractions proteins and a powerful tool in nutritional genomics.

  9. Isobaric Tags for Relative and Absolute Quantification-based Comparative Proteomics Reveals the Features of Plasma Membrane-Associated Proteomes of Pollen Grains and Pollen Tubes from Lilium davidii

    Institute of Scientific and Technical Information of China (English)

    Bing Han; Sixue Chen; Shaojun Dai; Ning Yang; Tai Wang

    2010-01-01

    Mature pollen grains (PGs) from most plant species are metabolically quiescent. However, once pollinated onto stigma, they quickly hydrate and germinate. APG can give rise to a vegetative cell-derived polarized pollen tube (PT), which represents a specialized polar cell. The polarized PT grows by the tip and requires interaction of different signaling molecules localized in the apical plasma membrane and active membrane trafficking. The mechanisms underlying the interaction and membrane trafficking are not well understood. In this work, we purified PG and PT plasma-membrane vesicles from Lilium davidii Duch. using the aqueous two-phase partition technique, then enriched plasma membrane proteins by using Brij58 and KCl to remove loosely bound contaminants. We identified 223 integral and membrane-associated proteins in the plasma membrane of PGs and PTs by using isobaric tags for relative and absolute quantification (iTRAQ) and 2-D high-performance liquid chromatography-tandem mass spectrometry. More than 68% of the proteins have putative transmembrane domains and/or lipid-modified motifs. Proteins involved in signal transduction, membrane trafficking and transport are predominant in the plasma-membrane proteome. We revealed most components of the clathrin-dependent endocytosis pathway. Statistical analysis revealed 14 proteins differentially expressed in the two development stages: in PTs, six upregulated and eight downregulated are mainly involved in signaling, transport and membrane trafficking. These results provide novel insights into polarized PT growth.

  10. 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...

  11. Proteomic analysis of GPI-anchored membrane proteins

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Jensen, Ole Nørregaard

    2006-01-01

    Glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) represent a subset of post-translationally modified proteins that are tethered to the outer leaflet of the plasma membrane via a C-terminal GPI anchor. GPI-APs are found in a variety of eukaryote species, from pathogenic microorganisms...

  12. WallProtDB, a database resource for plant cell wall proteomics

    OpenAIRE

    San Clemente, Hélène; Jamet, Elisabeth

    2015-01-01

    Background During the last fifteen years, cell wall proteomics has become a major research field with the publication of more than 50 articles describing plant cell wall proteomes. The WallProtDB database has been designed as a tool to facilitate the inventory, the interpretation of cell wall proteomics data and the comparisons between cell wall proteomes. Results WallProtDB (http://www.polebio.lrsv.ups-tlse.fr/WallProtDB/) presently contains 2170 proteins and ESTs identified experimentally i...

  13. Plasma membrane proteomics and its application in clinical cancer biomarker discovery

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Lund, Rikke; Ditzel, Henrik J

    2010-01-01

    Plasma membrane proteins that are exposed on the cell surface have important biological functions, such as signaling into and out of the cells, ion transport, and cell-cell and cell-matrix interactions. The expression level of many of the plasma membrane proteins involved in these key functions i...

  14. The cell surface proteome of Staphylococcus aureus

    NARCIS (Netherlands)

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

    2011-01-01

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

  15. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins

    Science.gov (United States)

    Adachi, Jun; Kumar, Chanchal; Zhang, Yanling; Olsen, Jesper V; Mann, Matthias

    2006-01-01

    Background Urine is a desirable material for the diagnosis and classification of diseases because of the convenience of its collection in large amounts; however, all of the urinary proteome catalogs currently being generated have limitations in their depth and confidence of identification. Our laboratory has developed methods for the in-depth characterization of body fluids; these involve a linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer. Here we applied these methods to the analysis of the human urinary proteome. Results We employed one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography for protein separation and fractionation. Fractionated proteins were digested in-gel or in-solution, and digests were analyzed with the LTQ-FT and LTQ-Orbitrap at parts per million accuracy and with two consecutive stages of mass spectrometric fragmentation. We identified 1543 proteins in urine obtained from ten healthy donors, while essentially eliminating false-positive identifications. Surprisingly, nearly half of the annotated proteins were membrane proteins according to Gene Ontology (GO) analysis. Furthermore, extracellular, lysosomal, and plasma membrane proteins were enriched in the urine compared with all GO entries. Plasma membrane proteins are probably present in urine by secretion in exosomes. Conclusion Our analysis provides a high-confidence set of proteins present in human urinary proteome and provides a useful reference for comparing datasets obtained using different methodologies. The urinary proteome is unexpectedly complex and may prove useful in biomarker discovery in the future. PMID:16948836

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

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  18. 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.

  19. Differential membrane proteomic study of nasopharyngeal carcinoma cell lines with different metastatic potentials%不同转移潜能鼻咽癌细胞株的差异膜蛋白质组研究

    Institute of Scientific and Technical Information of China (English)

    陈瑜; 李娇阳; 李茂玉; 肖志强

    2012-01-01

    Objective: To screen metastasis-related membrane proteins of nasopharyngeal carcinoma (NPC). Methods: After enriching membrane proteins from high metastatic NPC cell line 5-8F and non-metastatic NPC cell line 6-10B, the post-digestion isotope 18O labeling method, followed by Q-TOF-MS/MS, was conducted to identify the differentially expressed membrane proteins in the 2 cell lines. The expression of differential membrane protein EphA2 was measured by Western blot. Bioinformatics analysis was used to reveal the biological functions and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Results: A total of 31 differential membrane proteins were identified, of which 25 proteins were up-regulated in 5-8 F cells and 6 proteins were down-regulated. One of the differential proteins, EphA2, was further confirmed by Western blot. Bioinformatics analysis showed that the functions of the differential membrane proteins were mainly involved in cell adhesion, receptor recycling, and cell junction. The differential membrane proteins were also involved in 14 KEGG pathways including cell adhesion, cell junction, and cell migration pathways. Conclusion: Thirty-one differentially expressed membrane proteins may play an important role in NPC invasion and metastasis, which will provide a novel clue for further investigating the molecular mechanisms of NPC metastasis.%目的:采用定量蛋白质组学技术筛选鼻咽癌转移相关的膜蛋白质.方法:在富集高转移鼻咽癌细胞株5-8F和不转移鼻咽癌细胞株6-10B膜蛋白质的基础上,采用稳定同位素18O标记结合串联质谱技术比较两株细胞膜蛋白质组的差异,采用Western印迹对差异表达膜蛋白质EphA2的表达水平进行验证,采用生物信息学方法对差异蛋白质进行GO(gene oncology)功能聚类和全基因组及代谢途径数据库(Kyoto encyclopedia of genes and genomes,KEGG)信号通路分析.结果:鉴定了31个5-8F与6-10B细胞的差异膜蛋白,其中25

  20. Dielectric breakdown of cell membranes.

    Science.gov (United States)

    Zimmermann, U; Pilwat, G; Riemann, F

    1974-11-01

    With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 . 10(6) V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector

  1. 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

  2. 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.

  3. Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

    Directory of Open Access Journals (Sweden)

    Marlena M Wilson

    Full Text Available Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

  4. 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.

  5. Immunoscreening of the extracellular proteome of colorectal cancer cells

    International Nuclear Information System (INIS)

    The release of proteins from tumors can trigger an immune response in cancer patients involving T lymphocytes and B lymphocytes, which results in the generation of antibodies to tumor-derived proteins. Many studies aim to use humoral immune responses, namely autoantibody profiles, directly, as clinical biomarkers. Alternatively, the antibody immune response as an amplification system for tumor associated alterations may be used to indicate putative protein biomarkers with high sensitivity. Aiming at the latter approach we here have implemented an autoantibody profiling strategy which particularly focuses on proteins released by tumor cells in vitro: the so-called secretome. For immunoscreening, the extracellular proteome of five colorectal cancer cell lines was resolved on 2D gels, immobilized on PVDF membranes and used for serological screening with individual sera from 21 colorectal cancer patients and 24 healthy controls. All of the signals from each blot were assigned to a master map, and autoantigen candidates were defined based of the pattern of immunoreactivities. The corresponding proteins were isolated from preparative gels, identified by MALDI-MS and/or by nano-HPLC/ESI-MS/MS and exemplarily confirmed by duplex Western blotting combining the human serum samples with antibodies directed against the protein(s) of interest. From 281 secretome proteins stained with autoantibodies in total we first defined the 'background patterns' of frequently immunoreactive extracellular proteins in healthy and diseased people. An assignment of these proteins, among them many nominally intracellular proteins, to the subset of exosomal proteins within the secretomes revealed a large overlap. On this basis we defined and consequently confirmed novel biomarker candidates such as the extreme C-terminus of the extracellular matrix protein agrin within the set of cancer-enriched immunorectivities. Our findings suggest, first, that autoantibody responses may be due, in

  6. 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

    Abstract The understanding of the early mammalian development is a prerequisite for the advancement of in vitro fertilization and improvement of derivation and culturing of embryonic stem cells. While, whole genome transcriptomic analysis on bovine blastocysts has identified genes active in early...... 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...... by micromanipulation. From two independent replicates, 23 proteins were identified in the blastocoel fluid while 803 proteins were identified in the remaining cell material. The proteins were grouped into categories according to their gene ontology (GO) terms by which proteins involved in cell differentiation, cell...

  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. Corrugated Membrane Fuel Cell Structures

    Energy Technology Data Exchange (ETDEWEB)

    Grot, Stephen [President, Ion Power Inc.

    2013-09-30

    One of the most challenging aspects of traditional PEM fuel cell stacks is the difficulty achieving the platinum catalyst utilization target of 0.2 gPt/kWe set forth by the DOE. Good catalyst utilization can be achieved with state-of-the-art catalyst coated membranes (CCM) when low catalyst loadings (<0.3 mg/cm2) are used at a low current. However, when low platinum loadings are used, the peak power density is lower than conventional loadings, requiring a larger total active area and a larger bipolar plate. This results in a lower overall stack power density not meeting the DOE target. By corrugating the fuel cell membrane electrode structure, Ion Power?s goal is to realize both the Pt utilization targets as well as the power density targets of the DOE. This will be achieved by demonstrating a fuel cell single cell (50 cm2) with a twofold increase in the membrane active area over the geometric area of the cell by corrugating the MEA structure. The corrugating structure must be able to demonstrate the target properties of < 10 mOhm-cm2 electrical resistance at > 20 psi compressive strength over the active area, in combination with offering at least 80% of power density that can be achieved by using the same MEA in a flat plate structure. Corrugated membrane fuel cell structures also have the potential to meet DOE power density targets by essentially packaging more membrane area into the same fuel cell volume as compared to conventional stack constructions.

  9. Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers.

    Science.gov (United States)

    Gubbens, Jacob; Vader, Pieter; Damen, J Mirjam A; O'Flaherty, Martina C; Slijper, Monique; de Kruijff, Ben; de Kroon, Anton I P M

    2007-05-01

    To analyze proteins interacting at the membrane interface, a phospholipid analogue was used with a photoactivatable headgroup (ASA-DLPE, N-(4-azidosalicylamidyl)-1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) for selective cross-linking. The peripheral membrane protein cytochrome c from the inner mitochondrial membrane was rendered carbonate wash-resistant by cross-linking to ASA-DLPE in a model membrane system, validating our approach. Cross-link products of cytochrome c and its precursor apocytochrome c were demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and were specifically detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), taking advantage of the intrinsic UV absorbance of the cross-linker. Application of the method to inner mitochondrial membranes from Saccharomyces cerevisae revealed cross-link products of both exogenously added apocytochrome c and endogenous proteins with molecular weights around 34 and 72 kDa. Liquid chromatograpy (LC)-MS/MS was performed to identify these proteins, resulting in a list of candidate proteins potentially cross-linked at the membrane interface. The approach described here provides methodology for capturing phospholipid-protein interactions in their native environment of the biomembrane using modern proteomics techniques. PMID:17375948

  10. Proteomic Analysis of the Rat Canalicular Membrane Reveals Expression of a Complex System of P4-ATPases in Liver.

    Science.gov (United States)

    Chaubey, Pururawa Mayank; Hofstetter, Lia; Roschitzki, Bernd; Stieger, Bruno

    2016-01-01

    Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes.

  11. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    Science.gov (United States)

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  12. Learning robust cell signalling models from high throughput proteomic data

    OpenAIRE

    Koch, Mitchell; Broom, Bradley M.; Subramanian, Devika

    2009-01-01

    We propose a framework for learning robust Bayesian network models of cell signalling from high-throughput proteomic data. We show that model averaging using Bayesian bootstrap resampling generates more robust structures than procedures that learn structures using all of the data. We also develop an algorithm for ranking the importance of network features using bootstrap resample data. We apply our algorithms to derive the T-cell signalling network from the flow cytometry data of Sachs et al....

  13. Proteome analysis of bronchoalveolar lavage in pulmonary langerhans cell histiocytosis

    OpenAIRE

    2011-01-01

    Background Pulmonary Langerhans-cell histiocytosis (PLCH) is a rare interstitial lung disease characterized by clusters of Langerhans cells, organized in granulomas, in the walls of distal bronchioles. It is a diffuse lung disease related to tobacco smoking but otherwise of unknown etiopathogenesis. Methods In this study we used a proteomic approach to analyze BAL protein composition of patients with PLCH and of healthy smoker and non-smoker controls to obtain insights into the pathogenetic m...

  14. Global Proteome Analysis of the NCI-60 Cell Line Panel

    Directory of Open Access Journals (Sweden)

    Amin Moghaddas Gholami

    2013-08-01

    Full Text Available The NCI-60 cell line collection is a very widely used panel for the study of cellular mechanisms of cancer in general and in vitro drug action in particular. It is a model system for the tissue types and genetic diversity of human cancers and has been extensively molecularly characterized. Here, we present a quantitative proteome and kinome profile of the NCI-60 panel covering, in total, 10,350 proteins (including 375 protein kinases and including a core cancer proteome of 5,578 proteins that were consistently quantified across all tissue types. Bioinformatic analysis revealed strong cell line clusters according to tissue type and disclosed hundreds of differentially regulated proteins representing potential biomarkers for numerous tumor properties. Integration with public transcriptome data showed considerable similarity between mRNA and protein expression. Modeling of proteome and drug-response profiles for 108 FDA-approved drugs identified known and potential protein markers for drug sensitivity and resistance. To enable community access to this unique resource, we incorporated it into a public database for comparative and integrative analysis (http://wzw.tum.de/proteomics/nci60.

  15. 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.

  16. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  17. Proteomic profiling of a robust Wolbachia infection in an Aedes albopictus mosquito cell line.

    Science.gov (United States)

    Baldridge, Gerald D; Baldridge, Abigail S; Witthuhn, Bruce A; Higgins, LeeAnn; Markowski, Todd W; Fallon, Ann M

    2014-11-01

    Wolbachia pipientis, a widespread vertically transmitted intracellular bacterium, provides a tool for insect control through manipulation of host-microbe interactions. We report proteomic characterization of wStr, a Wolbachia strain associated with a strong cytoplasmic incompatibility phenotype in its native host, Laodelphax striatellus. In the Aedes albopictus C/wStr1 mosquito cell line, wStr maintains a robust, persistent infection. MS/MS analyses of gel bands revealed a protein 'footprint' dominated by Wolbachia-encoded chaperones, stress response and cell membrane proteins, including the surface antigen WspA, a peptidoglycan-associated lipoprotein and a 73 kDa outer membrane protein. Functional classifications and estimated abundance levels of 790 identified proteins suggested that expression, stabilization and secretion of proteins predominate over bacterial genome replication and cell division. High relative abundances of cysteine desulphurase, serine/glycine hydroxymethyl transferase, and components of the α-ketoglutarate dehydrogenase complex in conjunction with above average abundances of glutamate dehydrogenase and proline utilization protein A support Wolbachia genome-based predictions for amino acid metabolism as a primary energy source. wStr expresses 15 Vir proteins of a Type IV secretion system and its transcriptional regulator. Proteomic characterization of a robust insect-associated Wolbachia strain provides baseline information that will inform further development of in vitro protocols for Wolbachia manipulation. PMID:25155417

  18. Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique

    Directory of Open Access Journals (Sweden)

    Appleton Hazel

    2010-02-01

    Full Text Available Abstract Background Salmonella enterica serovar Typhimurium (S. Typhimurium is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI™ FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS and identified from mass spectral database searches. Results In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX. Conclusion Using a multi-step digest approach the LPI™ technique enables the incorporation of a

  19. What Is Cancer Proteomics?

    Science.gov (United States)

    ... What is Proteomics? Video Tutorial What is Cancer Proteomics? Print This Page The term "proteome" refers to ... that a cell or organism undergoes. The term "proteomics" is a large-scale comprehensive study of a ...

  20. 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.

  1. Identification of salt-tolerant Sinorhizobium sp. strain BL3 membrane proteins based on proteomics

    DEFF Research Database (Denmark)

    Tanthanuch, Waraporn; Tittabutr, Panlada; Mohammed, Shabaz;

    2010-01-01

    Sinorhizobium sp. BL3 is a salt-tolerant strain that can fix atmospheric nitrogen in symbiosis with leguminous host plants under salt-stress conditions. Since cell membranes are the first barrier to environmental change, it is interesting to explore the membrane proteins within this protective...... functional categories, the two biggest of which were energy production and conversion, and proteins not in clusters of orthologous groups (COGs). In addition, a comparative analysis of membrane proteins between salt-stressed and non-stressed BL3 cells was conducted using a membrane enrichment method and off...... barrier under salt stress. The protein contents of membrane-enriched fractions obtained from BL3 were analyzed by nanoflow liquid chromatography interfaced with electrospray ionization tandem mass spectrometry. A total of 105 membrane proteins were identified. These proteins could be classified into 17...

  2. Plasma membrane proteome analysis of the early effect of alcohol on liver:implications for alcoholic liver disease

    Institute of Scientific and Technical Information of China (English)

    Lijun Zhang; Ye Zheng; Pengyuan Yang; Zhenghong Yuan; Xiaofang Jia; Yanling Feng; Xia Peng; Zhiyong Zhang; Wenjiang Zhou; Zhanqing Zhang; Fang Ma; Xiaohui Liu

    2011-01-01

    In humans, the over-consumption of alcohol can lead to serious liver disease. To examine the early effects of alcohol on liver disease, rats were given sufficient ethanol to develop liver cirrhosis. Rats before the onset of fibrosis were studied in this work. Plasma membranes (PM) of liver were extracted by twice sucrose density gradient centrifugation. The proteome profiles of PM from ethanol-treated rats and the controls were analyzed using two-dimensional gel electrophoresis (2-DE) and isobaric tag for relative and absolute quantitation (iTRAQ) tech-nology. Ethanol treatment altered the amount of 15 differ-ent liver proteins: 10 of them were detected by 2-DE and 5 by iTRAQ. Keratin 8 was detected by both methods.Gene ontology analysis of these differentially detected proteins indicated that most of them were involved in important cell functions such as binding activity (includ-ing ion, DNA, ATP binding, etc.), cell structure, or enzyme activity. Among these, annexin A2, keratin 8, and keratin 18 were further verified using western blot analy-sis and annexin A2 was verified by immunohistochemis-try. Our results suggested that alcohol has the potential to affect cell structure, adhesion and enzyme activity by altering expression levels of several relevant proteins in the PM. To the best of our knowledge, this is the first time to study the effect of alcohol on the liver PM pro-teome and it might be helpful for understanding the poss-ible mechanisms of alcohol-induced liver disease.

  3. Mass spectrometry based proteomics in cell biology and signaling research

    International Nuclear Information System (INIS)

    Full text: Proteomics is one of the most powerful post-genomics technologies. Recently accomplishments include large scale protein-protein interaction mapping, large scale mapping of phosphorylation sites and the cloning of key signaling molecules. In this talk, current state of the art of the technology will be reviewed. Applications of proteomics to the mapping of multiprotein complexes will be illustrated with recent work on the spliceosome and the nucleolus. More than 300 proteins have been mapped to each of these complexes. Quantitative techniques are becoming more and more essential in proteomics. They are usually performed by the incorporation of stable isotopes - a light form in cell state 'A' and a heavy form in cell state 'E' - and subsequent comparison of mass spectrometric peak heights. A new technique called, SILAC for Stable isotope Incorporation by Amino acids in Cell culture, has been applied to studying cell differentiation and mapping secreted proteins from adipocytes. A number of known and novel proteins important in adipocyte differentiation have been identified by this technique. Some of these proved to be upregulated at the 1 mRNA level, too, whereas others appear to be regulated post-translationally. We have also applied the SILAC method to protein-protein interaction mapping. For example, we compared immunoprecipitates from stimulated and non-stimulated cells to find binding partners recruited to the bait due to the stimulus. Several novel substrates in the EGF pathway were found in this way. An important application of proteomics in the signaling field is the mapping of post-translational modifications. In particular, there are a number of techniques for phosphotyrosine phosphorylation mapping which have proven very useful. Making use of the mass deficiency of the phosphogroup, 'parent ion scans' con be performed, which selectively reveal phosphotyrosine peptides from complex peptides mixtures. This technique has been used to clone several

  4. The Genomic and Proteomic Content of Cancer Cell-Derived Exosomes

    Directory of Open Access Journals (Sweden)

    Meredith C Henderson

    2012-04-01

    Full Text Available Exosomes are secreted membrane vesicles that have been proposed as an effective means to detect a variety of disease states, including cancer. The properties of exosomes, including stability in biological fluids, allow for their efficient isolation and make them an ideal vehicle for studies on early disease detection and evaluation. Much data has been collected over recent years regarding the mRNA, miRNA, and protein contents of exosomes. In addition, many studies have described the functional role that exosomes play in disease initiation and progression. Tumor cells have been shown to secrete exosomes, often in increased amounts compared to normal cells, and these exosomes can carry the genomic and proteomic signatures characteristic of the tumor cells from which they were derived. While these unique signatures make exosomes ideal for cancer detection, exosomes derived from cancer cells have also been shown to play a functional role in cancer progression. Here, we review the unique genomic and proteomic contents of exosomes originating from cancer cells as well as their functional effects to promote tumor progression.

  5. 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.

  6. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    Science.gov (United States)

    Zhang, Haizhen; Brown, Roslyn N.; Qian, Wei-Jun; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Gritsenko, Marina A.; Shi, Liang; Romine, Margaret F; Fredrickson, James K.; Paša-Tolić, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2010-01-01

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope 18O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level 16O and 18O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in ΔgspD mutant cells of many outer membrane proteins including the outer membrane c-cype cytochromes OmcA and MtrC, in agreement with previously investigation demonstrating that these proteins are substrates of the type II secretion system. PMID:20380418

  7. STUDYING MEMBRANE ANCHOR ORGANIZATION IN LIVING CELL MEMBRANES

    OpenAIRE

    Huang, Hector Han-Li

    2011-01-01

    The cell membrane is a complex mixture of various lipids, proteins and other biomolecules that are all organized into a fluid 2-dimensional bilayer. A rather unique trait of this organelle is the lateral mobility of the component molecules. Surprisingly, these molecules are not necessarily distributed homogeneously in the membrane. From a physical perspective, these inhomogeneities are interesting because they indicate some level of organization in the membrane. From a biological perspect...

  8. 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. PMID:25211708

  9. 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.

  10. Fuel-Cell Structure Prevents Membrane Drying

    Science.gov (United States)

    Mcelroy, J.

    1986-01-01

    Embossed plates direct flows of reactants and coolant. Membrane-type fuel-cell battery has improved reactant flow and heat removal. Compact, lightweight battery produces high current and power without drying of membranes.

  11. Comparative proteomics of milk fat globule membrane in different species reveals variations in lactation and nutrition.

    Science.gov (United States)

    Lu, Jing; Wang, Xinyu; Zhang, Weiqing; Liu, Lu; Pang, Xiaoyang; Zhang, Shuwen; Lv, Jiaping

    2016-04-01

    In present study, 312, 554, 175 and 143 proteins were identified and quantified by label-free quantitative proteomics in human, cow, goat and yak milk fat globule membrane (MFGM), respectively. Fifty proteins involved in vesicle mediate transport and milk fat globule secretion were conserved among species. Moreover, proteins involved in lipid synthesis and secretion (xanthine dehydrogenase/oxidase, stomatin and CD36), showed different expression pattern and the host defense proteins exhibited various profiles within species. Notably, the content and activity of lipid catabolic enzymes were significantly higher in human MFGM, which could be indicative of the superior fat utilization in breast fed infants. Our findings unraveled the significant differences in protein composition of human milk and conventionally used substitutes of it. The in-depth study of lipid metabolic enzymes in human MFGM will probably contribute to the improvement of the fat utilization through modulation of lipid catabolic enzymes in infant formula.

  12. Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

    Science.gov (United States)

    Mol, Juliana P. S.; Pires, Simone F.; Chapeaurouge, Alexander D.; Perales, Jonas; Santos, Renato L.; Andrade, Hélida M.; Lage, Andrey P.

    2016-01-01

    Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation. PMID:27104343

  13. 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

  14. POLYMER ELECTROLYTE MEMBRANE FUEL CELLS

    DEFF Research Database (Denmark)

    2001-01-01

    thermoplastic polymers for high temperature polymer electrolyte fuel cells have also been developed. Miscible blends are used for solution casting of polymer membranes (solid electrolytes). High conductivity and enhanced mechanical strength were obtained for the blend polymer solid electrolytes....... With the thermally resistant polymer, e.g., polybenzimidazole or a mixture of polybenzimidazole and other thermoplastics as binder, the carbon-supported noble metal catalyst is tape-cast onto a hydrophobic supporting substrate. When doped with an acid mixture, electrodes are assembled with an acid doped solid...

  15. Proteomic Analysis of the Rat Canalicular Membrane Reveals Expression of a Complex System of P4-ATPases in Liver.

    Directory of Open Access Journals (Sweden)

    Pururawa Mayank Chaubey

    Full Text Available Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes.

  16. Chemical degradation mechanisms of membranes for alkaline membrane fuel cells

    International Nuclear Information System (INIS)

    Chemical degradation mechanisms of membranes for alkaline membrane fuel cells have been investigated using density functional theory (DFT). We have elucidated that the aryl-ether moiety of membranes is one of the weakest site against attack of hydroxide ions. The results of DFT calculations for hydroxide initiated aryl-ether cleavage indicated that the aryl-ether cleavage occurred prior to degradation of cationic functional group. Such a weak nature of the aryl-ether group arises from the electron deficiency of the aryl group as well as the low bond dissociation energy. The DFT results suggests that removal of the aryl-ether group in the membrane should enhance the stability of membranes under alkaline conditions. In fact, an ether fee poly(phenylene) membrane exhibits excellent stability against the attack from hydroxide ions

  17. Chemical degradation mechanisms of membranes for alkaline membrane fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Choe, Yoong-Kee [National Institute of Advanced Industrial Science and Technology, Umezono 1-1-1, Tsukuba (Japan); Henson, Neil J.; Kim, Yu Seung [Los Alamos National Laboratory, Los Alamos, NM (United States)

    2015-12-31

    Chemical degradation mechanisms of membranes for alkaline membrane fuel cells have been investigated using density functional theory (DFT). We have elucidated that the aryl-ether moiety of membranes is one of the weakest site against attack of hydroxide ions. The results of DFT calculations for hydroxide initiated aryl-ether cleavage indicated that the aryl-ether cleavage occurred prior to degradation of cationic functional group. Such a weak nature of the aryl-ether group arises from the electron deficiency of the aryl group as well as the low bond dissociation energy. The DFT results suggests that removal of the aryl-ether group in the membrane should enhance the stability of membranes under alkaline conditions. In fact, an ether fee poly(phenylene) membrane exhibits excellent stability against the attack from hydroxide ions.

  18. In-membrane micro fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Omosebi, Ayokunle; Besser, Ronald

    2016-09-06

    An in-membrane micro fuel cell comprises an electrically-insulating membrane that is permissive to the flow of cations, such as protons, and a pair of electrodes deposited on channels formed in the membrane. The channels are arranged as conduits for fluids, and define a membrane ridge between the channels. The electrodes are porous and include catalysts for promoting the liberation of a proton and an electron from a chemical species and/or or the recombination of a proton and an electron with a chemical specie. The fuel cell may be provided a biosensor, an electrochemical sensor, a microfluidic device, or other microscale devices fabricated in the fuel cell membrane.

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

    Science.gov (United States)

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

    2012-06-01

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

  20. A Proteomic Study of Human Merkel Cell Carcinoma.

    Science.gov (United States)

    Shao, Qiang; Byrum, Stephanie D; Moreland, Linley E; Mackintosh, Samuel G; Kannan, Aarthi; Lin, Zhenyu; Morgan, Michael; Stack, Brendan C; Cornelius, Lynn A; Tackett, Alan J; Gao, Ling

    2013-11-25

    Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin. The incidence has been quadrupled with a 5-year mortality rate of 46%, presently there is no cure for metastatic disease. Despite the contribution of Merkel cell polyomavirus, the molecular events of MCC carcinogenesis are poorly defined. To better understand MCC carcinogensis, we have performed the first quantitative proteomic comparison of formalin-fixed, paraffin-embedded (FFPE) MCC tissues using another neuroendocrine tumor (carcinoid tumor of the lung) as controls. Bioinformatic analysis of the proteomic data has revealed that MCCs carry distinct protein expression patterns. Further analysis of significantly over-expressed proteins suggested the involvement of MAPK, PI3K/Akt/mTOR, wnt, and apoptosis signaling pathways. Our previous study and that from others have shown mTOR activation in MCCs. Therefore, we have focused on two downstream molecules of the mTOR pathway, lactate dehydrogenase B (LDHB) and heterogeneous ribonucleoprotein F (hnRNPF). We confirm over-expression of LDHB and hnRNPF in two primary human MCC cell lines, 16 fresh tumors, and in the majority of 80 tissue microarray samples. Moreover, mTOR inhibition suppresses LDHB and hnRNPF expression in MCC cells. The results of the current study provide insight into MCC carcinogenesis and provide rationale for mTOR inhibition in pre-clinical studies. PMID:25284964

  1. 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

  2. Quantitative proteomic profiling of membrane proteins from the mouse brain cortex, hippocampus, and cerebellum using the HysTag reagent: mapping of neurotransmitter receptors and ion channels

    DEFF Research Database (Denmark)

    Olsen, Jesper V; Nielsen, Peter Aa; Andersen, Jens R;

    2007-01-01

    Analysis of the brain proteome and studying brain diseases through clinical biopsies and animal disease models require methods of quantitative proteomics that are sensitive and allow identification and quantification of low abundant membrane proteins from minute amount of tissue. Taking advantage...

  3. Intriguing bronchoalveolar lavage proteome in a case of pulmonary langerhans cell histiocytosis

    OpenAIRE

    Ghafouri, Bijar; Persson, H Lennart; Tagesson, Christer

    2013-01-01

    Background: Pulmonary Langerhans cell histiocytosis (PLCH) is a rare interstitial lung disease associated with tobacco smoke exposure. New insights into its pathogenesis and how it differs from that of chronic obstructive pulmonary disease (COPD) may be provided by proteomic studies on bronchoalveolar lavage fluid (BALF). Case Report: We present the BALF proteome in a biopsy-proven case of PLCH and compare it with typical proteomes of COPD and of the healthy lung. The BALF proteins were separ...

  4. Characterization of the Sclerotinia sclerotiorum cell wall proteome.

    Science.gov (United States)

    Liu, Longzhou; Free, Stephen J

    2016-08-01

    We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)-anchored cell wall proteins and 30 non-GPI-anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes. PMID:26661933

  5. Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yong; Liu, Liguo; Fu, Hua; Wei, Candong, E-mail: weicando@ipbcams.ac.cn; Jin, Qi, E-mail: zdsys@vip.sina.com

    2014-10-31

    Highlights: • We utilized mTRAQ-based quantification to study protein changes in Congo red-induced OMVs. • A total of 148 proteins were identified in S. flexneri-derived OMVs. • Twenty-eight and five proteins are significantly up- and down-regulated in the CR-induced OMV, respectively. • The result implied that a special sorting mechanism of particular proteins into OMVs may exist. • Key node proteins in the protein interaction network might be important for pathogenicity. - Abstract: The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein–protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria.

  6. Fuel cell and membrane therefore

    Energy Technology Data Exchange (ETDEWEB)

    Aindow, Tai-Tsui

    2016-08-09

    A fuel cell includes first and second flow field plates, and an anode electrode and a cathode electrode between the flow field plates. A polymer electrolyte membrane (PEM) is arranged between the electrodes. At least one of the flow field plates influences, at least in part, an in-plane anisotropic physical condition of the PEM that varies in magnitude between a high value direction and a low value direction. The PEM has an in-plane physical property that varies in magnitude between a high value direction and a low value direction. The PEM is oriented with its high value direction substantially aligned with the high value direction of the flow field plate.

  7. Live cell imaging of membrane / cytoskeleton interactions and membrane topology

    Science.gov (United States)

    Chierico, Luca; Joseph, Adrian S.; Lewis, Andrew L.; Battaglia, Giuseppe

    2014-09-01

    We elucidate the interaction between actin and specific membrane components, using real time live cell imaging, by delivering probes that enable access to components, that cannot be accessed genetically. We initially investigated the close interplay between Phosphatidylinositol 4,5-bisphosphate (PIP2) and the F-actin network. We show that, during the early stage of cell adhesion, PIP2 forms domains within the filopodia membrane. We studied these domains alongside cell spreading and observed that these very closely follow the actin tread-milling. We show that this mechanism is associated with an active transport of PIP2 rich organelles from the cell perinuclear area to the edge, along actin fibers. Finally, mapping other phospholipids and membrane components we observed that the PIP2 domains formation is correlated with sphingosine and cholesterol rafts.

  8. Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots

    Science.gov (United States)

    Zhu, Yingde; Li, Hui; Bhatti, Sarabjit; Zhou, Suping; Yang, Yong; Fish, Tara; Thannhauser, Theodore W

    2016-01-01

    Single-cell-type proteomics provides the capability to revealing the genomic and proteomics information at cell-level resolution. However, the methodology for this type of research has not been well-developed. This paper reports developing a workflow of laser capture microdissection (LCM) followed by gel-liquid chromatography-tandem mass spectrometry (GeLC-MS/MS)-based proteomics analysis for the identification of proteomes contained in individual cell layers of tomato roots. Thin-sections (~10-μm thick, 10 sections per root tip) were prepared for root tips of tomato germinating seedlings. Epidermal and cortical cells (5000–7000 cells per tissue type) were isolated under a LCM microscope. Proteins were isolated and then separated by SDS–polyacrylamide gel electrophoresis followed by in-gel-tryptic digestion. The MS and MS/MS spectra generated using nanoLC-MS/MS analysis of the tryptic peptides were searched against ITAG2.4 tomato protein database to identify proteins contained in each single-cell-type sample. Based on the biological functions, proteins with proven functions in root hair development were identified in epidermal cells but not in the cortical cells. Several of these proteins were found in Al-treated roots only. The results demonstrated that the cell-type-specific proteome is relevant for tissue-specific functions in tomato roots. Increasing the coverage of proteomes and reducing the inevitable cross-contamination from adjacent cell layers, in both vertical and cross directions when cells are isolated from slides prepared using intact root tips, are the major challenges using the technology in proteomics analysis of plant roots. PMID:27280026

  9. Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

    Science.gov (United States)

    Sidhu, Vishaldeep K; Huang, Bill X; Desai, Abhishek; Kevala, Karl; Kim, Hee-Yong

    2016-05-01

    Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function. PMID:27103520

  10. Comparative proteomics of inner membrane fraction from carbapenem-resistant Acinetobacter baumannii with a reference strain.

    Directory of Open Access Journals (Sweden)

    Vishvanath Tiwari

    Full Text Available Acinetobacter baumannii has been identified by the Infectious Diseases Society of America as one of the six pathogens that cause majority of hospital infections. Increased resistance of A.baumannii even to the latest generation of β-lactams like carbapenem is an immediate threat to mankind. As inner-membrane fraction plays a significant role in survival of A.baumannii, we investigated the inner-membrane fraction proteome of carbapenem-resistant strain of A.baumannii using Differential In-Gel Electrophoresis (DIGE followed by DeCyder, Progenesis and LC-MS/MS analysis. We identified 19 over-expressed and 4 down-regulated proteins (fold change>2, p<0.05 in resistant strain as compared to reference strain. Some of the upregulated proteins in resistant strain and their association with carbapenem resistance in A.baumannii are: i β-lactamases, AmpC and OXA-51: cleave and inactivate carbapenem ii metabolic enzymes, ATP synthase, malate dehydrogenase and 2-oxoglutarate dehydrogenase: help in increased energy production for the survival and iii elongation factor Tu and ribosomal proteins: help in the overall protein production. Further, entry of carbapenem perhaps is limited by controlled production of OmpW and low levels of surface antigen help to evade host defence mechanism in developing resistance in A.baumannii. Present results support a model for the importance of proteins of inner-membrane fraction and their synergistic effect in the mediation of resistance of A.baumannii to carbapenem.

  11. Differential proteomics of the synovial membrane between bilateral and unilateral knee osteoarthritis in surgery‑induced rabbit models.

    Science.gov (United States)

    Liu, Weilin; He, Jian; Lin, Ruhui; Liang, Jie; Luo, Qinglu

    2016-09-01

    The present study investigated the differential proteomics of synovial membranes between bilateral and unilateral anterior cruciate ligament transection (ACLT) in rabbits with knee osteoarthritis (KOA), in order to elucidate the pathological biomarkers of different degrees of KOA. A total of 6 New Zealand rabbits were randomly divided into groups A and B (three rabbits per group). The two groups were subjected to bilateral and unilateral ACLT, respectively. A total of 6 weeks following surgery, proteins were extracted from the knee joint synovial membranes of KOA rabbits and were separated by two‑dimensional polyacrylamide gel electrophoresis. The differentially expressed proteins in the OA synovial membranes were selected for further analysis by linear ion trap‑Fourier transform ion cyclotron resonance mass spectrometry. Ten protein spots were identified to be different between the synovial membranes of the bilateral and unilateral KOA rabbits. Protein disulfide‑isomerase and creatine kinase M‑type were identified in the unilateral KOA rabbit synovial membranes. Serum albumin (three spots), lumican, α‑2‑HS‑glycoprotein and three uncharacterized proteins were identified in the synovial membranes of the bilateral KOA rabbits. The differential proteomic expression demonstrated the different biomarkers associated with bilateral and unilateral KOA, and indicated that spontaneous and secondary KOA require diverse methods of treatment; thus the underlying mechanism of KOA requires further investigation. PMID:27430254

  12. Quantitative proteomic analysis of milk fat globule membrane (MFGM) proteins in human and bovine colostrum and mature milk samples through iTRAQ labeling.

    Science.gov (United States)

    Yang, Mei; Cong, Min; Peng, Xiuming; Wu, Junrui; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing

    2016-05-18

    Milk fat globule membrane (MFGM) proteins have many functions. To explore the different proteomics of human and bovine MFGM, MFGM proteins were separated from human and bovine colostrum and mature milk, and analyzed by the iTRAQ proteomic approach. A total of 411 proteins were recognized and quantified. Among these, 232 kinds of differentially expressed proteins were identified. These differentially expressed proteins were analyzed based on multivariate analysis, gene ontology (GO) annotation and KEGG pathway. Biological processes involved were response to stimulus, localization, establishment of localization, and the immune system process. Cellular components engaged were the extracellular space, extracellular region parts, cell fractions, and vesicles. Molecular functions touched upon were protein binding, nucleotide binding, and enzyme inhibitor activity. The KEGG pathway analysis showed several pathways, including regulation of the actin cytoskeleton, focal adhesion, neurotrophin signaling pathway, leukocyte transendothelial migration, tight junction, complement and coagulation cascades, vascular endothelial growth factor signaling pathway, and adherens junction. These results enhance our understanding of different proteomes of human and bovine MFGM across different lactation phases, which could provide important information and potential directions for the infant milk powder and functional food industries. PMID:27159491

  13. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    Science.gov (United States)

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  14. Membrane Organization and Dynamics in Cell Polarity

    OpenAIRE

    Orlando, Kelly; Guo, Wei

    2009-01-01

    The establishment and maintenance of cell polarity is important to a wide range of biological processes ranging from chemotaxis to embryogenesis. An essential feature of cell polarity is the asymmetric organization of proteins and lipids in the plasma membrane. In this article, we discuss how polarity regulators such as small GTP-binding proteins and phospholipids spatially and kinetically control vesicular trafficking and membrane organization. Conversely, we discuss how membrane trafficking...

  15. 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

  16. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian;

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...

  17. Interaction of Defensins with Model Cell Membranes

    Science.gov (United States)

    Sanders, Lori K.; Schmidt, Nathan W.; Yang, Lihua; Mishra, Abhijit; Gordon, Vernita D.; Selsted, Michael E.; Wong, Gerard C. L.

    2009-03-01

    Antimicrobial peptides (AMPs) comprise a key component of innate immunity for a wide range of multicellular organisms. For many AMPs, activity comes from their ability to selectively disrupt and lyse bacterial cell membranes. There are a number of proposed models for this action, but the detailed molecular mechanism of selective membrane permeation remains unclear. Theta defensins are circularized peptides with a high degree of selectivity. We investigate the interaction of model bacterial and eukaryotic cell membranes with theta defensins RTD-1, BTD-7, and compare them to protegrin PG-1, a prototypical AMP, using synchrotron small angle x-ray scattering (SAXS). The relationship between membrane composition and peptide induced changes in membrane curvature and topology is examined. By comparing the membrane phase behavior induced by these different peptides we will discuss the importance of amino acid composition and placement on membrane rearrangement.

  18. Does ATP cross the cell plasma membrane.

    OpenAIRE

    Chaudry, I. H.

    1982-01-01

    Although there is an abundance of evidence which indicates that ATP is released as well as taken up by cells, the concept that ATP cannot cross the cell membrane has tended to prevail. This article reviews the evidence for the release as well as uptake of ATP by cells. The evidence presented by various investigators clearly indicates that ATP can cross the cell membrane and suggests that the release and uptake of ATP are physiological processes.

  19. Functional dynamics of cell surface membrane proteins

    Science.gov (United States)

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

    2014-04-01

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

  20. Cell surface proteome analysis of human-hosted Trypanosoma cruzi life stages

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sébastien; Bastos, Izabela M D;

    2014-01-01

    addressed the analysis of the plasma membrane (PM) subproteome from T. cruzi human-hosted life stages, trypomastigote and axenic amastigote, by two complementary PM protein enrichment techniques followed by identification using an LC-MS/MS approach. The results revealed an extensive repertoire of proteins...... in the PM subproteomes, including enzymes that might be suitable candidates for drug intervention. The comparison of the cell surface proteome among the life forms revealed some potentially stage-specific enzymes, although the majority was shared by both stages. Bioinformatic analysis showed that the vast......Chagas' disease is a neglected infectious illness, caused by the protozoan Trypanosoma cruzi. It remains a challenging health issue in Latin America, where it is endemic, and so far there is no immunoprophylatic vaccine or satisfactory chemotherapic treatment for its chronic stage. The present work...

  1. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    OpenAIRE

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Ba...

  2. Label-Free Quantitative Proteomics and N-terminal Analysis of Human Metastatic Lung Cancer Cells

    OpenAIRE

    Min, Hophil; Han, Dohyun; Kim, Yikwon; Cho, Jee Yeon; Jin, Jonghwa; Kim, Youngsoo

    2014-01-01

    Proteomic analysis is helpful in identifying cancer-associated proteins that are differentially expressed and fragmented that can be annotated as dysregulated networks and pathways during metastasis. To examine meta-static process in lung cancer, we performed a proteomics study by label-free quantitative analysis and N-terminal analysis in 2 human non-small-cell lung cancer cell lines with disparate metastatic potentials—NCI-H1703 (primary cell, stage I) and NCI-H1755 (metastatic cell, stage ...

  3. Mass Spectrometry-based Quantitative Proteomic Profiling of Human Pancreatic and Hepatic Stellate Cell Lines

    OpenAIRE

    Paulo, Joao A.; Kadiyala, Vivek; Banks, Peter A; Conwell, Darwin L; Steen, Hanno

    2013-01-01

    The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immorta...

  4. 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.

  5. Mechanical tension drives cell membrane fusion

    OpenAIRE

    Kim, Ji Hoon; Ren, Yixin; Ng, Win Pin; Li, Shuo; Son, Sungmin; Kee, Yee-Seir; Zhang, Shiliang; Zhang, Guofeng; Fletcher, Daniel A.; Robinson, Douglas N.; Chen, Elizabeth H.

    2015-01-01

    Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an “attacking” cell drills finger-like protrusions into the “receiving” cell to promote cell fusion. Here we show that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its inv...

  6. Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics.

    Directory of Open Access Journals (Sweden)

    Mafumi Abiko

    Full Text Available In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms.

  7. 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.

  8. Proteomic identification of protein targets for 15-deoxy-Δ(12,14-prostaglandin J2 in neuronal plasma membrane.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Yamamoto

    Full Text Available 15-deoxy-Δ(12,14-prostaglandin J(2 (15d-PGJ(2 is one of factors contributed to the neurotoxicity of amyloid β (Aβ, a causative protein of Alzheimer's disease. Type 2 receptor for prostaglandin D(2 (DP2 and peroxysome-proliferator activated receptorγ (PPARγ are identified as the membrane receptor and the nuclear receptor for 15d-PGJ(2, respectively. Previously, we reported that the cytotoxicity of 15d-PGJ(2 was independent of DP2 and PPARγ, and suggested that 15d-PGJ(2 induced apoptosis through the novel specific binding sites of 15d-PGJ(2 different from DP2 and PPARγ. To relate the cytotoxicity of 15d-PGJ(2 to amyloidoses, we performed binding assay [(3H]15d-PGJ(2 and specified targets for 15d-PGJ(2 associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ(2 and fibrillar Aβ. Specific binding sites of [(3H]15d-PGJ(2 were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [(3H]15d-PGJ(2 were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ(2 in the plasma membrane. By using biotinylated 15d-PGJ(2, eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α, cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α. GAPDH, PKM1 and Tubulin β are Aβ-interacting proteins. Thus, the present study suggested that 15d-PGJ(2 plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues.

  9. Proteomic analysis of exosomes secreted by human mesothelioma cells

    NARCIS (Netherlands)

    J.P.J.J. Hegmans (Joost); A. Hemmes (Annabrita); T.M. Luider (Theo); M.J. Kleijmeer (Monique); J-B. Prins (Jan-Bas); L. Zitvogel; S.A. Burgers (Sjaak); H.C. Hoogsteden (Henk); B.N.M. Lambrecht (Bart); M.P.L. Bard (Martin)

    2004-01-01

    textabstractExosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins pre

  10. Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomics studies

    OpenAIRE

    Zhang, Ying; Bottinelli, Dario; Lisacek, Frédérique; Luban, Jeremy; De Castillia, Caterina Strambio; Varesio, Emmanuel; Hopfgartner, Gérard

    2015-01-01

    Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize mass spectrometry coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilisation and denaturation methods were ...

  11. Dataset of differential lipid raft and global proteomes of SILAC-labeled cystic fibrosis cells upon TNF -α stimulation.

    Science.gov (United States)

    Chhuon, C; Pranke, I; Borot, F; Tondelier, D; Lipecka, J; Fritsch, J; Chanson, M; Edelman, A; Ollero, M; Guerrera, I C

    2016-12-01

    Cystic fibrosis (CF) is a genetic disease due to mutations in the cystic fibrosis transmembrane regulator (CFTR), F508del-CFTR being the most frequent. Lipid raft-like microdomains (LRM) are regions of the plasma membrane that present a high cholesterol content and are insoluble to non-ionic detergents. LRM are essential functional and structural platforms that play an important role in the inflammatory response. CFTR is a known modulator of inflammation in LRM. Here we provide mass spectrometry data on the global impact of CFTR mutation and TNF-a stimulation on the LRM proteome. We used the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) approach to quantify and identify 332 proteins in LRM upon TNF-a stimulation in CF cells and 1381 for the global proteome. We report two detailed tables containing lists of proteins obtained by mass spectrometry and the immunofluorescence validation results for one of these proteins, the G-protein coupled receptor 5A. These results are associated with the article "Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells" (Chhuon et al., in press [1]).

  12. Nuclear myosin I regulates cell membrane tension

    Science.gov (United States)

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  13. Proton conducting membrane for fuel cells

    Science.gov (United States)

    Colombo, Daniel G.; Krumpelt, Michael; Myers, Deborah J.; Kopasz, John P.

    2007-03-27

    An ion conducting membrane comprising dendrimeric polymers covalently linked into a network structure. The dendrimeric polymers have acid functional terminal groups and may be covalently linked via linking compounds, cross-coupling reactions, or copolymerization reactions. The ion conducting membranes may be produced by various methods and used in fuel cells.

  14. Diffuse Charge Effects in Fuel Cell Membranes

    NARCIS (Netherlands)

    Biesheuvel, P.M.; Franco, A.A.; Bazant, M.Z.

    2009-01-01

    It is commonly assumed that electrolyte membranes in fuel cells are electrically neutral, except in unsteady situations, when the double-layer capacitance is heuristically included in equivalent circuit calculations. Indeed, the standard model for electron transfer kinetics at the membrane/electrode

  15. Membrane elastic properties and cell function.

    Directory of Open Access Journals (Sweden)

    Bruno Pontes

    Full Text Available Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.

  16. Advanced membrane electrode assemblies for fuel cells

    Science.gov (United States)

    Kim, Yu Seung; Pivovar, Bryan S

    2014-02-25

    A method of preparing advanced membrane electrode assemblies (MEA) for use in fuel cells. A base polymer is selected for a base membrane. An electrode composition is selected to optimize properties exhibited by the membrane electrode assembly based on the selection of the base polymer. A property-tuning coating layer composition is selected based on compatibility with the base polymer and the electrode composition. A solvent is selected based on the interaction of the solvent with the base polymer and the property-tuning coating layer composition. The MEA is assembled by preparing the base membrane and then applying the property-tuning coating layer to form a composite membrane. Finally, a catalyst is applied to the composite membrane.

  17. Cell wall proteomics of the green alga Haematococcus pluvialis (Chlorophyceae).

    Science.gov (United States)

    Wang, Sheng-Bing; Hu, Qiang; Sommerfeld, Milton; Chen, Feng

    2004-03-01

    The green microalga Haematococcus pluvialis can synthesize and accumulate large amounts of the ketocarotenoid astaxanthin, and undergo profound changes in cell wall composition and architecture during the cell cycle and in response to environmental stresses. In this study, cell wall proteins (CWPs) of H. pluvialis were systematically analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with peptide mass fingerprinting (PMF) and sequence-database analysis. In total, 163 protein bands were analyzed, which resulted in positive identification of 81 protein orthologues. The highly complex and dynamic composition of CWPs is manifested by the fact that the majority of identified CWPs are differentially expressed at specific stages of the cell cycle along with a number of common wall-associated 'housekeeping' proteins. The detection of cellulose synthase orthologue in the vegetative cells suggested that the biosynthesis of cellulose occurred during primary wall formation, in contrast to earlier observations that cellulose was exclusively present in the secondary wall of the organism. A transient accumulation of a putative cytokinin oxidase at the early stage of encystment pointed to a possible role in cytokinin degradation while facilitating secondary wall formation and/or assisting in cell expansion. This work represents the first attempt to use a proteomic approach to investigate CWPs of microalgae. The reference protein map constructed and the specific protein markers obtained from this study provide a framework for future characterization of the expression and physiological functions of the proteins involved in the biogenesis and modifications in the cell wall of Haematococcus and related organisms. PMID:14997492

  18. Proteomic analysis of nasal epithelial cells from cystic fibrosis patients.

    Directory of Open Access Journals (Sweden)

    Ludovic Jeanson

    Full Text Available The pathophysiology of cystic fibrosis (CF lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC. To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4, primary nasal polyps (NP, n = 8 and control mucosa (CTRL, n = 4 using isobaric tag for relative and absolute quantification (iTRAQ labeling coupled with liquid chromatography (LC-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology.

  19. 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.

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

    International Nuclear Information System (INIS)

    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. 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...... protein production has increased significantly because proteomics can track changes in protein levels for different cell lines over time, which can be advantageous for bioprocess development and optimization. Specifically, the identification of proteins that affect cell culture processes can aid efforts...... 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...

  2. Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

    Science.gov (United States)

    Billing, Anja M; Ben Hamidane, Hisham; Dib, Shaima S; Cotton, Richard J; Bhagwat, Aditya M; Kumar, Pankaj; Hayat, Shahina; Yousri, Noha A; Goswami, Neha; Suhre, Karsten; Rafii, Arash; Graumann, Johannes

    2016-01-01

    Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. PMID:26857143

  3. 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.

  4. Cell membrane softening in human breast and cervical cancer cells

    Science.gov (United States)

    Händel, Chris; Schmidt, B. U. Sebastian; Schiller, Jürgen; Dietrich, Undine; Möhn, Till; Kießling, Tobias R.; Pawlizak, Steve; Fritsch, Anatol W.; Horn, Lars-Christian; Briest, Susanne; Höckel, Michael; Zink, Mareike; Käs, Josef A.

    2015-08-01

    Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

  5. Metric dynamics for membrane transformation through regulated cell proliferation

    OpenAIRE

    Ito, Hiroshi C.

    2016-01-01

    This study develops an equation for describing three-dimensional membrane transformation through proliferation of its component cells regulated by morphogen density distributions on the membrane. The equation is developed in a two-dimensional coordinate system mapped on the membrane, referred to as the membrane coordinates. When the membrane expands, the membrane coordinates expand in the same manner so that the membrane is invariant in the coordinates. In the membrane coordinate system, the ...

  6. 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.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Ilona Turek

    2015-09-01

    Full Text Available 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. High temperature polymer electrolyte membrane fuel cells

    DEFF Research Database (Denmark)

    This book is a comprehensive review of high-temperature polymer electrolyte membrane fuel cells (PEMFCs). PEMFCs are the preferred fuel cells for a variety of applications such as automobiles, cogeneration of heat and power units, emergency power and portable electronics. The first 5 chapters...

  10. Proteomic differences in recombinant CHO cells producing two similar antibody fragments.

    Science.gov (United States)

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin; Meleady, Paula; Kunert, Renate

    2016-09-01

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. "Omics" studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label-free LC-MS proteomic analyses to investigate product-specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single-chain Fv-Fc homodimeric antibody fragments (scFv-Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase-mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label-free proteomic analysis. LC-MS-MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902-1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  11. Proteomics of loosely bound cell wall proteins of Arabidopsis thaliana cell suspension cultures: a critical analysis.

    OpenAIRE

    Borderies, Gisèle; Jamet, Elisabeth; Lafitte, Claude; Rossignol, Michel; Jauneau, Alain; Boudart, Georges; Monsarrat, Bernard; Esquerré-Tugayé, Marie-Thérèse; Boudet, Alain; Pont-Lezica, Rafael

    2003-01-01

    The complete sequencing of the Arabidopsis thaliana genome allows the use of the recently developed mass spectrometry techniques to identify the cell wall proteins (CWPs). Most proteomic approaches depend on the quality of sample preparation. Extraction of CWPs is particularly complex since the proteins may be free in the apoplast or are embedded in a polysaccharide matrix where they are retained by Van der Waals interactions, hydrogen bonds, hydrophobic or ionic interactions, or cross-linked...

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

    DEFF Research Database (Denmark)

    Pasini, Erica M; Kirkegaard, Morten; Salerno, Doris;

    2008-01-01

    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...... 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....

  13. Electrically Conductive, Hydrophilic Porous Membrane for Fuel Cell Applications Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This Phase I effort seeks to produce a conductive polyethersulfone (PES) microporous membrane for fuel cell water management applications. This membrane will...

  14. Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy.

    Science.gov (United States)

    Moon, Pyong-Gon; Lee, Jeong-Eun; You, Sungyong; Kim, Taek-Kyun; Cho, Ji-Hoon; Kim, In-San; Kwon, Tae-Hwan; Kim, Chan-Duck; Park, Sun-Hee; Hwang, Daehee; Kim, Yong-Lim; Baek, Moon-Chang

    2011-06-01

    To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level.

  15. Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling.

    Directory of Open Access Journals (Sweden)

    Anke Müller

    Full Text Available The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.

  16. Alternate Fuel Cell Membranes for Energy Independence

    Energy Technology Data Exchange (ETDEWEB)

    Storey, Robson, F.; Mauritz, Kenneth, A.; Patton, Derek, L.; Savin, Daniel, A.

    2012-12-18

    The overall objective of this project was the development and evaluation of novel hydrocarbon fuel cell (FC) membranes that possess high temperature performance and long term chemical/mechanical durability in proton exchange membrane (PEM) fuel cells (FC). The major research theme was synthesis of aromatic hydrocarbon polymers of the poly(arylene ether sulfone) (PAES) type containing sulfonic acid groups tethered to the backbone via perfluorinated alkylene linkages and in some cases also directly attached to the phenylene groups along the backbone. Other research themes were the use of nitrogen-based heterocyclics instead of acid groups for proton conduction, which provides high temperature, low relative humidity membranes with high mechanical/thermal/chemical stability and pendant moieties that exhibit high proton conductivities in the absence of water, and synthesis of block copolymers consisting of a proton conducting block coupled to poly(perfluorinated propylene oxide) (PFPO) blocks. Accomplishments of the project were as follows: 1) establishment of a vertically integrated program of synthesis, characterization, and evaluation of FC membranes, 2) establishment of benchmark membrane performance data based on Nafion for comparison to experimental membrane performance, 3) development of a new perfluoroalkyl sulfonate monomer, N,N-diisopropylethylammonium 2,2-bis(p-hydroxyphenyl) pentafluoropropanesulfonate (HPPS), 4) synthesis of random and block copolymer membranes from HPPS, 5) synthesis of block copolymer membranes containing high-acid-concentration hydrophilic blocks consisting of HPPS and 3,3'-disulfonate-4,4'-dichlorodiphenylsulfone (sDCDPS), 6) development of synthetic routes to aromatic polymer backbones containing pendent 1H-1,2,3-triazole moieties, 7) development of coupling strategies to create phase-separated block copolymers between hydrophilic sulfonated prepolymers and commodity polymers such as PFPO, 8) establishment of basic

  17. Resting microglial cells exhibit tubular membrane protrusions

    Directory of Open Access Journals (Sweden)

    Ulrike Gimsa

    2002-11-01

    Full Text Available Nano- and microtubular structures have recently become a subject of increasing interest due to their importance in biology and medicine as well as their technological potential. Such structures have been observed in anorganic (Iijima, 1991 as well as in organic (Schnur 1993; Oda et al. 1991 systems. Micro- and nanotubular protrusions of bilayer membranes have been found in cells (Kralj-Iglic et al. 1998; Kralj-Iglic et al. 2001a and phospholipid vesicles (Kralj-Iglic et al. 2002; Kralj-Iglic et al. 2001b. In this work we describe membrane protrusions in microglial cells.

  18. Functional Proteomics Screen Enables Enrichment of Distinct Cell Types from Human Pancreatic Islets

    OpenAIRE

    Revital Sharivkin; Walker, Michael D.; Yoav Soen

    2015-01-01

    The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates sp...

  19. Proteome alteration induced by hTERT transfection of human fibroblast cells

    Directory of Open Access Journals (Sweden)

    Riou Jean-François

    2008-04-01

    Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

  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. Lithium. Effects on excitable cell membranes

    NARCIS (Netherlands)

    Ploeger, Egbert Johan

    1974-01-01

    LITHIUM: Effects on excitable cell membranes. Lithium salts have been used in the treatment of manic-depressive psychosis for many years but their mechanism of action is not well understood. Many workers assume that the action of lithium on catecholamine metabolism and/or on electrolyte distribution

  2. 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...

  3. 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.

  4. Staphylococcus aureus produces membrane-derived vesicles that induce host cell death.

    Directory of Open Access Journals (Sweden)

    Mamata Gurung

    Full Text Available Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

  5. Microfluidic microbial fuel cells: from membrane to membrane free

    Science.gov (United States)

    Yang, Yang; Ye, Dingding; Li, Jun; Zhu, Xun; Liao, Qiang; Zhang, Biao

    2016-08-01

    Microfluidic microbial fuel cells (MMFCs) are small carbon-neutral devices that use self-organized bacteria to degrade organic substrates and harness energy from the waste water. Conventional MMFCs have made great strides in the past decade and have overcome some limitations, such as high capital costs and low energy output. A co-laminar flow MFC has been first proposed in 2011 with the potential to be an attractively power source to niche applications. Co-laminar MFCs typically operate without any physical membranes separating the reactants, and bacterial ecosystems can be easily manipulated by regulating the inlet conditions. This paper highlights recent accomplishments in the development of co-laminar MFCs, emphasizing basic principles, mass transport and fluid dynamics including boundary layer theory, entrance conditions and mixing zone issues. Furthermore, the development of current techniques, major challenges and the potential research directions are discussed.

  6. Comparative proteomics of chloroplasts envelopes from bundle sheath and mesophyll chloroplasts reveals novel membrane proteins with a possible role in C4-related metabolite fluxes and development.

    Directory of Open Access Journals (Sweden)

    Kalpana eManandhar-Shrestha

    2013-03-01

    Full Text Available As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second green revolution will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M and bundle sheath (BS chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts (cp. 74% have a known or predicted membrane association. 21 membrane proteins were 2-15 times more abundant in BS cells, while 36 proteins were more abundant in M cp envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of thirteen candidate genes. Cp association was confirmed using GFP labeling. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is increased in the immature part of the leaf and was lower in the fully developed parts of the leaf, suggesting a need for/incorporation of the protein during chloroplast

  7. Interaction of peptides with cell membranes: insights from molecular modeling

    International Nuclear Information System (INIS)

    The investigation of the interaction of peptides with cell membranes is the focus of active research. It can enhance the understanding of basic membrane functions such as membrane transport, fusion, and signaling processes, and it may shed light on potential applications of peptides in biomedicine. In this review, we will present current advances in computational studies on the interaction of different types of peptides with the cell membrane. Depending on the properties of the peptide, membrane, and external environment, the peptide–membrane interaction shows a variety of different forms. Here, on the basis of recent computational progress, we will discuss how different peptides could initiate membrane pores, translocate across the membrane, induce membrane endocytosis, produce membrane curvature, form fibrils on the membrane surface, as well as interact with functional membrane proteins. Finally, we will present a conclusion summarizing recent progress and providing some specific insights into future developments in this field. (topical review)

  8. Interaction of peptides with cell membranes: insights from molecular modeling

    Science.gov (United States)

    Li, Zhen-lu; Ding, Hong-ming; Ma, Yu-qiang

    2016-03-01

    The investigation of the interaction of peptides with cell membranes is the focus of active research. It can enhance the understanding of basic membrane functions such as membrane transport, fusion, and signaling processes, and it may shed light on potential applications of peptides in biomedicine. In this review, we will present current advances in computational studies on the interaction of different types of peptides with the cell membrane. Depending on the properties of the peptide, membrane, and external environment, the peptide-membrane interaction shows a variety of different forms. Here, on the basis of recent computational progress, we will discuss how different peptides could initiate membrane pores, translocate across the membrane, induce membrane endocytosis, produce membrane curvature, form fibrils on the membrane surface, as well as interact with functional membrane proteins. Finally, we will present a conclusion summarizing recent progress and providing some specific insights into future developments in this field.

  9. Proton exchange membrane fuel cells modeling

    CERN Document Server

    Gao, Fengge; Miraoui, Abdellatif

    2013-01-01

    The fuel cell is a potential candidate for energy storage and conversion in our future energy mix. It is able to directly convert the chemical energy stored in fuel (e.g. hydrogen) into electricity, without undergoing different intermediary conversion steps. In the field of mobile and stationary applications, it is considered to be one of the future energy solutions.Among the different fuel cell types, the proton exchange membrane (PEM) fuel cell has shown great potential in mobile applications, due to its low operating temperature, solid-state electrolyte and compactness.This book pre

  10. Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells.

    Directory of Open Access Journals (Sweden)

    Lucie Lorkova

    Full Text Available Mantle cell lymphoma (MCL is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino. We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine and to an inhibitor of Bruton tyrosine kinase (BTK ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib or remained unaffacted (cisplatin, bendamustine. The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib, but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.

  11. Fuel cell membranes and crossover prevention

    Science.gov (United States)

    Masel, Richard I.; York, Cynthia A.; Waszczuk, Piotr; Wieckowski, Andrzej

    2009-08-04

    A membrane electrode assembly for use with a direct organic fuel cell containing a formic acid fuel includes a solid polymer electrolyte having first and second surfaces, an anode on the first surface and a cathode on the second surface and electrically linked to the anode. The solid polymer electrolyte has a thickness t:.gtoreq..times..times..times..times. ##EQU00001## where C.sub.f is the formic acid fuel concentration over the anode, D.sub.f is the effective diffusivity of the fuel in the solid polymer electrolyte, K.sub.f is the equilibrium constant for partition coefficient for the fuel into the solid polymer electrolyte membrane, I is Faraday's constant n.sub.f is the number of electrons released when 1 molecule of the fuel is oxidized, and j.sub.f.sup.c is an empirically determined crossover rate of fuel above which the fuel cell does not operate.

  12. Proton-exchange membrane regenerative fuel cells

    Science.gov (United States)

    Swette, Larry L.; LaConti, Anthony B.; McCatty, Stephen A.

    This paper will update the progress in developing electrocatalyst systems and electrode structures primarily for the positive electrode of single-unit solid polymer proton-exchange membrane (PEM) regenerative fuel cells. The work was done with DuPont Nafion 117 in complete fuel cells (40 cm 2 electrodes). The cells were operated alternately in fuel cell mode and electrolysis mode at 80°C. In fuel cell mode, humidified hydrogen and oxygen were supplied at 207 kPa (30 psi); in electrolysis mode, water was pumped over the positive electrode and the gases were evolved at ambient pressure. Cycling data will be presented for Pt-Ir catalysts and limited bifunctional data will be presented for Pt. Ir, Ru. Rh and Na xPt 3O 4 catalysts as well as for electrode structure variations.

  13. The application of Dow Chemical's perfluorinated membranes in proton-exchange membrane fuel cells

    Science.gov (United States)

    Eisman, G. A.

    1990-02-01

    Dow Chemical's research activities in fuel cells revolve around the development of perfluorosulfonic acid membranes, useful as the proton transport medium and separator. The following work will outline some of the performance characteristics which are typical for such membranes.

  14. Integration of genomics, proteomics, and imaging for cardiac stem cell therapy

    International Nuclear Information System (INIS)

    Cardiac stem cell therapy is beginning to mature as a valid treatment for heart disease. As more clinical trials utilizing stem cells emerge, it is imperative to establish the mechanisms by which stem cells confer benefit in cardiac diseases. In this paper, we review three methods - molecular cellular imaging, gene expression profiling, and proteomic analysis - that can be integrated to provide further insights into the role of this emerging therapy. (orig.)

  15. 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

  16. 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...

  17. Proteomics analysis of human endothelial cells after shortterm exposure to mobile phone radiation

    International Nuclear Information System (INIS)

    Mobile phones have been a part of our everyday life in the developed world since the late 1990s. This has raised concerns over the potential health risks of mobile phone use. Biological and health effects potentially caused by mobile phone radiation have been extensively studied and several biological and medical endpoints have been examined. So far, results have not been conclusive on the potential effects of mobile phone radiation. Mobile phones generate a modulated radio frequency electromagnetic field (RF-EMF), which is a form of non-ionizing radiation. This means that mobile phone radiation does not have enough energy to ionize atoms and it cannot break chemical bonds directly (e.g., in DNA strands). There could, however, be other mechanisms by which mobile phone radiation may affect cellular and physiological functions. Whether these mechanisms exist is unknown. In this thesis, large-scale screening techniques, such as proteomics, were applied to examine changes on the proteome level after exposure to mobile phone radiation. Proteomics techniques allow the screening of several hundreds, and even thousands, of proteins simultaneously, and are thus more efficient than single endpoint techniques. Four different types of human endothelial cells (two cell lines, two types of primary cells) were exposed to two types of mobile phone radiation (900 and 1800 MHz GSM). The proteome of these cells was examined immediately after short-term exposure using two-dimensional gel electrophoresis (2DE). Two protein detection/analysis techniques were used: silver staining for the cell line samples and difference gel electrophoresis (DIGE) for the primary cells. 2DE-DIGE technology is currently a state-of-the-art technique in 2DE studies. Several changes were found in the proteome of the human endothelial cell line EA.hy926 after exposure to 900 MHz GSM mobile phone radiation. In addition, the proteome of a variant of the same cell line, the EA.hy926v1, was affected after 900 MHz

  18. Proteomic Analysis of Outer Membrane Proteins from Salmonella Enteritidis Strains with Different Sensitivity to Human Serum

    Science.gov (United States)

    Dudek, Bartłomiej; Krzyżewska, Eva; Kapczyńska, Katarzyna; Rybka, Jacek; Pawlak, Aleksandra; Korzekwa, Kamila; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

    2016-01-01

    Differential analysis of outer membrane composition of S. Enteritidis strains, resistant to 50% normal human serum (NHS) was performed in order to find factors influencing the resistance to higher concentrations of NHS. Ten S. Enteritidis clinical strains, resistant to 50% NHS, all producing very long lipopolysaccharide, were subjected to the challenge of 75% NHS. Five extreme strains: two resistant and three sensitive to 75% NHS, were chosen for the further analysis of outer membrane proteins composition. Substantial differences were found in the levels of particular outer membrane proteins between resistant and sensitive strains, i.e. outer membrane protease E (PgtE) was present mainly in resistant strains, while sensitive strains possessed a high level of flagellar hook-associated protein 2 (FliD) and significantly higher levels of outer membrane protein A (OmpA). PMID:27695090

  19. Sodium selectivity of Reissner's membrane epithelial cells

    Directory of Open Access Journals (Sweden)

    Kim Kyunghee X

    2011-02-01

    Full Text Available Abstract Background Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC, which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196, RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3. By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath. Conclusions These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala

  20. Cell membrane-camouflaged nanoparticles for drug delivery.

    Science.gov (United States)

    Luk, Brian T; Zhang, Liangfang

    2015-12-28

    Nanoparticles can preferentially accumulate at sites of action and hold great promise to improve the therapeutic index of many drugs. While conventional methods of nanocarrier-mediated drug delivery have focused on primarily synthetic approaches, engineering strategies that combine synthetic nanoparticles with natural biomaterials have recently gained much attention. In particular, cell membrane-camouflaged nanoparticles are a new class of biomimetic nanoparticles that combine the unique functionalities of cellular membranes and engineering versatility of synthetic nanomaterials for effective delivery of therapeutic agents. Herein, we report on the recent progress on cell membrane-coated nanoparticles for drug delivery. In particular, we highlight three areas: (i) prolonging systemic circulation via cell membrane coating, (ii) cell-specific targeting via cell membrane coating, and (iii) applications of cell membrane coating for drug delivery. The cell membrane-camouflaged nanoparticle platform has emerged as a novel delivery strategy with the potential to improve the therapeutic efficacy for the treatment of a variety of diseases.

  1. Comparative proteomic analysis of a membrane-enriched fraction from flag leaves reveals responses to chemical hybridization agent SQ-1 in wheat.

    Science.gov (United States)

    Song, Qilu; Wang, Shuping; Zhang, Gaisheng; Li, Ying; Li, Zheng; Guo, Jialin; Niu, Na; Wang, Junwei; Ma, Shoucai

    2015-01-01

    The induction of wheat male fertile lines by using the chemical hybridizing agent SQ-1 (CHA-SQ-1) is an effective approach in the utilization of heterosis; however, the molecular basis of male fertility remains unknown. Wheat flag leaves are the initial receptors of CHA-SQ-1 and their membrane structure plays a vital role in response to CHA-SQ-1 stress. To investigate the response of wheat flag leaves to CHA-SQ-1 stress, we compared their quantitative proteomic profiles in the absence and presence of CHA-SQ-1. Our results indicated that wheat flag leaves suffered oxidative stress during CHA-SQ-1 treatments. Leaf O2 (-), H2O2, and malonaldehyde levels were significantly increased within 10 h after CHA-SQ-1 treatment, while the activities of major antioxidant enzymes such as superoxide dismutase, catalase, and guaiacol peroxidase were significantly reduced. Proteome profiles of membrane-enriched fraction showed a change in the abundance of a battery of membrane proteins involved in multiple biological processes. These variable proteins mainly impaired photosynthesis, ATP synthesis protein mechanisms and were involved in the response to stress. These results provide an explanation of the relationships between membrane proteomes and anther abortion and the practical application of CHA for hybrid breeding. PMID:26379693

  2. Comparative proteomic analysis of a membrane-enriched fraction from flag leaves reveals responses to chemical hybridization agent SQ-1 in wheat

    Directory of Open Access Journals (Sweden)

    Qilu eSong

    2015-08-01

    Full Text Available The induction of wheat male fertile lines by using the chemical hybridizing agent SQ-1 (CHA-SQ-1 is an effective approach in the utilization of heterosis; however, the molecular basis of male fertility remains unknown. Wheat flag leaves are the initial receptors of CHA-SQ-1 and their membrane structure plays a vital role in response to CHA-SQ-1 stress. To investigate the response of wheat flag leaves to CHA-SQ-1 stress, we compared their quantitative proteomic profiles in the absence and presence of CHA-SQ-1. Our results indicated that wheat flag leaves suffered oxidative stress during CHA-SQ-1 treatments. Leaf O2-, H2O2, and malonaldehyde levels were significantly increased within 10 h after CHA-SQ-1 treatment, while the activities of major antioxidant enzymes such as superoxide dismutase, catalase, and guaiacol peroxidase were significantly reduced. Proteome profiles of membrane-enriched fraction showed a change in the abundance of a battery of membrane proteins involved in multiple biological processes. These variable proteins mainly impaired photosynthesis, ATP synthesis protein mechanisms and were involved in the response to stress. These results provide an explanation of the relationships between membrane proteomes and anther abortion and the practical application of CHA for hybrid breeding.

  3. Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics

    Science.gov (United States)

    Serwa, Remigiusz A.; Abaitua, Fernando; Krause, Eberhard; Tate, Edward W.; O’Hare, Peter

    2015-01-01

    Summary Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a generalized reduction in myristoylation of host proteins, whereas palmitoylation of host proteins, including regulators of interferon and tetraspanin family proteins, was selectively repressed. Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions. PMID:26256475

  4. High temperature polymer electrolyte membrane fuel cell

    Institute of Scientific and Technical Information of China (English)

    K.Scott; M. Mamlouk

    2006-01-01

    One of the major issues limiting the introduction of polymer electrolyte membrane fuel cells (PEMFCs) is the low temperature of operation which makes platinum-based anode catalysts susceptible to poisoning by the trace amount of CO, inevitably present in reformed fuel. In order to alleviate the problem of CO poisoning and improve the power density of the cell, operating at temperature above 100 ℃ is preferred. Nafion(R) -type perfluorosulfonated polymers have been typically used for PEMFC. However, the conductivity of Nafion(R) -type polymers is not high enough to be used for fuel cell operations at higher temperature ( > 90 ℃) and atmospheric pressure because they dehydrate under these condition.An additional problem which faces the introduction of PEMFC technology is that of supplying or storing hydrogen for cell operation,especially for vehicular applications. Consequently the use of alternative fuels such as methanol and ethanol is of interest, especially if this can be used directly in the fuel cell, without reformation to hydrogen. A limitation of the direct use of alcohol is the lower activity of oxidation in comparison to hydrogen, which means that power densities are considerably lower. Hence to improve activity and power output higher temperatures of operation are preferable. To achieve this goal, requires a new polymer electrolyte membrane which exhibits stability and high conductivity in the absence of liquid water.Experimental data on a polybenzimidazole based PEMFC were presented. A simple steady-state isothermal model of the fuel cell is also used to aid in fuel cell performance optimisation. The governing equations involve the coupling of kinetic, ohmic and mass transport. This paper also considers the advances made in the performance of direct methanol and solid polymer electrolyte fuel cells and considers their limitations in relation to the source and type of fuels to be used.

  5. Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells

    NARCIS (Netherlands)

    Silveira, da M.G.; Golovina, E.A.; Hoekstra, F.A.; Rombouts, F.M.; Abee, T.

    2003-01-01

    The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells.

  6. Origin of subdiffusion of water molecules on cell membrane surfaces

    CERN Document Server

    Yamamoto, Eiji; Yasui, Masato; Yasuoka, Kenji

    2014-01-01

    Water molecules play an important role in providing unique environments for biological reactions on cell membranes. It is widely believed that water molecules form bridges that connect lipid molecules and stabilize cell membranes. Using all-atom molecular dynamics simulations, we show that translational and rotational diffusion of water molecules on lipid membrane surfaces exhibit subdiffusion. Moreover, we provide evidence that both divergent mean trapping time (continuous-time random walk) and long-correlated noise (fractional Brownian motion) contribute to this subdiffusion. These results suggest that subdiffusion on cell membranes causes the water retardation, an enhancement of cell membrane stability, and a higher reaction efficiency.

  7. 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. PMID:27327609

  8. 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.

  9. Preparation of cell membranes for high resolution imaging by AFM

    International Nuclear Information System (INIS)

    Studies of cell membrane structure by atomic force microscopy (AFM) have been limited because of the softness of cell membranes. Here, we utilize a new technique of sample preparation to lay red blood cell membranes on the top of a mica surface to obtain high resolution images by in-situ AFM on both sides of cell membranes. Our results indicate that the location of oligosaccharides and proteins in red blood cell membranes might be different from the current membrane model. The inner membrane leaflet is covered by dense proteins with fewer free lipids than expected. In contrast, the outer membrane leaflet is quite smooth; oligosaccharides and peptides supposed to protrude out of the outer membrane leaflet surface might be actually hidden in the middle of hydrophilic lipid heads; transmembrane proteins might form domains in the membranes revealed by PNGase F and trypsin digestion. Our result could be significant to interpret some functions about red blood cell membranes and guide to heal the blood diseases related to cell membranes.

  10. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen

    OpenAIRE

    Jodie Lopez; Amina Bittame; Céline Massera; Virginie Vasseur; Grégory Effantin; Anne Valat; Célia Buaillon; Sophie Allart; Barbara A. Fox; Leah M. Rommereim; David J. Bzik; Guy Schoehn; Winfried Weissenhorn; Jean-François Dubremetz; Jean Gagnon

    2015-01-01

    Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effecto...

  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. Polybenzimidazole and sulfonated polyhedral oligosilsesquioxane composite membranes for high temperature polymer electrolyte membrane fuel cells

    DEFF Research Database (Denmark)

    Aili, David; Allward, Todd; Alfaro, Silvia Martinez;

    2014-01-01

    Composite membranes based on poly(2,2′(m-phenylene)-5,5́bibenzimidazole) (PBI) and sulfonated polyhedral oligosilsesquioxane (S-POSS) with S-POSS contents of 5 and 10wt.% were prepared by solution casting as base materials for high temperature polymer electrolyte membrane fuel cells. With membran...

  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. Comparative proteomics of exosomes secreted by tumoral Jurkat T cells and normal human T cell blasts unravels a potential tumorigenic role for valosin-containing protein

    Science.gov (United States)

    Sanclemente, Manuel; Iturralde, María; Naval, Javier; Alava, María Angeles; Martínez-Lostao, Luis; Thierse, Hermann-Josef; Anel, Alberto

    2016-01-01

    We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion. PMID:27086912

  15. Resting microglial cells exhibit tubular membrane protrusions

    OpenAIRE

    Ulrike Gimsa; Veronika Kralj-Iglic; Jan Gimsa; Ales Iglic

    2002-01-01

    Nano- and microtubular structures have recently become a subject of increasing interest due to their importance in biology and medicine as well as their technological potential. Such structures have been observed in anorganic (Iijima, 1991) as well as in organic (Schnur 1993; Oda et al. 1991) systems. Micro- and nanotubular protrusions of bilayer membranes have been found in cells (Kralj-Iglic et al. 1998; Kralj-Iglic et al. 2001a) and phospholipid vesicles (Kralj-Iglic et al. 2002; Kralj-Igl...

  16. Proteomic analysis of cancer stem cells in human prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun-Kyung; Cho, Hyungdon [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Chan-Wha, E-mail: cwkim@korea.ac.kr [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of)

    2011-08-26

    Highlights: {yields} DU145 prostate cancer cell line was isolated into CD44+ or CD44- cells. {yields} We confirmed CD44+ DU145 cells are more proliferative and tumorigenic than CD44- DU145 cells. {yields} We analyzed and identified proteins that were differentially expressed between CD44+ and CD44- DU145 cells. {yields} Cofilin and Annexin A5 associated with cancer were found to be positively correlated with CD44 expression. -- Abstract: Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44- cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44- using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.

  17. Nanocomposite Membranes based on Perlfuorosulfonic Acid/Ceramic for Proton Exchange Membrane Fuel Cells

    Institute of Scientific and Technical Information of China (English)

    LI Qiong; WANG Guangjin; YE Hong; YAN Shilin

    2015-01-01

    Perlfuorosulfonic acid/ceramic nanocomposite membranes were investigated as electrolytes for polymer electrolyte membrane fuel cell applications under low relative humidity. Different nanosized ceramics (SiO2, ZrO2, TiO2) with diameters in the range of 2-6 nm were synthesized in situ in Nafion solution through a sol-gel process and the formed nanosized ceramics were well-dispersed in the solution. The nanocomposite membranes were formed through a casting process. The nanocomposite membrane showes enhanced water retention ability and improved proton conductivity compared to those of pure Naifon membrane. The mechanical strength of the formed nanocomposite membranes is slightly less than that of pure Naifon membrane. The experimental results demonstrate that the polymer ceramic nanocompsite membranes are potential electrolyte for fuel cells operating at elevated temperature.

  18. Roles of membrane trafficking in plant cell wall dynamics

    OpenAIRE

    Ebine, Kazuo; Ueda, Takashi

    2015-01-01

    The cell wall is one of the characteristic components of plant cells. The cell wall composition differs among cell types and is modified in response to various environmental conditions. To properly generate and modify the cell wall, many proteins are transported to the plasma membrane or extracellular space through membrane trafficking, which is one of the key protein transport mechanisms in eukaryotic cells. Given the diverse composition and functions of the cell wall in plants, the transpor...

  19. Plasma membranes from insect midgut cells

    Directory of Open Access Journals (Sweden)

    Walter R. Terra

    2006-06-01

    Full Text Available Plasma membranes from insect midgut cells are separated into apical and basolateral domains. The apical domain is usually modified into microvilli with a molecular structure similar to other animals. Nevertheless, the microvillar structure should differ in some insects to permit the traffic inside them of secretory vesicles that may budd laterally or pinch-off from the tips of microvilli. Other microvillar modifications are associated with proton-pumping or with the interplay with an ensheathing lipid membrane (the perimicrovilllar membrane observed in the midgut cells of hemipterans (aphids and bugs. The perimicrovillar membranes are thought to be involved in amino acid absorption from diluted diets. The microvillar and perimicrovillar membranes have densities (and protein content that depend on the insect taxon. The role played by the microvillar and perimicrovillar proteins in insect midgut physiology is reviewed here trying to provide a coherent picture of data and highlighting further research areas.As membranas plasmáticas das células intestinais dos insetos apresentam um domínio apical e outro basal. O domínio apical é geralmente modificado em microvilosidades com organização molecular similar a de outros animais, embora possam diferir naqueles insetos que apresentam vesículas secretoras em trânsito que brotam lateralmente ou destacam-se das extremidades das microvilosidades. Outras modificações microvilares estão associadas a bombeamento de prótons ou a interrelações com uma membrana lipídica (a membrana perimicrovilar que reveste as microvilosidades de células intestinais de hemípteros (pulgões e percevejos. Admite-se que as membranas perimicrovilares estejam envolvidas na absorção de aminoácidos a partir de dietas diluídas. As membranas microvilares e perimicrovilares tem densidades distintas (e conteúdo protéico que dependem do táxon do inseto. O papel desempenhado pelas proteínas microvilares e

  20. A Quaternary Polybenzimidazole Membrane for Intermediate Temperature Polymer Electrolyte Membrane Fuel Cells

    DEFF Research Database (Denmark)

    Xu, C.; Scott, K.; Li, Qingfeng;

    2013-01-01

    A quaternary ammonium polybenzimidazole (QPBI) membrane was synthesized for applications in intermediate temperature (100–200 °C) hydrogen fuel cells. The QPBI membrane was imbibed with phosphoric acid to provide suitable proton conductivity. The proton conductivity of the membrane was 0.051 S cm–1...... at 150 °C with the PA acid loading level of 3.5 PRU (amount of H3PO4 per repeat unit of polymer QPBI). The QPBI membrane was characterized in terms of composition, structure and morphology by NMR, FTIR, SEM, and EDX. The fuel cell performance with the membrane gave peak power densities of 440 and 240 m...

  1. The Proteome of Native Adult Müller Glial Cells From Murine Retina.

    Science.gov (United States)

    Grosche, Antje; Hauser, Alexandra; Lepper, Marlen Franziska; Mayo, Rebecca; von Toerne, Christine; Merl-Pham, Juliane; Hauck, Stefanie M

    2016-02-01

    To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial

  2. Cytocompatibility of Three Corneal Cell Types with Amniotic Membrane

    Institute of Scientific and Technical Information of China (English)

    CHENJian-su; CHENRui; XUJin-tang; DINGYong; ZHAOSong-bin; LISui-lian

    2004-01-01

    Rabbit limbal corneal epithelial cells, corneal endothelial cells and keratocytes were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Histological and scan electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. Results showed that three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints which could form networks. In conclusion, amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea.

  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. The relevance of membrane models to understand nanoparticles-cell membrane interactions

    Science.gov (United States)

    Rascol, Estelle; Devoisselle, Jean-Marie; Chopineau, Joël

    2016-02-01

    Over the past two decades, numerous types of nanoparticles (NPs) have been developed for medical applications; however only a few nanomedicines are actually available on the market. One reason is the lack of understanding and data concerning the NP fate and their behavior upon contact with biological media and cell membranes. Biomimetic membrane models are interesting tools to approach and understand NPs-cell membrane interactions. The use of these models permits one to control physical and chemical parameters and to rapidly compare membrane types and the influence of different media conditions. The interactions between NPs and cell membranes can be qualified and quantified using analytical and modeling methods. In this review, the major studies concerning NPs-cell membrane models and associated methods are described. The advantages and drawbacks for each method are compared for the different models. The key mechanisms of interactions between NPs and cell membranes are revealed using cell membrane models and are interrogated in comparison with the NP behavior in cellulo or in vivo. Investigating the interactions between NPs and cell membrane models is now proposed as an intermediate step between physicochemical characterization of NPs and biological assays.

  5. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

    Science.gov (United States)

    Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

    2015-12-15

    Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation. PMID:26628378

  6. Exocytosis and endocytosis in neurodocrine cells: inseparable membranes !

    Directory of Open Access Journals (Sweden)

    Sébastien eHouy

    2013-10-01

    Full Text Available Although much has been learned concerning the mechanisms of secretory vesicle formation and fusion at donor and acceptor membrane compartments, relatively little attention has been paid towards understanding how cells maintain a homeostatic membrane balance through vesicular trafficking. In neurons and neuroendocrine cells, release of neurotransmitters, neuropeptides and hormones occurs through calcium-regulated exocytosis at the plasma membrane. To allow recycling of secretory vesicle components and to preserve organelles integrity, cells must initiate and regulate compensatory membrane uptake. This review relates the fate of secretory granule membranes after full fusion exocytosis in neuroendocrine cells. In particular, we focus on the potential role of lipids in preserving and sorting secretory granule membranes after exocytosis and we discuss the potential mechanisms of membrane retrieval.

  7. 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.

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

    OpenAIRE

    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 wa...

  9. Proteomic and genomic analysis reveals novel Campylobacter jejuni outer membrane proteins and potential heterogeneity

    Directory of Open Access Journals (Sweden)

    Eleanor Watson

    2014-09-01

    Full Text Available Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC–ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith–Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.

  10. 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 on...

  11. Development of composite membranes of PVA-TEOS doped KOH for alkaline membrane fuel cell

    International Nuclear Information System (INIS)

    Anion exchange membranes (AEMs) play an important role in separating fuel and oxygen (or air) in the Alkaline Membrane Fuel Cells. Preparation of hybrid organic inorganic materials of Polyvinylalcohol (PVA) - Tetraethylorthosilicate (TEOS) composite membrane doped KOH for direct alcohol alkaline fuel cell application has been investigated. The sol-gel method has been used to prepare the composite membrane of PVA-TEOS through crosslinking step and catalyzed by concentrated of hydrochloric acid. The gel solution was cast on the membrane plastic plate to obtain membrane sheets. The dry membranes were then doped by immersing in various concentrations of KOH solutions for about 4 hours. Investigations of the cross-linking process and the presence of hydroxyl group were conducted by FTIR as shown for frequency at about 1600 cm−1 and 3300 cm−1 respectively. The degree of swelling in ethanol decreased as the KOH concentration for membrane soaking process increased. The ion exchange capacity (IEC) of the membrane was 0.25meq/g. This composite membranes display significant ionic conductivity of 3.23 x 10−2 S/cm in deionized water at room temperature. In addition, the morphology observation by scanning electron microscope (SEM) of the membrane indicates that soaking process of membrane in KOH increased thermal resistant

  12. Development of composite membranes of PVA-TEOS doped KOH for alkaline membrane fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Haryadi,, E-mail: haryadi@polban.ac.id; Sugianto, D.; Ristopan, E. [Department of Chemical Engineering, Politeknik Negeri Bandung Jl. Gegerkalong Hilir, Ds. Ciwaruga, Bandung West Java (Indonesia)

    2015-12-29

    Anion exchange membranes (AEMs) play an important role in separating fuel and oxygen (or air) in the Alkaline Membrane Fuel Cells. Preparation of hybrid organic inorganic materials of Polyvinylalcohol (PVA) - Tetraethylorthosilicate (TEOS) composite membrane doped KOH for direct alcohol alkaline fuel cell application has been investigated. The sol-gel method has been used to prepare the composite membrane of PVA-TEOS through crosslinking step and catalyzed by concentrated of hydrochloric acid. The gel solution was cast on the membrane plastic plate to obtain membrane sheets. The dry membranes were then doped by immersing in various concentrations of KOH solutions for about 4 hours. Investigations of the cross-linking process and the presence of hydroxyl group were conducted by FTIR as shown for frequency at about 1600 cm{sup −1} and 3300 cm{sup −1} respectively. The degree of swelling in ethanol decreased as the KOH concentration for membrane soaking process increased. The ion exchange capacity (IEC) of the membrane was 0.25meq/g. This composite membranes display significant ionic conductivity of 3.23 x 10{sup −2} S/cm in deionized water at room temperature. In addition, the morphology observation by scanning electron microscope (SEM) of the membrane indicates that soaking process of membrane in KOH increased thermal resistant.

  13. Development of composite membranes of PVA-TEOS doped KOH for alkaline membrane fuel cell

    Science.gov (United States)

    Haryadi, Sugianto, D.; Ristopan, E.

    2015-12-01

    Anion exchange membranes (AEMs) play an important role in separating fuel and oxygen (or air) in the Alkaline Membrane Fuel Cells. Preparation of hybrid organic inorganic materials of Polyvinylalcohol (PVA) - Tetraethylorthosilicate (TEOS) composite membrane doped KOH for direct alcohol alkaline fuel cell application has been investigated. The sol-gel method has been used to prepare the composite membrane of PVA-TEOS through crosslinking step and catalyzed by concentrated of hydrochloric acid. The gel solution was cast on the membrane plastic plate to obtain membrane sheets. The dry membranes were then doped by immersing in various concentrations of KOH solutions for about 4 hours. Investigations of the cross-linking process and the presence of hydroxyl group were conducted by FTIR as shown for frequency at about 1600 cm-1 and 3300 cm-1 respectively. The degree of swelling in ethanol decreased as the KOH concentration for membrane soaking process increased. The ion exchange capacity (IEC) of the membrane was 0.25meq/g. This composite membranes display significant ionic conductivity of 3.23 x 10-2 S/cm in deionized water at room temperature. In addition, the morphology observation by scanning electron microscope (SEM) of the membrane indicates that soaking process of membrane in KOH increased thermal resistant.

  14. Extensive determination of glycan heterogeneity reveals an unusual abundance of high mannose glycans in enriched plasma membranes of human embryonic stem cells.

    Science.gov (United States)

    An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T; Schaffer, David V; Bertozzi, Carolyn R; Lebrilla, Carlito B

    2012-04-01

    Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine

  15. Selective effect of cell membrane on synaptic neurotransmission

    Science.gov (United States)

    Postila, Pekka A.; Vattulainen, Ilpo; Róg, Tomasz

    2016-01-01

    Atomistic molecular dynamics simulations were performed with 13 non-peptidic neurotransmitters (NTs) in three different membrane environments. The results provide compelling evidence that NTs are divided into membrane-binding and membrane-nonbinding molecules. NTs adhere to the postsynaptic membrane surface whenever the ligand-binding sites of their synaptic receptors are buried in the lipid bilayer. In contrast, NTs that have extracellular ligand-binding sites do not have a similar tendency to adhere to the membrane surface. This finding is a seemingly simple yet important addition to the paradigm of neurotransmission, essentially dividing it into membrane-independent and membrane-dependent mechanisms. Moreover, the simulations also indicate that the lipid composition especially in terms of charged lipids can affect the membrane partitioning of NTs. The revised paradigm, highlighting the importance of cell membrane and specific lipids for neurotransmission, should to be of interest to neuroscientists, drug industry and the general public alike.

  16. Identification of Thalidomide-Specific Transcriptomics and Proteomics Signatures during Differentiation of Human Embryonic Stem Cells

    OpenAIRE

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

    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 prof...

  17. Proteomic profiling of a robust Wolbachia infection in an Aedes albopictus mosquito cell line

    OpenAIRE

    Baldridge, Gerald D; Baldridge, Abigail S.; Witthuhn, Bruce A.; Higgins, LeeAnn; Markowski, Todd W.; FALLON, ANN M.

    2014-01-01

    Wolbachia pipientis a widespread vertically transmitted intracellular bacterium, provides a tool for insect control through manipulation of host-microbe interactions. We report proteomic characterization of wStr, a Wolbachia strain associated with a strong cytoplasmic incompatibility phenotype in its native host, Laodelphax striatellus. In the Aedes albopictus C/wStr1 mosquito cell line, wStr maintains a robust, persistent infection. MS/MS analyses of gel bands revealed a protein “footprint” ...

  18. 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...

  19. Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase

    Directory of Open Access Journals (Sweden)

    Deckwer Wolf-Dieter

    2006-10-01

    Full Text Available Abstract High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320, both carrying a heterologous dextransucrase (dsrS gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available. High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319, not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses. The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the

  20. Finite element analysis of microelectrotension of cell membranes

    OpenAIRE

    Bae, Chilman; Butler, Peter J.

    2007-01-01

    Electric fields can be focused by micropipette-based electrodes to induce stresses on cell membranes leading to tension and poration. To date, however, these membrane stress distributions have not been quantified. In this study, we determine membrane tension, stress, and strain distributions in the vicinity of a microelectrode using finite element analysis of a multiscale electro-mechanical model of pipette, media, membrane, actin cortex, and cytoplasm. Electric field forces are coupled to me...

  1. Membrane Fouling in Microfiltration used for Cell Harvesting

    Institute of Scientific and Technical Information of China (English)

    Tahereh Kaghazchi; Farzin Zokaee; Abbas Zare

    2001-01-01

    In the present study the membrane fouling in microfiltration used for cell harvesting in a deadend system has been investigated. Experimental results were analysed in terms of existing membrane filtration models and membrane resistances. The cake filtration model (CFM) and standard blocking model (SBM) have been considered in this study.Various membrane resistances were determined at different processing time, feed concentration and stirring speed. Resistances to permeation in this system include filter medium, pore blocking, adsorption, cake layer and concentration polarization.

  2. Proteome of human stem cells from periodontal ligament and dental pulp.

    Directory of Open Access Journals (Sweden)

    Enrica Eleuterio

    Full Text Available BACKGROUND: Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs including bone marrow stem cells (BMSCs, dental pulp stem cells (DPSCs and periodontal ligament stem cells (PDLSCs have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4-7 and 6-9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin. CONCLUSION/SIGNIFICANCE: This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.

  3. Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

    Directory of Open Access Journals (Sweden)

    Ruili Ma

    Full Text Available The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV-infected swine testicular (ST cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1, caspase-8, and heat shock protein 90 alpha (HSP90α were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

  4. Synergistic effects of retinoic acid and tamoxifen on human breast cancer cells: Proteomic characterization

    International Nuclear Information System (INIS)

    The anti-estrogen tamoxifen and vitamin A-related compound, all-trans retinoic acid (RA), in combination act synergistically to inhibit the growth of MCF-7 human breast cancer cells. In the present study, we applied two-dimensional gel electrophoresis based proteomic approach to globally analyze this synergistic effect of RA and tamoxifen. Proteomic study revealed that multiple clusters of proteins were involved in RA and tamoxifen-induced apoptosis in MCF-7 breast cancer cells, including post-transcriptional and splicing factors, proteins related to cellular proliferation or differentiation, and proteins related to energy production and internal degradation systems. The negative growth factor-transforming growth factor β (TGFβ) was secreted by RA and/or tamoxifen treatment and was studies as a potential mediator of the synergistic effects of RA and tamoxifen in apoptosis. By comparing protein alterations in treatments of RA and tamoxifen alone or in combination to those of TGFβ treatment, or co-treatment with TGFβ inhibitor SB 431542, proteomic results showed that a number of proteins were involved in TGFβ signaling pathway. These results provide valuable insights into the mechanisms of RA and tamoxifen-induced TGFβ signaling pathway in breast cancer cells

  5. 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.

  6. 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.

  7. Phosphoric acid doped imidazolium polysulfone membranes for high temperature proton exchange membrane fuel cells

    DEFF Research Database (Denmark)

    Yang, Jingshuai; Li, Qingfeng; Jensen, Jens Oluf;

    2012-01-01

    A novel acid–base polymer membrane is prepared by doping of imidazolium polysulfone with phosphoric acid for high temperature proton exchange membrane fuel cells. Polysulfone is first chloromethylated, followed by functionalization of the chloromethylated polysulfone with alkyl imidazoles i.......e. methyl (MePSU), ethyl (EtPSU) and butyl (BuPSU) imidazoliums, as revealed by 1H NMR spectra. The imidazolium polysulfone membranes are then doped with phosphoric acid and used as a proton exchange membrane electrolyte in fuel cells. An acid doping level of about 10–11mol H3PO4 per mole of the imidazolium...

  8. 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.

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

    International Nuclear Information System (INIS)

    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

  10. Engineered nanoparticles mimicking cell membranes for toxin neutralization.

    Science.gov (United States)

    Fang, Ronnie H; Luk, Brian T; Hu, Che-Ming J; Zhang, Liangfang

    2015-08-01

    Protein toxins secreted from pathogenic bacteria and venomous animals rely on multiple mechanisms to overcome the cell membrane barrier to inflict their virulence effect. A promising therapeutic concept toward developing a broadly applicable anti-toxin platform is to administer cell membrane mimics as decoys to sequester these virulence factors. As such, lipid membrane-based nanoparticulates are an ideal candidate given their structural similarity to cellular membranes. This article reviews the virulence mechanisms employed by toxins at the cell membrane interface and highlights the application of cell-membrane mimicking nanoparticles as toxin decoys for systemic detoxification. In addition, the implication of particle/toxin nanocomplexes in the development of toxoid vaccines is discussed. PMID:25868452

  11. Membrane tension and cytoskeleton organization in cell motility

    International Nuclear Information System (INIS)

    Cell membrane shape changes are important for many aspects of normal biological function, such as tissue development, wound healing and cell division and motility. Various disease states are associated with deregulation of how cells move and change shape, including notably tumor initiation and cancer cell metastasis. Cell motility is powered, in large part, by the controlled assembly and disassembly of the actin cytoskeleton. Much of this dynamic happens in close proximity to the plasma membrane due to the fact that actin assembly factors are membrane-bound, and thus actin filaments are generally oriented such that their growth occurs against or near the membrane. For a long time, the membrane was viewed as a relatively passive scaffold for signaling. However, results from the last five years show that this is not the whole picture, and that the dynamics of the actin cytoskeleton are intimately linked to the mechanics of the cell membrane. In this review, we summarize recent findings concerning the role of plasma membrane mechanics in cell cytoskeleton dynamics and architecture, showing that the cell membrane is not just an envelope or a barrier for actin assembly, but is a master regulator controlling cytoskeleton dynamics and cell polarity. (topical review)

  12. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

    Science.gov (United States)

    Zuccala, Elizabeth S; Satchwell, Timothy J; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C; Heesom, Kate J; Baum, Jake

    2016-01-01

    The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture. PMID:26830761

  13. 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.

  14. Anatomy of the red cell membrane skeleton: unanswered questions.

    Science.gov (United States)

    Lux, Samuel E

    2016-01-14

    The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias. PMID:26537302

  15. 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.

  16. Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

    OpenAIRE

    Friedberg, I.; Kaback, H R

    1980-01-01

    Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+...

  17. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    OpenAIRE

    Laura-Roxana Stingaciu; Hugh O’Neill; Michelle Liberton; Urban, Volker S.; Himadri B. Pakrasi; Michael Ohl

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membran...

  18. 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...

  19. 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.

  20. S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure.

    Science.gov (United States)

    Koo, Sue-Jie; Spratt, Heidi M; Soman, Kizhake V; Stafford, Susan; Gupta, Shivali; Petersen, John R; Zago, Maria P; Kuyumcu-Martinez, Muge N; Brasier, Allan R; Wiktorowicz, John E; Garg, Nisha Jain

    2016-01-01

    Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure. PMID:27635260

  1. Polybenzimidazole and sulfonated polyhedral oligosilsesquioxane composite membranes for high temperature polymer electrolyte membrane fuel cells

    International Nuclear Information System (INIS)

    Composite membranes based on poly(2,2′(m-phenylene)-5,5′bibenzimidazole) (PBI) and sulfonated polyhedral oligosilsesquioxane (S-POSS) with S-POSS contents of 5 and 10 wt.% were prepared by solution casting as base materials for high temperature polymer electrolyte membrane fuel cells. With membranes based on pure PBI as a reference point, the composite membranes were characterized with respect to spectroscopic and physicochemical properties. After doping with phosphoric acid, the composite membranes showed considerably improved ex situ proton conductivity under anhydrous as well as under fully humidified conditions in the 120-180 °C temperature range. The conductivity improvements were also confirmed by in situ fuel cell tests at 160 °C and further supported by the electrochemical impedance spectroscopy data based on the operating membrane electrode assemblies, demonstrating the technical feasibility of the novel electrolyte materials

  2. 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.

  3. 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. PMID:27508218

  4. 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.

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

    OpenAIRE

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

    2009-01-01

    International audience 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 ...

  6. Modulation of the proteome of peripheral blood mononuclear cells from HIV-1 infected patients by drugs of abuse

    OpenAIRE

    Jessica L. Reynolds; Supriya D Mahajan; Aalinkeel, Ravikunar; Nair, Bindukumar; Sykes, Donald E; Agosto-Mujica, Arnadri; Hsiao, Chiu Bin; Schwartz, Stanley A.

    2009-01-01

    We used proteomic analyses to assess how drug abuse modulates immunologic responses to infections with the human immunodeficiency virus type 1 (HIV-1). Two dimensional (2D) difference gel electrophoresis was utilized to determine changes in the proteome of peripheral blood mononuclear cells (PBMC) isolated from HIV-1 positive donors that occurred after treatment with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins. We fu...

  7. Comparative proteomics of milk fat globule membrane in goat colostrum and mature milk.

    Science.gov (United States)

    Lu, Jing; Liu, Lu; Pang, Xiaoyang; Zhang, Shuwen; Jia, Zhenhu; Ma, Changlu; Zhao, Lili; Lv, Jiaping

    2016-10-15

    As an important nutrient source in large area of world, the composition and nutritional value of goat milk are not well deliberated. Detailed annotation of protein composition is essential to address the physiological and nutritional value of goat milk. In the present study, 423 colostrum and mature goat milk fat globule membrane (MFGM) proteins were identified. The abundance of 189 proteins was significantly different between colostrums and mature milk MFGM. The acute phase proteins were higher in colostrums MFGM than those in mature milk MFGM which protected newborns at the beginning of life. Proteins related to synthesis and secretion were conserved through lactation to ensure the milk production. Of note, long term depression (LTD) proteins were observed in colostrum and mature milk MFGM. Milk LTD proteins could be potential biomarkers for diagnosis of lactation related depressive syndromes and should be taken into considerations of their effects on newborns. PMID:27173528

  8. 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...

  9. Kinomic and phospho-proteomic analysis of breast cancer stem-like cells

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Christensen, Anne Geske Lindhard; Ehmsen, Sidse;

    Kinomic and phospho-proteomic analysis of breast cancer stem-like cells Rikke Leth-Larsen1, Anne G Christensen1, Sidse Ehmsen1, Mark Møller1, Giuseppe Palmisano2, Martin R Larsen2, Henrik J Ditzel1,3 1Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark 2Institute...... cell death, while the bulk of a tumor lacks these capacities. The resistance mechanisms may cause these cells to survive and become the source of later tumor recurrence, highlighting the need for therapeutic strategies that specifically target pathways central to these cancer stem cells. The CD44hi....../CD24-/low compartment of human breast cancer is enriched in tumor-initiating cells; however the functional heterogeneity within this subpopulation remains poorly defined. From a triple-negative breast cancer cell line we isolated and cloned CD44hi single-cells that exhibited functional heterogeneity...

  10. The Stirred Tank Reactor Polymer Electrolyte Membrane Fuel Cell

    CERN Document Server

    Benziger, J; Karnas, E; Moxley, J; Teuscher, C; Kevrekidis, Yu G; Benziger, Jay

    2003-01-01

    The design and operation of a differential Polymer Electrolyte Membrane (PEM) fuel cell is described. The fuel cell design is based on coupled Stirred Tank Reactors (STR); the gas phase in each reactor compartment was well mixed. The characteristic times for reactant flow, gas phase diffusion and reaction were chosen so that the gas compositions at both the anode and cathode are uniform. The STR PEM fuel cell is one-dimensional; the only spatial gradients are transverse to the membrane. The STR PEM fuel cell was employed to examine fuel cell start- up, and its dynamic responses to changes in load, temperature and reactant flow rates. Multiple time scales in systems response are found to correspond to water absorption by the membrane, water transport through the membrane and stress-related mechanical changes of the membrane.

  11. Cell volume and membrane stretch independently control K+ channel activity

    DEFF Research Database (Denmark)

    Bomholtz, Sofia Hammami; Willumsen, Niels J; Olsen, Hervør L;

    2009-01-01

    A number of potassium channels including members of the KCNQ family and the Ca(2+) activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch....... To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (approximately 50 microm(2) macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current...... was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude...

  12. Changes in the proteomic profile of adipose tissue-derived mesenchymal stem cells during passages

    Directory of Open Access Journals (Sweden)

    Capra Emanuele

    2012-07-01

    Full Text Available Abstract Background Human mesenchymal stem cells (hMSC have recently raised the attention because of their therapeutic potential in the novel context of regenerative medicine. However, the safety of these new and promising cellular products should be carefully defined before they can be used in the clinical setting, as. The protein expression profile of these cells might reveal potential hazards associated with senescence and tumoral transformation which may occur during culture. Proteomic is a valuable tool for hMSC characterization and identification of possible changes during expansion. Results We used Surface Enhanced Laser Desorption/Ionization-Time Of Flight-Mass Spectrometry (SELDI-ToF-MS to evaluate the presence of stable molecular markers in adipose tissue-derived mesenchymal stem cells (AD-MSC produced under conditions of good manufacturing practices (GMP. Proteomic patterns of cells prepared were consistent, with 4 up-regulated peaks (mass-to-charge ratio (m/z 8950, 10087, 10345, and 13058 through subculture steps (P0-P7 with similar trend in three donors. Among the differentially expressed proteins found in the cytoplasmic and nuclear fractions, a cytoplasmic 10.1 kDa protein was upregulated during culture passages and was identified as S100A6 (Calcyclin. Conclusions This study suggests for the first time that common variation could occur in AD-MSC from different donors, with the identification of S100A6, a protein prevalently related to cell proliferation and cell culture condition. These results support the hypothesis of common proteomic changes during MSCs expansion and could give important insight in the knowledge of molecular mechanisms intervening during MSC expansion.

  13. Studying the Nucleated Mammalian Cell Membrane by Single Molecule Approaches

    Science.gov (United States)

    Wang, Feng; Wu, Jiazhen; Gao, Jing; Liu, Shuheng; Jiang, Junguang; Jiang, Shibo; Wang, Hongda

    2014-01-01

    The cell membrane plays a key role in compartmentalization, nutrient transportation and signal transduction, while the pattern of protein distribution at both cytoplasmic and ectoplasmic sides of the cell membrane remains elusive. Using a combination of single-molecule techniques, including atomic force microscopy (AFM), single molecule force spectroscopy (SMFS) and stochastic optical reconstruction microscopy (STORM), to study the structure of nucleated cell membranes, we found that (1) proteins at the ectoplasmic side of the cell membrane form a dense protein layer (4 nm) on top of a lipid bilayer; (2) proteins aggregate to form islands evenly dispersed at the cytoplasmic side of the cell membrane with a height of about 10–12 nm; (3) cholesterol-enriched domains exist within the cell membrane; (4) carbohydrates stay in microdomains at the ectoplasmic side; and (5) exposed amino groups are asymmetrically distributed on both sides. Based on these observations, we proposed a Protein Layer-Lipid-Protein Island (PLLPI) model, to provide a better understanding of cell membrane structure, membrane trafficking and viral fusion mechanisms. PMID:24806512

  14. Durability of symmetrically and asymmetrically porous polybenzimidazole membranes for high temperature proton exchange membrane fuel cells

    Science.gov (United States)

    Jheng, Li-Cheng; Chang, Wesley Jen-Yang; Hsu, Steve Lien-Chung; Cheng, Po-Yang

    2016-08-01

    Two types of porous polybenzimidazole (PBI) membranes with symmetric and asymmetric morphologies were fabricated by the template-leaching method and characterized by scanning electron microscope (SEM). Their physicochemical properties were compared in terms of acid-doping level, proton conductivity, mechanical strength, and oxidative stability. The durability of fuel cell operation is one of the most challenging for the PBI based membrane electrode assembly (MEA) used in high-temperature proton exchange membrane fuel cells (HT-PEMFCs). In the present work, we carried out a long-term steady-state fuel cell test to compare the effect of membrane structure on the cell voltage degradation. It has also been demonstrated that the asymmetrically porous PBI could bring some notable improvements on the durability of fuel cell operation, the fuel crossover problem, and the phosphoric acid leakage.

  15. Radiation-Grafted Polymer Electrolyte Membranes for Water Electrolysis Cells: Evaluation of Key Membrane Properties.

    Science.gov (United States)

    Albert, Albert; Barnett, Alejandro O; Thomassen, Magnus S; Schmidt, Thomas J; Gubler, Lorenz

    2015-10-14

    Radiation-grafted membranes can be considered an alternative to perfluorosulfonic acid (PFSA) membranes, such as Nafion, in a solid polymer electrolyte electrolyzer. Styrene, acrylonitrile, and 1,3-diisopropenylbenzene monomers are cografted into preirradiated 50 μm ethylene tetrafluoroethylene (ETFE) base film, followed by sulfonation to introduce proton exchange sites to the obtained grafted films. The incorporation of grafts throughout the thickness is demonstrated by scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX) analysis of the membrane cross-sections. The membranes are analyzed in terms of grafting kinetics, ion-exchange capacity (IEC), and water uptake. The key properties of radiation-grafted membranes and Nafion, such as gas crossover, area resistance, and mechanical properties, are evaluated and compared. The plot of hydrogen crossover versus area resistance of the membranes results in a property map that indicates the target areas for membrane development for electrolyzer applications. Tensile tests are performed to assess the mechanical properties of the membranes. Finally, these three properties are combined to establish a figure of merit, which indicates that radiation-grafted membranes obtained in the present study are promising candidates with properties superior to those of Nafion membranes. A water electrolysis cell test is performed as proof of principle, including a comparison to a commercial membrane electrode assembly (MEA). PMID:26393461

  16. Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands

    Science.gov (United States)

    Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G.

    2016-01-01

    The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands. PMID:27576083

  17. Nano thermo-hydrodynamics method for investigating cell membrane fluidity

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    As a barrier to compartmentalize cells,mem-branes form the interface between a cell and its surround-ings.The essential function of a membrane is to maintain a relatively stable environment in the cell,exchange sub-stances selectively and transfer energy and information continually from the outside.It is intriguing that above the phase transition temperature,the membrane lipid molecule will have three modes-lateral diffusion,rotational movement and flip-flop activity.These thermodynamic processes are vital to cell existence,growth,division,differentiation and are also responsible for hundreds of thousands of phenomena in life.Previously,species transport across the membrane was interpreted mainly from a phenomenological view using a lumped system model.Therefore,detailed flow processes occurred in the membrane domain and clues related to life mechanism were not sufficiently tackled.Such important issues can be clarifled by modeling nano scale thermal hydrodynamics over the gap space of a cell membrane.Previously observed complex membrane behaviors will be shown in this paper and explained by the thermally induced fluidic convections inside the membrane.A correlation between nano scale hydrodynamics,non-equilibrium thermodynamics and eell membrane activities is set up.The disclosed mechanisms are expected to provide a new viewpoint on the interaction between intracellular and extracellular processes through the membrane.

  18. 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...

  19. 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.

  20. Oncostatin M regulates membrane traffic and stimulates bile canalicular membrane biogenesis in HepG2 cells

    NARCIS (Netherlands)

    Van der Wouden, Johanna M.; Van IJzendoorn, Sven C.D.; Hoekstra, Dick

    2002-01-01

    Hepatocytes are the major epithelial cells of the liver and they display membrane polarity: the sinusoidal membrane representing the basolateral surface, while the bile canalicular membrane is typical of the apical membrane. In polarized HepG2 cells an endosomal organelle, SAC, fulfills a prominent

  1. Catalyst Degradation in High Temperature Proton Exchange Membrane Fuel Cells Based on Acid Doped Polybenzimidazole Membranes

    DEFF Research Database (Denmark)

    Cleemann, Lars Nilausen; Buazar, F.; Li, Qingfeng;

    2013-01-01

    Degradation of carbon supported platinum catalysts is a major failure mode for the long term durability of high temperature proton exchange membrane fuel cells based on phosphoric acid doped polybenzimidazole membranes. With Vulcan carbon black as a reference, thermally treated carbon black...

  2. Proteome array identification of bioactive soluble proteins/peptides in matrigel; relevance to stem cell responses

    Science.gov (United States)

    Matrigel and similar commercial products are extracts of the Engelbreth-Holm-Swarm sarcoma that provide a basement-membrane-like attachment factor or gel that is used to grow cells on or in. To ascertain further what proteins may be present in Matrigel, besides its major basement-membrane constitue...

  3. 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

  4. 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.

  5. Unique proteome signature of post-chemotherapy ovarian cancer ascites-derived tumor cells.

    Science.gov (United States)

    Ahmed, Nuzhat; Greening, David; Samardzija, Chantel; Escalona, Ruth M; Chen, Maoshan; Findlay, Jock K; Kannourakis, George

    2016-01-01

    Eighty % of ovarian cancer patients diagnosed at an advanced-stage have complete remission after initial surgery and chemotherapy. However, most patients die within identification of 353 proteins. There were significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism pathways in CR tumor cells. In conclusion, this is the first proteomics study to comprehensively analyze ascites-derived tumor cells from CN and CR ovarian cancer patients. PMID:27470985

  6. Electron Spin Resonance Study of Fuel Cell Polymer Membrane Degradation

    Institute of Scientific and Technical Information of China (English)

    Alexander Panchenko; Elena Aleksandrova; Emil Roduner

    2005-01-01

    @@ 1Introduction The long term stability of the membrane is an important factor limiting the fuel cell lifetime. During extended use the membrane degrades, probably via reaction with hydroxyl and superoxide radicals which are regular intermediates of the oxygen reduction at the cathode. Only extremely stable membranes can withstand the aggressive chemical and physical environment in an operating fuel cell. Within a given set of operating conditions, intrinsic chemical and mechanical properties of the membrane as well as its water content impact its durability dramatically.

  7. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    Science.gov (United States)

    Stingaciu, Laura-Roxana; O'Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  8. Proteomic signature of Arabidopsis cell cultures exposed to magnetically induced hyper- and microgravity environments.

    Science.gov (United States)

    Herranz, Raul; Manzano, Ana I; van Loon, Jack J W A; Christianen, Peter C M; Medina, F Javier

    2013-03-01

    Earth-based microgravity simulation techniques are required due to space research constraints. Using diamagnetic levitation, we exposed Arabidopsis thaliana in vitro callus cultures to environments with different levels of effective gravity and magnetic field strengths (B) simultaneously. The environments included simulated 0 g* at B=10.1 T, an internal 1 g* control (B=16.5 T), and hypergravity (2 g* at B=10.1 T). Furthermore, samples were also exposed to altered gravity environments that were created with mechanical devices, such as the Random Positioning Machine (simulated μg) and the Large Diameter Centrifuge (2 g). We have determined the proteomic signature of cell cultures exposed to these altered-gravity environments by means of the difference gel electrophoresis (DiGE) technique, and we have compared the results with microarray-based transcriptomes from the same samples. The magnetic field itself produced a low number of proteomic alterations, but the combination of gravitational alteration and magnetic field exposure produced synergistic effects on the proteome of plants (the number of significant changes is 3-7 times greater). Tandem mass spectrometry identification of 19 overlapping spots in the different conditions corroborates a major role of abiotic stress and secondary metabolism proteins in the molecular adaptation of plants to unusual environments, including microgravity.

  9. 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.

  10. A draft map of the mouse pluripotent stem cell spatial proteome.

    Science.gov (United States)

    Christoforou, Andy; Mulvey, Claire M; Breckels, Lisa M; Geladaki, Aikaterini; Hurrell, Tracey; Hayward, Penelope C; Naake, Thomas; Gatto, Laurent; Viner, Rosa; Martinez Arias, Alfonso; Lilley, Kathryn S

    2016-01-01

    Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data. PMID:26754106

  11. SILAC-Based Quantitative Proteomic Analysis of Diffuse Large B-Cell Lymphoma Patients

    Directory of Open Access Journals (Sweden)

    Ulla Rüetschi

    2015-01-01

    Full Text Available Diffuse large B-cell lymphoma (DLBCL, the most common lymphoma, is a heterogeneous disease where the outcome for patients with early relapse or refractory disease is very poor, even in the era of immunochemotherapy. In order to describe possible differences in global protein expression and network patterns, we performed a SILAC-based shotgun (LC-MS/MS quantitative proteomic analysis in fresh-frozen tumor tissue from two groups of DLBCL patients with totally different clinical outcome: (i early relapsed or refractory and (ii long-term progression-free patients. We could identify over 3,500 proteins; more than 1,300 were quantified in all patients and 87 were significantly differentially expressed. By functional annotation analysis on the 66 proteins overexpressed in the progression-free patient group, we found an enrichment of proteins involved in the regulation and organization of the actin cytoskeleton. Also, five proteins from actin cytoskeleton regulation, applied in a supervised regression analysis, could discriminate the two patient groups. In conclusion, SILAC-based shotgun quantitative proteomic analysis appears to be a powerful tool to explore the proteome in DLBCL tumor tissue. Also, as progression-free patients had a higher expression of proteins involved in the actin cytoskeleton protein network, such a pattern indicates a functional role in the sustained response to immunochemotherapy.

  12. Detecting Nanodomains in Living Cell Membrane by Fluorescence Correlation Spectroscopy

    Science.gov (United States)

    He, Hai-Tao; Marguet, Didier

    2011-05-01

    Cell membranes actively participate in numerous cellular functions. Inasmuch as bioactivities of cell membranes are known to depend crucially on their lateral organization, much effort has been focused on deciphering this organization on different length scales. Within this context, the concept of lipid rafts has been intensively discussed over recent years. In line with its ability to measure diffusion parameters with great precision, fluorescence correlation spectroscopy (FCS) measurements have been made in association with innovative experimental strategies to monitor modes of molecular lateral diffusion within the plasma membrane of living cells. These investigations have allowed significant progress in the characterization of the cell membrane lateral organization at the suboptical level and have provided compelling evidence for the in vivo existence of raft nanodomains. We review these FCS-based studies and the characteristic structural features of raft nanodomains. We also discuss the findings in regards to the current view of lipid rafts as a general membrane-organizing principle.

  13. Comparative proteomics of milk fat globule membrane proteins from transgenic cloned cattle.

    Science.gov (United States)

    Sui, Shunchao; Zhao, Jie; Wang, Jianwu; Zhang, Ran; Guo, Chengdong; Yu, Tian; Li, Ning

    2014-01-01

    The use of transgenic livestock is providing new methods for obtaining pharmaceutically useful proteins. However, the protein expression profiles of the transgenic animals, including expression of milk fat globule membrane (MFGM) proteins, have not been well characterized. In this study, we compared the MFGM protein expression profile of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle, i.e., expressing recombinant human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland, with those from cloned non-transgenic (C) and conventionally bred normal animals (N). We identified 1, 225 proteins in milk MFGM, 166 of which were specifically expressed only in the TC-LA group, 265 only in the TC-LF group, and 184 only in the TC-LZ group. There were 43 proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analysis also showed that the 43 proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed that MFGM proteins were up- or down-regulated more than twofold in the TC and C groups compared to N group: 126 in colostrum and 77 in mature milk of the TC-LA group; 157 in colostrum and 222 in mature milk of the TC-LF group; 49 in colostrum and 98 in mature milk of the TC-LZ group; 98 in colostrum and 132 in mature milk in the C group. These up- and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway, which appears that expression of certain exogenous proteins has no general deleterious effects on the cattle mammary gland.

  14. 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.

  15. 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.

  16. Proteomics study of progeny of normal human liver cells irradiated by 60Co γ-rays

    International Nuclear Information System (INIS)

    Objective: To characterize the differential protein expression in the progeny of human liver cells surviving from ionizing radiation by the proteomic analysis. Methods: Two-dimensional electrophoresis gel coupled with mass spectrometry was used to explore the specific protein expression in the progeny of 7702 human liver cells surviving from ionizing radiation. Alterations in expression level of protein spots between the control and the progeny groups were statistically analyzed by ImageMaster 2D Platinum software and mass spectrometry was used to identify the protein spots with significantly altered expression-level. Results: The progeny of irradiated ceils were derived from human liver cell line exposed to 0, 2, 4, 6 Gy of 60Co γ-irradiation. A total of 42 differentially expressed proteins between the control and the progeny of the irradiated cells groups were screened, of which 17 were identified by matrix assistant laser desorption ion-top off light-mass spectrometry (MALDI-TOF MS) analysis, including 4 up-regulated and 13 down-regnlated proteins. Conclusions: The differentially expressed proteins profile could be significantly altered in the progeny of irradiated cells. The proteomics approach has the potential to detect the protein changes relevant to radiatian-induced genomic instability (RIGI). Further study of differentially expressed proteins would likely reveal the molecular mechanisms of gene expression in RIGI. (authors)

  17. Label-free and spike-in standard-free mass spectrometry in the proteomic analysis of plasma membrane proteins and membrane-associated protein networks

    OpenAIRE

    Niehage, Christian

    2014-01-01

    Mass spectrometry is the primary technology of proteomics. For the analysis of complex proteomes, protein identities and quantities are inferred from their peptides that are generated by cleaving all proteins with the endopeptidase trypsin. But there is one major disadvantage that is due to biophysical differences, different peptides cause different intensities. Miscellaneous approaches have been developed to circumvent this problem based on the chemical or metabolic introduction of heavy sta...

  18. 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.

  19. Functional proteomics screen enables enrichment of distinct cell types from human pancreatic islets.

    Directory of Open Access Journals (Sweden)

    Revital Sharivkin

    Full Text Available The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates specific cell-surface markers with particular cell functionality by coupling cell capture on antibody arrays with immunofluorescent labeling. Using this approach in an iterative manner, we discovered marker combinations capable of enriching for discrete pancreatic cell subtypes from human islets of Langerhans: insulin-producing beta cells (CD9high/CD56+, glucagon-producing alpha cells (CD9-/CD56+ and trypsin-producing acinar cells (CD9-/CD56-. This strategy may assist future beta cell research and the development of diagnostic tools for diabetes. It can also be applied more generally for function-based purification of desired cell types from other limited and heterogeneous biological samples.

  20. How the antimicrobial peptides destroy bacteria cell membrane: Translocations vs. membrane buckling

    Science.gov (United States)

    Golubovic, Leonardo; Gao, Lianghui; Chen, Licui; Fang, Weihai

    2012-02-01

    In this study, coarse grained Dissipative Particle Dynamics simulation with implementation of electrostatic interactions is developed in constant pressure and surface tension ensemble to elucidate how the antimicrobial peptide molecules affect bilayer cell membrane structure and kill bacteria. We find that peptides with different chemical-physical properties exhibit different membrane obstructing mechanisms. Peptide molecules can destroy vital functions of the affected bacteria by translocating across their membranes via worm-holes, or by associating with membrane lipids to form hydrophilic cores trapped inside the hydrophobic domain of the membranes. In the latter scenario, the affected membranes are strongly corrugated (buckled) in accord with very recent experimental observations [G. E. Fantner et al., Nat. Nanotech., 5 (2010), pp. 280-285].

  1. Measurement of the nonlinear elasticity of red blood cell membranes

    Science.gov (United States)

    Park, Yongkeun; Best, Catherine A.; Kuriabova, Tatiana; Henle, Mark L.; Feld, Michael S.; Levine, Alex J.; Popescu, Gabriel

    2011-05-01

    The membranes of human red blood cells (RBCs) are a composite of a fluid lipid bilayer and a triangular network of semiflexible filaments (spectrin). We perform cellular microrheology using the dynamic membrane fluctuations of the RBCs to extract the elastic moduli of this composite membrane. By applying known osmotic stresses, we measure the changes in the elastic constants under imposed strain and thereby determine the nonlinear elastic properties of the membrane. We find that the elastic nonlinearities of the shear modulus in tensed RBC membranes can be well understood in terms of a simple wormlike chain model. Our results show that the elasticity of the spectrin network can mostly account for the area compression modulus at physiological osmolality, suggesting that the lipid bilayer has significant excess area. As the cell swells, the elastic contribution from the now tensed lipid membrane becomes dominant.

  2. Membrane curvature in cell biology: An integration of molecular mechanisms.

    Science.gov (United States)

    Jarsch, Iris K; Daste, Frederic; Gallop, Jennifer L

    2016-08-15

    Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists.

  3. 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.

  4. Oxidative degradation of polybenzimidazole membranes as electrolytes for high temperature proton exchange membrane fuel cells

    DEFF Research Database (Denmark)

    Liao, J.H.; Li, Qingfeng; Rudbeck, H.C.;

    2011-01-01

    Polybenzimidazole membranes imbibed with acid are emerging as a suitable electrolyte material for high-temperature polymer electrolyte fuel cells. The oxidative stability of polybenzimidazole has been identified as an important issue for the long-term durability of such cells. In this paper...... the oxidative degradation of the polymer membrane was studied under the Fenton test conditions by the weight loss, intrinsic viscosity, size exclusion chromatography, scanning electron microscopy and Fourier transform infrared spectroscopy. During the Fenton test, significant weight losses depending...

  5. The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

    Science.gov (United States)

    Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

    2015-04-21

    A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

  6. Nafion-layered silicate nanocomposite membrane for fuel cell application

    OpenAIRE

    Thomassin, Jean-Michel; Pagnoulle, Christophe; Bizzari, Didier; Caldarella, Giuseppe; Germain, Albert; Jérôme, Robert

    2004-01-01

    Direct methanol fuel cells (DMFCs) using a proton exchange membrane as electrolyte is an attractive option for electricity generation. The most widely used membrane in the DMFC system is based on a perfluorinated polymer bearing sulfonic acid functions like Nafion®. The latter combines chemical, mechanical and thermal stability and high protonic conductivity but shows elevated methanol permeability. We propose the preparation of a novel type of hybrid membranes to tentatively solve this probl...

  7. Improved Membrane Materials for PEM Fuel Cell Application

    Energy Technology Data Exchange (ETDEWEB)

    Kenneth A. Mauritz; Robert B. Moore

    2008-06-30

    The overall goal of this project is to collect and integrate critical structure/property information in order to develop methods that lead to significant improvements in the durability and performance of polymer electrolyte membrane fuel cell (PEMFC) materials. This project is focused on the fundamental improvement of PEMFC membrane materials with respect to chemical, mechanical and morphological durability as well as the development of new inorganically-modified membranes.

  8. Nanoscale cell membrane organization : a near-field optical view

    NARCIS (Netherlands)

    Koopman, Marjolein

    2006-01-01

    The cell plasma membrane of eukaryotic cells is a lipid bi-layer that separates the cell cytosol from the extracellular environment. The composition and organization of proteins and lipids within this bi-layer have a direct impact on many cellular processes, since they form the senses of the cell. T

  9. Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Dun [Population Council, 1230 York Avenue, New York, NY 10065 (United States); Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000 (China); Chen, Hai-Xiao, E-mail: Hxchen-1@163.net [Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000 (China); Yu, Hai-Qiang [Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Liang, Yong; Wang, Carrie [Population Council, 1230 York Avenue, New York, NY 10065 (United States); Lian, Qing-Quan [The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000 (China); Deng, Hai-Teng, E-mail: dengh@mail.rockefeller.edu [Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ge, Ren-Shan, E-mail: rge@popcbr.rockefeller.edu [Population Council, 1230 York Avenue, New York, NY 10065 (United States); The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000 (China)

    2010-08-15

    Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.

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

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian;

    2009-01-01

    of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B...

  11. Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomic studies.

    Science.gov (United States)

    Zhang, Ying; Bottinelli, Dario; Lisacek, Frédérique; Luban, Jeremy; Strambio-De-Castillia, Caterina; Varesio, Emmanuel; Hopfgartner, Gérard

    2015-09-01

    Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry (MS)-based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize MS coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilization and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA (radioimmunoprecipitation assay) buffer, was shown to be the method of choice based on total protein extraction and on the solubilization and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than that by 10% trichloroacetic acid (TCA)/acetone, allowing in excess of 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate into the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6 to 11% more distinct peptides and 14 to 19% more total proteins identified than using 0.5M triethylammonium bicarbonate alone, with the greatest increase (34%) for hydrophobic proteins. PMID:25983236

  12. Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomics studies

    Science.gov (United States)

    Zhang, Ying; Bottinelli, Dario; Lisacek, Frédérique; Luban, Jeremy; De Castillia, Caterina Strambio; Varesio, Emmanuel; Hopfgartner, Gérard

    2016-01-01

    Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize mass spectrometry coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilisation and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA buffer, was shown to be the method of choice based on total protein extraction and on the solubilisation and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than by 10% TCA/acetone, allowing greater than 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone-wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate in the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6-11% more distinct peptides and 14-19% more total proteins identified than using 0.5M triethylammonium bicarbonate alone with the greatest increase (34%) for hydrophobic proteins. PMID:25983236

  13. 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.

  14. Characterization of Plasma Membrane Proteins from Ovarian Cancer Cells Using Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    David L. Springer

    2004-01-01

    Full Text Available To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsin digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap. Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane-associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.

  15. 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. PMID:26945515

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

    Directory of Open Access Journals (Sweden)

    Meike Herget

    Full Text Available 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 <3% at the protein level and <1% at the peptide level. 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.

  17. Investigating cell membrane structure and dynamics with TCSPC-FLIM

    Science.gov (United States)

    Le Marois, Alix; Owen, Dylan M.; Suhling, Klaus

    2015-03-01

    We report the use of Time-Correlated Single Photon Counting (TCSPC) in a polarization-resolved Fluorescence Lifetime Imaging (FLIM) setup for the investigation of cell membrane structural and dynamic properties. This technique allows us to study the orientation and mobility of fluorescent membrane dyes, namely di-4-ANEPPDHQ and DiO, in model bilayers of different lipid compositions. Dipole alignment and extent of rotational motion can be linked to membrane order and fluidity. Comparison of the time-resolved anisotropy decays of the two fluorescent dyes suggests that rotational motion of membrane constituents is restricted in liquid-ordered phases, and appears to be limited to the region of aliphatic tails in liquid-disordered phases. In living cells, understanding the membrane structure provides crucial information on its functional properties, such as exo- and endocytosis, cell mobility and signal transduction.

  18. 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.

  19. Toughness of membranes applied in polymer electrolyte fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Kiefer, J.; Brack, H.P.; Scherer, G.G. [Paul Scherrer Inst. (PSI), Villigen (Switzerland)

    1999-08-01

    Since several years we apply the radiation-grafting technique to prepare polymeric membranes for application in polymer electrolyte fuel cells (PEFCs). Our investigations presented here focus on changes in toughness of these materials after the various synthesis steps and the importance of membrane toughness for their application in PEFCs. (author) 2 figs., 4 refs.

  20. 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...

  1. 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

    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...... analyzed by LC and electrospray QTOF-MS. The methods were evaluated according to efficiency, purity, transmembrane protein recovery, as well as for suitability to large-scale preparations. Our data clearly demonstrate that mucosal shaving is by far the best-suited method for in-depth MS analysis in terms...

  2. Prism-patterned Nafion membrane for enhanced water transport in polymer electrolyte membrane fuel cell

    Science.gov (United States)

    Kim, Sang Moon; Kang, Yun Sik; Ahn, Chiyeong; Jang, Segeun; Kim, Minhyoung; Sung, Yung-Eun; Yoo, Sung Jong; Choi, Mansoo

    2016-06-01

    Here, we report a simple and effective strategy to enhance the performance of the polymer electrolyte membrane fuel cell by imprinting prism-patterned arrays onto the Nafion membrane, which provides three combined effects directly related to the device performance. First, a locally thinned membrane via imprinted micro prism-structures lead to reduced membrane resistance, which is confirmed by electrochemical impedance spectroscopy. Second, increments of the geometrical surface area of the prism-patterned Nafion membrane compared to a flat membrane result in the increase in the electrochemical active surface area. Third, the vertically asymmetric geometry of prism structures in the cathode catalyst layer lead to enhanced water transport, which is confirmed by oxygen gain calculation. To explain the enhanced water transport, we propose a simple theoretical model on removal of water droplets existing in the asymmetric catalyst layer. These three combined effects achieved via incorporating prism patterned arrays into the Nafion membrane effectively enhance the performance of the polymer electrolyte membrane fuel cell.

  3. 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

  4. 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

    Ferritin, the major intracellular iron-storage protein, is made of 24 subunits of two types, H and L. Besides regulating intracellular iron homeostasis, it has been found that ferritin, in particular the H subunit (FHC), is involved in different biological events such as cell differentiation...... in metabolic pathways related to tumor progression and metastasis. In vitro assays confirmed that the FHC-silenced MM07(m) cells are characterized by a decreased growth activity, a reduced invasiveness, and a reduced cell adhesion capability. Moreover, nude mice (CD1 nu/nu), subcutaneously injected with FHC......-silenced MM07(m) cells, showed a remarkable 4-fold reduction of their tumor growth capacity compared to those who received the FHC-unsilenced MM07(m) counterpart. In conclusion, these data indicate that gene silencing technology, coupled to proteomic analysis, is a powerful tool for a better understanding...

  5. BLEND MEMBRANES FOR DIRECT METHANOL AND PROTON EXCHANGE MEMBRANE FUEL CELLS

    Institute of Scientific and Technical Information of China (English)

    Perurnal Bhavani; Dharmalingam Sangeetha

    2012-01-01

    Sulphonated polystyrene ethylene butylene polystyrene (SPSEBS) prepared with 35% sulphonation was found to be highly elastic and enlarged up to 300%-400% of its initial length.It absorbed over 110% of water by weight.A major drawback of this membrane is its poor mechanical properties which are not adequate for use as polymer electrolytes in fuel cells.To overcome this,SPSEBS was blended with poly(vinylidene fluoride) (PVDF),a hydrophobic polymer.The blend membranes showed better mechanical properties than the base polymer.The effect of PVDF content on water uptake,ion exchange capacity and proton conductivity of the blend membranes was investigated.This paper presents the results of recent studies applied to develop an optimized in-house membrane electrode assembly (MEA) preparation technique combining catalyst ink spraying and assembly hot pressing.Easy steps were chosen in this preparation technique in order to simplify the method,aiming at cost reduction.The open circuit voltage for the cell with SPSEBS is 0.980 V which is higher compared to that of the cell with Nafion 117 (0.790 V).From this study,it is concluded that a polymer electrolyte membrane suitable for proton exchange membrane fuel cell (PEMFC) and direct methanol fuel cell (DMFC) application can be obtained by blending SPSEBS and PVDF in appropriate proportions.The methanol permeability and selectivity showed a strong influence on DMFC performance.

  6. Sulfated Titania-Silica Reinforced Nafion Nanocomposite Membranes for Proton Exchange Membrane Fuel Cells.

    Science.gov (United States)

    Abu Sayeed, M D; Kim, Hee Jin; Gopalan, A I; Kim, Young Ho; Lee, Kwang-Pill; Choi, Sang-June

    2015-09-01

    Sulfated titania-silica (SO4(2-)-/TiO2-SiO2) composites were prepared by a sol-gel method with sulfate reaction and characterized by X-ray diffraction (XRD) and energy-dispersive X-ray spectroscopy (EDS). The nanometric diameter and geometry of the sulfated titania-silica (STS) was investigated by transmission electron microscopy (TEM). A small amount of the STS composite in the range of 0.5-3 wt% was then added as reinforcing into the Nafion membrane by water-assisted solution casting method to prepare STS reinforced Nafion nanocomposite membranes (STS-Nafion nanocomposite membranes). The additional functional groups, sulfate groups, of the nanocomposite membrane having more surface oxygenated groups enhanced the fuel cell membrane properties. The STS-Nafion nanocomposite membranes exhibited improved water uptake compared to that of neat Nafion membranes, whereas methanol uptake values were decreased dramatically improved thermal property of the prepared nanocomposite membranes were measured by thermogravimetric analysis (TGA). Furthermore, increased ion exchange capacity values were obtained by thermoacidic pretreatment of the nanocomposite membranes. PMID:26716283

  7. Gangliosides in cell recognition and membrane protein regulation

    OpenAIRE

    Lopez, Pablo H. H.; Schnaar, Ronald L.

    2009-01-01

    Gangliosides, sialic acid-bearing glycosphingolipids, are expressed on all vertebrate cells, and are the major glycans on nerve cells. They are anchored to the plasma membrane through their ceramide lipids with their varied glycans extending into the extracellular space. Through sugar-specific interactions with glycan binding proteins on apposing cells, gangliosides function as receptors in cell-cell recognition, regulating natural killer cell cytotoxicity via Siglec-7 binding, myelin-axon in...

  8. 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.

  9. 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.

  10. Proteomic study via a non-gel based approach of meningococcal outer membrane vesicle vaccine obtained from strain CU385: a road map for discovering new antigens.

    Science.gov (United States)

    Gil, Jeovanis; Betancourt, L Zaro H; Sardiñas, Gretel; Yero, Daniel; Niebla, Olivia; Delgado, Maité; García, Darien; Pajón, Rolando; Sánchez, Aniel; González, Luis J; Padrón, Gabriel; Campa, Concepción; Sotolongo, Franklin; Barberó, Ramón; Guillén, Gerardo; Herrera, Luis; Besada, Vladimir

    2009-05-01

    This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatographymass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.

  11. 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

    2016-02-01

    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.

  12. 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).

  13. Effect of irradiation on cell transcriptome and proteome of rat submandibular salivary glands.

    Directory of Open Access Journals (Sweden)

    Raluca Stiubea-Cohen

    Full Text Available Salivary glands (SGs are irreversibly damaged by irradiation (IR treatment in head and neck cancer patients. Here, we used an animal irradiation model to investigate and define the molecular mechanisms affecting SGs following IR, focusing on saliva proteome and global transcription profile of submandibular salivary gland (SSG tissue.We show that saliva secretion was gradually reduced to 50% of its initial level 12 weeks post-IR. Saliva protein composition was further examined by proteomic analysis following mass spectrometry (MS analysis that revealed proteins with reduced expression originating from SSGs and proteins with increased expression derived from the serum, both indicating salivary tissue damage. To examine alterations in mRNA expression levels microarray analysis was performed. We found significant alterations in 95 genes, including cell-cycle arrest genes, SG functional genes and a DNA repair gene.Tissue damage was seen by confocal immunofluorescence of α-amylase and c-Kit that showed an increase and decrease, respectively, in protein expression. This was coherent with real-time PCR results.This data indicates that IR damages the SSG cells' ability to produce and secrete saliva and proteins, and maintain the physiological barrier between serum and saliva. The damage does not heal due to cell-cycle arrest, which prevents tissue regeneration. Taken together, our results reveal a new insight into IR pathobiology.

  14. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    Science.gov (United States)

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  15. 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

  16. 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.

  17. 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

  18. Novel Membrane for Highly Efficient Fuel Cells Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Proton Exchange Membrane (PEM) fuel cells and electrolyzers are key technologies for NASA space systems utilizing hydrogen, oxygen, or water as reactants. In order...

  19. Novel High Temperature Membrane for PEM Fuel Cells Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The innovation proposed in this STTR program is a high temperature membrane to increase the efficiency and power density of PEM fuel cells. The NASA application is...

  20. Bipolar membranes in forward bias region for fuel cell reactors

    International Nuclear Information System (INIS)

    Three bipolar membranes, two home-made composed of commercial cation (DuPont) and anion (FuMA-Tech) exchange membranes (called Nafion/FT-FAA and Nafion/FT-FAS) and a commercial one, BP-1 from FuMA-Tech, were investigated in order to characterize their suitability to use in a H2/O2 fuel cell intended to produce hydrogen peroxide on the cathode instead of water. The Nafion/FT-FAA and Nafion/FT-FAS membranes were prepared using a hot-pressing method. The optimal hot-pressing conditions were determined by measuring the ionic conductivity of the membranes. The latter was observed to depend on the relative humidity of the bipolar membrane. Of the studied bipolar membranes, Nafion/FT-FAA showed the best performance. The transport number of protons measured in a concentration cell was observed to depend on the direction of the proton diffusion flux through these membranes so that transport numbers of ca. unity were obtained when the cation exchange side faced the solution with higher proton concentration. In the opposite case, when the higher concentration faced anion exchange side, the transport number of proton was clearly lower, indicating the usefulness of the bipolar membranes for hydrogen peroxide production in the fuel cell

  1. 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.

  2. 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. PMID:23525364

  3. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen

    Directory of Open Access Journals (Sweden)

    Jodie Lopez

    2015-12-01

    Full Text Available Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV, resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation.

  4. Electrospun nanofibre composite polymer electrolyte fuel cell and electrolysis membranes

    OpenAIRE

    Sood, Rakhi; Cavaliere, Sara; Rozière, Jacques; Jones, Deborah

    2016-01-01

    International audience Large-scale commercialisation of Proton Exchange Membrane Fuel Cell (PEMFC) technology for automotive and stationary applications demands the development of a robust, durable and cost-effective materials. In this regard, ionomer membranes being present at the core of PEMFCs are required to maintain elevated proton conductivity, high mechanical strength and low gas permeability during the lifespan of the fuel cell. These challenges are addressed by investigating novel...

  5. 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

  6. Effects of altered gravity on the cell cycle, actin cytoskeleton and proteome in Physarum polycephalum

    Science.gov (United States)

    He, Jie; Zhang, Xiaoxian; Gao, Yong; Li, Shuijie; Sun, Yeqing

    Some researchers suggest that the changes of cell cycle under the effect of microgravity may be associated with many serious adverse physiological changes. In the search for underlying mechanisms and possible new countermeasures, we used the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony to study the effects of altered gravity on the cell cycle, actin cytoskeleton and proteome. In parallel, the cell cycle was analyzed in Physarum incubated (1) in altered gravity for 20 h, (2) in altered gravity for 40 h, (3) in altered gravity for 80 h, and (4) in ground controls. The cell cycle, the actin cytoskeleton, and proteome in the altered gravity and ground controls were examined. The results indicated that the duration of the G2 phase was lengthened 20 min in high aspect ratio vessel (HARV) for 20 h, and prolonged 2 h in altered gravity either for 40 h or for 80 h, whereas the duration of other phases in the cell cycle was unchanged with respect to the control. The microfilaments in G2 phase had a reduced number of fibers and a unique abnormal morphology in altered gravity for 40 h, whereas the microfilaments in other phases of cell cycle were unchanged when compared to controls. Employing classical two-dimensional electrophoresis (2-DE), we examined the effect of the altered gravity on P. polycephalum proteins. The increase in the duration of G2 phase in altered gravity for 40 h was accompanied by changes in the 2-DE protein profiles, over controls. Out of a total of 200 protein spots investigated in G2 phase, which were reproducible in repeated experiments, 72 protein spots were visually identified as specially expressed, and 11 proteins were up-regulated by 2-fold and 28 proteins were down-regulated by 2-fold over controls. Out of a total of three low-expressed proteins in G2 phase in altered gravity for 40 h, two proteins were unknown proteins, and one protein was spherulin 3b by MALDI-TOF mass spectrometry (MS

  7. 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...

  8. Proteomic response analysis of endothelial cells of human coronary artery to stimulation with carbachol

    Institute of Scientific and Technical Information of China (English)

    Min YU; Dong-mei CHEN; Gang HU; Hai WANG

    2004-01-01

    AIM: To identify the molecular basis of the endothelial target for acetylcholine (ETA). METHODS: Proteomic methods were used to monitor changes in protein expression in the first 10 h following the stimulation of human coronary endothelial cells with carbachol 100 μmol/L. Thirty proteins showing the largest changes were identified by mass spectrometry. RESULTS: Based on analysis with Image Master 2-D Elite software, about 623 protein spots were detected in control cells and 825 protein spots in carbachol-treated cells, the matching rate was 68.1%.Among all the detected spots, 39 were up-regulated and 29 were down-regulated, showing detectable changes varied from 1.7-3.8 folds. Forty one spots in the peptide mass fingerprints were successfully obtained. The most interesting feature was that all the four newly synthesized proteins belonged to the heat shock protein family.CONCLUSION: These identified proteins played key roles in the molecular mechanism of ETA.

  9. Surface-enhanced Raman spectroscopy of the endothelial cell membrane.

    Directory of Open Access Journals (Sweden)

    Simon W Fogarty

    Full Text Available We applied surface-enhanced Raman spectroscopy (SERS to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces.

  10. Spray deposition of Nafion membranes: Electrode-supported fuel cells

    Science.gov (United States)

    Bayer, Thomas; Pham, Hung Cuong; Sasaki, Kazunari; Lyth, Stephen Matthew

    2016-09-01

    Fuel cells are a key technology for the successful transition towards a hydrogen society. In order to accelerate fuel cell commercialization, improvements in performance are required. Generally, polymer electrolyte membrane fuel cells (PEFCs) are membrane-supported; the electrocatalyst layer is sprayed onto both sides of the membrane, and sandwiched between carbon-based gas diffusion layers (GDLs). In this work we redesign the membrane electrode assembly (MEA) and fabricate an electrode-supported PEFC. First the electrocatalyst layer is sprayed onto the GDL, and then Nafion dispersion is sprayed over the top of this to form a thin membrane. This method has the advantage of simplifying the fabrication process, allowing the fabrication of extremely thin electrolyte layers (down to ∼10 μm in this case), and reducing the amount of ionomer required in the cell. Electrode-supported PEFCs operate at significantly increased power density compared to conventional membrane-supported PEFCs, with a maximum of 581 mW/cm2 at 80 °C (atmospheric pressure, air at the cathode). Impedance spectroscopy confirmed that the origin of the improved performance was an 80% reduction in the membrane resistance due the thinner Nafion layer. This novel fabrication method is a step towards cheaper, thinner, fully printable PEFCs with high power density and efficiency.

  11. 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.

  12. Identifying cytotoxic T cell epitopes from genomic and proteomic information: "The human MHC project."

    DEFF Research Database (Denmark)

    Lauemøller, S L; Kesmir, C; Corbet, S L;

    2000-01-01

    Complete genomes of many species including pathogenic microorganisms are rapidly becoming available and with them the encoded proteins, or proteomes. Proteomes are extremely diverse and constitute unique imprints of the originating organisms allowing positive identification and accurate discrimin...

  13. 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.

  14. Membrane transport of anandamide through resealed human red blood cell membranes

    DEFF Research Database (Denmark)

    Bojesen, I.N.; Hansen, Harald S.

    2005-01-01

    of unidirectional flux from inside to outside is 0.361 ± 0.023 s. The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]) increases with the square root of [BSA] in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane...... very rapidly, within seconds. At a molar ratio of anandamide to BSA of...

  15. Classification of Cells with Membrane Staining and/or Fixation Based on Cellular Specific Membrane Capacitance and Cytoplasm Conductivity

    Directory of Open Access Journals (Sweden)

    Song-Bin Huang

    2015-01-01

    Full Text Available Single-cell electrical properties (e.g., specific membrane capacitance (Cspecific membrane and cytoplasm conductivity (σcytoplasm have been regarded as potential label-free biophysical markers for the evaluation of cellular status. However, whether there exist correlations between these biophysical markers and cellular status (e.g., membrane-associate protein expression is still unknown. To further validate the utility of single-cell electrical properties in cell type classification, Cspecific membrane and σcytoplasm of single PC-3 cells with membrane staining and/or fixation were analyzed and compared in this study. Four subtypes of PC-3 cells were prepared: untreated PC-3 cells, PC-3 cells with anti-EpCAM staining, PC-3 cells with fixation, and fixed PC-3 cells with anti-EpCAM staining. In experiments, suspended single cells were aspirated through microfluidic constriction channels with raw impedance data quantified and translated to Cspecific membrane and σcytoplasm. As to experimental results, significant differences in Cspecific membrane were observed for both live and fixed PC-3 cells with and without membrane staining, indicating that membrane staining proteins can contribute to electrical properties of cellular membranes. In addition, a significant decrease in σcytoplasm was located for PC-3 cells with and without fixation, suggesting that cytoplasm protein crosslinking during the fixation process can alter the cytoplasm conductivity. Overall, we have demonstrated how to classify single cells based on cellular electrical properties.

  16. 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.

  17. Cytotoxicity of bovine and porcine collagen membranes in mononuclear cells.

    Science.gov (United States)

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Carneiro, Karine Fernandes; Souza, Maria Aparecida de; Magalhães, Denildo

    2012-01-01

    This study compared the cytotoxicity and the release of nitric oxide induced by collagen membranes in human mononuclear cells. Peripheral blood was collected from each patient and the separation of mononuclear cells was performed by Ficoll. Then, 2x10(5) cells were plated in 48-well culture plates under the membranes in triplicate. The polystyrene surface was used as negative control. Cell viability was assessed by measuring mitochondrial activity (MTT) at 4, 12 and 24 h, with dosage levels of nitrite by the Griess method for the same periods. Data had non-normal distribution and were analyzed by the Kruskal-Wallis test (p<0.05). Statistically significant differences (p<0.05) were observed between the membranes and the control in the experimental period, although there was a significant reduction in viability over time (p<0.01). At 4 and 12 h, the porcine membrane induced a higher release of nitrite compared with the control and bovine membrane, respectively (p<0.01), and this difference was maintained at 24 h (p<0.05). This in vitro study showed that the porcine collagen membrane induces an increased production of proinflammatory mediators by mononuclear cells in the first hours of contact, decreasing with time. PMID:22460313

  18. Proteins upregulated by mild and severe hypoxia in squamous cell carcinomas in vitro identified by proteomics

    International Nuclear Information System (INIS)

    Background: Solid malignant tumours are characterised by an inadequate vascular system, which can give rise to micro-regional hypoxic areas. As the negative impact of tumour hypoxia is believed largely to depend on dynamic changes in gene expression, it is important to identify the genes regulated by hypoxia to further enlighten the biology behind the cellular response to hypoxia. Previous studies have demonstrated that hypoxia has an impact not only on the gene transcription, but also on gene-specific mRNA translation. Therefore, proteomics is a suitable approach to understand the complexity of gene regulation under hypoxia at protein level. In this in vitro study we have studied the proteome of cells under intermediate hypoxia (1% O2) and anoxia and compared these to normoxic (21% O2) cells to identify proteins upregulated by mild and severe hypoxia. Materials and methods: A human cervix cancer cell line (SiHa) and a human head and neck cancer cell line (FaDuDD) were used. Total cell lysate from hypoxic and normoxic cells was separated by 2-dimensional gel electrophoresis, and images were analysed using Quantity One software. Proteins from significant spots (difference in intensity by more than a factor 2) were identified by Liquid chromatography-mass spectrometry (LC-MS/MS). In order to confirm the hypoxic regulation of the identified proteins, immunoblotting and qPCR were employed when possible. Results: All together 32 spots were found to be upregulated in the hypoxic gels. Of these, 11 different proteins were successfully identified and largely confirmed by Western blotting and qPCR. Amongst these proteins are protein disulfide isomerase family A, member 6 (PDIA6) and dynein light chain roadblock-type 1 (DynLRB1). Both 2D gels and Western blots revealed that PDAI6 exhibited a cell line specific pattern; in FaDuDD there was upregulation at 1% and further upregulated at 0% compared to atmospheric air, whereas there was no upregulation in SiHa cells. DynLRB1 was

  19. Proteomic analysis of oil body membrane proteins accompanying the onset of desiccation phase during sunflower seed development.

    Science.gov (United States)

    Thakur, Anita; Bhatla, Satish C

    2015-01-01

    A noteworthy metabolic signature accompanying oil body (OB) biogenesis during oilseed development is associated with the modulation of the oil body membranes proteins. Present work focuses on 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based analysis of the temporal changes in the OB membrane proteins analyzed by LC-MS/MS accompanying the onset of desiccation (20-30 d after anthesis; DAA) in the developing seeds of sunflower (Helianthus annuus L.). Protein spots unique to 20-30 DAA stages were picked up from 2-D gels for identification and the identified proteins were categorized into 7 functional classes. These include proteins involved in energy metabolism, reactive oxygen scavenging, proteolysis and protein turnover, signaling, oleosin and oil body biogenesis-associated proteins, desiccation and cytoskeleton. At 30 DAA stage, exclusive expressions of enzymes belonging to energy metabolism, desiccation and cytoskeleton were evident which indicated an increase in the metabolic and enzymatic activity in the cells at this stage of seed development (seed filling). Increased expression of cruciferina-like protein and dehydrin at 30 DAA stage marks the onset of desiccation. The data has been analyzed and discussed to highlight desiccation stage-associated metabolic events during oilseed development.

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

    Science.gov (United States)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas; Lyon, David; Mullari, Meeli; Madsen, Maria V; Daniel, Jeremy A; Jensen, Lars J; Nielsen, Michael L

    2016-01-01

    The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic 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 by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast 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 regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known 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 arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation. PMID:27577262

  1. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  2. 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.

  3. Proton Exchange Membrane Fuel Cell Characterization for Electric Vehicle Applications

    OpenAIRE

    Swan, D.H.; Dickinson, B.E.; Arikara, M.P.

    1994-01-01

    This paper presents experimental data and an analysis of a proton exchange membrane fuel cell system for electric vehicle applications. The dependence of the fuel cell system's performance on air stoichiometry, operating temperature, and reactant gas pressure was assessed in terms of the fuel cell's polarity and power density-efficiency graphs. All the experiments were performed by loading the fuel cell with resistive heater coils which could be controlled to provide a constant current or con...

  4. Organellar proteome analyses of ricin toxin-treated HeLa cells.

    Science.gov (United States)

    Liao, Peng; Li, Yunhu; Li, Hongyang; Liu, Wensen

    2016-07-01

    Apoptosis triggered by ricin toxin (RT) has previously been associated with certain cellular organellar compartments, but the diversity in the composition of the organellar proteins remains unclear. Here, we applied a shotgun proteomics strategy to examine the differential expression of proteins in the mitochondria, nuclei, and cytoplasm of HeLa cells treated and not treated with RT. Data were combined with a global bioinformatics analysis and experimental confirmations. A total of 3107 proteins were identified. Bioinformatics predictors (Proteome Analyst, WoLF PSORT, TargetP, MitoPred, Nucleo, MultiLoc, and k-nearest neighbor) and a Bayesian model that integrated these predictors were used to predict the locations of 1349 distinct organellar proteins. Our data indicate that the Bayesian model was more efficient than the individual implementation of these predictors. Additionally, a Biomolecular Interaction Network (BIN) analysis was used to identify 149 BIN subnetworks. Our experimental confirmations indicate that certain apoptosis-related proteins (e.g. cytochrome c, enolase, lamin B, Bax, and Drp1) were found to be translocated and had variable expression levels. These results provide new insights for the systematic understanding of RT-induced apoptosis responses. PMID:25227225

  5. 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...

  6. Polybenzimidazole Membranes Containing Benzimidazole Side Groups for High Temprature Polymer Electrolyte Membrane Fuel Cells

    DEFF Research Database (Denmark)

    Yang, Jingshuai; Li, Xueyuan; Xu, Yizin;

    2013-01-01

    Polybenzimidazole (PBI) with a high molecular weight of 69,000 was first synthesized. It was afterwards grafted with benzimidazole pendant groups on the backbones. The acid doped benzimidaozle grafted PBI membranes were investigated and characterized including fuel cell tests at elevated...... temperatures without humidification. At an acid doping level of 13.1 mol H3PO4 per average molar repeat unit, the PBI membranes with a benzimidazole grafting degree of 10.6% demonstrated a conductivity of 0.15 S cm-1 and a H2-air fuel cell peak power density of 378 mW cm-2 at 180 oC at ambient pressure without...

  7. Coating nanofiber scaffolds with beta cell membrane to promote cell proliferation and function

    Science.gov (United States)

    Chen, Wansong; Zhang, Qiangzhe; Luk, Brian T.; Fang, Ronnie H.; Liu, Younian; Gao, Weiwei; Zhang, Liangfang

    2016-05-01

    The cell membrane cloaking technique has emerged as an intriguing strategy in nanomaterial functionalization. Coating synthetic nanostructures with natural cell membranes bestows the nanostructures with unique cell surface antigens and functions. Previous studies have focused primarily on development of cell membrane-coated spherical nanoparticles and the uses thereof. Herein, we attempt to extend the cell membrane cloaking technique to nanofibers, a class of functional nanomaterials that are drastically different from nanoparticles in terms of dimensional and mechanophysical characteristics. Using pancreatic beta cells as a model cell line, we demonstrate successful preparation of cell membrane-coated nanofibers and validate that the modified nanofibers possess an antigenic exterior closely resembling that of the source beta cells. When such nanofiber scaffolds are used to culture beta cells, both cell proliferation rate and function are significantly enhanced. Specifically, glucose-dependent insulin secretion from the cells is increased by near five-fold compared with the same beta cells cultured in regular, unmodified nanofiber scaffolds. Overall, coating cell membranes onto nanofibers could add another dimension of flexibility and controllability in harnessing cell membrane functions and offer new opportunities for innovative applications.

  8. Transcriptome and proteome characterization of surface ectoderm cells differentiated from human iPSCs.

    Science.gov (United States)

    Qu, Ying; Zhou, Bo; Yang, Wei; Han, Bingchen; Yu-Rice, Yi; Gao, Bowen; Johnson, Jeffery; Svendsen, Clive N; Freeman, Michael R; Giuliano, Armando E; Sareen, Dhruv; Cui, Xiaojiang

    2016-01-01

    Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair, eye, and the mammary gland. In this study, we validate a protocol that utilizes BMP4 and the γ-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGFβ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGFβ-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development, studying disease pathogenesis, and development of regenerative medicine approaches. PMID:27550649

  9. Cell Membrane-Cloaked Nanoparticles for Targeted Therapeutics

    Science.gov (United States)

    Luk, Brian Tsengchi

    The advent of nanoparticle-based delivery systems has made a significant impact on clinical patient outcomes. In recent decades, myriad nanoparticle-based therapeutic agents have been developed for the treatment and management of ailments such as cancer, diabetes, pain, bacterial infections, and asthma, among many others. Nanotherapeutics offer many distinct advantages over conventional free drug formulations. For example, nanoparticles are able to accumulate at tumor sites by extravasation through leaky vasculature at tumor sites via the enhanced permeability and retention (EPR) effect; nanoparticles can also be tailored to have desirable characteristics, such as prolonged circulation in the blood stream, improved drug encapsulation, and sustained or triggered drug release. Currently, a growing number of nanoformulations with favorable pharmacological profiles and promising efficacy are being used in clinical trials for the treatment of various cancers. Building on the success of these encouraging clinical results, new engineering strategies have emerged that combine synthetic nanoparticles with natural biomaterials to create nature-inspired biomimetic delivery systems. The work presented in this dissertation focuses on the biointerfacing between synthetic and natural materials, namely in the manifestation of cell membrane-coated nanoparticles. By exploiting the natural functionalities of source cell membranes, cell membrane-cloaked nanoparticles have huge potential in the delivery of therapeutic agents for a variety of applications. The first portion of this thesis will focus on understanding the fundamentals underlying cell membrane coating on synthetic nanoparticles. First introduced in 2011, cell membrane-cloaked nanoparticles showed immediate promise in drug delivery applications, but further understanding was necessary to be able to harness the full potential of the membrane coating platform. The first section provides further insight into the interfacial

  10. Application of Proton Exchange Membrane Fuel Cell for Lift Trucks

    DEFF Research Database (Denmark)

    Hosseinzadeh, Elham; Rokni, Masoud

    2011-01-01

    in order to account for water back diffusion. Further Membrane water content is assumed to be a linear function of thickness. PEM fuel cell is working at rather low operating conditions which makes it suitable for the automotive systems. In this paper motive power part of a lift truck has been investigated......In this study a general PEMFC (Proton Exchange Membrane Fuel Cell) model has been developed to take into account the effect of pressure losses, water crossovers, humidity aspects and voltage over potentials in the cells. The model is zero dimensional and it is assumed to be steady state. The effect...

  11. Nonlinear electro-mechanobiological behavior of cell membrane during electroporation

    KAUST Repository

    Deng, Peigang

    2012-01-01

    A nonlinear electroporation (EP) model is proposed to study the electro-mechanobiological behavior of cell membrane during EP, by taking the nonlinear large deformation of the membrane into account. The proposed model predicts the critical transmembrane potential and the activation energy for EP, the equilibrium pore size, and the resealing process of the pore. Single-cell EP experiments using a micro EP chip were conducted on chicken red blood cells at different temperatures to determine the activation energy and the critical transmembrane potential for EP. The experimental results are in good agreement with the theoretical predictions. © 2012 American Institute of Physics.

  12. Attachment of killed Mycoplasma gallisepticum cells and membranes to erythrocytes

    International Nuclear Information System (INIS)

    To correlate viability with attachment capacity, Mycoplasma gallisepticum cells harvested at different growth phases and treated by various agents were tested for their capacity to attach to human erythrocytes. The results show that viability per se is not essential for M. gallisepticum attachment to erythrocytes, as cells killed by ultraviolet irradiation and membranes isolated by lysing M. gallisepticum cells by various means retained attachment capacity. However, treatment of the mycoplasmas by protein-denaturing agents, such as heart, glutaraldehyde, or prolonged exposure to low pH, drastically affected or even abolished attachment, supporting the protein nature of the mycoplasma membrane components responsible for specific binding to the sialoglycoprotein receptors on the erythrocytes

  13. Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells

    Science.gov (United States)

    Rodríguez-García, Ruddi; López-Montero, Iván; Mell, Michael; Egea, Gustavo; Gov, Nir S.; Monroy, Francisco

    2015-01-01

    Erythrocytes are flexible cells specialized in the systemic transport of oxygen in vertebrates. This physiological function is connected to their outstanding ability to deform in passing through narrow capillaries. In recent years, there has been an influx of experimental evidence of enhanced cell-shape fluctuations related to metabolically driven activity of the erythroid membrane skeleton. However, no direct observation of the active cytoskeleton forces has yet been reported to our knowledge. Here, we show experimental evidence of the presence of temporally correlated forces superposed over the thermal fluctuations of the erythrocyte membrane. These forces are ATP-dependent and drive enhanced flickering motions in human erythrocytes. Theoretical analyses provide support for a direct force exerted on the membrane by the cytoskeleton nodes as pulses of well-defined average duration. In addition, such metabolically regulated active forces cause global membrane softening, a mechanical attribute related to the functional erythroid deformability. PMID:26083919

  14. Interaction of injectable neurotropic drugs with the red cell membrane.

    Science.gov (United States)

    Reinhart, Walter H; Lubszky, Szabina; Thöny, Sandra; Schulzki, Thomas

    2014-10-01

    The normal red blood cell (RBC) shape is a biconcave discocyte. An intercalation of a drug in the outer half of the membrane lipid bilayer leads to echinocytosis, an intercalation in the inner half to stomatocytosis. We have used the shape transforming capacity of RBCs as a model to analyse the membrane interaction potential of various neurotropic drugs. Chlorpromazine, clomipramine, citalopram, clonazepam, and diazepam induced a reversible stomatocytosis, phenytoin induced echinocytosis, while the anticonvulsants levetiracetam, valproic acid and phenobarbital had no effect. This diversity of RBC shape transformations suggests that the pharmacological action is not linked to the membrane interaction. We conclude that this simple RBC shape transformation assay could be a useful tool to screen for potential drug interactions with cell membranes. PMID:24997296

  15. Membrane Mechanics of Endocytosis in Cells with Turgor

    CERN Document Server

    Dmitrieff, Serge

    2015-01-01

    Endocytosis is an essential process by which cells internalize a piece of plasma membrane and material from the outside. In cells with turgor, pressure opposes membrane defor- mations, and increases the amount of force that has to be generated by the endocytic machinery. To determine this force, and calculate the shape of the membrane, we used physical theory to model an elastic surface under pressure. Accurate fits of experimental profiles are obtained assuming that the coated membrane is highly rigid and preferentially curved at the endocytic site. The forces required from the actin machinery peaks at the onset of deformation, indicating that once invagination has been initiated, endocytosis is unlikely to stall before completion. Coat proteins do not lower the initiation force but may affect the process by the curvature they induce. In the presence of isotropic curvature inducers, pulling the tip of the invagination can trigger the formation of a neck at the base of the invagination. Hence direct neck cons...

  16. Altered Membrane Potential and Electrolyte in Sickle Cell Anemia

    Directory of Open Access Journals (Sweden)

    JK Nnodim

    2014-01-01

    Full Text Available Aim: This study has been to evaluate the level of membrane potential and electrolyte in sickle cell disease patients. Material and methods: 100 sickle cell patients in steady state ages 5 to 30 years attending General Hospital Owerri were used in the study while 100 normal subjects (HbAA were used as control. Also 30 HbSS in crisis have been involved. Results: The results obtained showed that the level of membrane potential was significantly lower in sickle cell anemia as compared to the controls. Also, the level of the electrolyte was found significantly decreased in HbSS when compared with HbAA at P<0.05. Conclusion: The membrane potential translates to energy which means that there is less energy in sickle cell disease which is linked to electrolyte imbalance. Hence people with sickle disease should be monitored closely for their electrolytes to avoid crisis.

  17. 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

  18. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

    Science.gov (United States)

    Menkhorst, Ellen Melaleuca; Lane, Natalie; Winship, Amy Louise; Li, Priscilla; Yap, Joanne; Meehan, Katie; Rainczuk, Adam; Stephens, Andrew; Dimitriadis, Evdokia

    2012-01-01

    Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8) M), medroxyprogesterone acetate (10(-7) M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel

  19. Endothelial monolayers on collagen-coated nanofibrous membranes: cell-cell and cell-ECM interactions.

    Science.gov (United States)

    Kang, Donggu; Kim, Jeong Hwa; Jeong, Young Hun; Kwak, Jong-Young; Yoon, Sik; Jin, Songwan

    2016-06-01

    Endothelial cells (ECs) form a monolayer lining over the entire vascular wall and play an important role in maintaining vascular homeostasis and cancer metastasis. Loss of proper endothelial function can lead to vascular diseases. Therefore, the endothelial monolayer is particularly important in tissue regeneration and mimicking vascular tissue in vitro. Numerous studies have described the effects of ECs on nanofibers made from a variety of synthetic polymer materials designed to mimic the extracellular matrix (ECM). However, little is known about maintaining the integrity of ECs in in vitro systems. Here we describe polycaprolactone nanofibrous membranes coated with collagen gel that overcome many limitations of conventional nanofibers used for engineering endothelia. We investigated cell-cell and cell-ECM junctional complexes using collagen-coated and conventional nanofibrous membranes. Conventional nanofibrous membranes alone did not form a monolayer with ECs, whereas collagen-coated nanofibrous membranes did. Several concentrations of collagen in the gel coating promoted the formation of cell-cell junctional complexes, facilitated the deposition of laminin, and increased the focal contact organization of ECs. These results suggest the possible use of collagen-coated nanofibrous membranes for vascular tissue engineering applications and a vascular platform for organ-on-a-chip systems. PMID:27186924

  20. Modeling Of Proton Exchange Membrane Fuel Cell Systems

    DEFF Research Database (Denmark)

    Nielsen, Mads Pagh

    The objective of this doctoral thesis was to develop reliable steady-state and transient component models suitable to asses-, develop- and optimize proton exchange membrane (PEM) fuel cell systems. Several components in PEM fuel cell systems were characterized and modeled. The developed component...

  1. Development of new membrane materials for direct methanol fuel cells

    NARCIS (Netherlands)

    Yildirim, Mustafa Hakan

    2009-01-01

    Development of new membrane materials for direct methanol fuel cells Direct methanol fuel cells (DMFCs) can convert the chemical energy of a fuel directly into electrical energy with high efficiency and low emission of pollutants. DMFCs can be used as the power sources to portable electronic devices

  2. A boron phosphate-phosphoric acid composite membrane for medium temperature proton exchange membrane fuel cells

    Science.gov (United States)

    Mamlouk, M.; Scott, K.

    2015-07-01

    A composite membrane based on a non-stoichiometric composition of BPO4 with excess of PO4 (BPOx) was synthesised and characterised for medium temperature fuel cell use (120-180 °C). The electrolyte was characterised by FTIR, SS-NMR, TGA and XRD and showed that the B-O is tetrahedral, in agreement with reports in the literature that boron phosphorus oxide compounds at B:P < 1 are exclusively built of borate and phosphate tetrahedra. Platinum micro electrodes were used to study the electrolyte compatibility and stability towards oxygen reduction at 150 °C and to obtain kinetic and mass transport parameters. The conductivities of the pure BPOx membrane electrolyte and a Polybenzimidazole (PBI)-4BPOx composite membrane were 7.9 × 10-2 S cm-1 and 4.5 × 10-2 S cm-1 respectively at 150 °C, 5%RH. Fuel cell tests showed a significant enhancement in performance of BPOx over that of typical 5.6H3PO4-PBI membrane electrolyte. The enhancement is due to the improved ionic conductivity (3×), a higher exchange current density of the oxygen reduction (30×) and a lower membrane gas permeability (10×). Fuel cell current densities at 0.6 V were 706 and 425 mA cm-2 for BPOx and 5.6H3PO4-PBI, respectively, at 150 °C with O2 (atm).

  3. Nanodomain stabilization dynamics in plasma membranes of biological cells

    Science.gov (United States)

    Das, Tamal; Maiti, Tapas K.; Chakraborty, Suman

    2011-02-01

    We discover that a synergistically amplifying role of stabilizing membrane proteins and continuous lipid recycling can explain the physics governing the stability, polydispersity, and dynamics of lipid raft domains in plasma membranes of biological cells. We establish the conjecture using a generalized order parameter based on theoretical formalism, endorsed by detailed scaling arguments and domain mapping. Quantitative agreements with morphological distributions of raft complexes, as obtained from Förster resonance energy transfer based visualization, support the present theoretical conjecture.

  4. Integration of genomic and proteomic data to identify candidate genes in HT-29 cells after incubation with Bifidobacterium bifidum ATCC 29521.

    Science.gov (United States)

    Wang, Bao-Gui; Wu, Yaoping; Qiu, Liang; Shah, Nagendra P; Xu, Feng; Wei, Hua

    2016-09-01

    As the predominant group inhabiting the human gastrointestinal tract, bifidobacteria play a vital role in human nutrition, therapeutics, and health by shaping and maintaining the gut ecosystem, reducing blood cholesterol, and promoting the supply of nutrients. The interaction between bacterial cells and human intestinal epithelial cell lines has been studied for decades in an attempt to understand the mechanisms of action. These studies, however, have been limited by lack of genomic and proteomic database to aid in achieving comprehensive understanding of these mechanisms at molecular levels. Microarray data (GSE: 74119) coupled with isobaric tags for relative and absolute quantitation (iTRAQ) were performed to detect differentially expressed genes and proteins in HT-29 cells after incubation with Bifidobacterium bifidum. Real-time quantitative PCR, gene ontology, and Kyoto Encyclopedia of Genes and Genomes analyses were further conducted for mRNA validation, functional annotation, and pathway identification, respectively. According to the results of microarray, 1,717 differentially expressed genes, including 1,693 upregulated and 24 downregulated genes, were selected and classified by the gene ontology database. The iTRAQ analysis identified 43 differentially expressed proteins, where 29 proteins were upregulated and 14 proteins were downregulated. Eighty-two candidate genes showing consistent differences with microarray and iTRAQ were further validated in HT-29 and Caco-2 cells by real-time quantitative PCR. Nine of the top genes showing interesting results with high confidence were further investigated in vivo in mice intestine samples. Integration of genomic and proteomic data provides an approach to identify candidate genes that are more likely to function in ubiquitin-mediated proteolysis, positive regulation of apoptosis, membrane proteins, and transferase catalysis. These findings might contribute to our understanding of molecular mechanisms regulating the

  5. Integration of genomic and proteomic data to identify candidate genes in HT-29 cells after incubation with Bifidobacterium bifidum ATCC 29521.

    Science.gov (United States)

    Wang, Bao-Gui; Wu, Yaoping; Qiu, Liang; Shah, Nagendra P; Xu, Feng; Wei, Hua

    2016-09-01

    As the predominant group inhabiting the human gastrointestinal tract, bifidobacteria play a vital role in human nutrition, therapeutics, and health by shaping and maintaining the gut ecosystem, reducing blood cholesterol, and promoting the supply of nutrients. The interaction between bacterial cells and human intestinal epithelial cell lines has been studied for decades in an attempt to understand the mechanisms of action. These studies, however, have been limited by lack of genomic and proteomic database to aid in achieving comprehensive understanding of these mechanisms at molecular levels. Microarray data (GSE: 74119) coupled with isobaric tags for relative and absolute quantitation (iTRAQ) were performed to detect differentially expressed genes and proteins in HT-29 cells after incubation with Bifidobacterium bifidum. Real-time quantitative PCR, gene ontology, and Kyoto Encyclopedia of Genes and Genomes analyses were further conducted for mRNA validation, functional annotation, and pathway identification, respectively. According to the results of microarray, 1,717 differentially expressed genes, including 1,693 upregulated and 24 downregulated genes, were selected and classified by the gene ontology database. The iTRAQ analysis identified 43 differentially expressed proteins, where 29 proteins were upregulated and 14 proteins were downregulated. Eighty-two candidate genes showing consistent differences with microarray and iTRAQ were further validated in HT-29 and Caco-2 cells by real-time quantitative PCR. Nine of the top genes showing interesting results with high confidence were further investigated in vivo in mice intestine samples. Integration of genomic and proteomic data provides an approach to identify candidate genes that are more likely to function in ubiquitin-mediated proteolysis, positive regulation of apoptosis, membrane proteins, and transferase catalysis. These findings might contribute to our understanding of molecular mechanisms regulating the

  6. A proteomic and genetic analysis of the Neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling.

    Science.gov (United States)

    Ao, Jie; Aldabbous, Mash'el; Notaro, Marysa J; Lojacono, Mark; Free, Stephen J

    2016-09-01

    A proteomic analysis of the conidial cell wall identified 35 cell wall proteins. A comparison with the proteome of the vegetative hyphae showed that 16 cell wall proteins were shared, and that these shared cell wall proteins were cell wall biosynthetic proteins or cell wall structural proteins. Deletion mutants for 34 of the genes were analyzed for phenotypes indicative of conidial cell wall defects. Mutants for two cell wall glycosyl hydrolases, the CGL-1 β-1,3-glucanase (NCU07523) and the NAG-1 exochitinase (NCU10852), were found to have a conidial separation phenotype. These two enzymes function in remodeling the cell wall between adjacent conidia to facilitate conidia formation and dissemination. Using promoter::RFP and promoter::GFP constructs, we demonstrated that the promoters for 15 of the conidia-specific cell wall genes, including cgl-1 and nag-1, provided for conidia-specific gene expression or for a significant increase in their expression during conidiation. PMID:27381444

  7. 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.

  8. Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Holger A. Russ

    2016-01-01

    Full Text Available Current approaches in human embryonic stem cell (hESC to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1, Neuroplastin (NPTN, and the Laminin α-2 subunit (LAMA2. Two of the three factors (LGALS1 and LAMA2 increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

  9. 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. PMID:21751360

  10. Proteomic analysis of apoptosis induction by lariciresinol in human HepG2 cells.

    Science.gov (United States)

    Ma, Zhan-Jun; Wang, Xue-Xi; Su, Gang; Yang, Jing-Jing; Zhu, Ya-Juan; Wu, You-Wei; Li, Jing; Lu, Li; Zeng, Long; Pei, Hai-Xia

    2016-08-25

    Lariciresinol (LA) is a traditional Chinese medicine possessing anticancer activity, but its mechanism of action remains unclear. The present study explored the effects of LA on human HepG2 cells and the underlying mechanism. Our data indicated that LA inhibited cell proliferation and induced cell cycle arrest in S phase, subsequently resulting in apoptosis in HepG2 cells. Using a proteomics approach, eight differentially expressed proteins were identified. Among them, three proteins, glyceraldehyde-3-phosphate, UDP-glucose 4-epimerase, and annexin A1, were upregulated, while the other five proteins, heat shock protein 27, haptoglobin, tropomodulin-2, tubulin alpha-1A chain, and brain acid soluble protein 1, were downregulated; all of these proteins are involved in cell proliferation, metabolism, cytoskeletal organization, and movement. Network analysis of these proteins suggested that the ubiquitin-conjugating enzyme (UBC) plays an important role in the mechanism of LA. Western blotting confirmed downregulation of heat shock protein 27 and upregulation of ubiquitin and UBC expression levels in LA-treated cells, consistent with the results of two-dimensional electrophoresis and a STRING software-based analysis. Overall, LA is a multi-target compound with anti-cancer effects potentially related to the ubiquitin-proteasome pathway. This study will increase our understanding of the anticancer mechanisms of LA. PMID:27417256

  11. Understanding the transport processes in polymer electrolyte membrane fuel cells

    Science.gov (United States)

    Cheah, May Jean

    Polymer electrolyte membrane (PEM) fuel cells are energy conversion devices suitable for automotive, stationary and portable applications. An engineering challenge that is hindering the widespread use of PEM fuel cells is the water management issue, where either a lack of water (resulting in membrane dehydration) or an excess accumulation of liquid water (resulting in fuel cell flooding) critically reduces the PEM fuel cell performance. The water management issue is addressed by this dissertation through the study of three transport processes occurring in PEM fuel cells. Water transport within the membrane is a combination of water diffusion down the water activity gradient and the dragging of water molecules by protons when there is a proton current, in a phenomenon termed electro-osmotic drag, EOD. The impact of water diffusion and EOD on the water flux across the membrane is reduced due to water transport resistance at the vapor/membrane interface. The redistribution of water inside the membrane by EOD causes an overall increase in the membrane resistance that regulates the current and thus EOD, thereby preventing membrane dehydration. Liquid water transport in the PEM fuel cell flow channel was examined at different gas flow regimes. At low gas Reynolds numbers, drops transitioned into slugs that are subsequently pushed out of the flow channel by the gas flow. The slug volume is dependent on the geometric shape, the surface wettability and the orientation (with respect to gravity) of the flow channel. The differential pressure required for slug motion primarily depends on the interfacial forces acting along the contact lines at the front and the back of the slug. At high gas Reynolds number, water is removed as a film or as drops depending on the flow channel surface wettability. The shape of growing drops at low and high Reynolds number can be described by a simple interfacial energy minimization model. Under flooding conditions, the fuel cell local current

  12. Corona discharge in electroporation of cell membranes

    Energy Technology Data Exchange (ETDEWEB)

    Cramariuc, R; Nisiparu, L [Competence Centre in Electrostatics and Electrotehchnologies (Romania); Tudorache, A; Branduse, E; Fotescu, L [Research Institute of Wine Processing, Valea Mantei Street, No.l, Valea Calugareasca (Romania); Popa, M E; Mitelut, A [Biotechnology Faculty, University of Agronomical Sciences and Veterinary Medicine (Romania); Turtoi, M O

    2008-12-01

    The objective of the present work is to demonstrate that electrical corona discharge is very efficient in cellular membrane electroporation due to current pulses with sharp front (2-5 ns) and to the fact that corona discharge is associated with UV radiation and micro particles emission. A comparison between DC and AC at 800 Hz and a special waveform to corona application is presented. The comparison is analyzed by means of applying all these in the maceration process (electroplasmolysis) of red wine production and in the processes of different types of the microbes.

  13. Estimation of membrane hydration status for standby proton exchange membrane fuel cell systems by impedance measurement

    DEFF Research Database (Denmark)

    Bidoggia, Benoit; Rugholt, Mark; Nielsen, Morten Busk;

    2014-01-01

    Fuel cells are getting growing interest in both backup systems and electric vehicles. Although these systems are characterized by long periods of inactivity, they must be able to start at any instant in the shortest time. However, the membrane of which PEMFCs are made tends to dry out when not in...

  14. 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.

  15. 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.

  16. Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

    Science.gov (United States)

    Blewett, Megan M; Xie, Jiji; Zaro, Balyn W; Backus, Keriann M; Altman, Amnon; Teijaro, John R; Cravatt, Benjamin F

    2016-01-01

    Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity of >2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase Cθ (PKCθ). DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology. PMID:27625306

  17. High-throughput quantitative proteomic analysis of dengue virus type 2 infected A549 cells.

    Directory of Open Access Journals (Sweden)

    Han-Chen Chiu

    Full Text Available Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC in combination with high-throughput mass spectrometry (MS. Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.

  18. 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. PMID:18778094

  19. 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.

  20. Nafion®/ODF-silica composite membranes for medium temperature proton exchange membrane fuel cells

    KAUST Repository

    Treekamol, Yaowapa

    2014-01-01

    A series of composite membranes were prepared by dispersing fluorinated polyoxadiazole oligomer (ODF)-functionalized silica nanoparticles in a Nafion matrix. Both melt-extrusion and solvent casting processes were explored. Ion exchange capacity, conductivity, water uptake and dimensional stability, thermal stability and morphology were characterized. The inclusion of functionalized nanoparticles proved advantageous, mainly due to a physical crosslinking effect and better water retention, with functionalized nanoparticles performing better than the pristine silica particles. For the same filler loading, better nanoparticle dispersion was achieved for solvent-cast membranes, resulting in higher proton conductivity. Filler agglomeration, however,was more severe for solvent-castmembranes at loadings beyond 5wt.%. The composite membranes showed excellent thermal stability, allowing for operation in medium temperature PEM fuel cells. Fuel cell performance of the compositemembranesdecreaseswithdecreasing relativehumidity, but goodperformance values are still obtained at 34% RHand 90 °C,with the best results obtained for solvent castmembranes loaded with 10 wt.% ODF-functionalized silica. Hydrogen crossover of the composite membranes is higher than that forpureNafion membranes,possiblydue toporosityresulting fromsuboptimalparticle- matrixcompatibility. © 2013 Crown Copyright and Elsevier BV. All rights reserved.

  1. Proteomic profiling of cellular targets of lipopolysaccharide-induced signalling in Nicotiana tabacum BY-2 cells.

    Science.gov (United States)

    Gerber, Isak B; Laukens, Kris; De Vijlder, Thomas; Witters, Erwin; Dubery, Ian A

    2008-11-01

    Plants constantly monitor for pathogen challenge and utilize a diverse array of adaptive defense mechanisms, including differential protein regulation, during pathogen attack. A proteomic analysis of Nicotiana tabacum BY-2 cells was performed in order to investigate the dynamic changes following perception of bacterial lipopolysaccharides. A multiplexed proteome analysis, employing two-dimensional difference-in-gel-electrophoresis with CyDye DIGE fluors, as well as Ruthenium II tris (bathophenanthroline disulfonate) fluorescence staining and Pro-Q Diamond phosphoprotein-specific gel staining, monitored over 1500 proteins and resulted in the identification of 88 differentially regulated proteins and phosphoproteins responsive to LPS(B.cep.)-elicitation. Functional clustering of the proteins both at the level of their abundance and phosphorylation status, revealed 9 proteins involved in transport, ion homeostasis and signal transduction. A large number of responsive proteins were found to be involved in metabolism- and energy-related processes (36), representing various metabolic pathways. Another abundant category corresponded to proteins classified as molecular chaperones and involved in protein destination/targeting (12). Other categories of proteins found to be LPS(B.cep.)-responsive and differentially regulated include cell structure- and cytoskeletal rearrangement proteins (8) and proteins involved in transcription and translation as well as degradation (11). The results indicate that LPS(B.cep.) induces metabolic reprogramming and changes in cellular activities supporting protein synthesis, -folding, vesicle trafficking and secretion; accompanied by changes to the cytoskeleton and proteosome function. Many of the identified proteins are known to be interconnected at various levels through a complex web of activation/deactivation, complex formation, protein-protein interactions, and chaperoning reactions. The presented data offers novel insights and further

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  3. Classification of Cells with Membrane Staining and/or Fixation Based on Cellular Specific Membrane Capacitance and Cytoplasm Conductivity

    OpenAIRE

    Song-Bin Huang; Yang Zhao; Deyong Chen; Shing-Lun Liu; Yana Luo; Tzu-Keng Chiu; Junbo Wang; Jian Chen; Min-Hsien Wu

    2015-01-01

    Single-cell electrical properties (e.g., specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm)) have been regarded as potential label-free biophysical markers for the evaluation of cellular status. However, whether there exist correlations between these biophysical markers and cellular status (e.g., membrane-associate protein expression) is still unknown. To further validate the utility of single-cell electrical properties in cell type classification, Cspe...

  4. 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:

  5. 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.

  6. Proteomic biomarkers of peripheral blood mononuclear cells obtained from postmenopausal women undergoing an intervention with soy isoflavones

    OpenAIRE

    Fuchs, D; Vafeiadou, K.; Hall, W.L.; Daniel, H; Williams, C.M.; Schroot, J.H.; Wenzel, U.

    2007-01-01

    Background: The incidence of cardiovascular diseases increases after menopause, and soy consumption is suggested to inhibit disease development. Objective: The objective was to identify biomarkers of response to a dietary supplementation with an isoflavone extract in postmenopausal women by proteome analysis of peripheral blood mononuclear cells. Design: The study with healthy postmenopausal woman was performed in a placebo-controlled sequential design. Peripheral mononuclear blood cells were...

  7. FABRICATION AND BIOCOMPATIBILITY OF CELL OUTER MEMBRANE MIMETIC SURFACES

    Institute of Scientific and Technical Information of China (English)

    Ming-ming Zong; Yong-kuan Gong

    2011-01-01

    The surface design used for improving biocompatibility is one of the most important issues for the fabrication of medical devices. For mimicking the ideal surface structure of cell outer membrane, a large number of polymers bearing phosphorylcholine (PC) groups have been employed to modify the surfaces of biomaterials and medical devices. It has been demonstrated that the biocompatibility of the modified materials whose surface is required to interact with a living organism has been obviously improved by introducing PC groups. In this review, the fabrication strategies of cell outer membrane mimetic surfaces and their resulted biocompatibilities were summarized.

  8. Microstructured Electrolyte Membranes to Improve Fuel Cell Performance

    Science.gov (United States)

    Wei, Xue

    Fuel cells, with the advantages of high efficiency, low greenhouse gas emission, and long lifetime are a promising technology for both portable power and stationary power sources. The development of efficient electrolyte membranes with high ionic conductivity, good mechanical durability and dense structure at low cost remains a challenge to the commercialization of fuel cells. This thesis focuses on exploring novel composite polymer membranes and ceramic electrolytes with the microstructure engineered to improve performance in direct methanol fuel cells (DMFCs) and solid oxide fuel cells (SOFCs), respectively. Polymer/particle composite membranes hold promise to meet the demands of DMFCs at lower cost. The structure of composite membranes was controlled by aligning proton conducting particles across the membrane thickness under an applied electric field. The field-induced structural changes caused the membranes to display an enhanced water uptake, proton conductivity, and methanol permeability in comparison to membranes prepared without an applied field. Although both methanol permeability and proton conductivity are enhanced by the applied field, the permeability increase is relatively lower than the proton conductivity improvement, which results in enhanced proton/methanol selectivity and improved DMFC performance. Apatite ceramics are a new class of fast ion conductors being studied as alternative SOFC electrolytes in the intermediate temperature range. An electrochemical/hydrothermal deposition method was developed to grow fully dense apatite membranes containing well-developed crystals with c-axis alignment to promote ion conductivity. Hydroxyapatite seed crystals were first deposited onto a metal substrate electrochemically. Subsequent ion substitution during the hydrothermal growth process promoted the formation of dense, fully crystalline films with microstructure optimal for ion transport. The deposition parameters were systematically investigated, such as

  9. iTRAQ-based proteomic analysis of dioscin on human HCT-116 colon cancer cells.

    Science.gov (United States)

    Chen, Hao; Xu, Lina; Yin, Lianhong; Xu, Youwei; Han, Xu; Qi, Yan; Zhao, Yanyan; Liu, Kexin; Peng, Jinyong

    2014-01-01

    Dioscin shows various pharmacological effects. However, its activity on colorectal cancer is still unknown. The present work showed that dioscin significantly inhibited cell proliferation on human HCT-116 colon cancer cells, and affected Ca(2+) release and ROS generation. The content of nitric oxide (NO) and its producer inducible NO synthase (iNOS) associated with DNA damage and aberrant cell signaling were assayed using the kits. DNA damage and cell apoptosis caused by dioscin were also analyzed through single-cell gel electrophoresis and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling assays. The results showed that dioscin increased the levels of NO and inducible NO synthase. The comet length in dioscin-treated groups was much longer than that of the control group, and the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells (apoptotic cells) was significantly increased by the compound (p dioscin caused mitochondrial damage and G2/M cell cycle arrest through transmission electron microscopy and flow cytometry analysis, respectively. To study the cytotoxic mechanism of dioscin, an iTRAQ-based proteomics approach was used. There were 288 significantly different proteins expressed in response to dioscin, which were connected with each other and were involved in different Kyoto Encyclopedia of Genes and Genomes pathways. Then, some differentially expressed proteins involved in oxidative phosphorylation, Wnt, p53, and calcium signaling pathways were validated by Western blotting and quantitative real-time PCR assays. Our work elucidates the molecular mechanism of dioscin-induced cytotoxicity in colon cancer cells, and the identified targets may be useful for treatment of colorectal cancer in future. PMID:24420967

  10. 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.

  11. Flavivirus cell entry and membrane fusion

    NARCIS (Netherlands)

    Smit, Jolanda M.; Moesker, Bastiaan; Rodenhuis-Zybert, Izabela; Wilschut, Jan

    2011-01-01

    Flaviviruses, such as dengue virus and West Nile virus, are enveloped viruses that infect cells through receptor-mediated endocytosis and fusion from within acidic endosomes. The cell entry process of flaviviruses is mediated by the viral E glycoprotein. This short review will address recent advance

  12. Proton Exchange Membranes for Fuel Cells Challenges and Recent Developments

    Institute of Scientific and Technical Information of China (English)

    Qingfeng Li; Jens Oluf Jensen; Pernille P. Noyé; Chao Pan; Niels J. Bjerrum

    2005-01-01

    @@ 1Introduction The current technology of proton exchange membrane fuel cells (PEMFC) is based on perfluorosulfonic acid (PFSA) membranes (e. g. Nafion(R)) as electrolyte. It operates on pure hydrogen and oxygen/air at typically 80℃ with high power density and long-term durability. For the membranes to be conductive, a minimum threshold of absorbed water molecules is about 6 to 7 mole per sulfonic site. The highest conductivity is only obtained under fully hydrated conductions, i.e. 21 - 22 mole water per sulfonic acid site. In other words, the proton conductivity is achieved by the locally liquid-like hydrophilic domain of the nanostructure.This strong dependence of conductivity on the water content in membranes limits the operational temperatureof PEMFC below 100℃.

  13. Smoking and red blood cell phospholipid membrane fatty acids.

    Science.gov (United States)

    Murff, H J; Tindle, H A; Shrubsole, M J; Cai, Q; Smalley, W; Milne, G L; Swift, L L; Ness, R M; Zheng, W

    2016-09-01

    Smoking is associated with lower n-3 long chain polyunsaturated fatty acids (LCPUFA) concentrations; however, limited studies have accounted for dietary PUFA intake or whether tobacco dose or smoking duration influences this association. We measured red blood cell phospholipid (RBC) membrane concentrations of fatty acids in 126 current smokers, 311 former smokers, and 461 never smokers using gas liquid chromatography and tandem mass spectrometry. Smokers had lower RBC membrane percentages of total n-3 LCPUFAs compared to former smokers or never smokers (median percent: 5.46, [interquartile range (IQR) 4.52, 6.28] versus 6.39; [IQR: 5.18, 7.85] versus 6.59; [IQR 5.34, 8.01]) (psmoking and cigarettes per day were not associated with RBC membrane n-3 LCPUFA differences. Smoking is associated with lower n-3 LCPUFA RBC membrane percentages and this association was not influenced by diet or smoking dose or duration. PMID:27637337

  14. Durability Issues of High Temperature Proton Exchange Membrane Fuel Cells Based on Acid Doped Polybenzimidazole Membranes

    DEFF Research Database (Denmark)

    To achieve high temperature operation of proton exchange membrane fuel cells (PEMFC), preferably under ambient pressure, phosphoric acid doped polybenzimidazole (PBI) membrane represents an effective approach, which in recent years has motivated extensive research activities with great progress...... or ionically cross-linking and structure modification With load, thermal or startup-shutdown cycling, the performance loss was found to be much bigger, about 300 µV per cycle or 40 µV per operating hour, due to the increased acid loss and catalyst support corrosion, particularly under open circuit voltage...

  15. 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.

  16. 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.

  17. 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.

  18. Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

    International Nuclear Information System (INIS)

    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.

  19. Electrospun fiber membranes enable proliferation of genetically modified cells

    Directory of Open Access Journals (Sweden)

    Borjigin M

    2013-02-01

    Full Text Available Mandula Borjigin*, Chris Eskridge*, Rohina Niamat, Bryan Strouse, Pawel Bialk, Eric B KmiecDepartment of Chemistry, Delaware State University, Dover, DE, USA *These authors contributed equally to this work Abstract: Polycaprolactone (PCL and its blended composites (chitosan, gelatin, and lecithin are well-established biomaterials that can enrich cell growth and enable tissue engineering. However, their application in the recovery and proliferation of genetically modified cells has not been studied. In the study reported here, we fabricated PCL-biomaterial blended fiber membranes, characterized them using physicochemical techniques, and used them as templates for the growth of genetically modified HCT116-19 colon cancer cells. Our data show that the blended polymers are highly miscible and form homogenous electrospun fiber membranes of uniform texture. The aligned PCL nanofibers support robust cell growth, yielding a 2.5-fold higher proliferation rate than cells plated on standard plastic plate surfaces. PCL-lecithin fiber membranes yielded a 2.7-fold higher rate of proliferation, while PCL-chitosan supported a more modest growth rate (1.5-fold higher. Surprisingly, PCL-gelatin did not enhance cell proliferation when compared to the rate of cell growth on plastic surfaces. Keywords: nanofibers, PCL-biomaterial blends, miscibility, gene editing, cell proliferation

  20. Antigenicity and immunogenicity of an extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells.

    OpenAIRE

    Gómez, A M; Rhodes, J C; Deepe, G S

    1991-01-01

    In order to identify T-cell antigens from Histoplasma capsulatum yeast cells, we prepared a detergent extract of the cell wall and cell membrane of yeast-phase H. capsulatum G217B and analyzed its antigenicity and immunogenicity. Mice injected with viable H. capsulatum yeast cells or with 500 or 1,000 micrograms of the extract mounted a delayed-type hypersensitivity response to solubilized cell wall and cell membrane. Vaccination with this antigenic preparation conferred a protective immune r...

  1. 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.

  2. Optical Trapping Techniques Applied to the Study of Cell Membranes

    Science.gov (United States)

    Morss, Andrew J.

    Optical tweezers allow for manipulating micron-sized objects using pN level optical forces. In this work, we use an optical trapping setup to aid in three separate experiments, all related to the physics of the cellular membrane. In the first experiment, in conjunction with Brian Henslee, we use optical tweezers to allow for precise positioning and control of cells in suspension to evaluate the cell size dependence of electroporation. Theory predicts that all cells porate at a transmembrane potential VTMof roughly 1 V. The Schwann equation predicts that the transmembrane potential depends linearly on the cell radius r, thus predicting that cells should porate at threshold electric fields that go as 1/r. The threshold field required to induce poration is determined by applying a low voltage pulse to the cell and then applying additional pulses of greater and greater magnitude, checking for poration at each step using propidium iodide dye. We find that, contrary to expectations, cells do not porate at a constant value of the transmembrane potential but at a constant value of the electric field which we find to be 692 V/cm for K562 cells. Delivering precise dosages of nanoparticles into cells is of importance for assessing toxicity of nanoparticles or for genetic research. In the second experiment, we conduct nano-electroporation—a novel method of applying precise doses of transfection agents to cells—by using optical tweezers in conjunction with a confocal microscope to manipulate cells into contact with 100 nm wide nanochannels. This work was done in collaboration with Pouyan Boukany of Dr. Lee's group. The small cross sectional area of these nano channels means that the electric field within them is extremely large, 60 MV/m, which allows them to electrophoretically drive transfection agents into the cell. We find that nano electroporation results in excellent dose control (to within 10% in our experiments) compared to bulk electroporation. We also find that

  3. 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.

  4. Effect of stress on the membrane capacitance of the auditory outer hair cell.

    OpenAIRE

    Iwasa, K H

    1993-01-01

    The membrane capacitance of the outer hair cell, which has unique membrane potential-dependent motility, was monitored during application of membrane tension. It was found that the membrane capacitance of the cell decreased when stress was applied to the membrane. This result is the opposite of stretching the lipid bilayer in the plasma membrane. It thus indicates the importance of some other capacitance component that decreases on stretching. It has been known that charge movement across the...

  5. 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

  6. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    Science.gov (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment. PMID:27312328

  7. Spatially selective sampling of single cells using optically trapped fusogenic emulsion droplets: a new single-cell proteomic tool

    OpenAIRE

    Lanigan, Peter M.P.; Chan, Karen; Ninkovic, Tanya; Templer, Richard H.; French, P. M. W.; de Mello, A.J.; Willison, K R; Parker, P.J.; Neil, M. A. A.; Ces, Oscar; Klug, D. R.

    2008-01-01

    We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5–5 μm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane...

  8. Durable, Low-cost, Improved Fuel Cell Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Chris Roger; David Mountz; Wensheng He; Tao Zhang

    2011-03-17

    The development of low cost, durable membranes and membranes electrode assemblies (MEAs) that operate under reduced relative humidity (RH) conditions remain a critical challenge for the successful introduction of fuel cells into mass markets. It was the goal of the team lead by Arkema, Inc. to address these shortages. Thus, this project addresses the following technical barriers from the fuel cells section of the Hydrogen Fuel Cells and Infrastructure Technologies Program Multi-Year Research, Development and Demonstration Plan: (A) Durability (B) Cost Arkema’s approach consisted of using blends of polyvinylidenefluoride (PVDF) and proprietary sulfonated polyelectrolytes. In the traditional approach to polyelectrolytes for proton exchange membranes (PEM), all the required properties are “packaged” in one macromolecule. The properties of interest include proton conductivity, mechanical properties, durability, and water/gas transport. This is the case, for example, for perfluorosulfonic acid-containing (PFSA) membranes. However, the cost of these materials is high, largely due to the complexity and the number of steps involved in their synthesis. In addition, they suffer other shortcomings such as mediocre mechanical properties and insufficient durability for some applications. The strength and originality of Arkema’s approach lies in the decoupling of ion conductivity from the other requirements. Kynar® PVDF provides an exceptional combination of properties that make it ideally suited for a membrane matrix (Kynar® is a registered trademark of Arkema Inc.). It exhibits outstanding chemical resistance in highly oxidative and acidic environments. In work with a prior grant, a membrane known as M41 was developed by Arkema. M41 had many of the properties needed for a high performance PEM, but had a significant deficiency in conductivity at low RH. In the first phase of this work, the processing parameters of M41 were explored as a means to increase its proton

  9. Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions.

    OpenAIRE

    Harder, Thomas

    2003-01-01

    Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, whi...

  10. Quantitative transporter proteomics by liquid chromatography with tandem mass spectrometry: addressing methodologic issues of plasma membrane isolation and expression-activity relationship.

    Science.gov (United States)

    Kumar, Vineet; Prasad, Bhagwat; Patilea, Gabriela; Gupta, Anshul; Salphati, Laurent; Evers, Raymond; Hop, Cornelis E C A; Unadkat, Jashvant D

    2015-02-01

    To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (overexpressing the transporter) and then scale these to via in vitro to in vivo extrapolation (IVIVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane. Second, that there is a linear correlation between expression level and activity of the transporter protein. The present study was conducted to test these two assumptions. We evaluated two commercially available kits that claimed to separate plasma membrane from other cell membranes. The Qiagen Qproteome kit yielded very little protein in the fraction purported to be the plasma membrane. The Abcam Phase Separation kit enriched the plasma membrane but did not separate it from other intracellular membranes. For the Abcam method, the expression level of organic anion-transporting polypeptides (OATP) 1B1/2B1 and breast cancer resistance protein (BCRP) proteins in all subcellular fractions isolated from cells or human liver tissue tracked that of Na⁺-K⁺ ATPase. Assuming that Na⁺-K⁺ ATPase is predominantly located in the plasma membrane, these data suggest that the transporters measured are also primarily located in the plasma membrane. Using short hairpin RNA, we created clones of cell lines with varying degrees of OATP1B1 or BCRP expression level. In these clones, transport activity of OATP1B1 or BCRP was highly correlated with protein expression level (r² > 0.9). These data support the use of transporter expression level data and activity data from transporter overexpressing cell lines for IVIVE of transporter-mediated disposition of drugs.

  11. The Succinated Proteome

    Energy Technology Data Exchange (ETDEWEB)

    Merkley, Eric D.; Metz, Thomas O.; Smith, Richard D.; Baynes, John; Frizell, Norma

    2014-03-30

    Succination is a chemical modification of cysteine in protein by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes. Increased succination of proteins is also detected in the kidney of a fumarase conditional knock-out mouse which develops renal tumors. Keap1, the gatekeeper of the antioxidant response, was identified as a major succinated protein in renal cancer cells, suggesting that succination may play a role in activation of the antioxidant response. A wide range of proteins is subject to succination, including enzymes, adipokines, cytoskeletal proteins and ER chaperones with functional cysteine residues. There is also significant overlap between succinated and glutathionylated proteins, and with proteins containing cysteine residues that are readily oxidized to the sulfenic (cysteic) acid. Succination of adipocyte proteins is inhibited by uncouplers, which discharge the mitochondrial membrane potential (Δψm) and by ER stress inhibitors. 2SC serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic post-translational modification of proteins by proteomics approaches.

  12. The Autohumidification Polymer Electrolyte Membrane Fuel Cell

    CERN Document Server

    Benziger, J B; Tulyani, S; Turner, A; Bocarsly, A B; Kevrekidis, Yu G

    2003-01-01

    A PEM fuel cell was specially constructed to determine kinetics under conditions of well-defined gas phase composition and cell temperature. Steady state multiplicity was discovered in the autohumidification PEM fuel cell, resulting from a balance between water production and water removal. Ignition was observed in the PEM fuel cell for a critical water activity of about 0.1. Ignition is a consequence of the exponential increase of proton conductivity with water activity, which creates an autocatalytic feedback between the water production and the proton conduction. The steady state current in the ignited state decreases with increasing temperature between 50 to 105 deg C. At temperatures greater than 70 deg C five steady states were observed in the PEM fuel cell. The steady state performance has been followed with variable load resistance and hysteresis loops have been mapped. The dynamics of transitions between steady states are slow about 10^3 to 10^4 s. These slow dynamics are suggested to result from a c...

  13. Comparative Proteomics Study on Human High-metastatic Large Cell Lung Cancer Cell Lines Before and After Transfecting with nm23-H1 Gene

    Directory of Open Access Journals (Sweden)

    Liwei GAO

    2010-10-01

    Full Text Available Background and objective As a tumor metastasis suppressor gene, the functions of nm23-H1 gene are still unclear. The aim of this study is to better understand the mechanism of lung cancer metastasis and to find new biomarkers for early diagnosis and new target for therapy by conducting comparative proteomics between the human high-metastatic large cell lung cancer cell lines (L9981 and L9981-nm23-H1 (constructed with transfecting nm23-H1 gene into the L9981 cell line. Methods The total proteins of L9981 and L9981-nm23-H1 were separated by immobilized pH gradient (IPG-based 2-dimensional electrophoresis (2-DE; the significantly differently expressed proteins were examined by mass spectrometry and analyzed by bioinformatics. Results It was observed that nm23-H1 gene transfection caused remarkable changes of the proteome of L9981 compared with L9981-nm23-H1 cells: 5 proteins were deleted, 9 proteins appeared, 16 proteins downregulated, and 12 proteins up-regulated. These proteins are involved in cell framework, signal transduction, metabolism, proliferation and metastasis. Conclusion After nm23-H1 gene is transfected into L9981, proteome in L9981 is remarkably changed. These changes of the proteome could serve as a basis for reversing the invasive and metastatic phenotype in lung cancer and elucidating the machanisms of the metastasis of lung cancer.

  14. Tandem cathode for proton exchange membrane fuel cells

    DEFF Research Database (Denmark)

    Siahrostami, Samira; Björketun, Mårten E.; Strasser, Peter;

    2013-01-01

    The efficiency of proton exchange membrane fuel cells is limited mainly by the oxygen reduction reaction at the cathode. The large cathodic overpotential is caused by correlations between binding energies of reaction intermediates in the reduction of oxygen to water. This work introduces a novel...

  15. Lipid signalling dynamics at the β-cell plasma membrane.

    Science.gov (United States)

    Wuttke, Anne

    2015-04-01

    Pancreatic β-cells are clustered in islets of Langerhans and secrete insulin in response to increased concentrations of circulating glucose. Insulin in turn acts on liver, muscle and fat tissue to store energy and normalize the blood glucose level. Inappropriate insulin release may lead to impaired glucose tolerance and diabetes. In addition to glucose, other nutrients, neural stimuli and hormonal stimuli control insulin secretion. Many of these signals are perceived at the plasma membrane, which is also the site where insulin granules undergo exocytosis. Therefore, it is not surprising that membrane lipids play an important role in the regulation of insulin secretion. β-cells release insulin in a pulsatile fashion. Signalling lipids integrate the nutrient and neurohormonal inputs to fine-tune, shape and co-ordinate the pulsatility. An important group of signalling lipids are phosphoinositides and their downstream messengers. This MiniReview will discuss new insights into lipid signalling dynamics in β-cells obtained from live-cell imaging experiments with fluorescent translocation biosensors. The plasma membrane concentration of several phosphoinositides and of their downstream messengers changes rapidly upon nutrient or neurohormonal stimulation. Glucose induces the most complex spatio-temporal patterns, typically involving oscillations of messenger concentrations, which sometimes are locally restricted. The tightly controlled levels of lipid messengers can mediate specific binding of downstream effectors to the plasma membrane, contributing to the appropriate regulation of insulin secretion.

  16. 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

  17. Cell-penetrating peptides for drug delivery across membrane barriers

    DEFF Research Database (Denmark)

    Foged, Camilla; Nielsen, Hanne Moerck

    2008-01-01

    , proteins and colloidal carriers such as liposomes and polymeric nanoparticles. Their ability to cross biological membranes in a non-disruptive way without apparent toxicity is highly desired for increasing drug bioavailability. This review provides an overview of the application of cell...

  18. 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.

  19. Proteomics of Rice Seed Germination

    Directory of Open Access Journals (Sweden)

    Dongli eHe

    2013-07-01

    Full Text Available Seed is a condensed form of plant. Under suitable environmental conditions, it can resume the metabolic activity from physiological quiescent status, and mobilize the reserves, biosynthesize new proteins, regenerate organelles and cell membrane, eventually protrude the radicle and enter into seedling establishment. So far, how these activities are regulated in a coordinated and sequential manner is largely unknown. With the availability of more and more genome sequence information and the development of mass spectrometry (MS technology, proteomics has been widely applied in analyzing the mechanisms of different biological processes, and proved to be very powerful. Regulation of rice seed germination is critical for rice cultivation. In recent years, a lot of proteomic studies have been conducted in exploring the gene expression regulation, reserves mobilization and metabolisms reactivation, which brings us new insights on the mechanisms of metabolism regulation during this process. Nevertheless, it also invokes a lot of questions. In this mini-review, we summarized the progress in the proteomic studies of rice seed germination. The current challenges and future perspectives were also discussed, which might be helpful for the following studies.

  20. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals “aging factors” and mechanism of lifespan asymmetry

    OpenAIRE

    Yang, Jing; McCormick, Mark A.; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K.; Li, Hao

    2015-01-01

    In this work, we took a proteome-centric view to analyze the cell division and lifespan asymmetry between mother and daughter cells in budding yeast. Using a flow cytometry-based, high-throughput approach, we quantified the partitioning of the proteome and identified 74 mother-enriched and 60 daughter-enriched proteins. Functional analysis of these proteins suggests mechanisms of asymmetric partitioning at an organelle/suborganelle level. We found that mother-enriched proteins are much more l...

  1. Dendronized Polymer Architectures for Fuel Cell Membranes

    DEFF Research Database (Denmark)

    Nielsen, Mads Møller; Dimitrov, Ivaylo; Takamuku, S.;

    2013-01-01

    evaluated as PEMs for use in fuel cells by proton conductivity measurements, and in the case of dendronized architectures: thermal stability. The proposed synthetic strategy facilitates exploration of a non‐fluorous system with various flexible side chains where IEC is tunable by the degree of substitution....

  2. How to Evaluate the Electric Noise in a Cell Membrane?

    Science.gov (United States)

    Bier, M.

    2006-05-01

    There has been considerable public anxiety about possible health effects of electromagnetic radiation emitted by high voltage power lines. Power frequencies (60 Hz in the US, 50 Hz in many other countries) are sufficiently slow for the associated electric fields to distribute themselves across the highly resistive cell membranes. To assess the ambient power frequency fields, researchers have compared the voltage that these fields induce across cell membranes to the strength of the electric noise that the membranes generate themselves through Brownian motion. However, there has been disagreement among researchers on how to evaluate this equilibrium membrane electric noise. I will review the different approaches and present an {ITALIC ab initio} modeling of membrane electric fields. I will show that different manifestations of Brownian noise lead to an electric noise intensity that is many times larger than what conventional estimates have yielded. Next, the legitimacy of gauging a nonequilibrium external signal against internal equilibrium noise is questioned and a more meaningful criterion is proposed. Finally, an estimate will be derived of the nonequilibrium noise intensity due to the driven ion traffic through randomly opening and closing ion channels.

  3. Experimental Investigation and Discussion on the Mechanical Endurance Limit of Nafion Membrane Used in Proton Exchange Membrane Fuel Cell

    OpenAIRE

    Yang Xiao; Chongdu Cho

    2014-01-01

    As a solution of high efficiency and clean energy, fuel cell technologies, especially proton exchange membrane fuel cell (PEMFC), have caught extensive attention. However, after decades of development, the performances of PEMFCs are far from achieving the target from the Department of Energy (DOE). Thus, further understanding of the degradation mechanism is needed to overcome this obstacle. Due to the importance of proton exchange membrane in a PEMFC, the degradation of the membrane, such as ...

  4. A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

    Science.gov (United States)

    Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

    2016-08-01

    In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency. PMID:27321140

  5. 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.

  6. 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.

  7. 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.

  8. The amniotic membrane as a source of stem cells.

    Science.gov (United States)

    Insausti, Carmen L; Blanquer, Miguel; Bleda, Patricia; Iniesta, Paqui; Majado, María J; Castellanos, Gregorio; Moraleda, José M

    2010-01-01

    Cellular therapy has emerged as a new potential tool for curing a wide range of degenerative diseases and tissue necrosis. Embryonic stem cells possess potential for differentiation into a wide range of cell lineages, but the ethical issues associated with establishment of this human cell line have to be resolved prior to any use. The bone marrow (BM) is the usual source of adult stem cells for hematopoietic stem cell transplants and cellular therapy, but the BM harvest is a surgical procedure that requires general anesthesia or sedation, and there seems to be a reduction of the proliferative potential and differentiation capacity of the marrow mesenchymal stem cells in older donors. For these reasons there is an increasing interest in other sources of stem cells from adult and fetal tissues. The amniotic membrane (AM) or amnion is a tissue of particular interest because its cells possess characteristics of stem cells with multipotent differentiation ability, and because of low immunogenicity and easy procurement from the placenta, which is a discarded tissue after parturition, thus avoiding the current controversies associated with the use of human embryonic stem cells. Therefore, amniotic membrane has been proposed as a good candidate to be used in cellular therapy and regenerative medicine. PMID:19924645

  9. 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.

  10. Chemical Imaging of the Cell Membrane by NanoSIMS

    Energy Technology Data Exchange (ETDEWEB)

    Weber, P K; Kraft, M L; Frisz, J F; Carpenter, K J; Hutcheon, I D

    2010-02-23

    The existence of lipid microdomains and their role in cell membrane organization are currently topics of great interest and controversy. The cell membrane is composed of a lipid bilayer with embedded proteins that can flow along the two-dimensional surface defined by the membrane. Microdomains, known as lipid rafts, are believed to play a central role in organizing this fluid system, enabling the cell membrane to carry out essential cellular processes, including protein recruitment and signal transduction. Lipid rafts are also implicated in cell invasion by pathogens, as in the case of the HIV. Therefore, understanding the role of lipid rafts in cell membrane organization not only has broad scientific implications, but also has practical implications for medical therapies. One of the major limitations on lipid organization research has been the inability to directly analyze lipid composition without introducing artifacts and at the relevant length-scales of tens to hundreds of nanometers. Fluorescence microscopy is widely used due to its sensitivity and specificity to the labeled species, but only the labeled components can be observed, fluorophores can alter the behavior of the lipids they label, and the length scales relevant to imaging cell membrane domains are between that probed by fluorescence resonance energy transfer (FRET) imaging (<10 nm) and the diffraction limit of light. Topographical features can be imaged on this length scale by atomic force microscopy (AFM), but the chemical composition of the observed structures cannot be determined. Immuno-labeling can be used to study the distribution of membrane proteins at high resolution, but not lipid composition. We are using imaging mass spectrometry by secondary ion mass spectrometry (SIMS) in concert with other high resolution imaging methods to overcome these limitations. The experimental approach of this project is to combine molecule-specific stable isotope labeling with high-resolution SIMS using a

  11. Durability aspects of polymer electrolyte membrane fuel cells

    Science.gov (United States)

    Sethuraman, Vijay Anand

    In order for the successful adoption of proton exchange membrane (PEM) fuel cell technology, it is imperative that durability is understood, quantified and improved. A number of mechanisms are known to contribute to PEMFC membrane electrode assembly (MEA) performance degradation. In this dissertation, we show, via experiments, some of the various processes that degrade the proton exchange membrane in a PEM fuel cell; and catalyst poisoning due to hydrogen sulfide (H2S) and siloxane. The effect of humidity on the chemical stability of two types of membranes, [i.e., perfluorosulfonic acid type (PFSA, NafionRTM 112) and biphenyl sulfone hydrocarbon type, (BPSH-35)] was studied by subjecting the MEAs to open-circuit voltage (OCV) decay and potential cycling tests at elevated temperatures and low inlet gas relative humidities. The BPSH-35 membranes showed poor chemical stability in ex situ Fenton tests compared to that of NafionRTM membranes. However, under fuel cell conditions, BPSH-35 MEAs outperformed NafionRTM 112 MEAs in both the OCV decay and potential cycling tests. For both membranes, (i) at a given temperature, membrane degradation was more pronounced at lower humidities and (ii) at a given relative humidity operation, increasing the cell temperature accelerated membrane degradation. Mechanical stability of these two types of membranes was also studied using relative humidity (RH) cycling. Hydrogen peroxide (H2O2) formation rates in a proton exchange membrane (PEM) fuel cell were estimated by studying the oxygen reduction reaction (ORR) on a rotating ring disc electrode (RRDE). Fuel cell conditions were replicated by depositing a film of Pt/Vulcan XC-72 catalyst onto the disk and by varying the temperature, dissolved O2 concentration and the acidity levels in HClO4. The HClO4 acidity was correlated to ionomer water activity and hence fuel cell humidity. H 2O2 formation rates showed a linear dependence on oxygen concentration and square dependence on water

  12. 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.

  13. Block copolymers for alkaline fuel cell membrane materials

    Science.gov (United States)

    Li, Yifan

    Alkaline fuel cells (AFCs) using anion exchange membranes (AEMs) as electrolyte have recently received considerable attention. AFCs offer some advantages over proton exchange membrane fuel cells, including the potential of non-noble metal (e.g. nickel, silver) catalyst on the cathode, which can dramatically lower the fuel cell cost. The main drawback of traditional AFCs is the use of liquid electrolyte (e.g. aqueous potassium hydroxide), which can result in the formation of carbonate precipitates by reaction with carbon dioxide. AEMs with tethered cations can overcome the precipitates formed in traditional AFCs. Our current research focuses on developing different polymer systems (blend, block, grafted, and crosslinked polymers) in order to understand alkaline fuel cell membrane in many aspects and design optimized anion exchange membranes with better alkaline stability, mechanical integrity and ionic conductivity. A number of distinct materials have been produced and characterized. A polymer blend system comprised of poly(vinylbenzyl chloride)-b-polystyrene (PVBC-b-PS) diblock copolymer, prepared by nitroxide mediated polymerization (NMP), with poly(2,6-dimethyl-1,4-phenylene oxide) (PPO) or brominated PPO was studied for conversion into a blend membrane for AEM. The formation of a miscible blend matrix improved mechanical properties while maintaining high ionic conductivity through formation of phase separated ionic domains. Using anionic polymerization, a polyethylene based block copolymer was designed where the polyethylene-based block copolymer formed bicontinuous morphological structures to enhance the hydroxide conductivity (up to 94 mS/cm at 80 °C) while excellent mechanical properties (strain up to 205%) of the polyethylene block copolymer membrane was observed. A polymer system was designed and characterized with monomethoxy polyethylene glycol (mPEG) as a hydrophilic polymer grafted through substitution of pendent benzyl chloride groups of a PVBC

  14. Quantitative proteomics as a tool to identify resistance mechanisms in erlotinib-resistant subclones of the non-small cell lung cancer cell line HCC827

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine

    line HCC827. Materials & Methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20, 30 µM erlotinib, respectively), and performed comparative quantitative proteomic analysis of these and the parental HCC827 cell line. The resistant subclones were examined both in absence and presence...

  15. Genistein and Daidzein Effects on Proliferation, Cell Membranes,Cell Cycles and Cell Apoptosis of Different Cell Lines

    Institute of Scientific and Technical Information of China (English)

    李重华; 王洪钟; 肖锐; 张勇; 于江涛; 谢莉萍; 张荣庆

    2001-01-01

    Genistein and daidzein are two principle isoflavonoids in soybeans. They have received increasing attention in the past few years because of their possible roles in cancer prevention. Here are provided experimental evidences that genistein could inhibit the growth of human bladder carcinoma cells (ECV-304), human colon cancer cells (HT29), human uterus cervix cancer cells (Hela), and murine transformed muscle cells (3T3). Different from genistein, daidzein could only inhibit the growth of ECV-304, HT29, and 3T3 cells. To elucidate the mechanisms of the anti-tumor effect of genistein and daidzein, fluorescent polarization, circular dichroism, and flow cytometric analysis were employed to study the influence of genistein and daidzein on membrane fluidity and membrane protein conformation of these cell lines. The results showed that genistein increased the order of membrane protein conformation and reduced the membrane fluidity of ECV-304, HT29, and Hela cells. Daidzein also increased the order of membrane protein conformation of ECV-304 and HT29, but had no effect on the membrane fluidity of all these four cell lines. Also demonstrated was that both compounds affected the apoptosis and cell cycle progression of some cell lines. However, the effects of genistein and daidzein were not the same. These evidences suggested that the effects of genistein and daidzein on malignant cells were multisites and multiapproaches, and there were differences between their functional mechanisms. The amelioration effect on cell conditions may represent one of the mechanisms of the effect of genistein and daidzein on the growth, differentiation, and transference of malignant cells.

  16. Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

    OpenAIRE

    Gama, José B.; Steffen Ohlmeier; Martins, Teresa G.; Fraga, Alexandra G.; Belém Sampaio-Marques; Carvalho, Maria A.; Fernanda Proença; Silva, Manuel T.; Jorge Pedrosa; Paula Ludovico

    2014-01-01

    Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This reveal...

  17. Proteomic Analysis of the Action of the Mycobacterium ulcerans Toxin Mycolactone: Targeting Host Cells Cytoskeleton and Collagen

    OpenAIRE

    Gama, José B.; Ohlmeier, S.; Martins, Teresa G.; Fraga, Alexandra G.; Marques, Belém Sampaio; Carvalho, M. Alice; Proença, M. Fernanda R. P.; Silva, Manuel T.; Pedrosa, Jorge; Ludovico, Paula

    2014-01-01

    Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This reveal...

  18. Proteomic Profiling of Recombinant Escherichia coli in High-Cell- Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

    OpenAIRE

    Aldor, Ilana S.; Krawitz, Denise C.; Forrest, William; Chen, Christina; Nishihara, Julie C.; Joly, John C.; Champion, Kathleen M.

    2005-01-01

    By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein sp...

  19. MEMBRANE LEc EXPRESSION IN BREAST CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Ya. A. Udalova

    2009-01-01

    Full Text Available Affine chromatography was used to isolate Lec antibodies from the sera of a healthy female donor with the high titers of these anti- bodies, which were labeled with biotin. The study enrolled 51 patients with primary breast cancer (BC. Antigen expression was found by immunohistochemistry and flow cytometry. With these two techniques being used, the detection rate of Lec expression in BC cells was 65% (33/51; the antigen was most frequently found by flow cytometry as compared with immunohistochemistry: 72 and 58% of cases, respectively.

  20. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Directory of Open Access Journals (Sweden)

    Shufeng Liu

    Full Text Available Hepatitis C virus (HCV poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP, UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1 and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection.

  1. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Science.gov (United States)

    Liu, Shufeng; Zhao, Ting; Song, BenBen; Zhou, Jianhua; Wang, Tony T

    2016-01-01

    Hepatitis C virus (HCV) poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP), UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1) and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection. PMID:26808496

  2. Comparison of Chloroflexus aurantiacus strain J-10-fl proteomes of cells grown chemoheterotrophically and photoheterotrophically

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Li; Bryant, Donald A.; Schepmoes, Athena A.; Vogl, Kajetan; Smith, Richard D.; Lipton, Mary S.; Callister, Stephen J.

    2012-01-17

    Chloroflexus aurantiacus J-10-fl is a thermophilic green bacterium, a filamentous anoxygenic phototroph, and the model organism of the phylum Chloroflexi. We applied high-throughput, liquid chromatography-mass spectrometry in a global quantitative proteomics investigation of C. aurantiacus cells grown under oxic (chemoorganoheterotrophically) and anoxic (photoorganoheterotrophically) redox states. Our global analysis identified 13,524 high-confidence peptides that matched to 1,286 annotated proteins, 242 of which were either uniquely identified or significantly increased in abundance under anoxic culture conditions. Fifty-three of the 242 proteins are previously characterized photosynthesis-related proteins, including chlorosome proteins, proteins involved in the bacteriochlorophyll biosynthesis, 3-hydroxypropionate (3-OHP) CO2 fixation pathway, and components of electron transport chains. The remaining 190 proteins have not previously been reported. Of these, five proteins were found to be encoded by genes from a novel operon and observed only in photoheterotrophically grown cells. These proteins candidates may prove useful in further deciphering the phototrophic physiology of C. aurantiacus and other filamentous anoxygenic phototrophs.

  3. Dihydroartemisinin-induced inhibition of proliferation in BEL-7402 cells: an analysis of the mitochondrial proteome.

    Science.gov (United States)

    Lu, Jin-Jian; Yang, Zhen; Lu, De-Zhao; Wo, Xing-De; Shi, Jia-Jie; Lin, Tao-Qi; Wang, Mi-Mi; Li, Yi; Tang, Li-Hua

    2012-08-01

    Artemisinin, the active ingredient of the Chinese medicinal herb Artemisia annua L., and its derivatives (ARTs) are currently widely used as anti-malarial drugs around the world. In this study, we found that dihydroartemisinin (DHA), one of the main active metabolites of ARTs, inhibited the proliferation of human hepatocarcinoma BEL-7402 cells in a concentration-dependent manner. To interpret the mechanisms involved, an analysis of the mitochondrial proteome was performed employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Seven mitochondrial proteins including fumarate hydratase, 60 kDa heat shock protein, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, two subunits of ATP synthase and NADPH:adrenodoxin oxidoreductase were identified to be differentially expressed between the control and DHA-treated groups. Our results indicate that the imbalance of energy metabolism induced by DHA may contribute, at least in part, to its anti-cancer potential in BEL-7402 cells. PMID:22580600

  4. Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

    KAUST Repository

    Janjanam, Jagadeesh

    2013-10-01

    Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. A Theory for the Membrane Potential of Living Cells

    CERN Document Server

    Endresen, L P; Høye, J S; Myrheim, Jan

    1998-01-01

    We give an explicit formula for the membrane potential of cells in terms of the intracellular and extracellular ionic concentrations, and derive equations for the ionic currents that flow through channels, exchangers and electrogenic pumps. We demonstrate that the work done by the pumps equals the change in potential energy of the cell, plus the energy lost in downhill ionic fluxes through the channels and exchangers. The theory is illustrated in a simple model of spontaneously active cells in the cardiac pacemaker. The model predicts the experimentally observed intracellular ionic concentration of potassium, calcium, and sodium. Likewise the shapes of the simulated action potential and five membrane currents are in good agreement with experiments. We do not see any drift in the values of the concentrations in a long time simulation, and we obtain the same asymptotic values when starting from the full equilibrium situation with equal intracellular and extracellular ionic concentrations.

  6. Gold Nanoparticles-Enhanced Proton Exchange Membrane (PEM) Fuel Cell

    Science.gov (United States)

    Li, Hongfei; Pan, Cheng; Liu, Ping; Zhu, Yimei; Adzic, Radoslav; Rafailovich, Miriam

    Proton exchange membrane fuel cells have drawn great attention and been taken as a promising alternated energy source. One of the reasons hamper the wider application of PEM fuel cell is the catalytic poison effect from the impurity of the gas flow. Haruta has predicted that gold nanoparticles that are platelet shaped and have direct contact with the metal oxide substrate to be the perfect catalysts of the CO oxidization, yet the synthesis method is difficult to apply in the Fuel Cell. In our approach, thiol-functionalized gold nanoparticles were synthesized through two-phase method developed by Brust et al. We deposit these Au particles with stepped surface directly onto the Nafion membrane in the PEM fuel cell by Langmuir-Blodgett method, resulting in over 50% enhancement of the efficiency of the fuel cell. DFT calculations were conducted to understand the theory of this kind of enhancement. The results indicated that only when the particles were in direct surface contact with the membrane, where AuNPs attached at the end of the Nafion side chains, it could reduce the energy barrier for the CO oxidation that could happen at T<300K.

  7. Membrane electrode assemblies for unitised regenerative polymer electrolyte fuel cells

    Science.gov (United States)

    Wittstadt, U.; Wagner, E.; Jungmann, T.

    Membrane electrode assemblies for regenerative polymer electrolyte fuel cells were made by hot pressing and sputtering. The different MEAs are examined in fuel cell and water electrolysis mode at different pressure and temperature conditions. Polarisation curves and ac impedance spectra are used to investigate the influence of the changes in coating technique. The hydrogen gas permeation through the membrane is determined by analysing the produced oxygen in electrolysis mode. The analysis shows, that better performances in both process directions can be achieved with an additional layer of sputtered platinum on the oxygen electrode. Thus, the electrochemical round-trip efficiency can be improved by more than 4%. Treating the oxygen electrode with PTFE solution shows better performance in fuel cell and less performance in electrolysis mode. The increase of the round-trip efficiency is negligible. A layer sputtered directly on the membrane shows good impermeability, and hence results in high voltages at low current densities. The mass transportation is apparently constricted. The gas diffusion layer on the oxygen electrode, in this case a titanium foam, leads to flooding of the cell in fuel cell mode. Stable operation is achieved after pretreatment of the GDL with a PTFE solution.

  8. 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.

  9. Inferring maps of forces inside cell membrane microdomains

    CERN Document Server

    Masson, J -B; Tuerkcan, S; Voisinne, G; Popoff, M R; Vergassola, M; Alexandrou, A

    2015-01-01

    Mapping of the forces on biomolecules in cell membranes has spurred the development of effective labels, e.g. organic fluorophores and nanoparticles, to track trajectories of single biomolecules. Standard methods use particular statistics, namely the mean square displacement, to analyze the underlying dynamics. Here, we introduce general inference methods to fully exploit information in the experimental trajectories, providing sharp estimates of the forces and the diffusion coefficients in membrane microdomains. Rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. The method is then applied to infer forces and potentials acting on the receptor of the $\\epsilon$-toxin labeled by lanthanide-ion nanoparticles. Our scheme is applicable to any labeled biomolecule and results show show its general relevance for membrane compartmentation.

  10. Nanoporous silicon membrane for fuel cells realized by electrochemical etching

    Energy Technology Data Exchange (ETDEWEB)

    Jaouadi, M., E-mail: mohja05.08@hotmail.com [Laboratoire de Photovoltaieque, Centre de Recherches et des Technologies de l' Energie, Technopole de Borj-Cedria, BP 95 Hammam-Lif 2050 (Tunisia); Dimassi, W.; Gaidi, M.; Chtourou, R.; Ezzaouia, H. [Laboratoire de Photovoltaieque, Centre de Recherches et des Technologies de l' Energie, Technopole de Borj-Cedria, BP 95 Hammam-Lif 2050 (Tunisia)

    2012-05-15

    In this work we propose a simple method to realize nanoporous silicon proton exchange membranes for fuel cells. The electrochemical etching allows in a single step, the grooving of the membrane and the realization of double porous silicon (PS) layer. We have studied the impact of the different electrochemical conditions: current density, the electrolyte concentration (ethanoic HF) and the anodization time leading to optimal thickness (of 50-90 {mu}m) and nanoporosity for silicon microstructures. The experimental techniques employed for surface studies and depths of groove in silicon are mainly scanning electronic microscopy (SEM) and atomic force microscopy (AFM) which give information of the silicon microstructures. Photoluminescence (PL) measurements of porous silicon membrane were carried out in order to investigate the optical properties of the PS.

  11. Benzimidazole grafted polybenzimidazoles for proton exchange membrane fuel cells

    DEFF Research Database (Denmark)

    Yang, Jingshuai; Aili, David; Li, Qingfeng;

    2013-01-01

    High molecular weight polybenzimidazole (PBI) was synthesized and grafted with benzimidazole pendant groups. The high molecular weight of PBI resulted in good film-forming properties and superior tensile strength. With a phosphoric acid doping level (ADL) of 13.1, a tensile strength of 16 MPa...... was achieved at room temperature. Grafting of benzimidazole moieties onto the PBI macromolecular chain introduced additional basic sites which allowed the membrane to achieve higher phosphoric acid uptakes. A molar acid conductivity, defined as the specific conductivity of each mole of doping acid......, was proposed to evaluate the effective conductivity contributed from the doping acids. With a grafting degree of 5.3% and an ADL of 13.1, the PBI membranes exhibited a total conductivity of 0.15 S cm-1. A H2-air fuel cell based on this membrane showed a peak power density of 378 mW cm-2 at 180 °C without...

  12. Nature of the elements transporting long-chain fatty acids through the red cell membrane

    DEFF Research Database (Denmark)

    Bojesen, Inge Norby; Bojesen, Eigil

    1998-01-01

    Docosahexaenoic acid, linoleic acid, red cell membrane, transporting elements, transport kinetics, fatty acid transport......Docosahexaenoic acid, linoleic acid, red cell membrane, transporting elements, transport kinetics, fatty acid transport...

  13. Inorganic-organic Composite Membranes with Novel Microstructure for High Temperature Proton Exchange Membrane Fuel Cells

    Institute of Scientific and Technical Information of China (English)

    Zhigang Ma; Jiandong Gao; Jing Guo; Zhenghua Deng; Jishuan Suo

    2007-01-01

    Nowadays,more and more fossil fuels are consumed and air pollurion has become a threat to the survival of people.Therefore,we need some other power sources to provide energy without damaging the environment.Proton exchange membrane fuel cells(PEMFCs)have received wide attention due to their advantages Such as high energy density and zero emission[1].Particularly, direct methanol fuel cells (DMFCs)were considered as the most suitable energy sources for electric vehicles(EVs)and portable electronics.

  14. Development of structured polymer electrolyte membranes for fuel cell applications

    Science.gov (United States)

    Gasa, Jeffrey

    The objective of this research was to explore structure-property relationships to develop the understanding needed for introduction of superior PEM materials. Polymer electrolyte membranes based on sulfonated poly(ether ketone ketone) (SPEKK) were fabricated using N-methyl pyrrolidone as casting solvent. The membranes were characterized in terms of properties that were relevant to fuel cell applications, such as proton conductivity, methanol permeability, and swelling properties, among others. It was found in this study that the proton conductivity of neat SPEKK membranes could reach the conductivity of commercial membranes such as NafionRTM. However, when the conductivity of SPEKK was comparable to NafionRTM, the swelling of SPEKK in water was quite excessive. The swelling problem was remedied by modifying the microstructure of SPEKK using different techniques. One of them involved blending of lightly sulfonated PEKK with highly acidic particles (sulfonated crosslinked polystyrene-SXLPS). Low sulfonation level of SPEKK was used to reduce the swelling of the membrane in water and the role of the highly acidic particles was to enhance the proton conductivity of the membrane. Because of the residual crystallinity in SPEKK with low sulfonation levels (IEC blending with non-conductive polymers (poly(ether imide) and poly(ether sulfone)) to act as mechanical reinforcement. It was found that miscibility behavior of the blends had a significant impact on the transport and swelling properties of these blends, which could be explained by the blend microstructure. The miscibility behavior was found to be strongly dependent on the sulfonation level of SPEKK. The conductivities of the blends were enhanced by as much as two orders of magnitude when the morphology was modified by electric field. The last approach was ionic crosslinking of the sulfonate groups in SPEKK using divalent cations, specifically barium ions. The crosslinking treatment has greatly improved the thermal

  15. Creating transient cell membrane pores using a standard inkjet printer.

    Science.gov (United States)

    Owczarczak, Alexander B; Shuford, Stephen O; Wood, Scott T; Deitch, Sandra; Dean, Delphine

    2012-03-16

    Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication. Recently, thermal inkjet printing has also been used for gene transfection. The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm. The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells. Cell viability after printing has been shown to be similar to standard cell plating methods. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. A standard HP DeskJet 500 printer was modified to allow for cell printing. The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the

  16. Modeling and Simulation for Fuel Cell Polymer Electrolyte Membrane

    Directory of Open Access Journals (Sweden)

    Takahiro Hayashi

    2013-01-01

    Full Text Available We have established methods to evaluate key properties that are needed to commercialize polyelectrolyte membranes for fuel cell electric vehicles such as water diffusion, gas permeability, and mechanical strength. These methods are based on coarse-graining models. For calculating water diffusion and gas permeability through the membranes, the dissipative particle dynamics–Monte Carlo approach was applied, while mechanical strength of the hydrated membrane was simulated by coarse-grained molecular dynamics. As a result of our systematic search and analysis, we can now grasp the direction necessary to improve water diffusion, gas permeability, and mechanical strength. For water diffusion, a map that reveals the relationship between many kinds of molecular structures and diffusion constants was obtained, in which the direction to enhance the diffusivity by improving membrane structure can be clearly seen. In order to achieve high mechanical strength, the molecular structure should be such that the hydrated membrane contains narrow water channels, but these might decrease the proton conductivity. Therefore, an optimal design of the polymer structure is needed, and the developed models reviewed here make it possible to optimize these molecular structures.

  17. Modified SPEEK membranes for direct ethanol fuel cell

    KAUST Repository

    Maab, Husnul

    2010-07-01

    Membranes with low ethanol crossover were prepared aiming their application for direct ethanol fuel cell (DEFC). They were based on (1) sulfonated poly(ether ether ketone) (SPEEK) coated with carbon molecular sieves (CMS) and (2) on SPEEK/PI homogeneous blends. The membranes were characterized concerning their water and ethanol solution uptake, water and ethanol permeability in pervaporation experiments and their performance in DEFC tests. The ethanol permeabilities for the CMS-coated (180 nm and 400 nm thick layers) SPEEK were 8.5 and 3.1 x 10(-10) kg m s(-1) m(-2) and for the homogeneous SPEEK/PI blends membranes with 10, 20 and 30 wt.% of PI were 4.4, 1.0 and 0.4 x 10(-10) kg m s(-1) m(-2) respectively, which is 2- to 50-fold lower than that for plain SPEEK (19 x 10(-10) kg m s(-1) m(-2)). Particularly the SPEEK/PI membranes had substantially better performance than Nafion 117 membranes in DEFC tests at 60 degrees C and 90 degrees C. (C) 2010 Elsevier B.V. All rights reserved.

  18. Assessing CMT cell line stability by two dimensional polyacrylamide gel electrophoresis and mass spectrometry based proteome analysis

    DEFF Research Database (Denmark)

    Zhang, Kelan; Wrzesinski, Krzysztof; Fey, Stephen J;

    2008-01-01

    Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometric identification of the proteins in the protein spots has become a central tool in proteomics. CMT167(H), CMT64(M) and CMT170(L) cell lines, selected from a spontaneous mouse lung adenocarcinoma, with high-...... to be a useful tool for assessing differences in cell line stability. This approach provided a tool to select the best cell line and optimal subculture period for studies of cancer related phenomena and for testing the effect of potential anticancer drugs....

  19. Lowering the platinum loading of high temperature polymer electrolyte membrane fuel cells with acid doped polybenzimidazole membranes

    DEFF Research Database (Denmark)

    Fernandez, Santiago Martin; Li, Qingfeng; Jensen, Jens Oluf

    2015-01-01

    Membrane electrode assemblies (MEAs) with ultra-low Pt loading electrodes were prepared for high temperature polymer electrolyte membrane fuel cells (HT-PEMFCs) based on acid doped polybenzimidazole. With no electrode binders or ionomers, the triple phase boundary of the catalyst layer was establ......Membrane electrode assemblies (MEAs) with ultra-low Pt loading electrodes were prepared for high temperature polymer electrolyte membrane fuel cells (HT-PEMFCs) based on acid doped polybenzimidazole. With no electrode binders or ionomers, the triple phase boundary of the catalyst layer...

  20. Comparison of functional proteomic analyses of human breast cancer cell lines T47D and MCF7.

    Directory of Open Access Journals (Sweden)

    Juliette Adjo Aka

    Full Text Available T47D and MCF7 are two human hormone-dependent breast cancer cell lines which are widely used as experimental models for in vitro and in vivo (tumor xenografts breast cancer studies. Several proteins involved in cancer development were identified in these cell lines by proteomic analyses. Although these studies reported the proteomic profiles of each cell line, until now, their differential protein expression profiles have not been established. Here, we used two-dimensional gel and mass spectrometry analyses to compare the proteomic profiles of the two cell lines, T47D and MCF7. Our data revealed that more than 164 proteins are differentially expressed between them. According to their biological functions, the results showed that proteins involved in cell growth stimulation, anti-apoptosis mechanisms and cancerogenesis are more strongly expressed in T47D than in MCF7. These proteins include G1/S-specific cyclin-D3 and prohibitin. Proteins implicated in transcription repression and apoptosis regulation, including transcriptional repressor NF-X1, nitrilase homolog 2 and interleukin-10, are, on the contrary, more strongly expressed in MCF7 as compared to T47D. Five proteins that were previously described as breast cancer biomarkers, namely cathepsin D, cathepsin B, protein S100-A14, heat shock protein beta-1 (HSP27 and proliferating cell nuclear antigen (PCNA, are found to be differentially expressed in the two cell lines. A list of differentially expressed proteins between T47D and MCF7 was generated, providing useful information for further studies of breast cancer mechanisms with these cell lines as models.