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Sample records for blood cell proteome

  1. 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....

  2. 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

  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. 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.

  5. 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...

  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. Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease

    Science.gov (United States)

    Soman, Kizhake V.; Zago, Maria P.; Koo, Sue-Jie; Spratt, Heidi; Stafford, Susan; Blell, Zinzi N.; Gupta, Shivali; Nuñez Burgos, Julio; Barrientos, Natalia; Brasier, Allan R.

    2016-01-01

    Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30–40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy. PMID:26919708

  8. Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.

    Directory of Open Access Journals (Sweden)

    Nisha Jain Garg

    2016-02-01

    Full Text Available Trypanosoma cruzi (Tc infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs of normal healthy controls (N/H, n = 30 and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25 or clinically symptomatic (C/S, n = 28 with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol and resolved by two-dimensional gel electrophoresis (2D-GE. After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.

  9. Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.

    Science.gov (United States)

    Garg, Nisha Jain; Soman, Kizhake V; Zago, Maria P; Koo, Sue-Jie; Spratt, Heidi; Stafford, Susan; Blell, Zinzi N; Gupta, Shivali; Nuñez Burgos, Julio; Barrientos, Natalia; Brasier, Allan R; Wiktorowicz, John E

    2016-02-01

    Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, psurvival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy. PMID:26919708

  10. 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.

  11. 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

  12. Proteomic responses of peripheral blood mononuclear cells in the European eel (Anguilla anguilla) after perfluorooctane sulfonate exposure.

    Science.gov (United States)

    Roland, Kathleen; Kestemont, Patrick; Hénuset, Laurence; Pierrard, Marie-Aline; Raes, Martine; Dieu, Marc; Silvestre, Frédéric

    2013-03-15

    Since the 1980s, the stocks of European eel have been declining in most of their geographical distribution area. Many factors can be attributed to this decline such as pollution by xenobiotics like perfluorooctane sulfonate (PFOS). This study aimed at evaluating the in vitro toxicity of eel peripheral blood mononuclear cells (PBMC) exposed to PFOS. Exposure time and two concentrations were chosen to avoid cell mortality (48 h exposure at 10 μg PFOS/L and 1mg PFOS/L). After in vitro contaminations, the post-nuclear fraction was isolated and a proteomic analysis using 2D-DIGE was performed to compare PBMC from the control group with cells exposed to the pollutant. On the 158 spots that were significantly affected by PFOS exposure, a total of 48 different proteins were identified using nano-LCESI-MS/MS and the Peptide and Protein Prophet of Scaffold software. These proteins can be categorized into diverse functional classes, related to cytoskeleton, protein folding, cell signaling, proteolytic pathway and carbohydrate and energy metabolism, which provide clues on the cellular pathways mainly affected by PFOS. Some of the identified proteins are rarely found in other ecotoxicological proteomic studies and could constitute potential biomarkers of exposure to PFOS in fish. PMID:23261670

  13. 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

  14. Accessing to the minor proteome of red blood cells through the influence of the nanoparticle surface properties on the corona composition

    Directory of Open Access Journals (Sweden)

    Zaccaria A

    2015-03-01

    Full Text Available Affif Zaccaria,1,* Florence Roux-Dalvai,2,3,* Ali Bouamrani,1 Adrien Mombrun,1 Pascal Mossuz,4 Bernard Monsarrat,2,3 François Berger1 1Clinatec CEA-LETI, Grenoble, 2CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale, 3Université de Toulouse, UPS, IPBS, Toulouse, 4TIMC-Therex UMR 5525 CNRS, UJF, CHU Grenoble, Grenoble, France *These authors contributed equally to this work Abstract: Nanoparticle (NP–protein interactions in complex samples have not yet been clearly understood. Nevertheless, several studies demonstrated that NP’s physicochemical features significantly impact on the protein corona composition. Taking advantage of the NP potential to harvest different subsets of proteins, we assessed for the first time the capacity of three kinds of superparamagnetic NPs to highlight the erythrocyte minor proteome. Using both qualitative and quantitative proteomics approaches, nano-liquid chromatography–tandem mass spectrometry allowed the identification of 893 different proteins, confirming the reproducible capacity of NPs to increase the number of identified proteins, through a reduction of the sample concentration range and the capture of specific proteins on the three different surfaces. These NP-specific protein signatures revealed significant differences in their isoelectric point and molecular weight. Moreover, this NP strategy offered a deeper access to the erythrocyte proteome highlighting several signaling pathways implicated in important erythrocyte functions. The automated potentiality, the reproducibility, and the low-consuming sample demonstrate the strong compatibility of our strategy for large-scale clinical studies and may become a standardized sample preparation in future erythrocyte-associated proteomics studies. Keywords: nanoparticles, red blood cells, mass spectrometry, quantitative proteomics, protein corona, minor proteome 

  15. 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...

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

    NARCIS (Netherlands)

    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

  17. 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...

  18. 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.

  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. Blood plasma reference material: a global resource for proteomic research.

    Science.gov (United States)

    Malm, Johan; Danmyr, Pia; Nilsson, Rolf; Appelqvist, Roger; Végvári, Akos; Marko-Varga, György

    2013-07-01

    There is an ever-increasing awareness and interest within the clinical research field, creating a large demand for blood fraction samples as well as other clinical samples. The translational research area is another field that is demanding for blood samples, used widely in proteomics, genomics, as well as metabolomics. Blood samples are globally the most common biological samples that are used in a broad variety of applications in life science. We hereby introduce a new reference blood plasma standard (heparin) that is aimed as a global resource for the proteomics community. We have developed these reference plasma standards by defining the Control group as those with C-reactive protein levels 30 mg/L. In these references we have used both newborn children 1-2 weeks, as well as youngsters 15-30 years, and middle aged 30-50 years, and elderly patients at the ages of 65+. In total, there were 80 patients in each group in the reference plasma pools. We provide data on the developments and characteristics of the reference blood plasma standards, as well as what is used by the team members at the respective laboratories. The standards have been evaluated by pilot sample processing in biobanking operations and are currently a resource that allows the Proteomic society to perform quantitative proteomic studies. By the use of high quality reference plasma samples, global initiatives, such as the Chromosome Human Proteome Project (C-HPP), will benefit as one scientific program when the entire human proteome is mapped and linked to human diseases. The plasma reference standards are a global resource and can be accessed upon request. PMID:23701512

  1. Functional proteomic analysis of Ankaferd® Blood Stopper

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    Duygu Özel Demiralp

    2010-06-01

    Full Text Available Objective: Ankaferd® Blood Stopper (ABS comprises a standardized mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. The basic mechanism of action for ABS is the formation of an encapsulated protein network that provides focal points for vital erythrocyte aggregation. ABS–induced protein network formation with blood cells, particularly erythrocytes, covers the primary and secondary hemostatic system without disturbing individual coagulation factors. Materials and Methods: To understand the effect mechanisms of ABS on hemostasis, a proteomic analysis using 2D gel electrophoresis and mass spectrometer was performed. Results: Proteins of plant origin in Ankaferd® were NADP-dependent-malic enzyme, ribulose bisphosphate-carboxylase-large chain, maturase K, ATP synthase subunit-beta, ATP synthase subunit-alpha, chalcone-flavanone isomerase-1, chalcone-flavanone isomerase-2, and actin-depolymerizing factor. Furthermore, functional proteomic studies revealed that proteins resembling human peptides have been detected within Ankaferd®, including ATP synthase, mucin-16 (CD164 sialomucin-like 2 protein, coiled-coil domain containing 141 hypothetical protein LOC283638 isoform 1, hypothetical protein LOC283638 isoform 2, dynactin 5, complex I intermediate-associated protein 30, mitochondrial, NADH dehydrogenase (ubiquinone 1 alpha subcomplex, TP synthase, H+ transporting, mitochondrial actin binding 1 isoform, LIM domain and actin binding 1 isoform a, LIM domain and actin binding 1 isoform b, spectrin alpha non erythrocytic 1, prolactin releasing hormone receptor, utrophin, tet oncogene family member 2 isoform b, protein phosphatase 1 regulatory subunit 12A, NIMA (never in mitosis gene a-related kinase, ATP-binding cassette protein C12, Homo sapiens malic enzyme 1, mitochondrial NADP(+-dependent malic enzyme 3, ME2 protein, nuclear factor 1 B-type, abhydrolase domain-containing protein 12B, E

  2. 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

  3. 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

  4. 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

  5. Effect of IL-6R Inhibition with Tocilizumab on the Proteome of Peripheral Blood Mononuclear Cells from a Rheumatoid Arthritis Patient

    DEFF Research Database (Denmark)

    Meyer, Michael Kruse; Andersen, Marlene; Bennike, Tue Bjerg;

    2015-01-01

    monoclonal antibody against the IL-6 receptor. Methods: Peripheral blood was obtained from one rheumatoid arthritis patient with poor response to conventional DMARD, before biological treatment and 4 months after. Peripheral blood mononuclear cells were extracted and separated into CD14+, CD4+, CD8+, CD19...... regulators, including Interleukin (IL)-6 are intimately associated with rheumatoid arthritis disease progression. In this proof-of-concept case study we assess the immunological changes in multiple important immune cell populations to treatment with the commonly applied bDMARD tocilizumab, a humanized...... the most responsive to therapy, and proteins involved in the JAK/STAT and MAPK signaling pathways were less abundant. Conclusion: Our data support that effective treatment of rheumatoid arthritis with tocilizumab causes decrease in the JAK/STAT and modulation of the MAPK signaling pathways, increases...

  6. Proteomic responses of peripheral blood mononuclear cells in the European eel (Anguilla anguilla) after perfluorooctane sulfonate exposure

    OpenAIRE

    Roland, K.; Kestemont, P.; Henuset, L.; Pierrard, M.-A.; Raes, M.; Dieu, M.; Silvestre, F.

    2013-01-01

    Since the 1980s, the stocks of European eel have been declining in most of their geographical distribution area. Many factors can be attributed to this decline such as pollution by xenobiotics like perfluorooctane sulfonate (PFOS). This study aimed at evaluating the in vitro toxicity of eel peripheral blood mononuclear cells (PBMC) exposed to PFOS. Exposure time and two concentrations were chosen to avoid cell mortality (48 h exposure at 10 mu g PFOS/L and 1 mg PFOS/L). After in vitro contami...

  7. 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...

  8. Blood-Brain Barrier Genomics, Proteomics, and New Transporter Discovery

    OpenAIRE

    Shusta, Eric V.

    2005-01-01

    Summary: The blood-brain barrier (BBB) is an impermeable cellular interface that physically separates the blood from the interstices of the brain. The endothelial cells lining the brain blood vessels form the principle barrier, and their unique phenotype is a consequence of dynamic interactions with several perivascular cell types present in the brain parenchyma. In addition, BBB dysfunction has been observed in the large majority of neurological diseases, but the causes of aberrant vascular ...

  9. Proteomic analysis of blood level of proteins before and after operation in patients with esophageal squamous cell carcinoma at high-incidence area in Henan Province

    Institute of Scientific and Technical Information of China (English)

    Ji-Ye An; Zong-Min Fan; Ze-Hao Zhuang; Yan-Ru Qin; Shan-Shan Gao; Ji-Lin Li; Li-Dong Wang

    2004-01-01

    AIM: To characterize the protein files in blood from same patients with esophageal squamous cell carcinoma (ESCC)before and after operation at the high-incidence area for ESCC in Henan Province, China.METHODS: Two-dimensional electrophoresis, silver staining and ImageMaster 2-DE analysis software were applied to the determination of protein files in the blood obtained from normal controls and ESCC patients before and after operation.RESULTS: A total of 655, 662 and 677 protein spots were identified, respectively, from the normal controls and ESCC patients before and after operation. No significant difference in the number of protein spots was observed between the normal group and ESCC patients. A total of seven protein spots were identified with a dramatic difference among the samples before and after operation. Six protein spots were up-regulated and one protein spot was down-regulated in the group after operation compared with those in normal and before operation. Three protein spots were further characterized by matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS). The proteins from these three spots were identified as serum amyloid A(SAA), amyloid related serum protein and haptoglobin.CONCLUSION: Serum amyloid A, amyloid related serum protein and haptoglobin may be related with ESCC and/or surgery. The significance of these proteins needs to be further characterized. The present study provides informative data for the establishment of serum protein profiles related with ESCC.

  10. 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

  11. 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...

  12. 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...

  13. Functional proteomic analysis of Ankaferd® Blood Stopper

    OpenAIRE

    Duygu Özel Demiralp; İbrahim C. Haznedaroğlu; Nejat Akar

    2010-01-01

    Objective: Ankaferd® Blood Stopper (ABS) comprises a standardized mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. The basic mechanism of action for ABS is the formation of an encapsulated protein network that provides focal points for vital erythrocyte aggregation. ABS–induced protein network formation with blood cells, particularly erythrocytes, covers the primary and secondary hemostatic system without disturbing individual ...

  14. 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

  15. 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...

  16. 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...

  17. 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

  18. Proteomics meets blood banking: identification of protein targets for the improvement of platelet quality.

    Science.gov (United States)

    Schubert, Peter; Devine, Dana V

    2010-01-01

    Proteomics has brought new perspectives to the fields of hematology and transfusion medicine in the last decade. The steady improvement of proteomic technology is propelling novel discoveries of molecular mechanisms by studying protein expression, post-translational modifications and protein interactions. This review article focuses on the application of proteomics to the identification of molecular mechanisms leading to the deterioration of blood platelets during storage - a critical aspect in the provision of platelet transfusion products. Several proteomic approaches have been employed to analyse changes in the platelet protein profile during storage and the obtained data now need to be translated into platelet biochemistry in order to connect the results to platelet function. Targeted biochemical applications then allow the identification of points for intervention in signal transduction pathways. Once validated and placed in a transfusion context, these data will provide further understanding of the underlying molecular mechanisms leading to platelet storage lesion. Future aspects of proteomics in blood banking will aim to make use of protein markers identified for platelet storage lesion development to monitor proteome changes when alterations such as the use of additive solutions or pathogen reduction strategies are put in place in order to improve platelet quality for patients.

  19. 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

  20. 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.

  1. 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.

  2. Identification of Residual Blood Proteins in Ticks by Mass Spectrometry Proteomics

    OpenAIRE

    Wickramasekara, Samanthi; Bunikis, Jonas; Wysocki, Vicki; Barbour, Alan G.

    2008-01-01

    Mass spectrometry–based proteomics of individual ticks demonstrated persistence of mammalian host blood components, including α- and β-globin chains, histones, and mitochondrial enzymes, in Ixodes scapularis and Amblyomma americanum ticks for months after molting. Residual host proteins may identify sources of infection for ticks.

  3. Blood-brain barrier proteomics: towards the understanding of neurodegenerative diseases.

    Science.gov (United States)

    Karamanos, Yannis; Gosselet, Fabien; Dehouck, Marie-Pierre; Cecchelli, Roméo

    2014-11-01

    The blood-brain barrier (BBB) regulates the passage of endogenous and exogenous compounds and thus contributes to the brain homeostasis with the help of well-known proteins such as tight junction proteins, plasma membrane transporters and metabolic barrier proteins. In the last decade, proteomics have emerged as supplementary tools for BBB research. The development of proteomic technologies has provided several means to extend knowledge on the BBB and to investigate additional routes for the bypass of this barrier. Proteomics approaches have been used in vivo and also using in vitro BBB models to decipher the physiological characteristics and, under stress conditions, to understand the molecular mechanisms of brain diseases. This work has demonstrated that both quantitative global and targeted proteomics approaches are powerful and provide significant information on the brain microvessel endothelium. However, current knowledge is only partial and it is necessary to increase the studies using proteomics tools that will provide additional information concerning brain pathologies or BBB metabolism. Highly sensitive, accurate and specific protein quantification by quantitative targeted proteomics appears as an essential methodology for human BBB studies. PMID:25446619

  4. 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

  5. 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...

  6. 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...

  7. Platelet proteomics and its advanced application for research of blood stasis syndrome and activated blood circulation herbs of Chinese medicine.

    Science.gov (United States)

    Liu, Yue; Yin, Huijun; Chen, Keji

    2013-11-01

    The development of novel and efficient antiplatelet agents that have few adverse effects and methods that improve antiplatelet resistance has long been the focus of international research on the prevention and treatment of cardiovascular and cerebrovascular diseases. Recent advances in platelet proteomics have provided a technology platform for high-quality research of platelet pathophysiology and the development of new antiplatelet drugs. The study of blood stasis syndrome (BSS) and activated blood circulation of traditional Chinese medicine (TCM) is one of the most active fields where the integration of TCM and western medicine in China has been successful. Activated blood circulation herbs (ABC herbs) of Chinese medicine are often used in the treatment of BSS. Most ABC herbs have antiplatelet and anti-atherosclerosis activity, but knowledge about their targets is lacking. Coronary heart disease (CHD), BSS, and platelet activation are closely related. By screening and identifying activated platelet proteins that are differentially expressed in BSS of CHD, platelet proteomics has helped researchers interpret the antiplatelet mechanism of action of ABC herbs and provided many potential biomarkers for BSS that could be used to evaluate the clinical curative effect of new antiplatelet drugs. In this article the progress of platelet proteomics and its advanced application for research of BSS and ABC herbs of Chinese medicine are reviewed.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. Characterization of monoclonal gammopathy of undetermined significance by calorimetric analysis of blood serum proteome.

    Directory of Open Access Journals (Sweden)

    Francisca Barceló

    Full Text Available Monoclonal gammopathy of undetermined significance (MGUS is a premalignant proliferative disorder that may progress to multiple myeloma, a malignant plasma cell neoplasia. We evaluated differential scanning calorimetry (DSC as an experimental tool for differentiating serum samples of MGUS patients from healthy individuals. DSC thermograms can be used for monitoring changes in the serum proteome associated with MGUS. MGUS patients showed great variability in serum thermogram characteristics, which depended on the IgG, IgA or IgM isotypes and/or the κ or λ light chains. Thermogram feature parameters distinguished patients with MGUS from healthy people. Serum samples, named as non-MGUS, were also collected from patients with subjacent immunological pathologies who were discarded of having MGUS through serum immunofixation. They were used to verify the sensitivity of DSC for discriminating MGUS from related blood dyscrasias. Only some DSC thermogram feature parameters differentiated, to a lesser extent, between MGUS and non-MGUS individuals. We contemplate DSC as a tool for early diagnosis and monitoring of MGUS.

  14. 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

  15. 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.

  16. White Blood Cell Disorders

    Science.gov (United States)

    ... where they are needed, and then kill and digest the harmful organism or substance (see White blood ... Patel Hello Everyone! Hello to all of you readers! I know you will be seeing my biography, ...

  17. 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...

  18. 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.

  19. 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....

  20. 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...

  1. 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.

  2. 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.

  3. 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:

  4. 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 ...

  5. 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.

  6. Characterization of monoclonal gammopathy of undetermined significance by calorimetric analysis of blood serum proteome

    OpenAIRE

    Barceló, Francisca; Cerdà, Joan J.; Gutiérrez, Antonio; Jiménez-Marco, Teresa; Durán, Maria Antònia; Novo, Andrés; Ros, Teresa; Sampol, Antonia; Portugal, José

    2015-01-01

    © 2015 Barceló et al. Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant proliferative disorder thatmay progress to multiplemyeloma, a malignant plasma cell neoplasia.We evaluated differential scanning calorimetry (DSC) as an experimental tool for differentiating serum samples of MGUS patients from healthy individuals. DSC thermograms can be used for monitoring changes in the serum proteome associated with MGUS. MGUS patients showed great variability in serum thermogr...

  7. Characterization of Monoclonal Gammopathy of Undetermined Significance by Calorimetric Analysis of Blood Serum Proteome

    OpenAIRE

    Barceló, Francisca; Cerdà, Joan J.; Gutiérrez, Antonio; Jimenez-Marco, Teresa; Durán, M. Antonia; Novo, Andrés; Ros, Teresa; Sampol, Antonia; Portugal, José

    2015-01-01

    Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant proliferative disorder that may progress to multiple myeloma, a malignant plasma cell neoplasia. We evaluated differential scanning calorimetry (DSC) as an experimental tool for differentiating serum samples of MGUS patients from healthy individuals. DSC thermograms can be used for monitoring changes in the serum proteome associated with MGUS. MGUS patients showed great variability in serum thermogram characteristics,...

  8. 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.

  9. 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

  10. 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.

  11. Towards an animal model of ovarian cancer: cataloging chicken blood proteins using combinatorial peptide ligand libraries coupled with shotgun proteomic analysis for translational research.

    Science.gov (United States)

    Ma, Yingying; Sun, Zeyu; de Matos, Ricardo; Zhang, Jing; Odunsi, Kunle; Lin, Biaoyang

    2014-05-01

    Epithelial ovarian cancer is the most deadly gynecological cancer around the world, with high morbidity in industrialized countries. Early diagnosis is key in reducing its morbidity rate. Yet, robust biomarkers, diagnostics, and animal models are still limited for ovarian cancer. This calls for broader omics and systems science oriented diagnostics strategies. In this vein, the domestic chicken has been used as an ovarian cancer animal model, owing to its high rate of developing spontaneous epithelial ovarian tumors. Chicken blood has thus been considered a surrogate reservoir from which cancer biomarkers can be identified. However, the presence of highly abundant proteins in chicken blood has compromised the applicability of proteomics tools to study chicken blood owing to a lack of immunodepletion methods. Here, we demonstrate that a combinatorial peptide ligand library (CPLL) can efficiently remove highly abundant proteins from chicken blood samples, consequently doubling the number of identified proteins. Using an integrated CPLL-1DGE-LC-MSMS workflow, we identified a catalog of 264 unique proteins. Functional analyses further suggested that most proteins were coagulation and complement factors, blood transport and binding proteins, immune- and defense-related proteins, proteases, protease inhibitors, cellular enzymes, or cell structure and adhesion proteins. Semiquantitative spectral counting analysis identified 10 potential biomarkers from the present chicken ovarian cancer model. Additionally, many human homologs of chicken blood proteins we have identified have been independently suggested as diagnostic biomarkers for ovarian cancer, further triangulating our novel observations reported here. In conclusion, the CPLL-assisted proteomic workflow using the chicken ovarian cancer model provides a feasible platform for translational research to identify ovarian cancer biomarkers and understand ovarian cancer biology. To the best of our knowledge, we report here

  12. 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

  13. 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.

  14. A proteomic approach for the rapid, multi-informative and reliable identification of blood.

    Science.gov (United States)

    Patel, E; Cicatiello, P; Deininger, L; Clench, M R; Marino, G; Giardina, P; Langenburg, G; West, A; Marshall, P; Sears, V; Francese, S

    2016-01-01

    Blood evidence is frequently encountered at the scene of violent crimes and can provide valuable intelligence in the forensic investigation of serious offences. Because many of the current enhancement methods used by crime scene investigators are presumptive, the visualisation of blood is not always reliable nor does it bear additional information. In the work presented here, two methods employing a shotgun bottom up proteomic approach for the detection of blood are reported; the developed protocols employ both an in solution digestion method and a recently proposed procedure involving immobilization of trypsin on hydrophobin Vmh2 coated MALDI sample plate. The methods are complementary as whilst one yields more identifiable proteins (as biomolecular signatures), the other is extremely rapid (5 minutes). Additionally, data demonstrate the opportunity to discriminate blood provenance even when two different blood sources are present in a mixture. This approach is also suitable for old bloodstains which had been previously chemically enhanced, as experiments conducted on a 9-year-old bloodstain deposited on a ceramic tile demonstrate. PMID:26596622

  15. 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

  16. 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

  17. 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

  18. 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.

  19. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... Find out why Close Becoming a Blood Stem Cell Donor NCIcancertopics Subscribe Subscribed Unsubscribe 351 351 Loading... ... considered becoming a bone marrow or blood stem cell donor? Follow this true story of a former ...

  20. Becoming a Blood Stem Cell Donor

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    Full Text Available ... total__ Find out why Close Becoming a Blood Stem Cell Donor NCIcancertopics Subscribe Subscribed Unsubscribe 351 351 Loading... ... Ever considered becoming a bone marrow or blood stem cell donor? Follow this true story of a former ...

  1. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... Find out why Close Becoming a Blood Stem Cell Donor NCIcancertopics Subscribe Subscribed Unsubscribe 352 352 Loading... ... considered becoming a bone marrow or blood stem cell donor? Follow this true story of a former ...

  2. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... on Jul 19, 2011 Ever considered becoming a bone marrow or blood stem cell donor? Follow this true ... Institutes of Health Clinical Center in Bethesda, MD. Bone marrow transplantation (BMT) and peripheral blood stem cell transplantation ( ...

  3. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... on Jul 19, 2011 Ever considered becoming a bone marrow or blood stem cell donor? Follow this ... Institutes of Health Clinical Center in Bethesda, MD. Bone marrow transplantation (BMT) and peripheral blood stem cell ...

  4. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... total__ Find out why Close Becoming a Blood Stem Cell Donor NCIcancertopics Subscribe Subscribed Unsubscribe 361 361 Loading... ... Ever considered becoming a bone marrow or blood stem cell donor? Follow this true story of a former ...

  5. Becoming a Blood Stem Cell Donor

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    Full Text Available ... total__ Find out why Close Becoming a Blood Stem Cell Donor NCIcancertopics Subscribe Subscribed Unsubscribe 350 350 Loading... ... Ever considered becoming a bone marrow or blood stem cell donor? Follow this true story of a former ...

  6. 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...

  7. 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

  8. 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).

  9. 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

  10. 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

  11. 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

  12. 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...

  13. 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 ...

  14. 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...

  15. 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.

  16. 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.

  17. 21 CFR 640.10 - Red Blood Cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  18. Avoiding Anemia: Boost Your Red Blood Cells

    Science.gov (United States)

    ... link, please review our exit disclaimer . Subscribe Avoiding Anemia Boost Your Red Blood Cells If you’re ... and sluggish, you might have a condition called anemia. Anemia is a common blood disorder that many ...

  19. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... cell donation experience at the National Institutes of Health Clinical Center in Bethesda, MD. Bone marrow transplantation ( ... About Cord Blood Banking - Duration: 49:19. Children's Health 27,845 views 49:19 Scott: Donating Blood ...

  20. 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 ...

  1. MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes

    DEFF Research Database (Denmark)

    Zhang, Yanling; Zhang, Yong; Adachi, Jun;

    2007-01-01

    Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several...... body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS......://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic...

  2. Understanding the rules of the road: proteomic approaches to interrogate the blood brain barrier

    OpenAIRE

    Torbett, Bruce E.; Baird, Andrew; Eliceiri, Brian P

    2015-01-01

    The blood brain barrier (BBB) is often regarded as a passive barrier that protects brain parenchyma from toxic substances, circulating leukocytes, while allowing the passage of selected molecules. Recently, a combination of molecular profiling techniques have characterized the constituents of the BBB based on in vitro models using isolated endothelial cells and ex vivo models analyzing isolated blood vessels. Characterization of gene expression profiles that are specific to the endothelium of...

  3. When Blood Cells Bend: Understanding Sickle Cell Disease

    Science.gov (United States)

    ... please review our exit disclaimer . Subscribe When Blood Cells Bend Understanding Sickle Cell Disease For people who don’t suspect they ... Cells Bend Wise Choices Links Living with Sickle Cell Disease See a sickle cell disease expert regularly. ...

  4. 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

  5. Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Yansheng Liu

    Full Text Available Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA and Multiple reaction monitoring (MRM assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG and Leucine-rich alpha-2-glycoprotein (LRG1, two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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...

  11. Proteomics-Based Metabolic Modeling Reveals That Fatty Acid Oxidation (FAO) Controls Endothelial Cell (EC) Permeability*

    Science.gov (United States)

    Patella, Francesca; Schug, Zachary T.; Persi, Erez; Neilson, Lisa J.; Erami, Zahra; Avanzato, Daniele; Maione, Federica; Hernandez-Fernaud, Juan R.; Mackay, Gillian; Zheng, Liang; Reid, Steven; Frezza, Christian; Giraudo, Enrico; Fiorio Pla, Alessandra; Anderson, Kurt; Ruppin, Eytan; Gottlieb, Eyal; Zanivan, Sara

    2015-01-01

    Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability. PMID:25573745

  12. Immune Cells in Blood Recognize Tumors

    Science.gov (United States)

    NCI scientists have developed a novel strategy for identifying immune cells circulating in the blood that recognize specific proteins on tumor cells, a finding they believe may have potential implications for immune-based therapies.

  13. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... Institutes of Health Clinical Center in Bethesda, MD. Bone marrow transplantation (BMT) and peripheral blood stem cell transplantation (PBSCT) are most commonly used in the treatment of cancers like leukemia and lymphoma to restore stem cells ...

  14. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... Institutes of Health Clinical Center in Bethesda, MD. Bone marrow transplantation (BMT) and peripheral blood stem cell transplantation (PBSCT) ... Medicine Clinics 225,676 views 6:18 Alicia's bone marrow donation - Duration: 8:33. ... Peripheral Blood Stem Cell Transplant - Duration: 15:50. Dartmouth-Hitchcock 2,764 views ...

  15. Cadmium uptake by rat red blood cells

    International Nuclear Information System (INIS)

    Rat red blood cells were employed to study the uptake of cadmium (109Cd). Suspensions of red blood cells were exposed to Cd concentrations (both bound and free) observed following in vivo Cd administration. Cd uptake was biphasic with an initial rapid phase (0C was one-fourth of that at 370C. The metabolic inhibitors: sodium fluoride (1mM), potassium cyanide (1mM) and carbonyl cyanide-m-chlorophenyl hydrazone (2μM) and the Na+-K+-ATPase inhibitor, ouabain (1mM) did not reduce Cd (50μM) uptake into red blood cells. This suggests that the uptake of Cd into red blood cells was not an active process. Incubation of Cd (10μM) with an equimolar concentration of Zn did not alter uptake of Cd into red blood cells, but at 5 and 10 times higher concentrations of Zn, Cd uptake was enhanced 5-fold. Mercury at one-tenth and equimolar concentrations of Cd increased Cd uptake by red blood cells 2-fold. N-Ethylmaleimide (0.5-5mM), which irreversibly inactivates membrane sulfhydryl groups, decreased Cd uptake. The data indicate that Cd uptake into rat red blood cells occurs by passive transport and that alterations of sulfhydryls of red blood cell membrane may modulate the process. (author)

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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...

  2. 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.

  3. 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...

  4. 21 CFR 864.9245 - Automated blood cell separator.

    Science.gov (United States)

    2010-04-01

    ... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated... automatically withdraw whole blood from a donor, separate the whole blood into blood components, collect one or more of the blood components, and return to the donor the remainder of the whole blood and...

  5. 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

  6. IBCIS:Intelligent blood cell identification system

    Institute of Scientific and Technical Information of China (English)

    Adnan Khashman

    2008-01-01

    The analysis of blood cells in microscope images can provide useful information concerning the health of patients.There are three major blood cell types,namely,erythrocytes (red),leukocytes (white),and platelets.Manual classification is time consuming and susceptible to error due to the different morphological features of the cells.This paper presents an intelligent system that simulates a human visual inspection and classification of the three blood cell types.The proposed system comprises two phases:The image preprocessing phase where blood cell features are extracted via global pattern averaging,and the neural network arbitration phase where training is the first and then classification is carried out.Experimental results suggest that the proposed method performs well in identifying blood cell types regardless of their irregular shapes,sizes and orientation,thus providing a fast,simple and efficient rotational and scale invariant blood cell identification system which can be used in automating laboratory reporting.

  7. Blood-Forming Stem Cell Transplants

    Science.gov (United States)

    ... Health Professionals Questions to Ask about Your Treatment Research Blood-Forming Stem Cell Transplants On This Page What are bone marrow ... are evaluating BMT and PBSCT in clinical trials (research studies) for the treatment ... are the donor’s stem cells matched to the patient’s stem cells in allogeneic ...

  8. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... Queue __count__/__total__ Find out why Close Becoming a Blood Stem Cell Donor NCIcancertopics Subscribe Subscribed Unsubscribe ... later? Sign in to add this video to a playlist. Sign in Share More Report Need to ...

  9. 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.

  10. 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

  11. 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...

  12. The saliva proteome of the blood-feeding insect Triatoma infestans is rich in platelet-aggregation inhibitors

    Science.gov (United States)

    Charneau, Sébastien; Junqueira, Magno; Costa, Camila M.; Pires, Daniele L.; Fernandes, Ellen S.; Bussacos, Ana C.; Sousa, Marcelo V.; Ricart, Carlos André O.; Shevchenko, Andrej; Teixeira, Antonio R. L.

    2007-12-01

    The saliva of the bloodsucking bug Triatoma infestans vector of Chagas disease contains an anti-hemostatic molecular cocktail that prevents coagulation, vasoconstriction and platelet aggregation in a vertebrate prey. In order to characterize T. infestans saliva proteome, we separated the secreted saliva by two-dimensional gel electrophoresis (2-DE). More than 200 salivary proteins were detected on the 2-DE map, mainly in the alkaline region. By nanoLC-MS/MS analysis using a LTQ-Orbitrap equipment followed by a combination of conventional and sequence-similarity searches, we identified 58 main protein spots. Most of such proteins possess potential blood-feeding associated functions, particularly anti-platelet aggregation proteins belonging to lipocalin and apyrase families. The saliva protein composition indicates a highly specific molecular mechanism of early response to platelet aggregation. This first proteome analysis of the T. infestans secreted saliva provides a basis for a better understanding of this fluid protein composition highly directed to counterpart hemostasis of the prey, thus promoting the bug's blood-feeding.

  13. Feasibility study on blood sample investigations from former Wismut employees with respect to possible biomarkers for arsenic or radiation exposure using proteomics and cDNA microarray technologies. Final report

    International Nuclear Information System (INIS)

    The final report on the feasibility of blood sample investigations from former Wismut employees with respect to possible biomarkers for arsenic or radiation exposure using proteomics and cDNA microarray technologies covers the following topics: blood samples; methodologies: 2D gel electrophoresis; protein identification using MALDI-MS; accomplishment and evaluation of the proteomics and cDNA microarray analysis.

  14. Single-cell measurement of red blood cell oxygen affinity

    CERN Document Server

    Caprio, Di; Higgins, John M; Schonbrun, Ethan

    2015-01-01

    Oxygen is transported throughout the body by hemoglobin in red blood cells. While the oxygen affinity of blood is well understood and is routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of red blood cell volume and hemoglobin concentration are taken millions of times per day by clinical hematology analyzers and are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume and hemoglobin concentration for individual red blood cells in high-throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.5%, which corresponds to the maximum slope of the oxygen-hemoglobin dissociation curve. In addition, single-cell oxygen affinity is positively correlated with hemoglobin concentr...

  15. Combined blood/tissue analysis for cancer biomarker discovery: application to renal cell carcinoma.

    Science.gov (United States)

    Johann, Donald J; Wei, Bih-Rong; Prieto, DaRue A; Chan, King C; Ye, Xiaying; Valera, Vladimir A; Simpson, R Mark; Rudnick, Paul A; Xiao, Zhen; Issaq, Haleem J; Linehan, W Marston; Stein, Stephen E; Veenstra, Timothy D; Blonder, Josip

    2010-03-01

    A method that relies on subtractive tissue-directed shot-gun proteomics to identify tumor proteins in the blood of a patient newly diagnosed with cancer is described. To avoid analytical and statistical biases caused by physiologic variability of protein expression in the human population, this method was applied on clinical specimens obtained from a single patient diagnosed with nonmetastatic renal cell carcinoma (RCC). The proteomes extracted from tumor, normal adjacent tissue and preoperative plasma were analyzed using 2D-liquid chromatography-mass spectrometry (LC-MS). The lists of identified proteins were filtered to discover proteins that (i) were found in the tumor but not normal tissue, (ii) were identified in matching plasma, and (iii) whose spectral count was higher in tumor tissue than plasma. These filtering criteria resulted in identification of eight tumor proteins in the blood. Subsequent Western-blot analysis confirmed the presence of cadherin-5, cadherin-11, DEAD-box protein-23, and pyruvate kinase in the blood of the patient in the study as well as in the blood of four other patients diagnosed with RCC. These results demonstrate the utility of a combined blood/tissue analysis strategy that permits the detection of tumor proteins in the blood of a patient diagnosed with RCC. PMID:20121140

  16. Blood cell morphology : controversies and alternatives

    NARCIS (Netherlands)

    Meer, Wim van der

    2006-01-01

    In this thesis we describe controversial morphologic features in both microscopic and automated differentiation of blood cells. In addition, we have investigated alternative methods to overcome these shortcomings. Furthermore we describe the variance of microscopic counting of band cells and variant

  17. Haemopoietic progenitor cells in human peripheral blood

    International Nuclear Information System (INIS)

    The purpose of the investigation reported is to purify haemopoietic progenitor cells from human peripheral blood using density gradient centrifugation in order to isolate a progenitor cell fraction without immunocompetent cells. The purification technique of peripheral blood flow colony forming unit culture (CFU-c) by means of density gradient centrifugation and a combined depletion of various rosettes is described. The results of several 'in vitro' characteristics of purified CFU-c suspensions and of the plasma clot diffusion chamber culture technique are presented. Irradiation studies revealed that for both human bone marrow and peripheral blood the CFU-c were less radioresistant than clusters. Elimination of monocytes (and granulocytes) from the test suspensions induced an alteration in radiosensitivity pararmeters. The results obtained with the different techniques are described by analysing peripheral progenitor cell activity in myeloproliferative disorders. (Auth.)

  18. 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.

  19. Automated red blood cell analysis compared with routine red blood cell morphology by smear review

    OpenAIRE

    Dr.Poonam Radadiya; Dr.Nandita Mehta; Dr.Hansa Goswami; Dr.R.N.Gonsai

    2015-01-01

    The RBC histogram is an integral part of automated haematology analysis and is now routinely available on all automated cell counters. This histogram and other associated complete blood count (CBC) parameters have been found abnormal in various haematological conditions and may provide major clues in the diagnosis and management of significant red cell disorders. Performing manual blood smears is important to ensure the quality of blood count results an...

  20. Whole Blood Cell Staining Device

    Science.gov (United States)

    Sams, Clarence F.; Clift, Vaughan L.; McDonald, Kelly E.

    2000-01-01

    An apparatus and method for staining particular cell markers is disclosed. The apparatus includes a flexible tube that is reversibly pinched into compartments with one or more clamps. Each compartment of the tube contains a separate reagent and is in selective fluid communication with adjoining compartments.

  1. White blood cell deformation and firm adhesion

    Science.gov (United States)

    Szatmary, Alex; Eggleton, Charles

    2011-11-01

    For a white blood cell (WBC) to arrive at infection sites, it forms chemical attachments with activated endothelial cells. First, it bonds with P-selectin, which holds it to the wall, but weakly; this allows the WBC to roll under the shear flow of the blood around it. Later, the WBCs bond with the stronger intracellular adhesion molecule-1 (ICAM-1); it is these ICAM bonds that allow the WBCs to fully resist the flow and stop rolling, allowing them to crawl through the endothelial wall. We model this numerically. Our model uses the immersed boundary method to represent the interaction of the shear flow with the deformable cell membrane. Receptors are on the tips of microvilli-little fingers sticking off of the cell membrane. The microvilli also deform. The receptors stochastically form and break bonds with molecules on the wall. Using this method, the history of each microvillus and its bonds can be found, as well as the distribution of the adhesion traction forces and how all of these vary with the deformability of the white blood cell. At higher shear rates, the white blood cell membrane deforms more, increasing its contact area with the surface; this effect is larger for softer membranes. We investigate how the deformability of the WBC affects the ease with which it forms firm adhesion.

  2. 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.)

  3. 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...

  4. 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

  5. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... blood stem cell (PBSC) donor, explains the donation process - Duration: 3:28. Be The Match 23,393 ... Copyright Creators Advertise Developers +YouTube Terms Privacy Policy & Safety Send feedback Try something new! Loading... Working... Sign ...

  6. Colour measurement and white blood cell recognition

    CERN Document Server

    Gelsema, E S

    1972-01-01

    As a part of a collaboration with NEMCH aimed at the automation of the differential white blood cell count, studies have been made of the different possibilities for using colour to help in the recognition process. Results are presented comparing data obtained with a microspectrophotometer and with a simulated three-colour scanner.

  7. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... be donors at http://www.marrow.org . Category Science & Technology License Standard YouTube License Show more Show ... Monks 3,700 views 4:41 Stem Cell Basics - How Blood is Made. - Duration: 10:58. Vernon ...

  8. Salivary Gland Proteome during Adult Development and after Blood Feeding of Female Anopheles dissidens Mosquitoes (Diptera: Culicidae)

    Science.gov (United States)

    Phattanawiboon, Benjarat; Jariyapan, Narissara; Mano, Chonlada; Roytrakul, Sittiruk; Paemanee, Atchara; Sor-Suwan, Sriwatapron; Sriwichai, Patchara; Saeung, Atiporn; Bates, Paul A.

    2016-01-01

    Understanding changes in mosquito salivary proteins during the time that sporozoite maturation occurs and after blood feeding may give information regarding the roles of salivary proteins during the malarial transmission. Anopheles dissidens (formerly Anopheles barbirostris species A1) is a potential vector of Plasmodium vivax in Thailand. In this study, analyses of the proteomic profiles of female An. dissidens salivary glands during adult development and after blood feeding were carried out using two-dimensional gel electrophoresis coupled with nano-liquid chromatography-mass spectrometry. Results showed at least 17 major salivary gland proteins present from day one to day 21 post emergence at 8 different time points sampled. Although there was variation observed, the patterns of protein expression could be placed into one of four groups. Fifteen protein spots showed significant depletion after blood feeding with the percentages of the amount of depletion ranging from 8.5% to 68.11%. The overall results identified various proteins, including a putative mucin-like protein, an anti-platelet protein, a long form D7 salivary protein, a putative gVAG protein precursor, a D7-related 3.2 protein, gSG7 salivary proteins, and a gSG6 protein. These results allow better understanding of the changes of the salivary proteins during the adult mosquito development. They also provide candidate proteins to investigate any possible link or not between sporozoite maturation, or survival of skin stage sporozoites, and salivary proteins. PMID:27669021

  9. 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

  10. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-01

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

  12. 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)

  13. 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.

  14. 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.

  15. 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.

  16. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  17. The antibody approach of labeling blood cells

    International Nuclear Information System (INIS)

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated

  18. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  19. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  20. Red blood cell replacement, or nanobiotherapeutics with enhanced red blood cell functions?

    Science.gov (United States)

    Chang, Thomas Ming Swi

    2015-06-01

    Why is this important? Under normal circumstances, donor blood is the best replacement for blood. However, there are exceptions: During natural epidemics (e.g., HIV, Ebola, etc.) or man-made epidemics (terrorism, war, etc.), there is a risk of donor blood being contaminated, and donors being disqualified because they have contracted disease. Unlike red blood cells (RBCs), blood substitutes can be sterilized to remove infective agents. Heart attack and stroke are usually caused by obstruction of arterial blood vessels. Unlike RBCs, which are particulate, blood substitutes are in the form of a solution that can perfuse through obstructed vessels with greater ease to reach the heart and brain, as has been demonstrated in animal studies. Severe blood loss from injuries sustained during accidents, disasters, or war may require urgent blood transfusion that cannot wait for transportation to the hospital for blood group testing. Unlike RBCs, blood substitutes do not have specific blood groups, and can be administered on the spot. RBCs have to be stored under refrigeration for up to 42 days, and are thus difficult to transport and store in times of disaster and at the battlefront. Blood substitutes can be stored at room temperature for more than 1 year, compared to the RBC shelf life of 1 day, at room temperature. In cases of very severe hemorrhagic shock, there is usually a safety window of 60 min for blood replacement, beyond which there could be problems related to irreversible shock. Animal studies show that a particular type of blood substitute, with enhanced RBC enzymes, may be able to prolong the duration of the safety window. PMID:26096663

  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. Automated microscopy system for peripheral blood cells

    Science.gov (United States)

    Boev, Sergei F.; Sazonov, Vladimir V.; Kozinets, Gennady I.; Pogorelov, Valery M.; Gusev, Alexander A.; Korobova, Farida V.; Vinogradov, Alexander G.; Verdenskaya, Natalya V.; Ivanova, Irina A.

    2000-11-01

    The report describes the instrument ASPBS (Automated Screening of Peripheral Blood Cells) designed for an automated analysis of dry blood smears. The instrument is based on computer microscopy and uses dry blood smears prepared according to the standard Romanovskii-Giemza procedure. In comparison with the well-known flow cytometry systems, our instrument provides more detailed information and offers an opporunity of visualizing final results. The basic performances of the instrument are given. Software of this instrument is based on digital image processing and image recognition procedures. It is pointed out that the instrument can be used as a fairly universal tool in scientific research, public demonstrations, in medical treatment, and in medical education. The principle used as the basis of the instrument appeared adequate for creating an instrument version serviceable even during space flights where standard manual procedures and flow cytometry systems fail. The benefit of the use of the instrument in clinical laboratories is described.

  3. 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.

  4. 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

  5. Blood cells and endothelial barrier function.

    Science.gov (United States)

    Rodrigues, Stephen F; Granger, D Neil

    2015-01-01

    The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of soluble mediators from resident cells (e.g., mast cells, macrophages) and/or recruited blood cells. The interaction of the mediators with receptors expressed on the surface of endothelial cells diminishes barrier function either by altering the expression of adhesive proteins in the inter-endothelial junctions, by altering the organization of the cytoskeleton, or both. Reactive oxygen species (ROS), proteolytic enzymes (e.g., matrix metalloproteinase, elastase), oncostatin M, and VEGF are part of a long list of mediators that have been implicated in endothelial barrier failure. In this review, we address the role of blood borne cells, including, neutrophils, lymphocytes, monocytes, and platelets, in the regulation of endothelial barrier function in health and disease. Attention is also devoted to new targets for therapeutic intervention in disease states with morbidity and mortality related to endothelial barrier dysfunction. PMID:25838983

  6. 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.

  7. Responder individuality in red blood cell alloimmunization.

    Science.gov (United States)

    Körmöczi, Günther F; Mayr, Wolfgang R

    2014-11-01

    Many different factors influence the propensity of transfusion recipients and pregnant women to form red blood cell alloantibodies (RBCA). RBCA may cause hemolytic transfusion reactions, hemolytic disease of the fetus and newborn and may be a complication in transplantation medicine. Antigenic differences between responder and foreign erythrocytes may lead to such an immune answer, in part with suspected specific HLA class II associations. Biochemical and conformational characteristics of red blood cell (RBC) antigens, their dose (number of transfusions and pregnancies, absolute number of antigens per RBC) and the mode of exposure impact on RBCA rates. In addition, individual circumstances determine the risk to form RBCA. Responder individuality in terms of age, sex, severity of underlying disease, disease- or therapy-induced immunosuppression and inflammation are discussed with respect to influencing RBC alloimmunization. For particular high-risk patients, extended phenotype matching of transfusion and recipient efficiently decreases RBCA induction and associated clinical risks. PMID:25670932

  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. 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

    infectious lung injury, 96 proteins were differentially expressed. Gene ontology enrichment analysis showed that these proteins participate in biological processes involved in the development of lung injury after HSCT. These include acute phase response signaling, complement system, coagulation system, liver X receptor (LXR)/retinoid X receptor (RXR), and farsenoid X receptor (FXR)/RXR modulation. We identified 2 canonical pathways modulated by TNF-α, FXR/RXR activation, and IL2 signaling in macrophages. The proteins also mapped to blood coagulation, fibrinolysis, and wound healing-processes that participate in organ repair. Cell movement was identified as significantly over-represented by proteins with differential expression between IPS and infection. In conclusion, the BALF protein expression in IPS differed significantly from infectious lung injury in HSCT recipients. These differences provide insights into mechanisms that are activated in lung injury in HSCT recipients and suggest potential therapeutic targets to augment lung repair. PMID:27155584

  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. Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry

    Science.gov (United States)

    Rawal, Ritu; Vijay, Sonam; Kadian, Kavita; Singh, Jagbir; Pande, Veena; Sharma, Arun

    2016-01-01

    In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS) and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite transmission during the

  12. Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry.

    Science.gov (United States)

    Rawal, Ritu; Vijay, Sonam; Kadian, Kavita; Singh, Jagbir; Pande, Veena; Sharma, Arun

    2016-01-01

    In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS) and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite transmission during the

  13. 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...

  14. Erythropoietin reduces storage lesions and decreases apoptosis indices in blood bank red blood cells

    OpenAIRE

    Oscar Andrés Penuela; Fernando Palomino; Lina Andrea Gómez

    2015-01-01

    ABSTRACT Background: Recent evidence shows a selective destruction of the youngest circulating red blood cells (neocytolysis) trigged by a drop in erythropoietin levels. Objective: The aim of this study was to evaluate the effect of recombinant human erythropoietin beta on the red blood cell storage lesion and apoptosis indices under blood bank conditions. Methods: Each one of ten red blood cell units preserved in additive solution 5 was divided in two volumes of 100 mL and assigned to one...

  15. 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...

  16. 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” ...

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

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

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

  18. 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.

  19. 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.

  20. 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

  1. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Science.gov (United States)

    2010-04-01

    ... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  2. 21 CFR 660.30 - Reagent Red Blood Cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall...

  3. Isolation of mesenchymal stem cells from equine umbilical cord blood

    DEFF Research Database (Denmark)

    Koch, Thomas Gadegaard; Heerkens, Tammy; Thomsen, Preben Dybdahl;

    2007-01-01

    Background: There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC) have been isolated from fresh umbilical cord blood of humans collected non-i...

  4. 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.

  5. Red blood cell in simple shear flow

    Science.gov (United States)

    Chien, Wei; Hew, Yayu; Chen, Yeng-Long

    2013-03-01

    The dynamics of red blood cells (RBC) in blood flow is critical for oxygen transport, and it also influences inflammation (white blood cells), thrombosis (platelets), and circulatory tumor migration. The physical properties of a RBC can be captured by modeling RBC as lipid membrane linked to a cytoskeletal spectrin network that encapsulates cytoplasm rich in hemoglobin, with bi-concave equilibrium shape. Depending on the shear force, RBC elasticity, membrane viscosity, and cytoplasm viscosity, RBC can undergo tumbling, tank-treading, or oscillatory motion. We investigate the dynamic state diagram of RBC in shear and pressure-driven flow using a combined immersed boundary-lattice Boltzmann method with a multi-scale RBC model that accurately captures the experimentally established RBC force-deformation relation. It is found that the tumbling (TU) to tank-treading (TT) transition occurs as shear rate increases for cytoplasm/outer fluid viscosity ratio smaller than 0.67. The TU frequency is found to be half of the TT frequency, in agreement with experiment observations. Larger viscosity ratios lead to the disappearance of stable TT phase and unstable complex dynamics, including the oscillation of the symmetry axis of the bi-concave shape perpendicular to the flow direction. The dependence on RBC bending rigidity, shear modulus, the order of membrane spectrin network and fluid field in the unstable region will also be discussed.

  6. Mechanosensing Dynamics of Red blood Cells

    Science.gov (United States)

    Wan, Jiandi

    2015-11-01

    Mechanical stress-induced deformation of human red blood cells (RBCs) plays important physiopathological roles in oxygen delivery, blood rheology, transfusion, and malaria. Recent studies demonstrate that, in response to mechanical deformation, RBCs release adenosine-5'-triphosphate (ATP), suggesting the existence of mechanotransductive pathways in RBCs. Most importantly, the released ATP from RBCs regulates vascular tone and impaired release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. To date, however, the mechanisms of mechanotransductive release of ATP from RBCs remain unclear. Given that RBCs experience shear stresses continuously during the circulation cycle and the released ATP plays a central role in vascular physiopathology, understanding the mechanotransductive release of ATP from RBCs will provide not only fundamental insights to the role of RBCs in vascular homeostasis but also novel therapeutic strategies for red cell dysfunction and vascular disease. This talk describes the main research in my group on integrating microfluidic-based approaches to study the mechanosensing dynamics of RBCs. Specifically, I will introduce a micro?uidic approach that can probe the dynamics of shear-induced ATP release from RBCs with millisecond resolution and provide quantitative understandings of the mechanosensitive ATP release processes in RBCs. Furthermore, I will also describe our recent findings about the roles of the Piezo1 channel, a newly discovered mechanosensitive cation channel in the mechanotransductive ATP release in RBCs. Last, possible functions of RBCs in the regulation of cerebral blood flow will be discussed.

  7. 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.

  8. Automated red blood cell analysis compared with routine red blood cell morphology by smear review

    Directory of Open Access Journals (Sweden)

    Dr.Poonam Radadiya

    2015-01-01

    Full Text Available The RBC histogram is an integral part of automated haematology analysis and is now routinely available on all automated cell counters. This histogram and other associated complete blood count (CBC parameters have been found abnormal in various haematological conditions and may provide major clues in the diagnosis and management of significant red cell disorders. Performing manual blood smears is important to ensure the quality of blood count results and to make presumptive diagnosis. In this article we have taken 100 samples for comparative study between RBC histograms obtained by automated haematology analyzer with peripheral blood smear. This article discusses some morphological features of dimorphism and the ensuing characteristic changes in their RBC histograms.

  9. Radiolabeled blood cells: radiation dosimetry and significance

    International Nuclear Information System (INIS)

    Over the past few years blood cells labeled with In-111 have become increasingly useful in clinical diagnosis and biomedical research. Indium-111 by the virtue of its physical characteristics and ability to bind to cell cytoplasmic components, provides an excellent cell tracer and thereby, allows investigators to monitor in vivo cell distribution by external imaging and help determine a course of regimen in treating life threatening diseases. Due to natural phenomena such as margination, blood pool, and reticuloendothelial cell activity, in the normal state, depending upon the cell type and the quality of cell preparations, 30%-50% of the administered radioactivity is immediately distributed in the liver, spleen and bone marrow. Over a period of time the radioactivity in these organs slightly increases and decays with a physical half-life of In-111. The resulting radiation dose to these organs ranges between 1-25 rads/mCi In-111 administered. The authors have developed a new In-111 labeling technique which preserves platelet ultrastructure and shown that human lymphocytes labeled with In-111 in mixed leukocytes preparations a) are only 0.003% of the total -body lymphocytes population and b) are killed. The consequence if any may be considered insignificant, particularly because 5.6% metaphases from normal men and 6.5% metaphases from normal women in the US have at least one chromosome aberration. Calculations have shown that the risk of fatal hematological malignancy, over a 30 year period, in recipients of 100 million lymphocytes labeled with 100 μCi In-111 is 1/million patients studied. This risk is less than 0.025% of the 1981 spontaneous cancer patient rate in the country. 32 references, 10 tables

  10. Red blood cell-incompatible allogeneic hematopoietic progenitor cell transplantation.

    Science.gov (United States)

    Rowley, S D; Donato, M L; Bhattacharyya, P

    2011-09-01

    Transplantation of hematopoietic progenitor cells from red cell-incompatible donors occurs in 30-50% of patients. Immediate and delayed hemolytic transfusion reactions are expected complications of red cell-disparate transplantation and both ABO and other red cell systems such as Kidd and rhesus can be involved. The immunohematological consequences of red cell-incompatible transplantation include delayed red blood cell recovery, pure red cell aplasia and delayed hemolysis from viable lymphocytes carried in the graft ('passenger lymphocytes'). The risks of these reactions, which may be abrupt in onset and fatal, are ameliorated by graft processing and proper blood component support. Red blood cell antigens are expressed on endothelial and epithelial tissues in the body and could serve to increase the risk of GvHD. Mouse models indicate that blood cell antigens may function as minor histocompatibility antigens affecting engraftment. Similar observations have been found in early studies of human transplantation for transfused recipients, although current conditioning and immunosuppressive regimens appear to overcome this affect. No deleterious effects from the use of red cell-incompatible hematopoietic grafts on transplant outcomes, such as granulocyte and platelet engraftments, the incidences of acute or chronic GvHD, relapse risk or OS, have been consistently demonstrated. Most studies, however, include limited number of patients, varying diagnoses and differing treatment regimens, complicating the detection of an effect of ABO-incompatible transplantation. Classification of patients by ABO phenotype ignoring the allelic differences of these antigens also may obscure the effect of red cell-incompatible transplantation on transplant outcomes. PMID:21897398

  11. 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.

  12. 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

  13. Aortic wall proteomic analysis in spontaneously hypertensive rats with a blood pressure decrease induced by 6-week load-free swimming

    OpenAIRE

    Feng, Hong; Li, Haiying; ZHANG, DERONG; ZHAO, YUNGANG; Jiang, Ning; Zhao, Xiaoling; Zhang, Yu; TAN, JUNZHEN; Fang, Wen; Zhang, Yong; Liu, Wei

    2015-01-01

    Decreased arterial compliance is one of the earliest detectable manifestations of adverse structural and functional changes within the vessel wall in hypertension. The proteomic approach is a powerful technique to analyze a complex mixture of proteins in various settings. Physical activity level was negatively associated with blood pressure. Sixteen 4-week-old male spontaneously hypertensive rats (SHR) and 16 Wistar-Kyoto (WKY) rats were randomly divided into four groups: i) SHR exercise grou...

  14. 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.

  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. Proteomics Core

    Data.gov (United States)

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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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 ...

  2. 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...

  3. 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...

  4. Proteomic analysis of human skin treated with larval schistosome peptidases reveals distinct invasion strategies among species of blood flukes.

    Directory of Open Access Journals (Sweden)

    Jessica Ingram

    2011-09-01

    Full Text Available Skin invasion is the initial step in infection of the human host by schistosome blood flukes. Schistosome larvae have the remarkable ability to overcome the physical and biochemical barriers present in skin in the absence of any mechanical trauma. While a serine peptidase with activity against insoluble elastin appears to be essential for this process in one species of schistosomes, Schistosoma mansoni, it is unknown whether other schistosome species use the same peptidase to facilitate entry into their hosts.Recent genome sequencing projects, together with a number of biochemical studies, identified alternative peptidases that Schistosoma japonicum or Trichobilharzia regenti could use to facilitate migration through skin. In this study, we used comparative proteomic analysis of human skin treated with purified cercarial elastase, the known invasive peptidase of S. mansoni, or S. mansoni cathespin B2, a close homolog of the putative invasive peptidase of S. japonicum, to identify substrates of either peptidase. Select skin proteins were then confirmed as substrates by in vitro digestion assays.This study demonstrates that an S. mansoni ortholog of the candidate invasive peptidase of S. japonicum and T. regenti, cathepsin B2, is capable of efficiently cleaving many of the same host skin substrates as the invasive serine peptidase of S. mansoni, cercarial elastase. At the same time, identification of unique substrates and the broader species specificity of cathepsin B2 suggest that the cercarial elastase gene family amplified as an adaptation of schistosomes to human hosts.

  5. 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.

  6. 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...

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Alterations in cell surface area and deformability of individual human red blood cells in stored blood

    CERN Document Server

    Park, HyunJoo; Lee, SangYun; Kim, Kyoohyun; Sohn, Yong-Hak; Jang, Seongsoo; Park, YongKeun

    2015-01-01

    The functionality and viability of stored human red blood cells (RBCs) is an important clinical issue in transfusion. To systematically investigate changes in stored whole blood, the hematological properties of individual RBCs were quantified in blood samples stored for various periods with and without a preservation solution called CPDA-1. With 3-D quantitative phase imaging techniques, the optical measurements of the 3-D refractive index (RI) distributions and membrane fluctuations were done at the individual cell level. From the optical measurements, the morphological (volume, surface area and sphericity), biochemical (hemoglobin content and concentration), and mechanical parameters (dynamic membrane fluctuation) were simultaneously quantified to investigate the functionalities and their progressive alterations in stored RBCs. Our results show that the stored RBCs without CPDA-1 had a dramatic morphological transformation from discocytes to spherocytes within 2 weeks which was accompanied with significant ...

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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)

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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...

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Red blood cell transfusion in septic shock

    DEFF Research Database (Denmark)

    Rosland, Ragnhild G; Hagen, Marte U; Haase, Nicolai;

    2014-01-01

    BACKGROUND: Treating anaemia with red blood cell (RBC) transfusion is frequent, but controversial, in patients with septic shock. Therefore we assessed characteristics and outcome associated with RBC transfusion in this group of high risk patients. METHODS: We did a prospective cohort study at 7...... general intensive care units (ICUs) including all adult patients with septic shock in a 5-month period. RESULTS: Ninety-five of the 213 included patients (45%) received median 3 (interquartile range 2-5) RBC units during shock. The median pre-transfusion haemoglobin level was 8.1 (7.4-8.9) g....../dl and independent of shock day and bleeding. Patients with cardiovascular disease were transfused at higher haemoglobin levels. Transfused patients had higher Simplified Acute Physiology Score (SAPS) II (56 (45-69) vs. 48 (37-61), p = 0.0005), more bleeding episodes, lower haemoglobin levels days 1 to 5, higher...

  13. Red Blood Cells Estimation Using Hough Transform Technique

    Directory of Open Access Journals (Sweden)

    Nasrul Humaimi Mahmood

    2012-05-01

    Full Text Available The number of red blood cells contributes more to clinical diagnosis with respect to blood diseases. Theaim of this research is to produce a computer vision system that can detect and estimate the number of redblood cells in the blood sample image. Morphological is a very powerful tool in image processing, and it isbeen used to segment and extract the red blood cells from the background and other cells. The algorithmused features such as shape of red blood cells for counting process, and Hough transform is introduced inthis process. The result presented here is based on images with normal blood cells. The tested data consistsof 10 samples and produced the accurate estimation rate closest to 96% from manual counting.

  14. SBR-Blood: systems biology repository for hematopoietic cells.

    Science.gov (United States)

    Lichtenberg, Jens; Heuston, Elisabeth F; Mishra, Tejaswini; Keller, Cheryl A; Hardison, Ross C; Bodine, David M

    2016-01-01

    Extensive research into hematopoiesis (the development of blood cells) over several decades has generated large sets of expression and epigenetic profiles in multiple human and mouse blood cell types. However, there is no single location to analyze how gene regulatory processes lead to different mature blood cells. We have developed a new database framework called hematopoietic Systems Biology Repository (SBR-Blood), available online at http://sbrblood.nhgri.nih.gov, which allows user-initiated analyses for cell type correlations or gene-specific behavior during differentiation using publicly available datasets for array- and sequencing-based platforms from mouse hematopoietic cells. SBR-Blood organizes information by both cell identity and by hematopoietic lineage. The validity and usability of SBR-Blood has been established through the reproduction of workflows relevant to expression data, DNA methylation, histone modifications and transcription factor occupancy profiles. PMID:26590403

  15. 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...

  16. Evaluation of preparation methods for MS-based analysis of intestinal epithelial cell proteomes

    DEFF Research Database (Denmark)

    Hesselager, Marianne Overgaard; Codrea, Marius Cosmin; Bendixen, Emøke

    2015-01-01

    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...

  17. 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

  18. 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...

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Mechanical damage of red blood cells by rotary blood pumps: selective destruction of aged red blood cells and subhemolytic trauma.

    Science.gov (United States)

    Sakota, Daisuke; Sakamoto, Ryuki; Sobajima, Hideo; Yokoyama, Naoyuki; Waguri, Satoshi; Ohuchi, Katsuhiro; Takatani, Setsuo

    2008-10-01

    In this study, mean cell volume (MCV), mean cell hemoglobin concentration (MCHC), and mean cell hemoglobin (MCH) were measured to quantify RBC damage by rotary blood pumps. Six-hour hemolysis tests were conducted with a Bio-pump BPX-80, a Sarns 15200 roller pump, and a prototype mag-lev centrifugal pump (MedTech Heart) using fresh porcine blood circulated at 5 L/min against a 100 mm Hg head pressure. The temperature of the test and noncirculated control blood was maintained at 37 degrees C. The normalized index of hemolysis (NIH) of each pump was determined by measuring the plasma-free hemoglobin level. The MCV was measured with a Coulter counter, and MCHC was derived from total hemoglobin and hematocrit. MCH was derived from MCV and MCHC. A multivariance statistical analysis (ANOVA) revealed statistically significant differences (n = 15, P < 0.05) in MCV, MCHC, and MCH between the blood sheared by the rotary blood pumps and the nonsheared control blood. Normalized to the control blood, the Bio-pump BPX-80 showed an MCV of 1.04 +/- 0.03, an MCHC of 0.95 +/- 0.04, and an MCH of 0.98 +/- 0.02; the mag-lev MedTech Heart had an MCV of 1.02 +/- 0.02, an MCHC of 0.97 +/- 0.02, and an MCH of 0.99 +/- 0.01; and the roller pump exhibited an MCV of 1.03 +/- 0.03, an MCHC of 0.96 +/- 0.03, and an MCH of 0.99 +/- 0.01. Per 0.01 increase in NIH, the BPX-80 showed a normalized MCV change of +10.1% and a normalized MCHC change of -14.0%; the MedTech Heart demonstrated a +6.9% MCV and -9.5% MCHC change; and the roller pump had a +0.5% MCV and -0.6% MCHC change. Due to shear in the pump circuits, the RBC increased while the MCHC decreased. The likely mechanism is that older RBCs with smaller size and higher hemoglobin concentration were destroyed fast by the shear, leaving younger RBCs with larger size and lower hemoglobin concentration. Subhemolytic trauma caused the intracellular hemoglobin to decrease due to gradual hemoglobin leakage through the micropores formed in the thinned

  5. 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.

  6. 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

  7. The treatment of neurodegenerative disorders using umbilical cord blood and menstrual blood-derived stem cells.

    Science.gov (United States)

    Sanberg, Paul R; Eve, David J; Willing, Alison E; Garbuzova-Davis, Svitlana; Tan, Jun; Sanberg, Cyndy D; Allickson, Julie G; Cruz, L Eduardo; Borlongan, Cesar V

    2011-01-01

    Stem cell transplantation is a potentially important means of treatment for a number of disorders. Two different stem cell populations of interest are mononuclear umbilical cord blood cells and menstrual blood-derived stem cells. These cells are relatively easy to obtain, appear to be pluripotent, and are immunologically immature. These cells, particularly umbilical cord blood cells, have been studied as either single or multiple injections in a number of animal models of neurodegenerative disorders with some degree of success, including stroke, Alzheimer's disease, amyotrophic lateral sclerosis, and Sanfilippo syndrome type B. Evidence of anti-inflammatory effects and secretion of specific cytokines and growth factors that promote cell survival, rather than cell replacement, have been detected in both transplanted cells.

  8. 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.

  9. 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

  10. 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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  12. Proteomic 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.

  13. Segmentation and Analysis of Cancer Cells in Blood Samples

    Directory of Open Access Journals (Sweden)

    Arjun Nelikanti

    2015-10-01

    Full Text Available Blood cancer is an umbrella term for cancers that affect the blood, bone marrow and lymphatic system. Acute Lymphoblastic Leukemia (ALL is one of the kinds of blood cancer which can be affected at any age in the humans. The analysis of peripheral blood samples is an important test in the procedures for the diagnosis of leukemia. In this paper the blood sample images are used and implementing a clustering algorithm for detection of the cancer cells. This paper also implements morphological operations and feature extraction techniques using MATLAB for the analysis of cancer cells in the images.

  14. Leucocyte filtration of salvaged blood during cardiac surgery : effect on red blood cell function in concentrated blood compared with diluted blood

    NARCIS (Netherlands)

    Gu, Y. John; de Vries, Adrianus J.; Hagenaars, J. Ans M.; van Oeveren, Willem

    2009-01-01

    Objective: Leucocyte filtration of salvaged blood has been suggested to prevent patients from receiving activated leucocytes during autotransfusion in cardiac surgery. This study examines whether leucocyte filtration of salvaged blood affects the red blood cell (RBC) function and whether there is a

  15. 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...

  16. 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.

  17. Stem Cell Transplant (Peripheral Blood, Bone Marrow, and Cord Blood Transplants)

    Science.gov (United States)

    ... are studied in cloning and other types of research. These stem cells are blood-forming stem cells. Stem cells mostly ... Preventing and managing GVHD are major priorities for research. Chronic ... 90 to 600 days after the stem cell transplant. A rash on the palms of the ...

  18. A photonic crystal hydrogel suspension array for the capture of blood cells from whole blood

    Science.gov (United States)

    Zhang, Bin; Cai, Yunlang; Shang, Luoran; Wang, Huan; Cheng, Yao; Rong, Fei; Gu, Zhongze; Zhao, Yuanjin

    2016-02-01

    Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells.Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06368j

  19. CD163 positive subsets of blood dendritic cells

    DEFF Research Database (Denmark)

    Maniecki, Maciej Bogdan; Møller, Holger Jon; Moestrup, Søren Kragh;

    2006-01-01

    expression in dendritic cells (DCs) was investigated using multicolor flow cytometry in peripheral blood from 31 healthy donors and 15 HIV-1 patients in addition to umbilical cord blood from 5 newborn infants. Total RNA was isolated from MACS purified DCs and CD163 mRNA was determined with real-time reverse...... transcriptase polymerase chain reaction. The effect of glucocorticoid and phorbol ester stimulation on monocyte and dendritic cell CD163 and CD91 expression was investigated in cell culture of mononuclear cells using multicolor flow cytometry. We identified two CD163+ subsets in human blood with dendritic cell...

  20. Proteomic response to 5,6-dimethylxanthenone 4-acetic acid (DMXAA, vadimezan) in human non-small cell lung cancer A549 cells determined by the stable-isotope labeling by amino acids in cell culture (SILAC) approach.

    Science.gov (United States)

    Pan, Shu-Ting; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Yang, Yin-Xue; Wang, Dong; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    5,6-Dimethylxanthenone 4-acetic acid (DMXAA), also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC) and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC) approach. The proteomic data showed that treatment with DMXAA modulated the expression of 588 protein molecules in A549 cells, with 281 protein molecules being up regulated and 306 protein molecules being downregulated. Ingenuity pathway analysis (IPA) identified 256 signaling pathways and 184 cellular functional proteins that were regulated by DMXAA in A549 cells. These targeted molecules and signaling pathways were mostly involved in cell proliferation and survival, redox homeostasis, sugar, amino acid and nucleic acid metabolism, cell migration, and invasion and programed cell death. Subsequently, the effects of DMXAA on cell cycle distribution, apoptosis, autophagy, and reactive oxygen species (ROS) generation were experimentally verified. Flow cytometric analysis showed that DMXAA significantly induced G1 phase arrest in A549 cells. Western blotting assays demonstrated that DMXAA induced apoptosis via a mitochondria-dependent pathway and promoted autophagy, as indicated by the increased level of cytosolic cytochrome c, activation of caspase 3, and enhanced expression of beclin 1 and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) in A549 cells. Moreover, DMXAA significantly promoted intracellular ROS

  1. Hormones that Stimulate the Growth of Blood Cells.

    Science.gov (United States)

    Golde, David W.; Gasson, Judith C.

    1988-01-01

    Describes the nature and action of hematopoietic proteins which regulate the production of specific sets of blood cells. Discusses the production of these hematopoietins by recombinant-DNA methods in an effort to enable physicians to treat patients by eliciting production of specific types of blood cells. (CW)

  2. Multifactorial aspects of antibody-mediated blood cell destruction

    NARCIS (Netherlands)

    R. Kapur

    2014-01-01

    The research described in this thesis focuses on diseases of antibody-mediated blood cell destruction via FcγRs on phagocytes, in particular regarding platelets in fetal or neonatal alloimmune thrombocytopenia (FNAIT) and red blood cells (RBC) in hemolytic disease of the fetus and newborn (HDFN). Di

  3. 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

  4. Isolation of mesenchymal stem cells from equine umbilical cord blood

    Directory of Open Access Journals (Sweden)

    Thomsen Preben D

    2007-05-01

    Full Text Available Abstract Background There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC have been isolated from fresh umbilical cord blood of humans collected non-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low. The hypothesis of this study was that equine MSCs could be isolated from fresh whole equine cord blood. Results Cord blood was collected from 7 foals immediately after foaling. The mononuclear cell fraction was isolated by Ficoll density centrifugation and cultured in a DMEM low glucose based media at 38.5°C in humidified atmosphere containing 5% CO2. In 4 out of 7 samples colonies with MSC morphology were observed. Cellular morphology varied between monolayers of elongated spindle-shaped cells to layered cell clusters of cuboidal cells with shorter cytoplasmic extensions. Positive Alizarin Red and von Kossa staining as well as significant calcium deposition and alkaline phosphatase activity confirmed osteogenesis. Histology and positive Safranin O staining of matrix glycosaminoglycans illustrated chondrogenesis. Oil Red O staining of lipid droplets confirmed adipogenesis. Conclusion We here report, for the first time, the isolation of mesenchymal-like stem cells from fresh equine cord blood and their differentiation into osteocytes, chondrocytes and adipocytes. This novel isolation of equine cord blood MSCs and their preliminary in vitro differentiation positions the horse as the ideal pre-clinical animal model for proof-of-principle studies of cord blood derived MSCs.

  5. A Simulation of Blood Cells in Branching Capillaries

    CERN Document Server

    Isfahani, Amir H G; Freund, Jonathan B

    2008-01-01

    The multi-cellular hydrodynamic interactions play a critical role in the phenomenology of blood flow in the microcirculation. A fast algorithm has been developed to simulate large numbers of cells modeled as elastic thin membranes. For red blood cells, which are the dominant component in blood, the membrane has strong resistance to surface dilatation but is flexible in bending. Our numerical method solves the boundary integral equations built upon Green's functions for Stokes flow in periodic domains. This fluid dynamics video is an example of the capabilities of this model in handling complex geometries with a multitude of different cells. The capillary branch geometries have been modeled based upon observed capillary networks. The diameter of the branches varies between 10-20 mum. A constant mean pressure gradient drives the flow. For the purpose of this fluid dynamics video, the red blood cells are initiated as biconcave discs and white blood cells and platelets are initiated as spheres and ellipsoids resp...

  6. Establishment of outgrowth endothelial cells from peripheral blood.

    Science.gov (United States)

    Martin-Ramirez, Javier; Hofman, Menno; van den Biggelaar, Maartje; Hebbel, Robert P; Voorberg, Jan

    2012-09-01

    Blood outgrowth endothelial cells (BOECs) are important tools when investigating diagnostic and therapeutic approaches for vascular disease. In this protocol, mononuclear cells are isolated from peripheral blood and plated on type I collagen at ∼135,000 cells per cm(2) in endothelial cell differentiation medium. On average, 0.34 colonies of endothelial cells per milliliter of blood can be obtained. Colonies of endothelial cells become visible after 14-28 d. Upon confluence, these rapidly expanding colonies can be passaged and have been shown to propagate up to 10(18)-fold. Isolated BOECs are phenotypically similar to vascular endothelial cells, as revealed by their cobblestone morphology, the presence of endothelial cell-specific Weibel-Palade bodies and the expression of endothelial cell markers such as VE-cadherin. The protocol presented here also provides a particularly useful tool for the ex vivo assessment of endothelial cell function from patients with different vascular abnormalities. PMID:22918388

  7. Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells.

    Science.gov (United States)

    Reisz, Julie A; Wither, Matthew J; Dzieciatkowska, Monika; Nemkov, Travis; Issaian, Aaron; Yoshida, Tatsuro; Dunham, Andrew J; Hill, Ryan C; Hansen, Kirk C; D'Alessandro, Angelo

    2016-09-22

    Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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...

  14. Interpretation of automated blood cell counts

    OpenAIRE

    Zühre Kaya

    2013-01-01

    Complete blood count (CBC) tests are rapid, inexpensiveand universally available, and often aid primary clinicianswith decision making about patients with severaldisorders. Thus the rapid availability of the results of CBCcould provide considerable advantage for both patientsand clinicians. Furthermore, physicians can also avoidunnecessary peripheral blood smear examination usingCBC parameters. Many hematology analyzers, which enabledus simultaneously, measure several different CBCparameters,...

  15. 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

  16. 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.

  17. Red blood cells in sports: Effects of exercise and training on oxygen supply by red blood cells

    Directory of Open Access Journals (Sweden)

    Heimo eMairbäurl

    2013-11-01

    Full Text Available During exercise the cardiovascular system has to warrant substrate supply to working muscle. The main function of red blood cells in exercise is the transport of O2 from the lungs to the tissues and the delivery of metabolically produced CO2 to the lungs for expiration. Hemoglobin also contributes to the blood’s buffering capacity, and ATP and NO release from red blood cells contributes to vasodilation and improved blood flow to working muscle. These functions require adequate amounts of red blood cells in circulation. Trained athletes, particularly in endurance sports, have a decreased hematocrit, which is sometimes called sports anemia. This is not anemia in a clinical sense because athletes have in fact an increased total mass of red blood cells and hemoglobin in circulation relative to sedentary individuals. The slight decrease in hematocrit by training is brought about by an increased plasma volume. The mechanisms that increase total red blood cell mass by training are not understood fully. Despite stimulated erythropoiesis, exercise can decrease the red blood cell mass by intravascular hemolysis mainly of senescent red blood cells, which is caused by mechanical rupture when red blood cells pass through capillaries in contracting muscles, and by compression of red cells e.g. in foot soles during running or in hand palms in weightlifters. Together, these adjustments cause a decrease in the average age of the population of circulating red blood cells in trained athletes. These younger red cells are characterized by improved oxygen release and deformability, both of which also improve tissue oxygen supply during exercise.

  18. 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

  19. 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

  20. 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.

  1. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    Directory of Open Access Journals (Sweden)

    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

  2. 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.

  3. [Promising technologies of packed red blood cells production and storage].

    Science.gov (United States)

    Maksimov, A G; Golota, A S; Krassiĭ, A B

    2013-10-01

    The current article is dedicated to promising technologies of packed red blood cells production and storage. The following new technical approaches are presented: (1) erythrocytes storage in strict anaerobic argon-hydrogen environment, (2) lyophilization of erythrocyte suspension by its atomization in nitrogen gas, (3) lyophilization of erythrocytes by directional freezing under the influence of radio frequency radiation, (4) automated pharming of antigen free packed red blood cells from progenitor cell directly at the battlefield. PMID:24611298

  4. [Promising technologies of packed red blood cells production and storage].

    Science.gov (United States)

    Maksimov, A G; Golota, A S; Krassiĭ, A B

    2013-10-01

    The current article is dedicated to promising technologies of packed red blood cells production and storage. The following new technical approaches are presented: (1) erythrocytes storage in strict anaerobic argon-hydrogen environment, (2) lyophilization of erythrocyte suspension by its atomization in nitrogen gas, (3) lyophilization of erythrocytes by directional freezing under the influence of radio frequency radiation, (4) automated pharming of antigen free packed red blood cells from progenitor cell directly at the battlefield.

  5. 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

  6. Interpretation of automated blood cell counts

    Directory of Open Access Journals (Sweden)

    Zühre Kaya

    2013-09-01

    Full Text Available Complete blood count (CBC tests are rapid, inexpensiveand universally available, and often aid primary clinicianswith decision making about patients with severaldisorders. Thus the rapid availability of the results of CBCcould provide considerable advantage for both patientsand clinicians. Furthermore, physicians can also avoidunnecessary peripheral blood smear examination usingCBC parameters. Many hematology analyzers, which enabledus simultaneously, measure several different CBCparameters, are available for early diagnosis. Herein theimpact of both pre and post analytic variations on the interpretationof the CBC results with case reports are reviewedin the light of the latest literature.Key words: Complete blood count, interpretation

  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. A model for red blood cells in simulations of large-scale blood flows

    CERN Document Server

    Melchionna, Simone

    2011-01-01

    Red blood cells (RBCs) are an essential component of blood. A method to include the particulate nature of blood is introduced here with the goal of studying circulation in large-scale realistic vessels. The method uses a combination of the Lattice Boltzmann method (LBM) to account for the plasma motion, and a modified Molecular Dynamics scheme for the cellular motion. Numerical results illustrate the quality of the model in reproducing known rheological properties of blood as much as revealing the effect of RBC structuring on the wall shear stress, with consequences on the development of cardiovascular diseases.

  9. White blood cell count - series (image)

    Science.gov (United States)

    ... the hand. The puncture site is cleaned with antiseptic, and a tourniquet (an elastic band) or blood ... or young child: The area is cleansed with antiseptic and punctured with a sharp needle or a ...

  10. Measuring density and compressibility of white blood cells and prostate cancer cells by microchannel acoustophoresis

    DEFF Research Database (Denmark)

    Barnkob, Rune; Augustsson, Per; Magnusson, Cecilia;

    2011-01-01

    to determine the density and compressibility of individual cells enables the prediction and alteration of the separation outcome for a given cell mixture. We apply the method on white blood cells (WBCs) and DU145 prostate cancer cells (DUCs) aiming to improve isolation of circulating tumor cells from blood......, an emerging tool in the monitoring and characterizing of metastatic cancer....

  11. Net haemoglobin increase from reinfusion of refrigerated vs. frozen red blood cells after autologous blood transfusions

    DEFF Research Database (Denmark)

    Ashenden, M; Mørkeberg, Jakob Sehested

    2011-01-01

    freezing. Nevertheless, frozen storage allowed haemoglobin to fully recover before reinfusion, while the haemoglobin was 10% lower in the refrigerated group compared with baseline. After reinfusion, the haemoglobin levels were 11·5% higher than the baseline values in the group reinfused with frozen blood......BACKGROUND AND OBJECTIVES  Two main blood storage procedures can be used for storing red blood cells: refrigeration and freezing. Nevertheless, the efficiency of these procedures measured as the increase in haemoglobin after reinfusion compared with baseline has never been examined. The main...... objective was to examine which storage procedure yielded the largest increase in circulating haemoglobin after reinfusion compared to baseline. MATERIALS AND METHODS  Equal volumes of blood from 15 men were withdrawn and stored either frozen or refrigerated as packed red blood cells. Serial measures...

  12. 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.

  13. 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...

  14. 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.

  15. Viable Bacteria Associated with Red Blood Cells and Plasma in Freshly Drawn Blood Donations

    DEFF Research Database (Denmark)

    Damgaard, Christian; Magnussen, Karin; Enevold, Christian;

    2015-01-01

    OBJECTIVES: Infection remains a leading cause of post-transfusion mortality and morbidity. Bacterial contamination is, however, detected in less than 0.1% of blood units tested. The aim of the study was to identify viable bacteria in standard blood-pack units, with particular focus on bacteria from...... the oral cavity, and to determine the distribution of bacteria revealed in plasma and in the red blood cell (RBC)-fraction. DESIGN: Cross-sectional study. Blood were separated into plasma and RBC-suspensions, which were incubated anaerobically or aerobically for 7 days on trypticase soy blood agar (TSA......), self-reported medically healthy. RESULTS: Bacterial growth was observed on plates inoculated with plasma or RBCs from 62% of the blood donations. Growth was evident in 21 (35%) of 60 RBC-fractions and in 32 (53%) of 60 plasma-fractions versus 8 of 60 negative controls (p = 0.005 and p = 2.6x10...

  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. 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

  1. 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.

  2. Transfusion management of patients with red blood cell antibodies

    OpenAIRE

    Bujandrić Nevenka B.; Grujić Jasmina N.; Krga-Milanović Mirjana M.

    2013-01-01

    Introduction. Red blood cell antibodies may cause a positive result of pre-transfusion blood compatibility testing (crossmatch test). It can be a problem to provide suitable blood units for patients with clinically significant antibodies to high-frequency antigens as well as for those with multiple alloantibody specificities. This study was aimed at identifying transfused patients in the population of South-Backa who had developed clinically significant red...

  3. Human umbilical cord blood cells and diabetes mellitus: recent advances.

    Science.gov (United States)

    Reddi, Alluru S; Kothari, Neil; Kuppasani, Kishore; Ende, Norman

    2015-01-01

    Stem cell therapy for patients with diabetes is an area of great interest to both scientists and clinicians. Human umbilical cord blood cells (HUCBCs) are being increasingly used as a source of stem cells for cell-based therapy for diabetes because these cells can differentiate into pancreatic islet β-cells. Administration of HUCBCs has been shown to lower blood glucose levels in diabetic animal models. The use of autologous HUCBC transfusion in type 1 diabetic children has not shown any benefit. However, "Stem Cell Educator" therapy has shown promise in long term lowering of blood glucose levels in both type 1 and type 2 diabetic patients. In this review, we will briefly discuss recent advances in HUCBC therapy in the treatment of diabetes and some of its complications.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. Bacterial glycosidases for the production of universal red blood cells

    DEFF Research Database (Denmark)

    Liu, Qiyong P; Sulzenbacher, Gerlind; Yuan, Huaiping;

    2007-01-01

    Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this techno...

  10. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Atay, Safinur [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Gercel-Taylor, Cicek [Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States); Kesimer, Mehmet [Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC (United States); Taylor, Douglas D., E-mail: ddtaylor@louisville.edu [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States)

    2011-05-01

    Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.

  12. 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.

  13. 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.

  14. 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.

  15. Deep diving in the blood stem cell-ome

    OpenAIRE

    Kalaitzidis, Demetrios; Scadden, David T.

    2014-01-01

    Defining the functional distinctions between cells comprising the bone marrow has yielded fundamental insights into lineage ordering and drivers of blood cell production. A novel, highly granular and multi-dimensional molecular characterization of functional subsets of hematopoietic stem- and progenitor cells recently published in Cell Stem Cell (Cabezas-Wallscheid et al, 2014) will serve as a landmark and treasure trove for unanticipated insights into basic biology and the development of fut...

  16. Extracting Biological Meaning From Global Proteomic Data on Circulating-Blood Platelets: Effects of Diabetes and Storage Time

    Energy Technology Data Exchange (ETDEWEB)

    Miller, John H.; Suleiman, Atef; Daly, Don S.; Springer, David L.; Spinelli, Sherry L.; Blumberg, Neil; Phipps, Richard P.

    2008-11-25

    Transfusion of platelets into patients suffering from trauma and a variety of disease is a common medical practice that involves millions of units per year. Partial activation of platelets can result in the release of bioactive proteins and lipid mediators that increase the risk of adverse post-transfusion effects. Type-2 diabetes and storage are two factors known to cause partial activation of platelets. A global proteomic study was undertaken to investigate these effects. In this paper we discuss the methods used to interpret these data in terms of biological processes affected by diabetes and storage. The main emphasis is on the processing of proteomic data for gene ontology enrichment analysis by techniques originally designed for microarray data.

  17. Mechanisms Linking Red Blood Cell Disorders and Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Ioana Mozos

    2015-01-01

    Full Text Available The present paper aims to review the main pathophysiological links between red blood cell disorders and cardiovascular diseases, provides a brief description of the latest studies in this area, and considers implications for clinical practice and therapy. Anemia is associated with a special risk in proatherosclerotic conditions and heart disease and became a new therapeutic target. Guidelines must be updated for the management of patients with red blood cell disorders and cardiovascular diseases, and targets for hemoglobin level should be established. Risk scores in several cardiovascular diseases should include red blood cell count and RDW. Complete blood count and hemorheological parameters represent useful, inexpensive, widely available tools for the management and prognosis of patients with coronary heart disease, heart failure, hypertension, arrhythmias, and stroke. Hypoxia and iron accumulation cause the most important cardiovascular effects of sickle cell disease and thalassemia. Patients with congenital chronic hemolytic anemia undergoing splenectomy should be monitored, considering thromboembolic and cardiovascular risk.

  18. Safety and radiation risks in the labelling of blood cells

    International Nuclear Information System (INIS)

    Risk in the management of radioactive material and biological exposition to infectious agents. Protocols and normative to observe GOOD RADIOPHARMACY Practices. Main infectious agents that may be transmitted during preparation of a blood cell radiopharmaceutical. Problems of contamination

  19. 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.

  20. 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.

  1. 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

  2. Multifactorial aspects of antibody-mediated blood cell destruction

    OpenAIRE

    Schoot, van der, B.H.; Vidarsson, G.; Kapur, R.

    2014-01-01

    The research described in this thesis focuses on diseases of antibody-mediated blood cell destruction via FcγRs on phagocytes, in particular regarding platelets in fetal or neonatal alloimmune thrombocytopenia (FNAIT) and red blood cells (RBC) in hemolytic disease of the fetus and newborn (HDFN). Diagnostically, for HDFN laboratory tests are in place in order to predict risk for severe fetal RBC destruction and thereby initiate appropriate treatments. This test is sensitive, but has relativel...

  3. Is red blood cell rheology preserved during routine blood bank storage?

    NARCIS (Netherlands)

    Henkelman, Sandra; Dijkstra-Tiekstra, Margriet J.; de Wildt-Eggen, Janny; Graaff, Reindert; Rakhorst, Gerhard; van Oeveren, Willem

    2010-01-01

    BACKGROUND: Red blood cell (RBC) units stored for more than 2 weeks at 4 degrees C are currently considered of impaired quality. This opinion has primarily been based on altered RBC rheologic properties (i.e., enhanced aggregability, reduced deformability, and elevated endothelial cell interaction),

  4. Raman spectroscopy of stored red blood cells: evaluating clinically-relevant biochemical markers in donated blood

    Science.gov (United States)

    Atkins, Chad G.; Buckley, Kevin; Chen, Deborah; Schulze, H. G.; Devine, Dana V.; Blades, Michael W.; Turner, Robin F. B.

    2015-07-01

    Modern transfusion medicine relies on the safe, secure, and cost-effective delivery of donated red blood cells (RBCs). Once isolated, RBCs are suspended in a defined additive solution and stored in plastic blood bags in which, over time, they undergo chemical, physiological, and morphological changes that may have a deleterious impact on some patients. Regulations limit the storage period to 42 days and the cells do not routinely undergo analytical testing before use. In this study, we use Raman spectroscopy to interrogate stored RBCs and we identify metabolic and cell-breakdown products, such as haemoglobin and membrane fragments, that build-up in the blood bags as the cells age. Our work points the way to the development of an instrument which could quickly and easily assess the biochemical nature of stored RBC units before they are transfused.

  5. Supernatant of Bone Marrow Mesenchymal Stromal Cells Induces Peripheral Blood Mononuclear Cells Possessing Mesenchymal Features

    OpenAIRE

    Hu, Gang; Xu, Jun-jun; Deng, Zhi-Hong; Feng, Jie; Jin, Yan

    2011-01-01

    Increasing evidence shows that some cells from peripheral blood fibroblast-like mononuclear cells have the capacity to differentiate into mesenchymal lineages. However, the insufficiency of these cells in the circulation challenges the cell isolation and subsequently limits the clinical application of these cells. In the present study, the peripheral blood mononuclear cells (pbMNCs) were isolated from wound animals and treated with the supernatant of bone marrow mesenchymal stromal cells (bmM...

  6. Erythropoietin reduces storage lesions and decreases apoptosis indices in blood bank red blood cells

    Directory of Open Access Journals (Sweden)

    Oscar Andrés Penuela

    2016-02-01

    Full Text Available ABSTRACT Background: Recent evidence shows a selective destruction of the youngest circulating red blood cells (neocytolysis trigged by a drop in erythropoietin levels. Objective: The aim of this study was to evaluate the effect of recombinant human erythropoietin beta on the red blood cell storage lesion and apoptosis indices under blood bank conditions. Methods: Each one of ten red blood cell units preserved in additive solution 5 was divided in two volumes of 100 mL and assigned to one of two groups: erythropoietin (addition of 665 IU of recombinant human erythropoietin and control (isotonic buffer solution was added. The pharmacokinetic parameters of erythropoietin were estimated and the following parameters were measured weekly, for six weeks: Immunoreactive erythropoietin, hemolysis, percentage of non-discocytes, adenosine triphosphate, glucose, lactate, lactate dehydrogenase, and annexin-V/esterase activity. The t-test or Wilcoxon's test was used for statistical analysis with significance being set for a p-value 6 weeks under blood bank conditions, with persistent supernatant concentrations of erythropoietin during the entire storage period. Adenosine triphosphate was higher in the Erythropoietin Group in Week 6 (4.19 ± 0.05 µmol/L vs. 3.53 ± 0.02 µmol/L; p-value = 0.009. The number of viable cells in the Erythropoietin Group was higher than in the Control Group (77% ± 3.8% vs. 71% ± 2.3%; p-value <0.05, while the number of apoptotic cells was lower (9.4% ± 0.3% vs. 22% ± 0.8%; p-value <0.05. Conclusions: Under standard blood bank conditions, an important proportion of red blood cells satisfy the criteria of apoptosis. Recombinant human erythropoietin beta seems to improve storage lesion parameters and mitigate apoptosis.

  7. 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.

  8. 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.

  9. 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...

  10. Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus.

    Science.gov (United States)

    Soares, Filipa A C; Pedersen, Roger A; Vallier, Ludovic

    2016-01-01

    This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.

  11. A Framework for White Blood Cell Segmentation in Microscopic Blood Images Using Digital Image Processing

    OpenAIRE

    Seman Zainina; Abdul Kahar Badrul; Sadeghian Farnoosh; Ramli Abdul; Saripan M-Iqbal

    2009-01-01

    Abstract Evaluation of blood smear is a commonly clinical test these days. Most of the time, the hematologists are interested on white blood cells (WBCs) only. Digital image processing techniques can help them in their analysis and diagnosis. For example, disease like acute leukemia is detected based on the amount and condition of the WBC. The main objective of this paper is to segment the WBC to its two dominant elements: nucleus and cytoplasm. The segmentation is conducted using a proposed ...

  12. Quantification of depletion-induced adhesion of Red Blood Cells

    CERN Document Server

    Steffen, Patrick; Wagner, Christian

    2012-01-01

    Red blood cells (RBC) are known to form aggregates in the forms of rouleaux due to the presence of plasma proteins under physiological conditions. Rouleaux formation can be also induced in vitro by the addition of macromolecules to the RBC solution. Current data on the adhesion strength between red blood cells in their natural discocyte shapes mostly rely on indirect measurements like flow chamber experiments, but on the single cell level data is lacking. Here we present measurements on the dextran induced aggregation of red blood cells by use of atomic force microscopy based single cell force spectroscopy (SCFS). The effects of dextran concentration and molecular weight on the interaction energy of adhering RBCs was determined. The results are in good agreement with a model based on the depletion effect and former experimental studies.

  13. 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.

  14. Blood cell manufacture: current methods and future challenges.

    Science.gov (United States)

    Timmins, Nicholas E; Nielsen, Lars K

    2009-07-01

    Blood transfusion depends on availability of donor material, and concerns over supply and safety have spurred development of methods to manufacture blood from stem cells. Current methods could theoretically yield therapeutic doses of red blood cells (RBCs) and platelets. However, due to the very large number of cells required to have any impact on supply (currently 10(19) RBC/year in the US), realization of routine manufacture faces significant challenges. Current yields are orders of magnitude too low for production of meaningful quantities, and the physical scale of the problem is a challenge in itself. We discuss these challenges in relation to current methods and how it might be possible to realize limited 'blood pharming' of neutrophils in the near future. PMID:19500866

  15. Blood cell manufacture: current methods and future challenges.

    Science.gov (United States)

    Timmins, Nicholas E; Nielsen, Lars K

    2009-07-01

    Blood transfusion depends on availability of donor material, and concerns over supply and safety have spurred development of methods to manufacture blood from stem cells. Current methods could theoretically yield therapeutic doses of red blood cells (RBCs) and platelets. However, due to the very large number of cells required to have any impact on supply (currently 10(19) RBC/year in the US), realization of routine manufacture faces significant challenges. Current yields are orders of magnitude too low for production of meaningful quantities, and the physical scale of the problem is a challenge in itself. We discuss these challenges in relation to current methods and how it might be possible to realize limited 'blood pharming' of neutrophils in the near future.

  16. Laser-photophoretic migration and fractionation of human blood cells.

    Science.gov (United States)

    Monjushiro, Hideaki; Tanahashi, Yuko; Watarai, Hitoshi

    2013-05-13

    Laser photophoretic migration behavior of human blood cells in saline solution was investigated under the irradiation of Nd:YAG laser beam (532 nm) in the absence and the presence of the flow in a fused silica capillary. Red blood cells (RBC) were migrated faster than white blood cells (WBC) and blood pellets to the direction of propagation of laser light. The observed photophoretic velocity of RBC was about 11 times faster than those of others. This was understood from the larger photophoretic efficiency of RBC than that of WBC, which was simulated based on the Mie scattering theory. Furthermore, it was found that, during the photophoretic migration, RBCs spontaneously orientated parallel to the migration direction so as to reduce the drag force. Finally, it was demonstrated that RBC and WBC were separated in a micro-channel flow system by the laser photophoresis.

  17. Computational modeling of red blood cells: A symplectic integration algorithm

    Science.gov (United States)

    Schiller, Ulf D.; Ladd, Anthony J. C.

    2010-03-01

    Red blood cells can undergo shape transformations that impact the rheological properties of blood. Computational models have to account for the deformability and red blood cells are often modeled as elastically deformable objects. We present a symplectic integration algorithm for deformable objects. The surface is represented by a set of marker points obtained by surface triangulation, along with a set of fiber vectors that describe the orientation of the material plane. The various elastic energies are formulated in terms of these variables and the equations of motion are obtained by exact differentiation of a discretized Hamiltonian. The integration algorithm preserves the Hamiltonian structure and leads to highly accurate energy conservation, hence he method is expected to be more stable than conventional finite element methods. We apply the algorithm to simulate the shape dynamics of red blood cells.

  18. 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.

  19. 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.

  20. Following red blood cells in a pulmonary capillary

    CERN Document Server

    Mauroy, Benjamin

    2007-01-01

    The red blood cells or erythrocytes are biconcave shaped cells and consist mostly in a membrane delimiting a cytosol with a high concentration in hemoglobin. This membrane is highly deformable and allows the cells to go through narrow passages like the capillaries which diameters can be much smaller than red blood cells one. They carry oxygen thanks to hemoglobin, a complex molecule that have very high affinity for oxygen. The capacity of erythrocytes to load and unload oxygen is thus a determinant factor in their efficacy. In this paper, we will focus on the pulmonary capillary where red blood cells capture oxygen. We propose a camera method in order to numerically study the behavior of the red blood cell along a whole capillary. Our goal is to understand how erythrocytes geometrical changes along the capillary can affect its capacity to capture oxygen. The first part of this document presents the model chosen for the red blood cells along with the numerical method used to determine and follow their shapes a...

  1. 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.

  2. Hypoxia/hypercapnia-induced adaptation maintains functional capacity of cord blood stem and progenitor cells at 4°C.

    Science.gov (United States)

    Vlaski, Marija; Negroni, Luc; Kovacevic-Filipovic, Milica; Guibert, Christelle; Brunet de la Grange, Philippe; Rossignol, Rodrigue; Chevaleyre, Jean; Duchez, Pascale; Lafarge, Xavier; Praloran, Vincent; Schmitter, Jean-Marie; Ivanovic, Zoran

    2014-12-01

    We analyzed the effect of exposure to hypoxic/hypercapnic (HH) gas mixture (5% O2 /9% CO2 ) on the maintenance of functional cord blood CD34(+) hematopoietic stem and progenitor cells in severe hypothermia (4°C) employing the physiological and proteomic approaches. Ten-day exposure to HH maintained the Day 0 (D-0) level of hematopoietic stem cells as detected in vivo on the basis of hematopoietic repopulation of immunodeficient mice-short-term scid repopulating cells (SRC). Conversely, in the atmospheric air (20% O2 /0.05% CO2 ), usual condition used for cell storage at 4°C, stem cell activity was significantly decreased. Also, HH doubled the survival of CD34(+) cells and committed progenitors (CFCs) with respect to the atmospheric air (60% vs. 30%, respectively). Improved cell maintenance in HH was associated with higher proportion of aldehyde dehydrogenase (ALDH) positive cells. Cell-protective effects are associated with an improved maintenance of the plasma and mitochondrial membrane potential and with a conversion to the glycolytic energetic state. We also showed that HH decreased apoptosis, despite a sustained ROS production and a drop of ATP amount per viable cell. The proteomic study revealed that the global protein content was better preserved in HH. This analysis identified: (i) proteins sensitive or insensitive to hypothermia irrespective of the gas phase, and (ii) proteins related to the HH cell-protective effect. Among them are some protein families known to be implicated in the prolonged survival of hibernating animals in hypothermia. These findings suggest a way to optimize short-term cell conservation without freezing.

  3. 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.

  4. The homeostasis of Plasmodium falciparum-infected red blood cells.

    Directory of Open Access Journals (Sweden)

    Jakob M A Mauritz

    2009-04-01

    Full Text Available The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15-32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before approximately 48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis. However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.

  5. 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...

  6. Becoming a Blood Stem Cell Donor

    Science.gov (United States)

    ... 3:22. CTV News 322 views 3:22 Stem Cell Therapy Injections - Duration: 6:18. Caring Medical Regenerative Medicine Clinics 254,233 views 6:18 Bone Marrow/Stem Cell Transplant - Duration: 7:24. tannermom80 106,911 views ...

  7. 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...

  8. 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...

  9. 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...

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. A smart core-sheath nanofiber that captures and releases red blood cells from the blood

    Science.gov (United States)

    Shi, Q.; Hou, J.; Zhao, C.; Xin, Z.; Jin, J.; Li, C.; Wong, S.-C.; Yin, J.

    2016-01-01

    A smart core-sheath nanofiber for non-adherent cell capture and release is demonstrated. The nanofibers are fabricated by single-spinneret electrospinning of poly(N-isopropylacrylamide) (PNIPAAm), polycaprolactone (PCL) and nattokinase (NK) solution blends. The self-assembly of PNIPAAm and PCL blends during the electrospinning generates the core-sheath PCL/PNIPAAm nanofibers with PNIPAAm as the sheath. The PNIPAAm-based core-sheath nanofibers are switchable between hydrophobicity and hydrophilicity with temperature change and enhance stability in the blood. When the nanofibers come in contact with blood, the NK is released from the nanofibers to resist platelet adhesion on the nanofiber surface, facilitating the direct capture and isolation of red blood cells (RBCs) from the blood above phase-transition temperature of PNIPAAm. Meanwhile, the captured RBCs are readily released from the nanofibers with temperature stimuli in an undamaged manner. The release efficiency of up to 100% is obtained while maintaining cellular integrity and function. This work presents promising nanofibers to effectively capture non-adherent cells and release for subsequent molecular analysis and diagnosis of single cells.A smart core-sheath nanofiber for non-adherent cell capture and release is demonstrated. The nanofibers are fabricated by single-spinneret electrospinning of poly(N-isopropylacrylamide) (PNIPAAm), polycaprolactone (PCL) and nattokinase (NK) solution blends. The self-assembly of PNIPAAm and PCL blends during the electrospinning generates the core-sheath PCL/PNIPAAm nanofibers with PNIPAAm as the sheath. The PNIPAAm-based core-sheath nanofibers are switchable between hydrophobicity and hydrophilicity with temperature change and enhance stability in the blood. When the nanofibers come in contact with blood, the NK is released from the nanofibers to resist platelet adhesion on the nanofiber surface, facilitating the direct capture and isolation of red blood cells (RBCs) from

  16. Rapid white blood cell detection for peritonitis diagnosis

    Science.gov (United States)

    Wu, Tsung-Feng; Mei, Zhe; Chiu, Yu-Jui; Cho, Sung Hwan; Lo, Yu-Hwa

    2013-03-01

    A point-of-care and home-care lab-on-a-chip (LoC) system that integrates a microfluidic spiral device as a concentrator with an optical-coding device as a cell enumerator is demonstrated. The LoC system enumerates white blood cells from dialysis effluent of patients receiving peritoneal dialysis. The preliminary results show that the white blood cell counts from our system agree well with the results from commercial flow cytometers. The LoC system can potentially bring significant benefits to end stage renal disease (ESRD) patients that are on peritoneal dialysis (PD).

  17. Effect of Induced Pluripotent Stem Cell Technology in Blood Banking.

    Science.gov (United States)

    Focosi, Daniele; Pistello, Mauro

    2016-03-01

    Population aging has imposed cost-effective alternatives to blood donations. Artificial blood is still at the preliminary stages of development, and the need for viable cells seems unsurmountable. Because large numbers of viable cells must be promptly available for clinical use, stem cell technologies, expansion, and banking represent ideal tools to ensure a regular supply. Provided key donors can be identified, induced pluripotent stem cell (iPSC) technology could pave the way to a new era in transfusion medicine, just as it is already doing in many other fields of medicine. The present review summarizes the current state of research on iPSC technology in the field of blood banking, highlighting hurdles, and promises.

  18. 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]).

  19. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... are most commonly used in the treatment of cancers like leukemia and lymphoma to restore stem cells ... use of BMT and PBSCT, see http://www.cancer.gov/cancertopics/fa... If you are interested in ...

  20. 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....

  1. Filtration parameters influencing circulating tumor cell enrichment from whole blood.

    Directory of Open Access Journals (Sweden)

    Frank A W Coumans

    Full Text Available Filtration can achieve circulating tumor cell (CTC enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·10(4∶10(2∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm(2 track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.

  2. Transfusion management of patients with red blood cell antibodies

    Directory of Open Access Journals (Sweden)

    Bujandrić Nevenka B.

    2013-01-01

    Full Text Available Introduction. Red blood cell antibodies may cause a positive result of pre-transfusion blood compatibility testing (crossmatch test. It can be a problem to provide suitable blood units for patients with clinically significant antibodies to high-frequency antigens as well as for those with multiple alloantibody specificities. This study was aimed at identifying transfused patients in the population of South-Backa who had developed clinically significant red blood cell alloantibodies. Material and methods. We analyzed the records of crossmatch results and antibody screening performed at the Blood Transfusion Institute of Vojvodina during 2012. Results. Antibodies were found in 103 patients: A 63 patients with single antibodies: 1 16 with antibodies of unknown specificity (3 autoantibodies, 13 alloantibodies; 2 39 with clinically significant antibodies (23 from Rh system (2 anti-C, 2 anti-D, 12 anti-E, 7 anti-c, 4 anti-K, 3 anti-Fya, 7 anti-Jka, 2 anti-S; 3 8 with usually not significant antibodies (6 anti-M, 1 anti-A1, 1 anti- Cw; B 40 patients developed multiple antibodies: 1 all patients had at least one clinically significant antibody from various blood group system (44 Rh, 13 Kell, 7 Kidd, 7 MNSs (S, s; 2 3 patients had usually not significant antibodies (1 Lewis, 2 Lutheran; 3 3 patients occasionally had clinically significant antibody (3 anti- Yta; 4 3 patients had antibodies of unknown specificity (2 autoantibodies, 1alloantibody. Antibodies detected in the majority of patients (65-63.1% had a specificity of Rh and/or the Kell system. Conclusions. The main goal of pre-transfusion blood compatibility testing is to detect clinically significant antibodies. The provision of antigen negative blood units for those patients is a special challenge for blood establishments. Database with a sufficient number of typed blood donors can help to resolve this problem.

  3. 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.

  4. In-vitro red blood cell partitioning of doxycycline

    OpenAIRE

    Deshmukh, P.V.; Badgujar, P.C.; Gatne, M. M.

    2009-01-01

    Objective: In-vitro red blood cell (RBC) partitioning of doxycycline was studied to determine whether doxycycline penetrates RBC and its concentration was assayed keeping in view its high lipophilicity. Materials and Methods: Standardization of doxycycline was performed in whole blood and plasma of cattle by microbiological assay using Bacillus subtillis ATCC 6633 as indicator organizm. Actual concentration of the drug was obtained by comparing zone inhibition with standard graph and the exte...

  5. Proteomic analysis of prolactinoma cells by immuno-laser capture microdissection combined with online two-dimensional nano-scale liquid chromatography/mass spectrometry

    Directory of Open Access Journals (Sweden)

    Chen Luping

    2010-01-01

    Full Text Available Abstract Background Pituitary adenomas, the third most common intracranial tumor, comprise nearly 16.7% of intracranial neoplasm and 25%-44% of pituitary adenomas are prolactinomas. Prolactinoma represents a complex heterogeneous mixture of cells including prolactin (PRL, endothelial cells, fibroblasts, and other stromal cells, making it difficult to dissect the molecular and cellular mechanisms of prolactin cells in pituitary tumorigenesis through high-throughout-omics analysis. Our newly developed immuno-laser capture microdissection (LCM method would permit rapid and reliable procurement of prolactin cells from this heterogeneous tissue. Thus, prolactin cell specific molecular events involved in pituitary tumorigenesis and cell signaling can be approached by proteomic analysis. Results Proteins from immuno-LCM captured prolactin cells were digested; resulting peptides were separated by two dimensional-nanoscale liquid chromatography (2D-nanoLC/MS and characterized by tandem mass spectrometry. All MS/MS spectrums were analyzed by SEQUEST against the human International Protein Index database and a specific prolactinoma proteome consisting of 2243 proteins was identified. This collection of identified proteins by far represents the largest and the most comprehensive database of proteome for prolactinoma. Category analysis of the proteome revealed a widely unbiased access to various proteins with diverse functional characteristics. Conclusions This manuscript described a more comprehensive proteomic profile of prolactinomas compared to other previous published reports. Thanks to the application of immuno-LCM combined with online two-dimensional nano-scale liquid chromatography here permitted identification of more proteins and, to our best knowledge, generated the largest prolactinoma proteome. This enlarged proteome would contribute significantly to further understanding of prolactinoma tumorigenesis which is crucial to the management of

  6. Identification of malaria parasite-infected red blood cell surface aptamers by inertial microfluidic SELEX (I-SELEX)

    Science.gov (United States)

    Birch, Christina M.; Hou, Han Wei; Han, Jongyoon; Niles, Jacquin C.

    2015-07-01

    Plasmodium falciparum malaria parasites invade and remodel human red blood cells (RBCs) by trafficking parasite-synthesized proteins to the RBC surface. While these proteins mediate interactions with host cells that contribute to disease pathogenesis, the infected RBC surface proteome remains poorly characterized. Here we use a novel strategy (I-SELEX) to discover high affinity aptamers that selectively recognize distinct epitopes uniquely present on parasite-infected RBCs. Based on inertial focusing in spiral microfluidic channels, I-SELEX enables stringent partitioning of cells (efficiency ≥ 106) from unbound oligonucleotides at high volume throughput (~2 × 106 cells min-1). Using an RBC model displaying a single, non-native antigen and live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct, surface-displayed epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, var2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts.

  7. Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.

    Science.gov (United States)

    Su, Ruijun Jeanna; Neises, Amanda; Zhang, Xiao-Bing

    2016-01-01

    Peripheral blood is the easy-to-access, minimally invasive, and the most abundant cell source to use for cell reprogramming. The episomal vector is among the best approaches for generating integration-free induced pluripotent stem (iPS) cells due to its simplicity and affordability. Here we describe the detailed protocol for the efficient generation of integration-free iPS cells from peripheral blood mononuclear cells. With this optimized protocol, one can readily generate hundreds of iPS cell colonies from 1 ml of peripheral blood.

  8. Counting White Blood Cells from a Blood Smear Using Fourier Ptychographic Microscopy

    OpenAIRE

    Chung, Jaebum; Ou, Xiaoze; Kulkarni, Rajan P.; Yang, Changhuei

    2015-01-01

    White blood cell (WBC) count is a valuable metric for assisting with diagnosis or prognosis of various diseases such as coronary heart disease, type 2 diabetes, or infection. Counting WBCs can be done either manually or automatically. Automatic methods are capable of counting a large number of cells to give a statistically more accurate reading of the WBC count of a sample, but the specialized equipment tends to be expensive. Manual methods are inexpensive since they only involve a convention...

  9. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... a Patient-Centered Approach - Duration: 4:12. NCIcancertopics 3,087 views 4:12 The Truth About Cord ... 19 Stem cell donation: Step by step - Duration: 3:35. hemaquebec1998 1,127 views 3:35 Two ...

  10. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... be donors at http://www.marrow.org . Category Science & Technology License Standard YouTube License Show more Show ... views 4:25 Susan Solomon: The promise of research with stem cells - Duration: 14:59. TED 55, ...

  11. Becoming a Blood Stem Cell Donor

    Medline Plus

    Full Text Available ... playlist. Sign in Share More Report Need to report the video? Sign in to report inappropriate content. Sign in Transcript 6,983 views ... Stem Cell Therapy Injections - Duration: 6:18. Caring Medical Regenerative Medicine Clinics 234,106 views 6:18 ...

  12. Deep Proteomics of Breast Cancer Cells Reveals that Metformin Rewires Signaling Networks Away from a Pro-growth State.

    Science.gov (United States)

    Sacco, Francesca; Silvestri, Alessandra; Posca, Daniela; Pirrò, Stefano; Gherardini, Pier Federico; Castagnoli, Luisa; Mann, Matthias; Cesareni, Gianni

    2016-03-23

    Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin. PMID:27135362

  13. Time-course proteomics dataset monitoring HeLa cells subjected to DTT induced endoplasmic reticulum stress.

    Science.gov (United States)

    Cheng, Zhe; Rendleman, Justin; Vogel, Christine

    2016-09-01

    The data described here provide an analysis of the dynamic response of HeLa cell proteome to dithiothreitol (DTT) inducing stress of the endoplasmic reticulum (ER). During ER stress, accumulation of misfolded and unfolded proteins in the lumen of the ER initiates the Unfolded Protein Response (UPR), resulting in a large-scale redistribution of proteins. We used label-free mass spectrometry to monitor the proteomic changes of HeLa cells during a 30-h time course, monitoring eight time points (0, 0.5, 1, 2, 8, 16, 24, and 30 h). The data are associated with the research article "Differential dynamics of the mammalian mRNA and protein expression response to misfolding stress" [1], which discusses a core dataset of 1237 proteins. Here, we present the extended dataset of 2131 proteins. The raw mass spectrometry data and the analysis results have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PRIDE: PXD002039. PMID:27547793

  14. Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins.

    Science.gov (United States)

    Chan, Yuk-Kit; Zhang, Huoming; Liu, Pei; Tsao, Sai-Wah; Lung, Maria Li; Mak, Nai-Ki; Ngok-Shun Wong, Ricky; Ying-Kit Yue, Patrick

    2015-10-15

    Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1 and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future.

  15. Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

    KAUST Repository

    Chan, Yuk-kit

    2015-04-01

    Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient, and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1, and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. This article is protected by copyright. All rights reserved.

  16. Plasma proteome analysis of cervical intraepithelial neoplasia and cervical squamous cell carcinoma

    Indian Academy of Sciences (India)

    Mee Lee Looi; Saiful Anuar Karsani; Mariati Abdul Rahman; Ahmad Zailani Hatta Mohd Dali; Siti Aishah Md Ali; Wan Zurinah Wan Ngah; Yasmin Anum Mohd Yusof

    2009-12-01

    Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.

  17. Data from a comparative proteomic analysis of tumor-derived lung-cancer CD105(+) endothelial cells.

    Science.gov (United States)

    Jin, Hongwei; Cheng, Xiao; Pei, Yihua; Fu, Jianguo; Lyu, Zhi; Peng, Huifang; Yao, Qin; Jiang, Yu; Luo, Lianzhong; Zhuo, Huiqin

    2016-06-01

    Increasing evidence indicates that tumor-derived endothelial cells (TECs) are more relevant for the study of tumor angiogenesis and for screening antiangiogenic drugs than normal ECs (NECs). In this data article, high-purity (>98%) primary CD105(+) NECs and TECs purified from a mouse Lewis lung carcinoma model bearing 0.5 cm tumors were identified using 2D-PAGE and Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). All the identified proteins were categorized functionally by Gene Ontology (GO) analysis, and gene-pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, protein-protein interaction networks were also built. The proteomics and bioinformatics data presented here provide novel insights into the molecular characteristics and the early modulation of the TEC proteome in the tumor microenvironment. PMID:27081670

  18. Charting the interactome of PDE3A in human cells using an IBMX based chemical proteomics approach

    DEFF Research Database (Denmark)

    Corradini, Eleonora; Klaasse, Gruson; Leurs, Ulrike;

    2015-01-01

    proteomics characterization of this resin in HeLa cell lysates led to the capture of several different PDEs. Combining the IBMX-resin with in-solution competition with the available more selective PDE inhibitors, cilostamide and papaverine, allowed us to selectively probe the interactome of PDE3A in He...... processes inspiring the quest for and synthesis of selective PDE inhibitors, that unfortunately have led to very mixed successes in clinical trials. This may be partially caused by their pharmacological action. Accumulating data suggests that small differences between different PDE isoforms may already...... comprehensive way. Affinity based chemical proteomics is a relatively new tool to identify specific protein-protein interactions. Here, to study the interactome of PDEs, we synthesized a broad spectrum PDE-capturing resin based on the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Chemical...

  19. Proteomic response to 5,6-dimethylxanthenone 4-acetic acid (DMXAA, vadimezan in human non-small cell lung cancer A549 cells determined by the stable-isotope labeling by amino acids in cell culture (SILAC approach

    Directory of Open Access Journals (Sweden)

    Pan ST

    2015-02-01

    Full Text Available Shu-Ting Pan,1,* Zhi-Wei Zhou,2,3,* Zhi-Xu He,3 Xueji Zhang,4 Tianxin Yang,5 Yin-Xue Yang,6 Dong Wang,7 Jia-Xuan Qiu,1 Shu-Feng Zhou2 1Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou 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, 4Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 5Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 6Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 7Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China *These two authors contributed equally to this work Abstract: 5,6-Dimethylxanthenone 4-acetic acid (DMXAA, also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC approach. The proteomic data showed that treatment with DMXAA

  20. Proteomic Analyses of the Effects of Drugs of Abuse on Monocyte-Derived Mature Dendritic Cells

    OpenAIRE

    Jessica L. Reynolds; Supriya D Mahajan; Aalinkeel, Ravikunar; Nair, B.; Sykes, Donald E; Schwartz, Stanley A.

    2009-01-01

    Drug abuse has become a global health concern. Understanding how drug abuse modulates the immune system and how the immune system responds to pathogens associated with drug abuse, such hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1), can be assessed by an integrated approach comparing proteomic analyses and quantitation of gene expression. Two-dimensional (2D) difference gel electrophoresis was used to determine the molecular mechanisms underlying the proteomic changes that a...

  1. 2 D gel based analysis of biological variability of the human plasma proteome

    DEFF Research Database (Denmark)

    Rentsch, Maria Louise; Jessen, Flemming

    Human blood plasma is a valuable specimen for the biomarker discovery process, since it is easily accessible and contains proteins that are synthesised, secreted or lost from cells and tissue. In this way, changes in plasma proteome reflect the current state of the organism. The analysis of plasma...... proteome is yet challenging due to the huge dynamic range of protein abundance. When evaluating a potential biomarker, stable basal level of the protein is needed before it can be considered a functional biomarker. However, basal level differences of plasma proteins are naturally occurring between...... by one-week interval. Blood samples were drawn before the meal intake and five times during 24 hours for proteome analysis. Plasma was fractionated by use of IgY-12 spin column depleting the 12 highly abundant proteins and further processed for two-dimensional gel electrophoresis. The plasma proteome...

  2. Blood cells and endothelial barrier function

    OpenAIRE

    Rodrigues, Stephen F.; Granger, D Neil

    2015-01-01

    The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of solub...

  3. Detection of hepatitis B virus DNA in mononuclear blood cells.

    OpenAIRE

    Pontisso, P; Poon, M C; Tiollais, P.; Brechot, C

    1984-01-01

    The Southern transfer hybridisation technique was used to test mononuclear blood cells for hepatitis B virus DNA. Viral DNA sequences were detected in mononuclear cells of 10 out of 16 patients with hepatitis B virus infection and in none of 21 normal controls. Blood contamination was excluded by the absence of hepatitis B virus DNA in the corresponding serum samples in all cases. Free monomeric hepatitis B virus DNA was found in three patients positive for hepatitis Be antigen (HBeAg) and on...

  4. Histomorphometric study on blood cells in male adult ostrich

    Directory of Open Access Journals (Sweden)

    Mina Tadjalli

    2013-09-01

    Full Text Available In order to perform a histomorphometric study of blood cells in male adult ostrich, blood samples were obtained from jugular vein of 10 clinically healthy male adult ostriches (2 - 3 years old. The slides were stained with the Giemsa methods and the smears were evaluated for cellular morphology, with cellular size being determined by micrometry. The findings of this study revealed that the shape of the cell, cytoplasm and nucleus of erythrocytes in male adult ostriches were similar to those in other birds such as quails, chickens, Iranian green-head ducks.

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

    Directory of Open Access Journals (Sweden)

    Kirsch Roy

    2012-11-01

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

  6. Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation.

    Science.gov (United States)

    Gerashchenko, Bogdan I; Yamagata, Akira; Oofusa, Ken; Yoshizato, Katsutoshi; de Toledo, Sonia M; Howell, Roger W

    2007-06-01

    Recently (Cytometry 2003, 56A, 71-80), we reported that direct cell-to-cell contact is required for stimulating proliferation of bystander rat liver cells (WB-F344) cocultured with irradiated cells, and neither functional gap junction intercellular communication nor long-range extracellular factors appear to be involved in this proliferative bystander response (PBR). The molecular basis for this response is unknown. Confluent monolayers of WB-F344 cells were exposed to 5-Gray (Gy) of gamma-rays. Irradiated cells were mixed with unirradiated cells and co-cultured for 24 h. Cells were harvested and protein expression was examined using 2-DE. Protein expression was also determined in cultures of unirradiated and 5-Gy irradiated cells. Proteins were identified by MS. Nucleophosmin (NPM)-1, a multifunctional nucleolar protein, was more highly expressed in bystander cells than in either unirradiated or 5-Gy irradiated cells. Enolase-alpha, a glycolytic enzyme, was present in acidic and basic variants in unirradiated cells. In bystander and 5-Gy irradiated cells, the basic variant was weakly expressed, whereas the acidic variant was overwhelmingly present. These data indicate that the presence of irradiated cells can affect NPM-1 and enolase-alpha in adjacent bystander cells. These proteins appear to participate in molecular events related to the PBR and suggest that this response may involve cellular defense, proliferation, and metabolism.

  7. Comparative proteomic and phosphoproteomic profiling of pancreatic adenocarcinoma cells treated with CB1 or CB2 agonists.

    Science.gov (United States)

    Brandi, Jessica; Dando, Ilaria; Palmieri, Marta; Donadelli, Massimo; Cecconi, Daniela

    2013-05-01

    The pancreatic adenocarcinoma cell line Panc1 was treated with cannabinoid receptor ligands (arachidonylcyclopropylamide or GW405833) in order to elucidate the molecular mechanism of their anticancer effect. A proteomic approach was used to analyze the protein and phosphoprotein profiles. Western blot and functional data mining were also employed in order to validate results, classify proteins, and explore their potential relationships. We demonstrated that the two cannabinoids act through a widely common mechanism involving up- and down-regulation of proteins related to energetic metabolism and cell growth regulation. Overall, the results reported might contribute to the development of a therapy based on cannabinoids for pancreatic adenocarcinoma.

  8. Designer blood: creating hematopoietic lineages from embryonic stem cells

    Science.gov (United States)

    Olsen, Abby L.; Stachura, David L.; Weiss, Mitchell J.

    2006-01-01

    Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases. PMID:16254136

  9. Membranotropic photobiomodulation on red blood cell deformability

    Science.gov (United States)

    Luo, Gang-Yue; Zhao, Yan-Ping; Liu, Timon C.; Liu, Song-Hao

    2007-05-01

    To assess modulation of laser on erythrocyte permeability and deformability via cell morphology changes, healthy human echinocytes with shrinking size and high plasmic viscosity due to cellular dehydration were treated with 1 mW, 2 mW, 3 mW, and 5 mW laser power exposure respectively. Image analyzing system on single intact erythrocyte was applied for measuring comprehensive cell morphological parameters (surface area, external membrane perimeter, circle index and elongation index) that were determined by the modulation of erythrocyte water permeability and deformability to detect relationship between erythrocyte water permeability alteration and deformability. Our preliminary experiment showed that exposure under light dose of 5 mW for 5 min could induce more active erythrocyte swelling and deformation. water channel aquaporin-1(AQP-1) was inhibited by the incubation of HgCl II in the presence and absence of 5 mW laser irradiation. The result suggested that osmotic water permeability is a primary factor in the procedure of erythrocyte deformability. In addition, no modulation of laser(5mW) on erythrocyte deformability had been found when the echinocytes were cultured with GDP-β-S (G protein inhibitor).

  10. Separation of cancer cells from white blood cells by pinched flow fractionation

    DEFF Research Database (Denmark)

    Jensen, Marie Pødenphant; Ashley, Neil; Koprowska, Kamila;

    2015-01-01

    In this paper, the microfluidic size-separation technique pinched flow fractionation (PFF) is used to separate cancer cells from white blood cells (WBCs). The cells are separated at efficiencies above 90% for both cell types. Circulating tumor cells (CTCs) are found in the blood of cancer patients...... is challenged by the size overlap between cancer cells and the 106 times more abundant WBCs. The size overlap prevents high efficiency separation, however we demonstrate that cell deformability can be exploited in PFF devices to gain higher efficiencies than expected from the size distribution of the cells....

  11. Related Hematopoietic Stem Cell Transplantation (HSCT) for Genetic Diseases of Blood Cells

    Science.gov (United States)

    2016-05-11

    Stem Cell Transplantation; Bone Marrow Transplantation; Peripheral Blood Stem Cell Transplantation; Allogeneic Transplantation,; Genetic Diseases; Thalassemia; Pediatrics; Diamond-Blackfan Anemia; Combined Immune Deficiency; Wiskott-Aldrich Syndrome; Chronic Granulomatous Disease; X-linked Lymphoproliferative Disease; Metabolic Diseases

  12. Proteomic Profiling of Mesenchymal Stem Cell Responses to Mechanical Strain and TGF-B1

    Energy Technology Data Exchange (ETDEWEB)

    Kurpinski, Kyle; Chu, Julia; Wang, Daojing; Li, Song

    2009-10-12

    Mesenchymal stem cells (MSCs) are a potential source of smooth muscle cells (SMCs) for constructing tissue-engineered vascular grafts. However, the details of how specific combinations of vascular microenvironmental factors regulate MSCs are not well understood. Previous studies have suggested that both mechanical stimulation with uniaxial cyclic strain and chemical stimulation with transforming growth factor {beta}1 (TGF-{beta}1) can induce smooth muscle markers in MSCs. In this study, we investigated the combined effects of uniaxial cyclic strain and TGF-{beta}1 stimulation on MSCs. By using a proteomic analysis, we found differential regulation of several proteins and genes, such as the up-regulation of TGF-{beta}1-induced protein ig-h3 (BGH3) protein levels by TGF-{beta}1 and up-regulation of calponin 3 protein level by cyclic strain. At the gene expression level, BGH3 was induced by TGF-{beta}1, but calponin 3 was not significantly regulated by mechanical strain or TGF-{beta}1, which was in contrast to the synergistic up-regulation of calponin 1 gene expression by cyclic strain and TGF-{beta}1. Further experiments with cycloheximide treatment suggested that the up-regulation of calponin 3 by cyclic strain was at post-transcriptional level. The results in this study suggest that both mechanical stimulation and TGF-{beta}1 signaling play unique and important roles in the regulation of MSCs at both transcriptional and post-transcriptional levels, and that a precise combination of microenvironmental cues may promote MSC differentiation.

  13. Are clear cell carcinomas of the ovary and endometrium phenotypically identical? A proteomic analysis.

    Science.gov (United States)

    Fata, Cynthia R; Seeley, Erin H; Desouki, Mohamed M; Du, Liping; Gwin, Katja; Hanley, Krisztina Z; Hecht, Jonathan L; Jarboe, Elke A; Liang, Sharon X; Parkash, Vinita; Quick, Charles M; Zheng, Wenxin; Shyr, Yu; Caprioli, Richard M; Fadare, Oluwole

    2015-10-01

    Phenotypic differences between otherwise similar tumors arising from different gynecologic locations may be highly significant in understanding the underlying driver molecular events at each site and may potentially offer insights into differential responses to treatment. In this study, the authors sought to identify and quantify phenotypic differences between ovarian clear cell carcinoma (OCCC) and endometrial clear cell carcinoma (ECCC) using a proteomic approach. Tissue microarrays were constructed from tumor samples of 108 patients (54 ECCCs and 54 OCCCs). Formalin-fixed samples on microarray slides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry, and 730 spectral peaks were generated from the combined data set. A linear mixed-effect model with random intercept was used to generate 93 (12.7%) peaks that were significantly different between OCCCs and ECCCs at the fold cutoffs of 1.5 and 0.667 and an adjusted P value cutoff of 1.0 × 10(-10). Liquid chromatography-tandem mass spectrometry was performed on selected cores from each group, and peptides identified therefrom were compared with lists of statistically significant peaks from the aforementioned linear mixed-effects model to find matches within 0.2 Da. A total of 53 candidate proteins were thus identified as being differentially expressed in OCCCs and ECCCs, 45 (85%) of which were expressed at higher levels in ECCCs than OCCCs. These proteins were functionally diverse and did not highlight a clearly dominant cellular theme or molecular pathway. Although ECCCs and OCCCs are very similar, some phenotypic differences are demonstrable. Additional studies of these differentially expressed proteins may ultimately clarify the significance of these differences. PMID:26243671

  14. Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Anis Rageh Al-Maleki

    Full Text Available Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV] to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk, ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis.

  15. A proteomic perspective on the changes in milk proteins due to high somatic cell count.

    Science.gov (United States)

    Zhang, L; Boeren, S; van Hooijdonk, A C M; Vervoort, J M; Hettinga, K A

    2015-08-01

    Although cows with subclinical mastitis have no difference in the appearance of their milk, milk composition and milk quality are altered because of the inflammation. To know the changes in milk quality with different somatic cell count (SCC) levels, 5 pooled bovine milk samples with SCC from 10(5) to 10(6) cells/mL were analyzed qualitatively and quantitatively using both one-dimension sodium dodecyl sulfate PAGE and filter-aided sample preparation coupled with dimethyl labeling, both followed by liquid chromatography tandem mass spectrometry. Minor differences were found on the qualitative level in the proteome from milk with different SCC levels, whereas the concentration of milk proteins showed remarkable changes. Not only immune-related proteins (cathelicidins, IGK protein, CD59 molecule, complement regulatory protein, lactadherin), but also proteins with other biological functions (e.g., lipid metabolism: platelet glycoprotein 4, butyrophilin subfamily 1 member A1, perilipin-2) were significantly different in milk from cows with high SCC level compared with low SCC level. The increased concentration of protease inhibitors in the milk with higher SCC levels may suggest a protective role in the mammary gland against protease activity. Prostaglandin-H2 D-isomerase showed a linear relation with SCC, which was confirmed with an ELISA. However, the correlation coefficient was lower in individual cows compared with bulk milk. These results indicate that prostaglandin-H2 D-isomerase may be used as an indicator to evaluate bulk milk quality and thereby reduce the economic loss in the dairy industry. The results from this study reflect the biological phenomena occurring during subclinical mastitis and in addition provide a potential indicator for the detection of bulk milk with high SCC. PMID:26094216

  16. Magnetic nanoparticle effects on the red blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Creanga, D E; Nadejde, C; Curecheriu, L [' Al. I. Cuza' University, Faculty of Physics, 11A Blvd. Carol I, Iasi (Romania)], E-mail: dorinacreanga@yahoo.com; Culea, M [' Babes Bolyai' University, Cluj-Napoca (Romania); Oancea, S [University of Veterinary Medicine ' I. Ionescu de la Brad' , Iasi (Romania); Racuciu, M [' Lucian Blaga' University, Sibiu (Romania)

    2009-05-01

    In vitro tests on magnetite colloidal nanoparticles effects upon animal red blood cells were carried out. Magnetite cores were stabilized with citric acid in the form of biocompatible magnetic fluid administrated in different dilutions in the whole blood samples. The hemolysis extent was found increased up to 2.75 in horse blood and respectively up to 2.81 in the dog blood. The electronic transitions assigned to the heme group were found shifted with about 500 cm{sup -1} or, respectively, affected by supplementary vibronic structures. The Raman vibrations assigned to oxyhemoglobin were much diminished in intensity probably due to the bonding of OH group from citrate shell to the heme iron ion.

  17. The Redox Proteome*

    OpenAIRE

    Go, Young-Mi; Jones, Dean P.

    2013-01-01

    The redox proteome consists of reversible and irreversible covalent modifications that link redox metabolism to biologic structure and function. These modifications, especially of Cys, function at the molecular level in protein folding and maturation, catalytic activity, signaling, and macromolecular interactions and at the macroscopic level in control of secretion and cell shape. Interaction of the redox proteome with redox-active chemicals is central to macromolecular structure, regulation,...

  18. Concise review: programming human pluripotent stem cells into blood.

    Science.gov (United States)

    Easterbrook, Jennifer; Fidanza, Antonella; Forrester, Lesley M

    2016-06-01

    Blood disorders are treated with cell therapies including haematopoietic stem cell (HSC) transplantation as well as platelet and red blood cell transfusions. However the source of cells is entirely dependent on donors, procedures are susceptible to transfusion-transmitted infections and serious complications can arise in recipients due to immunological incompatibility. These problems could be alleviated if it was possible to produce haematopoietic cells in vitro from an autologous and renewable cell source. The production of haematopoietic cells in the laboratory from human induced pluripotent stem cells (iPSCs) may provide a route to realize this goal but it has proven challenging to generate long-term reconstituting HSCs. To date, the optimization of differentiation protocols has mostly relied on the manipulation of extrinsic signals to mimic the in vivo environment. We review studies that have taken an alternative approach to modulate intrinsic signals by enforced expression of transcription factors. Single and combinations of multiple transcription factors have been used in a variety of contexts to enhance the production of haematopoietic cells from human pluripotent stem cells. This programming approach, together with the recent advances in the production and use of synthetic transcription factors, holds great promise for the production of fully functional HSCs in the future. PMID:26996518

  19. Concise review: programming human pluripotent stem cells into blood.

    Science.gov (United States)

    Easterbrook, Jennifer; Fidanza, Antonella; Forrester, Lesley M

    2016-06-01

    Blood disorders are treated with cell therapies including haematopoietic stem cell (HSC) transplantation as well as platelet and red blood cell transfusions. However the source of cells is entirely dependent on donors, procedures are susceptible to transfusion-transmitted infections and serious complications can arise in recipients due to immunological incompatibility. These problems could be alleviated if it was possible to produce haematopoietic cells in vitro from an autologous and renewable cell source. The production of haematopoietic cells in the laboratory from human induced pluripotent stem cells (iPSCs) may provide a route to realize this goal but it has proven challenging to generate long-term reconstituting HSCs. To date, the optimization of differentiation protocols has mostly relied on the manipulation of extrinsic signals to mimic the in vivo environment. We review studies that have taken an alternative approach to modulate intrinsic signals by enforced expression of transcription factors. Single and combinations of multiple transcription factors have been used in a variety of contexts to enhance the production of haematopoietic cells from human pluripotent stem cells. This programming approach, together with the recent advances in the production and use of synthetic transcription factors, holds great promise for the production of fully functional HSCs in the future.

  20. A spectral and morphologic method for white blood cell classification

    Science.gov (United States)

    Wang, Qian; Chang, Li; Zhou, Mei; Li, Qingli; Liu, Hongying; Guo, Fangmin

    2016-10-01

    The identification of white blood cells is important as it provides an assay for diagnosis of various diseases. To overcome the complexity and inaccuracy of traditional methods based on light microscopy, we proposed a spectral and morphologic method based on hyperspectral blood images. We applied mathematical morphology-based methods to extract spatial information and supervised method is employed for spectral analysis. Experimental results show that white blood cells could be segmented and classified into five types with an overall accuracy of more than 90%. Moreover, the experiments including spectral features reached higher accuracy than the spatial-only cases, with a maximum improvement of nearly 20%. By combing both spatial and spectral features, the proposed method provides higher classification accuracy than traditional methods.

  1. Mechanopathology of red blood cell diseases—Why mechanics matters

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    During the onset of a disease a cell may experience alterations in both the composition and organization of its cellular and molecular structures.These alterations may eventually lead to changes in its geometrical and mechanical properties such as cell size and shape,deformability and adhesion.As such,knowing how diseased cells respond to mechanical forces can reveal ways by which they differ from healthy ones.Here,we will present biomechanistic insights into red blood cell related diseases that manifest...

  2. RBCs and Parasites Segmentation from Thin Smear Blood Cell Images

    Directory of Open Access Journals (Sweden)

    Vishal V. Panchbhai

    2012-09-01

    Full Text Available Manually examine the blood smear for the detection of malaria parasite consumes lot of time for trend pathologists. As the computational power increases, the role of automatic visual inspection becomes more important. An automated system is therefore needed to complete as much work as possible for the identification of malaria parasites. The given scheme based on used of RGB color space, G layer processing, and segmentation of Red Blood Cells (RBC as well as cell parasites by auto-thresholding with offset value and use of morphological processing. The work compare with the manual results obtained from the pathology lab, based on total RBC count and cells parasite count. The designed system successfully detects malaria parasites and RBC cells in thin smear image.

  3. Aggregation of Red Blood Cells: From Rouleaux to Clot Formation

    CERN Document Server

    Wagner, C; Svetina, S

    2013-01-01

    Red blood cells are known to form aggregates in the form of rouleaux. This aggregation process is believed to be reversible, but there is still no full understanding on the binding mechanism. There are at least two competing models, based either on bridging or on depletion. We review recent experimental results on the single cell level and theoretical analyses of the depletion model and of the influence of the cell shape on the binding strength. Another important aggregation mechanism is caused by activation of platelets. This leads to clot formation which is life saving in the case of wound healing but also a major cause of death in the case of a thrombus induced stroke. We review historical and recent results on the participation of red blood cells in clot formation.

  4. Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans

    OpenAIRE

    Maddi, Abhiram; Bowman, Shaun M.; Free, Stephen J.

    2009-01-01

    Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and 6 secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and 9 secreted proteins. Most ...

  5. Shear stress-induced improvement of red blood cell deformability

    OpenAIRE

    Meram, Ece; Yılmaz, Bahar D.; Bas, Ceren; Atac, Nazlı; Yalçın, Ö.; Başkurt, Oguz K.; Meiselman, Herbert J.

    2013-01-01

    Classically, it is known that red blood cell (RBC) deformability is determined by the geometric and material properties of these cells. Experimental evidence accumulated during the last decade has introduced the concept of active regulation of RBC deformability. This regulation is mainly related to altered associations between membrane skeletal proteins and integral proteins, with the latter serving to anchor the skeleton to the lipid matrix. It has been hypothesized that shear stress induces...

  6. Hypoxia, hormones, and red blood cell function in chick embryos.

    Science.gov (United States)

    Dragon, Stefanie; Baumann, Rosemarie

    2003-04-01

    The red blood cell function of avian embryos is regulated by cAMP. Adenosine A(2A) and beta-adrenergic receptor activation during hypoxic conditions cause changes in the hemoglobin oxygen affinity and CO(2) transport. Furthermore, experimental evidence suggests a general involvement of cAMP in terminal differentiation of avian erythroblasts.

  7. Red Blood Cell Spectrin Skeleton in the Spotlight.

    Science.gov (United States)

    Braun-Breton, Catherine; Abkarian, Manouk

    2016-02-01

    Das et al. recently reported a role for the major merozoite surface protein MSP1 in malarial parasite egress from the red blood cell (RBC). On the basis of these new data and physical considerations, we propose an updated model for the main steps of this essential process for parasite proliferation. PMID:26652974

  8. Red blood cell antibodies in pregnancy and their clinical consequences

    DEFF Research Database (Denmark)

    Nordvall, Maria; Dziegiel, Morten Hanefeld; Hegaard, Hanne Kristine;

    2009-01-01

    The objective was to determine clinical consequences of various specificities for the infant/fetus. The population was patients referred between 1998 and 2005 to the tertiary center because of detected red blood cell (RBC) alloimmunization. Altogether 455 infants were delivered by 390 alloimmunized...

  9. Automated counting of white blood cells in synovial fluid.

    NARCIS (Netherlands)

    R. de Jonge (Robert); R.W. Brouwer (Reinoud); M. Smit (Marij); M. de Frankrijker-Merkestijn; R.J. Dolhain; J.M.W. Hazes (Mieke); A.W. van Toorenenbergen (Albert); J. Lindemans (Jan)

    2004-01-01

    textabstractOBJECTIVES: To evaluate the performance of automated leucocyte (white blood cell; WBC) counting by comparison with manual counting. METHODS: The number of WBC was determined in heparinized synovial fluid samples by the use of (i) a standard urine cytometer (Kova) and a

  10. The effects of cryopreservation on red blood cell rheologic properties

    NARCIS (Netherlands)

    Henkelman, Sandra; Lagerberg, Johan W. M.; Graaff, Reindert; Rakhorst, Gerhard; van Oeveren, Willem

    2010-01-01

    BACKGROUND: In transfusion medicine, frozen red blood cells (RBCs) are an alternative for liquid-stored RBCs. Little is known about the rheologic properties (i.e., aggregability and deformability) of thawed RBCs. In this study the rheologic properties of high-glycerol frozen RBCs and postthaw stored

  11. Red blood cell transfusion during septic shock in the ICU

    DEFF Research Database (Denmark)

    Perner, A; Smith, S H; Carlsen, S;

    2012-01-01

    Transfusion of red blood cells (RBCs) remains controversial in patients with septic shock, but current practice is unknown. Our aim was to evaluate RBC transfusion practice in septic shock in the intensive care unit (ICU), and patient characteristics and outcome associated with RBC transfusion....

  12. Expansion of human cord blood hematopoietic stem cells for transplantation.

    Science.gov (United States)

    Chou, Song; Chu, Pat; Hwang, William; Lodish, Harvey

    2010-10-01

    A recent Science paper reported a purine derivative that expands human cord blood hematopoietic stem cells in culture (Boitano et al., 2010) by antagonizing the aryl hydrocarbon receptor. Major problems need to be overcome before ex vivo HSC expansion can be used clinically.

  13. Proteomic profile of aminoglutethimide-induced apoptosis in HL-60 cells: Role of myeloperoxidase and arylamine free radicals.

    Science.gov (United States)

    Khan, Saifur R; Baghdasarian, Argishti; Nagar, Prarthna H; Fahlman, Richard; Jurasz, Paul; Michail, Karim; Aljuhani, Naif; Siraki, Arno G

    2015-09-01

    In this study, the cellular effects resulting from the metabolism of aminoglutethimide by myeloperoxidase were investigated. Human promyelocytic leukemia (HL-60) cells were treated with aminoglutethimide (AG), an arylamine drug that has a risk of adverse drug reactions, including drug-induced agranulocytosis. HL-60 cells contain abundant amounts of myeloperoxidase (MPO), a hemoprotein, which catalyzes one-electron oxidation of arylamines using H2O2 as a cofactor. Previous studies have shown that arylamine metabolism by MPO results in protein radical formation. The purpose of this study was to determine if pathways associated with a toxic response could be determined from conditions that produced protein radicals. Conditions for AG-induced protein radical formation (with minimal cytotoxicity) were optimized, and these conditions were used to carry out proteomic studies. We identified 43 proteins that were changed significantly upon AG treatment among which 18 were up-regulated and 25 were down-regulated. The quantitative proteomic data showed that AG peroxidative metabolism led to the down-regulation of critical anti-apoptotic proteins responsible for inhibiting the release of pro-apoptotic factors from the mitochondria as well as cytoskeletal proteins such as nuclear lamina. This overall pro-apoptotic response was confirmed with flow cytometry which demonstrated apoptosis to be the main mode of cell death, and this was attenuated by MPO inhibition. This response correlated with the intensity of AG-induced protein radical formation in HL-60 cells, which may play a role in cell death signaling mechanisms.

  14. The white blood cell scan in orthopedics

    Energy Technology Data Exchange (ETDEWEB)

    Propst-Proctor, S.L.; Dillingham, M.F.; McDougall, I.R.; Goodwin, D.

    1982-08-01

    A new nuclear scanning technique was found more specific for bone, joint, and soft tissue infections than any previously described scanning technique. The leukocyte scan, whereby a patient's own cells are labeled with a radioactive tagging agent (/sup 111/In oxine), can distinguish an active infectious process from other pain-inducing conditions. Ninety-seven /sup 111/In labeled autologous leukocyte scans were performed in 88 patients. The findings in 17 of 40 patients scanned for possible acute osteomyelitis, six of nine for suspected septic arthritis, and six for possible soft tissue infections, were positive. Subsequent clinical courses verified the infectious nature of these processes in all patients. Patients who had chronic osteomyelitis (14), bony metastases (four patients), heterotopic ossification (three), and degenerative arthritis (two) demonstrated negative findings. Of the seven patients scanned for acute long-bone fractures, one demonstrated positive findings. Nine scans demonstrated positive findings without determined causes. The leukocyte scan is a useful addition to the diagnostic tools of the orthopedic surgeon.

  15. Natural Antioxidants Improve Red Blood Cell “Survival” in Non-Leukoreduced Blood Samples

    Directory of Open Access Journals (Sweden)

    Yuliya V Kucherenko

    2015-03-01

    Full Text Available Background: Blood collected in an anticoagulant can be kept refrigerated in an unmodified state within 5 - 6 weeks. Oxidative damage is considered to be a one of the major factors contributing to the development of storage lesions. Lipid and membrane proteins oxidation results in changes in cation gradients that affect the cell survival. Aim: In the present study we used the natural antioxidants and ion channels blockers (L-carnosine, spermine, phloretin and their mixtures to prolong “survival” of red blood cells (RBCs, measured as the lack of PS exposure and cell hemolysis, in the Alsever's preservative solution upon hypothermic storage. Results: We show that the mixture of carnosine (20 mM, spermine (20 µM and phloretin (100 µM effectively blunted phosphatidylserine (PS exposure, Ca2+ accumulation and RBCs hemolysis in non-leukoreduced low (∼2% hematocrit samples after 36 days of storage as well as after 1 day of post-storage incubation of the stored cells in physiological saline solution. In addition, a slight but significant decrease in PS exposure was observed in non-leukoreduced high (∼20% hematocrit samples after 36 days of storage with the mixture of substances. Conclusion: We conclude that the use of the mixture of natural antioxidants (carnosine, spermine, and phloretin as an additive to blood preservative solution provides better RBCs storage and “survival”.

  16. Multiscale modeling of red blood cell mechanics and blood flow in malaria.

    Directory of Open Access Journals (Sweden)

    Dmitry A Fedosov

    2011-12-01

    Full Text Available Red blood cells (RBCs infected by a Plasmodium parasite in malaria may lose their membrane deformability with a relative membrane stiffening more than ten-fold in comparison with healthy RBCs leading to potential capillary occlusions. Moreover, infected RBCs are able to adhere to other healthy and parasitized cells and to the vascular endothelium resulting in a substantial disruption of normal blood circulation. In the present work, we simulate infected RBCs in malaria using a multiscale RBC model based on the dissipative particle dynamics method, coupling scales at the sub-cellular level with scales at the vessel size. Our objective is to conduct a full validation of the RBC model with a diverse set of experimental data, including temperature dependence, and to identify the limitations of this purely mechanistic model. The simulated elastic deformations of parasitized RBCs match those obtained in optical-tweezers experiments for different stages of intra-erythrocytic parasite development. The rheological properties of RBCs in malaria are compared with those obtained by optical magnetic twisting cytometry and by monitoring membrane fluctuations at room, physiological, and febrile temperatures. We also study the dynamics of infected RBCs in Poiseuille flow in comparison with healthy cells and present validated bulk viscosity predictions of malaria-infected blood for a wide range of parasitemia levels (percentage of infected RBCs with respect to the total number of cells in a unit volume.

  17. Assessment of Blood Contamination in Biological Fluids Using MALDI-TOF MS.

    Science.gov (United States)

    Laks, Katrina; Kirsipuu, Tiina; Dmitrijeva, Tuuli; Salumets, Andres; Palumaa, Peep

    2016-06-01

    Biological fluid sample collection often includes the risk of blood contamination that may alter the proteomic profile of biological fluid. In proteomics studies, exclusion of contaminated samples is usually based on visual inspection and counting of red blood cells in the sample; analysis of specific blood derived proteins is less used. To fill the gap, we developed a fast and sensitive method for ascertainment of blood contamination in crude biological fluids, based on specific blood-derived protein, hemoglobin detection by MALDI-TOF MS. The MALDI-TOF MS based method allows detection of trace hemoglobin with the detection limit of 0.12 nM. UV-spectrometry, which was used as reference method, was found to be less sensitive. The main advantages of the presented method are that it is fast, effective, sensitive, requires very small sample amount and can be applied for detection of blood contamination in various biological fluids collected for proteomics studies. Method applicability was tested on human cerebrospinal and follicular fluid, which proteomes generally do not contain hemoglobin, however, which possess high risk for blood contamination. Present method successfully detected the blood contamination in 12 % of cerebrospinal fluid and 24 % of follicular fluid samples. High percentage of contaminated samples accentuates the need for initial inspection of proteomic samples to avoid incorrect results from blood proteome overlap.

  18. 78 FR 47714 - Advisory Council on Blood Stem Cell Transplantation; Notice of Meeting

    Science.gov (United States)

    2013-08-06

    ... HUMAN SERVICES Health Resources and Services Administration Advisory Council on Blood Stem Cell... Health Service Act, as amended), the Advisory Council on Blood Stem Cell Transplantation (ACBSCT) advises... Advancing Hematopoietic Stem Cell Transplantation for Hemoglobinopathies. The Council also will...

  19. 78 FR 23571 - Advisory Council on Blood Stem Cell Transplantation; Notice of Meeting

    Science.gov (United States)

    2013-04-19

    ... HUMAN SERVICES Health Resources and Services Administration Advisory Council on Blood Stem Cell... amended), the Advisory Council on Blood Stem Cell Transplantation (ACBSCT) advises the Secretary of the... Hematopoietic Stem Cell Transplantation for Hemoglobinopathies. The Council will also hear presentations...

  20. Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target

    Science.gov (United States)

    Toman, O.; Kabickova, T.; Vit, O.; Fiser, R.; Polakova, K. Machova; Zach, J.; Linhartova, J.; Vyoral, D.; Petrak, J.

    2016-01-01

    Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib-resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients. PMID:27430982

  1. The minotaur proteome

    DEFF Research Database (Denmark)

    Bunkenborg, Jakob; García, Guadalupe Espadas; Paz, Marcia Ivonne Peña;

    2010-01-01

    Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5-15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides...

  2. 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...

  3. 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...

  4. Untargeted Proteomics and Systems-Based Mechanistic Investigation of Artesunate in Human Bronchial Epithelial Cells.

    Science.gov (United States)

    Ravindra, Kodihalli C; Ho, Wanxing Eugene; Cheng, Chang; Godoy, Luiz C; Wishnok, John S; Ong, Choon Nam; Wong, W S Fred; Wogan, Gerald N; Tannenbaum, Steven R

    2015-10-19

    The antimalarial drug artesunate is a semisynthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua. It is hypothesized to attenuate allergic asthma via inhibition of multiple signaling pathways. We used a comprehensive approach to elucidate the mechanism of action of artesunate by designing a novel biotinylated dihydroartemisinin (BDHA) to identify cellular protein targets of this anti-inflammatory drug. By adopting an untargeted proteomics approach, we demonstrated that artesunate may exert its protective anti-inflammatory effects via direct interaction with multiple proteins, most importantly with a number of mitochondrial enzymes related to glucose and energy metabolism, along with mRNA and gene expression, ribosomal regulation, stress responses, and structural proteins. In addition, the modulatory effects of artesunate on various cellular transcription factors were investigated using a transcription factor array, which revealed that artesunate can simultaneously modulate multiple nuclear transcription factors related to several major pro- and anti-inflammatory signaling cascades in human bronchial epithelial cells. Artesunate significantly enhanced nuclear levels of nuclear factor erythroid-2-related factor 2 (Nrf2), a key promoter of antioxidant mechanisms, which is inhibited by the Kelch-like ECH-associated protein 1 (Keap1). Our results demonstrate that, like other electrophilic Nrf2 regulators, artesunate activates this system via direct molecular interaction/modification of Keap1, freeing Nrf2 for transcriptional activity. Altogether, the molecular interactions and modulation of nuclear transcription factors provide invaluable insights into the broad pharmacological actions of artesunate in inflammatory lung diseases and related inflammatory disorders. PMID:26340163

  5. Differentiation of Human Cord Blood and Stromal Derived Stem Cells into Neuron Cells

    Directory of Open Access Journals (Sweden)

    Özlem Pamukçu Baran

    2007-01-01

    Full Text Available The most basic properties of stem cells are the capacities to self-renew indefinitely and to differentiate into multiple cell or tissue types. Umbilical cord blood has been utilized for human hematopoietic stem cell transplantation as an alternative source to bone marrow.The experiments show that Wharton’s jelly cells are easily attainable and can be expanded in vitro, maintained in culture, and induced to differentiate into neural cells. Almost recent studies it has been discovered that the cord blood-derived cells can differantiate not only to blood cells but also to various somatic cells like neuron or muscle cell with the signals taken from the envoirenment.Interestingly, neural cells obtained from umbilical cord blood show a relatively high spontaneous differentiation into oligodendrocytes, Embryonic stem cells proliferate indefinitely and can differentiate spontaneously into all tissue types.It has been shown that embryonic stem cells can be induced to differentiate into neurons and glia by treatment with retinoic acid or basic fibroblast growth factor. It has been studied that the diseases as Motor Neuron Disease, Parkinson, Alzheimer and degeneration of medulla spinalis and also paralysises could be treated with transplantation of cord blood-dericed stem cells.

  6. Flow of Red Blood Cells in Stenosed Microvessels

    Science.gov (United States)

    Vahidkhah, Koohyar; Balogh, Peter; Bagchi, Prosenjit

    2016-06-01

    A computational study is presented on the flow of deformable red blood cells in stenosed microvessels. It is observed that the Fahraeus-Lindqvist effect is significantly enhanced due to the presence of a stenosis. The apparent viscosity of blood is observed to increase by several folds when compared to non-stenosed vessels. An asymmetric distribution of the red blood cells, caused by geometric focusing in stenosed vessels, is observed to play a major role in the enhancement. The asymmetry in cell distribution also results in an asymmetry in average velocity and wall shear stress along the length of the stenosis. The discrete motion of the cells causes large time-dependent fluctuations in flow properties. The root-mean-square of flow rate fluctuations could be an order of magnitude higher than that in non-stenosed vessels. Several folds increase in Eulerian velocity fluctuation is also observed in the vicinity of the stenosis. Surprisingly, a transient flow reversal is observed upstream a stenosis but not downstream. The asymmetry and fluctuations in flow quantities and the flow reversal would not occur in absence of the cells. It is concluded that the flow physics and its physiological consequences are significantly different in micro- versus macrovascular stenosis.

  7. Expert Assistant For A Clinical Hematology Blood Cell Analyzer

    Science.gov (United States)

    Young, Carole; Navlakha, Jainendra K.

    1989-03-01

    The COULTER COUNTER Model S Plus Series instruments are automated clinical hematology blood cell analyzers which measure the count, volume and population distribution of red blood cells, white blood cells and platelets, and hemoglobin from patient blood samples. In the clinical laboratory environment, instrument startup consists of a number of component and system checks to assure proper operation and calibration to insure reliable results are produced on patient samples. If a startup check fails, troubleshooting procedures are provided to assist the operator in determining the cause of the error. Troubleshooting requires expertise in instrument operation, troubleshooting procedures and evaluation of the data produced. This expert system is designed and developed to assist the startup diagnostics of COULTER COUNTER Model S Plus Series instruments. The system reads data produced by the instrument and validates it against expected values. If the values are not all correct, then the troubleshooting starts. Troubleshooting is handled for the most common subsystem problems and those which the operator has the equipment and knowledge to handle, problems that are cheapest to fix and problems that are quickest to fix. The expert system restarts the startup sequence whenever troubleshooting has been successful or recommends calling Customer Service when unsuccessful.

  8. Hemoglobin Aggregation in Single Red Blood Cells of Sickle Cell Anemia

    Science.gov (United States)

    Nishio, Izumi; Tanaka, Toyoichi; Sun, Shao-Tang; Imanishi, Yuri; Tsuyoshi Ohnishi, S.

    1983-06-01

    A laser light scattering technique was used to observe the extent of hemoglobin aggregation in solitary red blood cells of sickle cell anemia. Hemoglobin aggregation was confirmed in deoxygenated cells. The light scattering technique can also be applied to cytoplasmic studies of any biological cell.

  9. Generation of suppressive blood cells for control of allograft rejection.

    Science.gov (United States)

    Kleist, Christian; Sandra-Petrescu, Flavius; Jiga, Lucian; Dittmar, Laura; Mohr, Elisabeth; Greil, Johann; Mier, Walter; Becker, Luis E; Lang, Peter; Opelz, Gerhard; Terness, Peter

    2015-05-01

    Our previous studies in rats showed that incubation of monocytic dendritic cells (DCs) with the chemotherapeutic drug mitomycin C (MMC) renders the cells immunosuppressive. Donor-derived MMC-DCs injected into the recipient prior to transplantation prolonged heart allograft survival. Although the generation of DCs is labour-intensive and time-consuming, peripheral blood mononuclear cells (PBMCs) can be easily harvested. In the present study, we analyse under which conditions DCs can be replaced by PBMCs and examine their mode of action. When injected into rats, MMC-incubated donor PBMCs (MICs) strongly prolonged heart allograft survival. Removal of monocytes from PBMCs completely abrogated their suppressive effect, indicating that monocytes are the active cell population. Suppression of rejection was donor-specific. The injected MICs migrated into peripheral lymphoid organs and led to an increased number of regulatory T-cells (Tregs) expressing cluster of differentiation (CD) markers CD4 and CD25 and forkhead box protein 3 (FoxP3). Tolerance could be transferred to syngeneic recipients with blood or spleen cells. Depletion of Tregs from tolerogenic cells abrogated their suppressive effect, arguing for mediation of immunosuppression by CD4⁺CD25⁺FoxP3⁺ Tregs. Donor-derived MICs also prolonged kidney allograft survival in pigs. MICs generated from donor monocytes were applied for the first time in humans in a patient suffering from therapy-resistant rejection of a haploidentical stem cell transplant. We describe, in the present paper, a simple method for in vitro generation of suppressor blood cells for potential use in clinical organ transplantation. Although the case report does not allow us to draw any conclusion about their therapeutic effectiveness, it shows that MICs can be easily generated and applied in humans.

  10. Erythropoietic Potential of CD34+ Hematopoietic Stem Cells from Human Cord Blood and G-CSF-Mobilized Peripheral Blood

    Directory of Open Access Journals (Sweden)

    Honglian Jin

    2014-01-01

    Full Text Available Red blood cell (RBC supply for transfusion has been severely constrained by the limited availability of donor blood and the emergence of infection and contamination issues. Alternatively, hematopoietic stem cells (HSCs from human organs have been increasingly considered as safe and effective blood source. Several methods have been studied to obtain mature RBCs from CD34+ hematopoietic stem cells via in vitro culture. Among them, human cord blood (CB and granulocyte colony-stimulating factor-mobilized adult peripheral blood (mPB are common adult stem cells used for allogeneic transplantation. Our present study focuses on comparing CB- and mPB-derived stem cells in differentiation from CD34+ cells into mature RBCs. By using CD34+ cells from cord blood and G-CSF mobilized peripheral blood, we showed in vitro RBC generation of artificial red blood cells. Our results demonstrate that CB- and mPB-derived CD34+ hematopoietic stem cells have similar characteristics when cultured under the same conditions, but differ considerably with respect to expression levels of various genes and hemoglobin development. This study is the first to compare the characteristics of CB- and mPB-derived erythrocytes. The results support the idea that CB and mPB, despite some similarities, possess different erythropoietic potentials in in vitro culture systems.

  11. The Effect of Pulsatile Versus Nonpulsatile Blood Flow on Viscoelasticity and Red Blood Cell Aggregation in Extracorporeal Circulation

    OpenAIRE

    Ahn, Chi Bum; Kang, Yang Jun; Kim, Myoung Gon; Yang, Sung; Lim, Choon Hak; Son, Ho Sung; Kim, Ji Sung; Lee, So Young; Son, Kuk Hui; Sun, Kyung

    2016-01-01

    Background Extracorporeal circulation (ECC) can induce alterations in blood viscoelasticity and cause red blood cell (RBC) aggregation. In this study, the authors evaluated the effects of pump flow pulsatility on blood viscoelasticity and RBC aggregation. Methods Mongrel dogs were randomly assigned to two groups: a nonpulsatile pump group (n=6) or a pulsatile pump group (n=6). After ECC was started at a pump flow rate of 80 mL/kg/min, cardiac fibrillation was induced. Blood sampling was perfo...

  12. A statistical model for red blood cell survival.

    Science.gov (United States)

    Korell, Julia; Coulter, Carolyn V; Duffull, Stephen B

    2011-01-01

    A statistical model for the survival time of red blood cells (RBCs) with a continuous distribution of cell lifespans is presented. The underlying distribution of RBC lifespans is derived from a probability density function with a bathtub-shaped hazard curve, and accounts for death of RBCs due to senescence (age-dependent increasing hazard rate) and random destruction (constant hazard), as well as for death due to initial or delayed failures and neocytolysis (equivalent to early red cell mortality). The model yields survival times similar to those of previously published studies of RBC survival and is easily amenable to inclusion of drug effects and haemolytic disorders. PMID:20950630

  13. Atypical carcinoid and large cell neuroendocrine carcinoma of the lung: a proteomic dataset from formalin-fixed archival samples

    Science.gov (United States)

    Tanca, Alessandro; Addis, Maria Filippa; Pisanu, Salvatore; Abbondio, Marcello; Pagnozzi, Daniela; Eccher, Albino; Rindi, Guido; Cossu-Rocca, Paolo; Uzzau, Sergio; Fanciulli, Giuseppe

    2016-01-01

    Here we present a dataset generated using formalin-fixed paraffin-embedded archival samples from two rare lung neuroendocrine tumor subtypes (namely, two atypical carcinoids, ACs, and two large-cell neuroendocrine carcinomas, LCNECs). Samples were subjected to a shotgun proteomics pipeline, comprising full-length protein extraction, SDS removal through spin columns, in solution trypsin digestion, long gradient liquid chromatography peptide separation and LTQ-Orbitrap mass spectrometry analysis. A total of 1260 and 2436 proteins were identified in the AC and LCNEC samples, respectively, with FDR <1%. MS data are available in the PeptideAtlas repository at http://www.peptideatlas.org/PASS/PASS00375. PMID:27054153

  14. Atypical carcinoid and large cell neuroendocrine carcinoma of the lung: a proteomic dataset from formalin-fixed archival samples.

    Science.gov (United States)

    Tanca, Alessandro; Addis, Maria Filippa; Pisanu, Salvatore; Abbondio, Marcello; Pagnozzi, Daniela; Eccher, Albino; Rindi, Guido; Cossu-Rocca, Paolo; Uzzau, Sergio; Fanciulli, Giuseppe

    2016-06-01

    Here we present a dataset generated using formalin-fixed paraffin-embedded archival samples from two rare lung neuroendocrine tumor subtypes (namely, two atypical carcinoids, ACs, and two large-cell neuroendocrine carcinomas, LCNECs). Samples were subjected to a shotgun proteomics pipeline, comprising full-length protein extraction, SDS removal through spin columns, in solution trypsin digestion, long gradient liquid chromatography peptide separation and LTQ-Orbitrap mass spectrometry analysis. A total of 1260 and 2436 proteins were identified in the AC and LCNEC samples, respectively, with FDR <1%. MS data are available in the PeptideAtlas repository at http://www.peptideatlas.org/PASS/PASS00375.

  15. Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells

    DEFF Research Database (Denmark)

    Bursomanno, Sara; Beli, Petra; Khan, Asif M;

    2015-01-01

    subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock. Substantial evidence from studies in yeast has shown that SUMOylation plays an important role in the regulation of DNA replication and repair. Here, we report a proteomic...... analysis of proteins modified by SUMO2 in response to DNA replication stress in S phase in human cells. We have identified a panel of 22 SUMO2 targets with increased SUMOylation during DNA replication stress, many of which play key functions within the DNA replication machinery and/or in the cellular...

  16. Atypical carcinoid and large cell neuroendocrine carcinoma of the lung: a proteomic dataset from formalin-fixed archival samples

    Directory of Open Access Journals (Sweden)

    Alessandro Tanca

    2016-06-01

    Full Text Available Here we present a dataset generated using formalin-fixed paraffin-embedded archival samples from two rare lung neuroendocrine tumor subtypes (namely, two atypical carcinoids, ACs, and two large-cell neuroendocrine carcinomas, LCNECs. Samples were subjected to a shotgun proteomics pipeline, comprising full-length protein extraction, SDS removal through spin columns, in solution trypsin digestion, long gradient liquid chromatography peptide separation and LTQ-Orbitrap mass spectrometry analysis. A total of 1260 and 2436 proteins were identified in the AC and LCNEC samples, respectively, with FDR <1%. MS data are available in the PeptideAtlas repository at http://www.peptideatlas.org/PASS/PASS00375.

  17. Proteomic and meatbolomic analysis of smooth muscle cells derived from the arterial media and adventitial progenitors of apolipoprotein E-deficient mice

    NARCIS (Netherlands)

    Mayr, M; Zampetaki, A.; Sidibe, A.; Mayr, U.; Yin, X.; Souza, A.I. de; Chung, Y.L.; Madhu, B.; Quax, P.H.; Hu, Y.; Griffiths, J.R.; Xu, Q.

    2008-01-01

    We have recently demonstrated that stem cell antigen 1-positive (Sca-1(+)) progenitors exist in the vascular adventitia of apolipoprotein E-deficient (apoE(-/-)) mice and contribute to smooth muscle cell (SMC) accumulation in vein graft atherosclerosis. Using a combined proteomic and metabolomic app

  18. Genomic and proteomic analyses of Prdm5 reveal interactions with insulator binding proteins in embryonic stem cells

    DEFF Research Database (Denmark)

    Galli, Giorgio Giacomo; Carrara, Matteo; Francavilla, Chiara;

    2013-01-01

    find that Prdm5 is highly expressed in mouse embryonic stem cells (mES) and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next generation sequencing technologies we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that......-occupies genomic loci. In summary, our data indicate how Prdm5 may modulate transcription by interacting with factors involved in genome organization in mouse embryonic stem cells......., despite Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, Cohesin and TFIIIC and co...

  19. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics

    DEFF Research Database (Denmark)

    Ong, S.E.; Blagoev, B.; Kratchmarova, I.;

    2002-01-01

    . Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non......Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures......-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred...

  20. 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...

  1. Cell-cell interaction in blood flow in patients with coronary heart disease (in vitro study)

    Science.gov (United States)

    Malinova, Lidia I.; Simonenko, Georgy V.; Denisova, Tatyana P.; Tuchin, Valery V.

    2007-02-01

    Blood cell-cell and cell-vessel wall interactions are one of the key patterns in blood and vascular pathophysiology. We have chosen the method of reconstruction of pulsative blood flow in vitro in the experimental set. Blood flow structure was studied by PC integrated video camera with following slide by slide analysis. Studied flow was of constant volumetric blood flow velocity (1 ml/h). Diameter of tube in use was comparable with coronary arteries diameter. Glucose solution and unfractured heparin were used as the nonspecial irritants of studied flow. Erythrocytes space structure in flow differs in all groups of patients in our study (men with stable angina pectoris (SAP), myocardial infarction (MI) and practically healthy men (PHM). Intensity of erythrocytes aggregate formation was maximal in patients with SAP, but time of their "construction/deconstruction" at glucose injection was minimal. Phenomena of primary clotting formation in patients with SAP of high function class was reconstructed under experimental conditions. Heparin injection (10 000 ED) increased linear blood flow velocity both in patients with SAP, MI and PHP but modulated the cell profile in the flow. Received data correspond with results of animal model studies and noninvasive blood flow studies in human. Results of our study reveal differences in blood flow structure in patients with coronary heart disease and PHP under irritating conditions as the possible framework of metabolic model of coronary blood flow destabilization.

  2. Characterization of red blood cells (RBCs) using dual Brillouin/Raman micro-spectroscopy

    Science.gov (United States)

    Meng, Zhaokai; Bustamante-Lopez, Sandra C.; Yakovlev, Vladislav V.; Meissner, Kenith E.

    2016-04-01

    Erythrocytes, or red blood cells, transport oxygen to and carbon dioxide from the body's tissues and organs. Red blood cell mechanical properties are altered in a number of diseases such as sickle cell anaemia and malaria. Additionally, mechanically modified red blood cell ghosts are being considered as a long-term, biocompatible carrier for drug delivery and for blood analyte sensing. Brillouin spectroscopy enables viscoelastic characterization of samples at the microscale. In this report, Brillouin spectroscopy is applied to characterize the mechanical properties of red blood cells and red blood cell ghosts.

  3. Saving the leftovers: models for banking cord blood stem cells.

    Science.gov (United States)

    Cogdell, Kimberly J

    2009-01-01

    Each year there are over four million live births in the United States. Each birth produces umbilical cord blood stem cells, which are usually discarded. The author argues that rather than discarding the umbilical cord, this valuable resource of cord blood should be banked and used for research and therapeutic purposes. Umbilical cord blood could provide a solution to the critical need to find matching donors for hematopoietic transplants in patients who have no matching bone marrow donors. Creating a system of universal donation to a public bank will greatlyincrease the number of donors and therefore, the number of matches for patients. Such a system will facilitate the development and use of new technologies and transplant procedures, while providing an opportunity for treatment to individuals who would otherwise not be able to find suitable donors. PMID:20101907

  4. Saving the leftovers: models for banking cord blood stem cells.

    Science.gov (United States)

    Cogdell, Kimberly J

    2009-01-01

    Each year there are over four million live births in the United States. Each birth produces umbilical cord blood stem cells, which are usually discarded. The author argues that rather than discarding the umbilical cord, this valuable resource of cord blood should be banked and used for research and therapeutic purposes. Umbilical cord blood could provide a solution to the critical need to find matching donors for hematopoietic transplants in patients who have no matching bone marrow donors. Creating a system of universal donation to a public bank will greatlyincrease the number of donors and therefore, the number of matches for patients. Such a system will facilitate the development and use of new technologies and transplant procedures, while providing an opportunity for treatment to individuals who would otherwise not be able to find suitable donors.

  5. Image-based red cell counting for wild animals blood.

    Science.gov (United States)

    Mauricio, Claudio R M; Schneider, Fabio K; Dos Santos, Leonilda Correia

    2010-01-01

    An image-based red blood cell (RBC) automatic counting system is presented for wild animals blood analysis. Images with 2048×1536-pixel resolution acquired on an optical microscope using Neubauer chambers are used to evaluate RBC counting for three animal species (Leopardus pardalis, Cebus apella and Nasua nasua) and the error found using the proposed method is similar to that obtained for inter observer visual counting method, i.e., around 10%. Smaller errors (e.g., 3%) can be obtained in regions with less grid artifacts. These promising results allow the use of the proposed method either as a complete automatic counting tool in laboratories for wild animal's blood analysis or as a first counting stage in a semi-automatic counting tool. PMID:21096766

  6. Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Deok-Jin; Wang, Daojing

    2006-05-26

    Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

  7. Comparative Proteomic Analysis of Anti-Cancer Mechanism by Periplocin Treatment in Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Zejun Lu

    2014-03-01

    Full Text Available Background: Periplocin is used for treatment of rheumatoid arthritis, reinforcement of bones and tendons, palpitations or shortness of breath and lower extremity edema in traditional medicine. Our previous findings suggested that periplocin could inhibit the growth of lung cancer both in vitro and in vivo. But the biological processes and molecular pathways by which periplocin induces these beneficial effects remain largely undefined. Methods: To explore the molecular mechanisms of periplocin involved in anti-cancer activity, in the present study the protein profile changes of human lung cancer cell lines A549 in response to periplocin treatment were investigated using the proteomics approaches (2-DE combined with MS/MS. Western blot was employed to verify the changed proteins. Interactions between changed proteins were analyzed by STRING. Results: 29 down-regulated protein species named GTP-binding nuclear protein Ran (RAN, Rho GDP-dissociation inhibitor 1 (ARHGDIA, eukaryotic translation initiation factor 5A-1 (EIF5A and Profilin-1(PFN1, and 10 up-regulated protein species named Heat shock cognate 71 kDa protein (HSPA8,10 kDa heat shock protein (HSPE1, and Cofilin-1(CFL-1 were identified. Among them, GTP-binding nuclear protein Ran (RAN and Rho GDP-dissociation inhibitor 1 (ARHGDIA were the most significantly changed (over tenfold. The proteasome subunit beta type-6 (PSMB6, ATP synthase ecto-α-subunit (ATP5A1, Aldehyde dehydrogenase 1 (ALDH1 and EIF5A were verified by immunoblot assays to be dramatically down-regulated. By STRING bioinformatics analysis revealing interactions and signaling networks it became apparent that the proteins changed they are primarily involved in transcription and proteolysis. Conclusion: Periplocin inhibited growth of lung cancer by down-regulating proteins, such as ATP5A1, EIF5A, ALDH1 and PSMB6. These findings may improve our understanding of the molecular mechanisms underlying the anti-cancer effects of

  8. Transplantation proteomics

    OpenAIRE

    Traum, Avram Z.; Schachter, Asher D.

    2005-01-01

    The field of proteomics is developing at a rapid pace in the post-genome era. Translational proteomics investigations aim to apply a combination of established methods and new technologies to learn about protein expression profiles predictive of clinical events, therapeutic response, and underlying mechanisms. However, in contrast to genetic studies and in parallel with gene expression studies, the dynamic nature of the proteome in conjunction with the challenges of accounting for post-transl...

  9. Protein translation machinery holds a key for transition of planktonic cells to biofilm state in Enterococcus faecalis: A proteomic approach.

    Science.gov (United States)

    Qayyum, Shariq; Sharma, Divakar; Bisht, Deepa; Khan, Asad U

    2016-06-10

    Enterococcus faecalis is a member of human gut microflora causing nosocomial infection involving biofilm formation. Ethyl methyl sulfonate induced mutants were analysed using crystal violet assay, SEM and CLSM microscopy which confirmed AK-E12 as biofilm efficient and AK-F6 as biofilm deficient mutants. Growth curve pattern revealed AK-E12 was fast growing whereas, AK-F6 was found slow growing mutant. 2D-Electrophorosis and MALDI-TOF analysis revealed over and underexpression of many translation-elongation associated proteins in mutants compared to wild type. Protein translation elongation factor G, translation elongation factor Tu and ribosomal subunit interface proteins were underexpressed and UTP-glucose-1-phosphate uridylyl transferase and cell division protein divIVA were overexpressed in AK-E12 as compared to wild type. In AK-F6, except 10 kDa chaperonin which was over-expressed other selected proteins were found to be suppressed. RT-PCR confirmed proteomic data except for the translation elongation factor G which showed contradictory data of proteome expression in AK-E12. Protein-protein interaction networks were constructed using STRING 10.0 which demonstrated strong connection of translation-elongation proteins with other proteins. Hence, it concludes from the data that translation elongation factors are important in transition of planktonic cells to biofilm cells in Enterococcus faecalis. PMID:27144316

  10. Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria

    Science.gov (United States)

    Mitchell, Adam J.; Gray, Warren D.; Schroeder, Max; Yi, Hong; Taylor, Jeannette V.; Dillard, Rebecca S.; Ke, Zunlong; Wright, Elizabeth R.; Stephens, David; Roback, John D.; Searles, Charles D.

    2016-01-01

    Background Red blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria. Results Flow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents. Conclusions These studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators. PMID:27760197

  11. Pyruvate effects on red blood cells during in vitro cardiopulmonary bypass with dogs' blood.

    Science.gov (United States)

    Gou, DaMing; Tan, HongJing; Cai, HuiJun; Zhou, FangQiang

    2012-11-01

    To investigate the effects of pyruvate (Pyr) on adenosine triphosphate (ATP), endothelial nitric oxide synthase (eNOS), and nitric oxide (NO) in red blood cells (RBCs) during the cardiopulmonary bypass procedure (CPB), blood, 500 mL, was collected from each of 10 healthy dogs (weight 12-18 kg). The blood was divided into two parts (250 mL each) and randomly assigned into the control group (Group C, n = 10) or the Pyr group (Group P, n = 10). The blood was commingled with an equal volume of 0.9% NaCl and pyruvated isotonic solution (Pyr 50 mM) in the extracorporeal circuit in the two groups, respectively. The CPB procedure was fixed at 120 min, and the transferring flow was 4 L/min. Contents of ATP in RBCs, eNOS activities, and NO productions in plasma were measured before CPB and during CPB at 30, 60, 90, and 120 min in both groups. The ATP level, eNOS activity, and NO production were not different prior to CPB between the two groups. A decline of ATP levels was shown in both groups but remained significantly higher in Group P than in Group C at the same time points during in vitro CPB (P dogs' RBCs in the ATP level, eNOS activity, and NO production, in vitro, but Pyr effectively protected RBCs in these functions during CPB. Pyr would be clinically protective for RBCs during CPB.

  12. Red Cell Properties after Different Modes of Blood Transportation

    Science.gov (United States)

    Makhro, Asya; Huisjes, Rick; Verhagen, Liesbeth P.; Mañú-Pereira, María del Mar; Llaudet-Planas, Esther; Petkova-Kirova, Polina; Wang, Jue; Eichler, Hermann; Bogdanova, Anna; van Wijk, Richard; Vives-Corrons, Joan-Lluís; Kaestner, Lars

    2016-01-01

    Transportation of blood samples is unavoidable for assessment of specific parameters in blood of patients with rare anemias, blood doping testing, or for research purposes. Despite the awareness that shipment may substantially alter multiple parameters, no study of that extent has been performed to assess these changes and optimize shipment conditions to reduce transportation-related artifacts. Here we investigate the changes in multiple parameters in blood of healthy donors over 72 h of simulated shipment conditions. Three different anticoagulants (K3EDTA, Sodium Heparin, and citrate-based CPDA) for two temperatures (4°C and room temperature) were tested to define the optimal transportation conditions. Parameters measured cover common cytology and biochemistry parameters (complete blood count, hematocrit, morphological examination), red blood cell (RBC) volume, ion content and density, membrane properties and stability (hemolysis, osmotic fragility, membrane heat stability, patch-clamp investigations, and formation of micro vesicles), Ca2+ handling, RBC metabolism, activity of numerous enzymes, and O2 transport capacity. Our findings indicate that individual sets of parameters may require different shipment settings (anticoagulants, temperature). Most of the parameters except for ion (Na+, K+, Ca2+) handling and, possibly, reticulocytes counts, tend to favor transportation at 4°C. Whereas plasma and intraerythrocytic Ca2+ cannot be accurately measured in the presence of chelators such as citrate and EDTA, the majority of Ca2+-dependent parameters are stabilized in CPDA samples. Even in blood samples from healthy donors transported using an optimized shipment protocol, the majority of parameters were stable within 24 h, a condition that may not hold for the samples of patients with rare anemias. This implies for as short as possible shipping using fast courier services to the closest expert laboratory at reach. Mobile laboratories or the travel of the patients to

  13. Red cell properties after different modes of blood transportation

    Directory of Open Access Journals (Sweden)

    Asya Makhro

    2016-07-01

    Full Text Available Transportation of blood samples is unavoidable for assessment of specific parameters in blood of patients with rare anemias, blood doping testing or for research purposes. Despite the awareness that shipment may substantially alter multiple parameters, no study of that extend has been performed to assess these changes and optimize shipment conditions to reduce transportation-related artifacts. Here we investigate the changes in multiple parameters in blood of healthy donors over 72 hours of simulated shipment conditions. Three different anticoagulants (K3EDTA, Sodium Heparin and citrate-based CPDA for two temperatures (4oC and room temperature were tested to define the optimal transportation conditions. Parameters measured cover common cytology and biochemistry parameters (complete blood count, hematocrit, morphological examination, red blood cell (RBC volume, ion content and density, membrane properties and stability (hemolysis, osmotic fragility, membrane heat stability, patch-clamp investigations and formation of micro vesicles, Ca2+ handling, RBC metabolism, activity of numerous enzymes and O2 transport capacity. Our findings indicate that individual sets of parameter may require different shipment settings (anticoagulants, temperature. Most of the parameters except for ion (Na+, K+, Ca2+ handling and, possibly, reticulocytes counts, tend to favor transportation at 4oC. Whereas plasma and intraerythrocytic Ca2+ cannot be accurately measured in the presence of chelators such as citrate and EDTA, majority of Ca2+-dependent parameters are stabilized in CPDA samples. Even in blood samples from healthy donors transported using optimized shipment protocol the majority of parameters were stable within 24 hours, the condition that may not hold for the samples of patients with rare anemias. This implies for the as short as possible shipping using fast courier services to the closest expert laboratory at reach. Mobile laboratories or the travel of the

  14. Proteomic profiling of human colon cancer cells treated with the histone deacetylase inhibitor belinostat

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Petersen, Jørgen; Nielsen, Søren Jensby;

    2010-01-01

    The anticancer drug belinostat is a hydroxamate histone deacetylase inhibitor that has shown significant antitumour activity in various tumour models and also in clinical trials. In this study, we utilized a proteomic approach in order to evaluate the effect of this drug on protein expression...

  15. Red blood cell phenotype matching for various ethnic groups.

    Science.gov (United States)

    Badjie, Karafa S W; Tauscher, Craig D; van Buskirk, Camille M; Wong, Clare; Jenkins, Sarah M; Smith, Carin Y; Stubbs, James R

    2011-01-01

    Patients requiring chronic transfusion support are at risk of alloimmunization after red blood cell (RBC) transfusion because of a disparity between donor and recipient antigen profiles. This research explored the probability of obtaining an exact extended phenotype match between blood donors randomly selected from our institution and patients randomly selected from particular ethnic groups. Blood samples from 1,000 blood donors tested by molecular method were evaluated for the predicted phenotype distribution of Rh, Kell, Kidd, Duffy, and MNS. A random subsample of 800 donor phenotypes was then evaluated for the probability of obtaining an exact match with respect to phenotype with a randomly selected patient from a particular ethnic group. Overall, there was a greater than 80 percent probability of finding an exact donor-recipient match for the K/k alleles in the Kell system. The probability ranged from 3 percent to 38 percent, depending on the ethnicity and disparities in phenotypic profiles, for the Rh, Kidd, Duffy, and MNS systems. A significant donor-recipient phenotype mismatch ratio exists with certain blood group antigens such that, with current routine ABO and D matching practices, recipients of certain ethnic groups are predisposed to alloimmunization. PMID:22356481

  16. Blood cell telomere length is a dynamic feature.

    Directory of Open Access Journals (Sweden)

    Ulrika Svenson

    Full Text Available There is a considerable heterogeneity in blood cell telomere length (TL for individuals of similar age and recent studies have revealed that TL changes by time are dependent on TL at baseline. TL is partly inherited, but results from several studies indicate that e.g. life style and/or environmental factors can affect TL during life. Collectively, these studies imply that blood cell TL might fluctuate during a life time and that the actual TL at a defined time point is the result of potential regulatory mechanism(s and environmental factors. We analyzed relative TL (RTL in subsequent blood samples taken six months apart from 50 individuals and found significant associations between RTL changes and RTL at baseline. Individual RTL changes per month were more pronounced than the changes recorded in a previously studied population analyzed after 10 years' follow up. The data argues for an oscillating TL pattern which levels out at longer follow up times. In a separate group of five blood donors, a marked telomere loss was demonstrated within a six month period for one donor where after TL was stabilized. PCR determined RTL changes were verified by Southern blotting and STELA (single telomere elongation length analysis. The STELA demonstrated that for the donor with a marked telomere loss, the heterogeneity of the telomere distribution decreased considerably, with a noteworthy loss of the largest telomeres. In summary, the collected data support the concept that individual blood cell telomere length is a dynamic feature and this will be important to recognize in future studies of human telomere biology.

  17. Reduction of prion infectivity in packed red blood cells

    International Nuclear Information System (INIS)

    The link between a new variant form of Creutzfeldt-Jakob disease (vCJD) and the consumption of prion contaminated cattle meat as well as recent findings showing that vCJD can be transmitted by blood transfusion have raised public health concerns. Currently, a reliable test to identify prions in blood samples is not available. The purpose of this study was to evaluate the possibility to remove scrapie prion protein (PrPSc) and infectivity from red blood cell (RBC) suspensions by a simple washing procedure using a cell separation and washing device. The extent of prion removal was assessed by Western blot, PMCA and infectivity bioassays. Our results revealed a substantial removal of infectious prions (≥3 logs of infectivity) by all techniques used. These data suggest that a significant amount of infectivity present in RBC preparations can be removed by a simple washing procedure. This technology may lead to increased safety of blood products and reduce the risk of further propagation of prion diseases.

  18. A SILAC-Based Approach Elicits the Proteomic Responses to Vancomycin-Associated Nephrotoxicity in Human Proximal Tubule Epithelial HK-2 Cells.

    Science.gov (United States)

    Li, Zhi-Ling; Zhou, Shu-Feng

    2016-01-29

    Vancomycin, a widely used antibiotic, often induces nephrotoxicity, however, the molecular targets and underlying mechanisms of this side effect remain unclear. The present study aimed to examine molecular interactome and analyze the signaling pathways related to the vancomycin-induced nephrotoxicity in human proximal tubule epithelial HK-2 cells using the stable isotope labeling by amino acids in cell culture (SILAC) approach. The quantitative proteomic study revealed that there were at least 492 proteins interacting with vancomycin and there were 290 signaling pathways and cellular functions potentially regulated by vancomycin in HK-2 cells. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, EMT, and ROS generation. These findings suggest that vancomycin-induced proteomic responses in HK-2 cells involvefunctional proteins and pathways that regulate cell cycle, apoptosis, autophagy, and redox homeostasis. This is the first systemic study revealed the networks of signaling pathways and proteomic responses to vancomycin treatment in HK-2 cells, and the data may be used to discriminate the molecular and clinical subtypes and to identify new targets and biomarkers for vancomycin-induced nephrotoxic effect. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for drug-induced renal toxicity.

  19. Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

    DEFF Research Database (Denmark)

    Harkness, Linda; Prokhorova, Tatyana A; Kassem, Moustapha;

    2012-01-01

    The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass...... spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we...

  20. Isolation of human monoclonal antibodies from peripheral blood B cells.

    Science.gov (United States)

    Huang, Jinghe; Doria-Rose, Nicole A; Longo, Nancy S; Laub, Leo; Lin, Chien-Li; Turk, Ellen; Kang, Byong H; Migueles, Stephen A; Bailer, Robert T; Mascola, John R; Connors, Mark

    2013-10-01

    Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity. PMID:24030440

  1. 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

  2. Cinnamomum zeylanicum extract on the radiolabelling of blood constituents and the morphometry of red blood cells: In vitro assay

    Energy Technology Data Exchange (ETDEWEB)

    Benarroz, M.O. [Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, 4o Andar, Vila Isabel, 20551-030 Rio de Janeiro, RJ (Brazil); Universidade Federal do Rio Grande do Norte, Programa de Pos-Graduacao em Ciencias da Saude, Avenida General Gustavo Cordeiro de Farias, s/n, 59010-180 Natal, RN (Brazil); Fonseca, A.S. [Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, 4o Andar, Vila Isabel, 20551-030 Rio de Janeiro, RJ (Brazil)], E-mail: adenilso@uerj.br; Rocha, G.S. [Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, 4o Andar, Vila Isabel, 20551-030 Rio de Janeiro, RJ (Brazil); Frydman, J.N.G. [Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, 4o Andar, Vila Isabel, 20551-030 Rio de Janeiro, RJ (Brazil); Universidade Federal do Rio Grande do Norte, Programa de Pos-Graduacao em Ciencias da Saude, Avenida General Gustavo Cordeiro de Farias, s/n, 59010-180 Natal, RN (Brazil); Rocha, V.C.; Pereira, M.O. [Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, 4o Andar, Vila Isabel, 20551-030 Rio de Janeiro, RJ (Brazil)] (and others)

    2008-02-15

    Effects of Cinnamomum zeylanicum (cinnamon) on the labelling of blood constituents with technetium-99 m({sup 99m}Tc) and on the morphology of red blood cells were studied. Blood samples from Wistar rats were incubated with cinnamon extract for 1hour or with 0.9% NaCl, as control. Labelling of blood constituents with {sup 99m}Tc was performed. Plasma (P) and blood cells (BC), soluble (SF-P and SF-BC) and insoluble (IF-P and IF-BC) fractions were separated. The radioactivity in each fraction was counted and the percentage of radioactivity incorporated (%ATI) was calculated. Blood smears were prepared, fixed, stained and the qualitative and quantitative morphological analysis of the red blood cells was evaluated. The data showed that the cinnamon extract decreased significantly (p<0.05) the %ATI on BC, IF-P and IF-BC. No modifications were verified on shape of red blood cells. Cinnamon extracts could alter the labelling of blood constituents with {sup 99m}Tc, and although our results were obtained with animals, precaution is suggested in interpretations of nuclear medicine examinations involving the labelling of blood constituents in patients who are using cinnamon.

  3. Harvesting, processing and inventory management of peripheral blood stem cells

    Directory of Open Access Journals (Sweden)

    Mijovic Aleksandar

    2007-01-01

    Full Text Available By 2003, 97% autologous transplants and 65% of allogeneic transplants in Europe used mobilised peripheral blood stem cells (PBSC. Soon after their introduction in the early 1990′s, PBSC were associated with faster haemopoietic recovery, fewer transfusions and antibiotic usage, and a shorter hospital stay. Furthermore, ease and convenience of PBSC collection made them more appealing than BM harvests. Improved survival has hitherto been demonstrated in patients with high risk AML and CML. However, the advantages of PBSC come at a price of a higher incidence of extensive chronic GVHD. In order to be present in the blood, stem cells undergo the process of "mobilisation" from their bone marrow habitat. Mobilisation, and its reciprocal process - homing - are regulated by a complex network of molecules on the surface of stem cells and stromal cells, and enzymes and cytokines released from granulocytes and osteoclasts. Knowledge of these mechanisms is beginning to be exploited for clinical purposes. In current practice, stem cell are mobilised by use of chemotherapy in conjunction with haemopoietic growth factors (HGF, or with HGF alone. Granulocyte colony stimulating factor has emerged as the single most important mobilising agent, due to its efficacy and a relative paucity of serious side effects. Over a decade of use in healthy donors has resulted in vast experience of optimal dosing and administration, and safety matters. PBSC harvesting can be performed on a variety of cell separators. Apheresis procedures are nowadays routine, but it is important to be well versed in the possible complications in order to avoid harm to the patient or donor. To ensure efficient collection, harvesting must begin when sufficient stem cells have been mobilised. A rapid, reliable, standardized blood test is essential to decide when to begin harvesting; currently, blood CD34+ cell counting by flow cytometry fulfils these criteria. Blood CD34+ cell counts strongly

  4. Secretome of Peripheral Blood Mononuclear Cells Enhances Wound Healing

    OpenAIRE

    Mildner, Michael; Hacker, Stefan; Haider, Thomas; Gschwandtner, Maria; Werba, Gregor; Barresi, Caterina; Zimmermann, Matthias; Golabi, Bahar; Tschachler, Erwin; Ankersmit, Hendrik Jan

    2013-01-01

    Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SECPBMC). Six mm punch biopsy wounds were set on ...

  5. Flow of red blood cells in capillary networks

    OpenAIRE

    Couto, Ana; Teixeira, Lúcia; Leble, Vladimir; Lima, R.; Ribeiro, António E.; Dias, Ricardo

    2011-01-01

    In the present work we have studied the flow of red blood cells through a column packed with soda lime glass spheres with diameter of 337.5 micron (pore diameter 150 micron). The ratio between the average velocity of the RBCs and the average velocity of the carrying fluid (physiological saline) was close to 0.9. The RBCs migrated faster through the column than the carrying fluid mainly due to a hydrodynamic chromatographic effect.

  6. Effect of Irradiation on Microparticles in Red Blood Cell Concentrates

    OpenAIRE

    Cho, Chi Hyun; Yun, Seung Gyu; Koh, Young Eun; Lim, Chae Seung

    2016-01-01

    Changes in microparticles (MP) from red blood cell (RBC) concentrates in the context of irradiation have not been investigated. The aim of this study was to evaluate how irradiation affects the number of MPs within transfusion components. Twenty RBC concentrates, within 14 days after donation, were exposed to gamma rays (dose rate: 25 cGy) from a cesium-137 irradiator. Flow cytometry was used to determine the numbers of MPs derived from RBC concentrates before and 24 hr after irradiation. The...

  7. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    Science.gov (United States)

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  8. Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHEN XU; MIN XIONG SHEN; DONG ZHU MA; LI YING WANG; XI LIANG ZHA

    2003-01-01

    Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 × l06 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6~8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6~8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.

  9. Mitochondrial DNA mutations in single human blood cells.

    Science.gov (United States)

    Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

    2015-09-01

    Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. PMID:26149767

  10. Proteome complexity and the forces that drive proteome imbalance.

    Science.gov (United States)

    Harper, J Wade; Bennett, Eric J

    2016-01-01

    The cellular proteome is a complex microcosm of structural and regulatory networks that requires continuous surveillance and modification to meet the dynamic needs of the cell. It is therefore crucial that the protein flux of the cell remains in balance to ensure proper cell function. Genetic alterations that range from chromosome imbalance to oncogene activation can affect the speed, fidelity and capacity of protein biogenesis and degradation systems, which often results in proteome imbalance. An improved understanding of the causes and consequences of proteome imbalance is helping to reveal how these systems can be targeted to treat diseases such as cancer. PMID:27629639

  11. 77 FR 22791 - Advisory Council on Blood Stem Cell Transplantation; Notice of Meeting

    Science.gov (United States)

    2012-04-17

    ... HUMAN SERVICES Health Resources and Services Administration Advisory Council on Blood Stem Cell... Health Service Act, as amended), the Advisory Council on Blood Stem Cell Transplantation (ACBSCT) advises... Thawing and Washing, (4) Access to Transplantation, and (5) Advancing Hematopoietic Stem...

  12. Mapping the chromatographic behavior of a cell proteome utilizing orthogonal routines: the influence of feedstock pH

    Directory of Open Access Journals (Sweden)

    Rosa Cabrera

    2008-08-01

    Full Text Available Surface charge, molecular weight, and folding state are known to influence protein chromatographic behavior onto ion-exchangers. Experimentally, information related to such factors can be gathered via two-dimensional electrophoretic (2-DE methods. The separation behavior depicted by the insect cultured-cells proteome, which is an important host for recombinant protein production, was explored in this study. Experimental evidence showed a correlation between apparent isoelectric point distributions and the mobile phase conductivity. It was observed that the information contained in the isoelectric point (pI value(s obtained with a 2-DE routine showed a good correlation with the IEX chromatographic behavior, for a number of commercial adsorbents. This correlation was observed irrespective of the pH of the feedstock within the range 6 to 8. An initial prediction of protein ion-exchange chromatographic behavior could be possible utilizing an experimental approach based on the mentioned orthogonal methods. This technique is providing information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.Keywords: Insect cells, proteome, chromatography, ion-exchange, bioprocessingReceived: 3 August 2008 / Received in revised form: 15 August 2008, Accepted: 19 August 2008, Published online: 20 August 2008

  13. Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

    2012-04-06

    Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  15. Blood cell mitochondrial DNA content and premature ovarian aging.

    Directory of Open Access Journals (Sweden)

    Marco Bonomi

    Full Text Available Primary ovarian insufficiency (POI is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA content in a group of women undergoing ovarian hyperstimulation (OH, and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF and 42 poor responders (PR to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001 in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.

  16. Cost-effective and Rapid Blood Analysis on a Cell-phone

    OpenAIRE

    Zhu, Hongying; Sencan, Ikbal; Wong, Justin; Dimitrov, Stoyan; Tseng, Derek; Nagashima, Keita; Ozcan, Aydogan

    2013-01-01

    We demonstrate a compact and cost-effective imaging cytometry platform installed on a cell-phone for the measurement of the density of red and white blood cells as well as hemoglobin concentration in human blood samples. Fluorescent and bright-field images of blood samples are captured using separate optical attachments to the cell-phone and are rapidly processed through a custom-developed smart application running on the phone for counting of blood cells and determining hemoglobin density. W...

  17. Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

    Science.gov (United States)

    Xian, Feng; Zi, Jin; Wang, Quanhui; Lou, Xiaomin; Sun, Haidan; Lin, Liang; Hou, Guixue; Rao, Weiqiao; Yin, Changcheng; Wu, Lin; Li, Shuwei; Liu, Siqi

    2016-08-01

    Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible. PMID:27234506

  18. Interleukin-15 Promotes the Commitment of Cord Blood CD34+ Stem Cells into NK Cells

    Institute of Scientific and Technical Information of China (English)

    张建; 夏青; 孙汭; 田志刚

    2004-01-01

    To explore the effect of rhlL-15 on CB-CD34+ stem cells committing to NK cells, CD34+ stem cells were obtained from cord blood (CB) by magnetic-assisted cell sorting (MACS) method. CD3, CD16 and CD56 molecules expressed on cell surface were detected by flow cytometer. MTF method was used to test the cytotoxicity of NK cells. The results were that stem cell factor (SCF) alone has no effect on CD34+ stem cells. IL-15 stimulated CD34+ stem cells commit to NK cells, and SCF showed strong synergistic effect with IL-15. It was concluded that IL-15 and SCF played different roles during NK cell development, llr15 promoted CD34+ stem cells differentiate to NK cell precursor and SCF improved the effectsof IL-15 on NK cell differentiation.

  19. Some technetium complexes for labelling red blood cells

    International Nuclear Information System (INIS)

    A new approach to produce technetium labelled red blood cells, used routinely in diagnostic nuclear medicine, is reported. The enzyme Carbonic Anhydrase (CA), present in erythrocytes, is strongly inhibited by primary aromatic sulphonamides, which bind at the enzyme active site. Three types of ligand able to coordinate to technetium and suitable for modification to include a primary aromatic sulphonamide group were studied; bis(thiosemicarbazones), Schiff bases and some propylene amine oximes. The experimental conditions needed to label the ligands were determined. Both the thiosemicarbazone and propyleneamine oxime derivatives were labelled, but under no conditions attempted were the Schiff bases complexed by Technetium. The two major isozymes of Human Carbonic Anhydrase, HCA I and HCA II, were isolated from blood. The strength of binding of the free ligands SET, PN130 and PN135 with each of the isozymes was measured and expressed as the Dissociation Constant Kd. The rate of uptake of the technetium complexes into washed RBCs and whole blood was measured and found to be much slower in whole blood. The biodistribution of both TcPN130 and TcPN135 in rats was determined and scintigraphic images for the TcPN130 complex were recorded. Attempts to synthesise the Tc-99 analogues on the milligram scale to allow chemical characterisation of these complexes were unsuccessful. (author)

  20. Some technetium complexes for labelling red blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Emery, M.F.

    1988-01-01

    A new approach to produce technetium labelled red blood cells, used routinely in diagnostic nuclear medicine, is reported. The enzyme Carbonic Anhydrase (CA), present in erythrocytes, is strongly inhibited by primary aromatic sulphonamides, which bind at the enzyme active site. Three types of ligand able to coordinate to technetium and suitable for modification to include a primary aromatic sulphonamide group were studied; bis(thiosemicarbazones), Schiff bases and some propylene amine oximes. The experimental conditions needed to label the ligands were determined. Both the thiosemicarbazone and propyleneamine oxime derivatives were labelled, but under no conditions attempted were the Schiff bases complexed by Technetium. The two major isozymes of Human Carbonic Anhydrase, HCA I and HCA II, were isolated from blood. The strength of binding of the free ligands SET, PN130 and PN135 with each of the isozymes was measured and expressed as the Dissociation Constant K{sub d}. The rate of uptake of the technetium complexes into washed RBCs and whole blood was measured and found to be much slower in whole blood. The biodistribution of both TcPN130 and TcPN135 in rats was determined and scintigraphic images for the TcPN130 complex were recorded. Attempts to synthesise the Tc-99 analogues on the milligram scale to allow chemical characterisation of these complexes were unsuccessful. (author).

  1. Blood Flow through an Open-Celled Foam

    Science.gov (United States)

    Ortega, Jason; Maitland, Duncan

    2011-11-01

    The Hazen-Dupuit-Darcy (HDD) equation is commonly used in engineering applications to model the pressure gradient of flow through a porous media. One major advantage of this equation is that it simplifies the complex geometric details of the porous media into two coefficients: the permeability, K, and form factor, C. However through this simplification, the flow details within the porous media are no longer accessible, making it difficult to study the phenomena that contribute to changes in K and C due to clotting of blood flow. To obtain a more detailed understanding of blood flow through a porous media, a direct assessment of the complex interstitial geometry and flow is required. In this study, we solve the Navier-Stokes equations for Newtonian and non-Newtonian blood flow through an open-celled foam geometry obtained from a micro-CT scan. The nominal strut size of the foam sample is of O(10e-5) m and the corresponding Reynolds number based upon this length ranges up to O(10). Fitting the pressure gradient vs. Darcy velocity data with the HDD equation demonstrates that both viscous and inertial forces play an important role in the flow through the foam at these Reynolds numbers. Recirculation zones are observed to form in the wake of the pore struts, producing regions of flow characterized by both low shear rates and long fluid residence times, factors of which have been shown in previous studies to promote blood clotting.

  2. 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

  3. Utilization and quality of cryopreserved red blood cells in transfusion medicine

    NARCIS (Netherlands)

    Henkelman, S.; Noorman, F.; Badloe, J. F.; Lagerberg, J. W. M.

    2015-01-01

    Cryopreserved (frozen) red blood cells have been used in transfusion medicine since the Vietnam war. The main method to freeze the red blood cells is by usage of glycerol. Although the usage of cryopreserved red blood cells was promising due to the prolonged storage time and the limited cellular det

  4. Transplantation? Peripheral Stem Cell/Bone Marrow/Cord Blood

    Directory of Open Access Journals (Sweden)

    Itır Sirinoglu Demiriz

    2012-01-01

    Full Text Available The introduction of peripheral stem cell (PSC and cord blood (CB as an alternative to bone marrow (BM recently has caused important changes on hematopoietic stem cell transplantation (HSCT practice. According to the CIBMTR data, there has been a significant decrease in the use of bone marrow and increase in the use of PSC and CB as the stem cell source for HSCT performed during 1997–2006 period for patients under the age of 20. On the other hand, the stem cell source in 70% of the HSCT procedures performed for patients over the age of 20 was PSC and the second most preferred stem cell source was bone marrow. CB usage is very limited for the adult population. Primary disease, stage, age, time and urgency of transplantation, HLA match between the patient and the donor, stem cell quantity, and the experience of the transplantation center are some of the associated factors for the selection of the appropriate stem cell source. Unfortunately, there is no prospective randomized study aimed to facilitate the selection of the correct source between CB, PSC, and BM. In this paper, we would like to emphasize the data on stem cell selection in light of the current knowledge for patient populations according to their age and primary disease.

  5. Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

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    Reshu Saxena

    Full Text Available Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1 through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01 in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1, VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61 and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein

  6. Quantitative Proteomic Analysis of Bromotetrandrine and Tetrandrine in K562 Cell Line Using 18O-labeling Method

    Institute of Scientific and Technical Information of China (English)

    TAN Ying; GE Zhi-qiang; LIU Chang-xiao

    2012-01-01

    Objective To compare quantitative proteomic analysis of bromotetrandrine (W198) which was a Class Ⅰ new antitumor drug in China and tetrandrine (Tet) in K562 cell line using 18O-labeling method.Methods To illustrate its mechanism,a shotgun quantitative proteomic strategy employing 2D LC-MS-MS and trypsin catalyzed 18O-labeling quantification was carried out in this study.Compared to normal chronic leukemia cell line K562 and K562 induced by Tet,the proteomic changes of K562 induced by W198 were investigated.In order to validate the quantitation by the 18O-labeling,the analysis was done on an equivalent sample composed of the same amount of labeled and unlabeled proteins from normally cultured cells to act as a reference to the comparative sample.Results A threshold of ± 2-fold change for deciding whether a protein concentration was changed was settled for the following experiments.Comparing the 105 identified soluble proteins' expression levels of the apoptosis starting up K562 cells after W198 induction with the normally cultured cells,16 proteins were found with significantly altered expression levels after W198 treatment.Eight proteins were up-expressed including HMGB2,peroxiredoxin-2,and eIF4A-I,etc.Eight proteins were down-expressed including TCP-1,GRP94,GST-π,and SFGHs,etc.Compared to K562 induced by Tet,eight proteins of K562 were found with significantly altered expression levels after W198 treatment.Five proteins were up-expressed including HSP 90-β and 40S ribosomal protein S15a,etc.Three proteins were down-expressed including phosphoglycerate kinase 1,isoform 5 of interleukin enhancer-binding factor 3,etc.Conclusion The 18O-labeling MS-MS-based method is ideal as a discovery tool,but it is not suitable for validation using a large number of samples.Other more effective methods,such as Western blotting should be used for further validation of candidate cancer proteins discovered from 18O-labeling samples.In total,105 soluble proteins were discovered

  7. MEASUREMENT OF REGIONAL BONE BLOOD FLOW IN THE CANINE MANDIBULAR RAMUS USING RADIOLABELLED TOAD RED BLOOD CELLS

    Institute of Scientific and Technical Information of China (English)

    毛驰; 王翰章

    1994-01-01

    Toad red blood cells were used to measure regional bone blood flow in the canine mandibular ramus.The blood cells were labelled with sodium pertechnetate and fixed in 10% formalin;they were 22×15 μm in size and had a specific gravity close to that of dog red blood cells.These cells had no discernible effect on systemic hemody-namics after injection,did not agglutinate,were well mixed and evenly distributed throughout the body,and were completely extracted in one circulation through the mandible.The mandibular ramus was divided into six regions,and the blood flow rates in each were found to be similar to those reported in previous studies with radiolabelled carbonized,microspheres.Furthermore,the blood flow distribution pattern of the mandibular ramus determined in this study was identical to that of our previous study using the bone-seeking radionuclide method.We suggest that radiolabelled toad red blood cells are an ideal marker for measuring regional blood flow in the canine mandible.

  8. A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

    Directory of Open Access Journals (Sweden)

    Duban-Deweer Sophie

    2010-11-01

    Full Text Available Abstract Background Brain capillary endothelial cells (BCECs form the physiological basis of the blood-brain barrier (BBB. The barrier function is (at least in part due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein. Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

  9. Isolation of rare tumor cells from blood cells with buoyant immuno-microbubbles.

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    Guixin Shi

    Full Text Available Circulating tumor cells (CTCs are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs. MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM antibody. EpCAM-targeted MBs efficiently (85% and rapidly (within 15 minutes bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88% isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77% isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.

  10. Chaperone proteins identified from synthetic proteasome inhibitor-induced inclusions in PC12 cells by proteomic analysis

    Institute of Scientific and Technical Information of China (English)

    Xing'an Li; Yinjiu Zhang; Yihong Hu; Ming Chang; Tao Liu; Danping Wang; Yu Zhang; Lei Zhang; Linsen Hu

    2008-01-01

    Chaperone proteins are significant in Lewy bodies, but the profile of chaperone proteins is incompletely unraveled.Protcomic analysis is used to determine protein candidates for further study. Here, to identify potential chaperone proteins from agent-induced inclusions, we carried out proteomic analysis of artificially synthetic proteasome inhibitor (PSI)-induced inclusions formed in PC12 cells exposed to 10 μM PSI for 48 h. Using biochemical fractionation, 2-D electrophoresis, and identification through peptide mass fingerprints searched against multiple protein databases, we repeatedly identified eight reproducible chaperone proteins from the PSI-induced inclusions. Of these, 58 kDa glucose regulated protein, 75 kDa glucose regulated protein, and caldum-binding protein I were newly identified. The other five had been reported to be consistent components of Lewy bodies. These findings suggested that the three potential chaperone proteins might be recruited to PSI-induced inclusions in PC12 cells under proteasome inhibition.

  11. Environmental proteomics and metallomics.

    Science.gov (United States)

    López-Barea, Juan; Gómez-Ariza, José Luis

    2006-04-01

    Monitoring environmental pollution using biomarkers requires detailed knowledge about the markers, and many only allow a partial assessment of pollution. New proteomic methods (environmental proteomics) can identify proteins that, after validation, might be useful as alternative biomarkers, although this approach also has its limitations, derived mainly from their application to non-model organisms. Initial studies using environmental proteomics were carried out in animals exposed to model pollutants, and led to the concept of protein expression signatures. Experiments have been carried out in model organisms (yeast, Arabidopsis, rat cells, or mice) exposed to model contaminants. Over the last few years, proteomics has been applied to organisms from ecosystems with different pollution levels, forming the basis of an environmental branch in proteomics. Another focus is connected with the presence of metals bound to biomolecules, which adds an additional dimension to metal-biomolecule and metalloprotein characterization - the field of metallomics. The metallomic approach considers the metallome: a whole individual metal or metalloid species within a cell or tissue. A metallomic analytical approach (MAA) is proposed as a new tool to study and identify metalloproteins.

  12. Use of cryopreserved peripheral mononuclear blood cells in biomonitoring

    DEFF Research Database (Denmark)

    Risom, Lotte; Knudsen, Lisbeth E.

    1999-01-01

    This study was performed to investigate the effect of storing blood samples by freezing on selected biomarkers and possible implications for biomonitoring. Comparative measurements were performed in order to investigate the use of cryopreserved vs. freshly separated peripheral mononuclear blood c...... correlation of frequencies was seen when comparing fresh with cryopreserved samples. Furthermore we recommend fresh human plasma used in UDS incubation media........ We measured the DNA repair activity as dimethylsulfate induced unscheduled DNA synthesis (UDS) in PMBC incubated with either autologous plasma or fetal bovine serum (FBS). Comparison of the hprt mutant frequency by the T cell cloning assay was made in parallel. Finally the content of B....../T-lymphocytes and monocytes was measured in phytohemaglutinin (PHA)-stimulated cultures at different time intervals. The results showed a higher DNA repair activity in cryopreserved samples compared with fresh samples. We also found differences in mutant frequencies with higher values in fresh samples. A significant...

  13. Analysis of White Blood Cell Dynamics in Nailfold Capillaries

    Science.gov (United States)

    Bourquard, Aurélien; Butterworth, Ian; Sánchez-Ferro, Alvaro; Giancardo, Luca; Soenksen, Luis; Cerrato, Carolina; Flores, Rafael; Castro-González, Carlos

    2016-01-01

    Based on video data acquired with low-cost, portable microscopy equipment, we introduce a semi-automatic method to count visual gaps in the blood flow as a proxy for white blood cells (WBC) passing through nailfold capillaries. Following minimal user interaction and a pre-processing stage, our method consists in the spatio-temporal segmentation and analysis of capillary profiles. Besides the mere count information, it also estimates the speed associated with every WBC event. The accuracy of our algorithm is validated through the analysis of two capillaries acquired from one healthy subject. Results are compared with manual counts from four human raters and confronted with related physiological data reported in literature. PMID:26738019

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

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    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

  15. Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target.

    Science.gov (United States)

    Toman, O; Kabickova, T; Vit, O; Fiser, R; Polakova, K Machova; Zach, J; Linhartova, J; Vyoral, D; Petrak, J

    2016-09-01

    Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib‑resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients. PMID:27430982

  16. Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

    Energy Technology Data Exchange (ETDEWEB)

    Joo, Hyung Joon; Seo, Ha-Rim [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of); Jeong, Hyo Eun [Department of Mechanical Engineering, Korea University, Seoul (Korea, Republic of); Choi, Seung-Cheol; Park, Jae Hyung; Yu, Cheol Woong; Hong, Soon Jun [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of); Chung, Seok [Department of Mechanical Engineering, Korea University, Seoul (Korea, Republic of); Lim, Do-Sun, E-mail: dslmd@kumc.or.kr [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of)

    2014-07-11

    Highlights: • Two distinct vascular progenitor cells are induced from adult peripheral blood. • ECFCs induce vascular structures in vitro and in vivo. • SMPCs augment the in vitro and in vivo angiogenic potential of ECFCs. • Both cell types have synergistic therapeutic potential in ischemic hindlimb model. - Abstract: Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood.

  17. Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

    Directory of Open Access Journals (Sweden)

    Schriemer David C

    2010-11-01

    Full Text Available Abstract Background Papaver somniferum (opium poppy is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. Results A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. Conclusions The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a

  18. Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

    Directory of Open Access Journals (Sweden)

    José B Gama

    2014-08-01

    Full Text Available 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 revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1 and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1. In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

  19. Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

    Science.gov (United States)

    Gama, José B; Ohlmeier, Steffen; Martins, Teresa G; Fraga, Alexandra G; Sampaio-Marques, Belém; Carvalho, Maria A; Proença, Fernanda; Silva, Manuel T; Pedrosa, Jorge; Ludovico, Paula

    2014-08-01

    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 revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis. PMID:25101965

  20. Effect of adipose-derived stromal cells and BMP12 on intrasynovial tendon repair: A biomechanical, biochemical, and proteomics study.

    Science.gov (United States)

    Gelberman, Richard H; Shen, Hua; Kormpakis, Ioannis; Rothrauff, Benjamin; Yang, Guang; Tuan, Rocky S; Xia, Younan; Sakiyama-Elbert, Shelly; Silva, Matthew J; Thomopoulos, Stavros

    2016-04-01

    The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor- and cell-based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin-based scaffold. Control groups consisted of repair-only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC + BMP12 treatment for range of motion or tensile properties outcomes versus repair-only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC + BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC + BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC + BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions. PMID:26445383