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Sample records for plasma protein expression

  1. Expression of Recombinant Pregnancy-associated Plasma Protein-A

    Institute of Scientific and Technical Information of China (English)

    CHENG; Bin-yan; LI; Zi-ying; ZHANG; Xue-feng; LIU; Yi-bing

    2013-01-01

    Pregnancy-associated plasma protein-A(PAPP-A)is producted by the syntrophoblast tissue of the placenta and decidual cells.It belongs to macromolecular glycoprotein.PAPP-A is a sensitive serum marker of Down’s syndrome and has clinical valuable in the early identification of acute coronary syndrome(ACS).According to the structure of PAPP-A,PAPP-A DNA is divided into five segments(S1-S5)for

  2. Expression of Bovine Leukemia Virus Genome is Blocked by a Nonimmunoglobulin Protein in Plasma from Infected Cattle

    Science.gov (United States)

    Gupta, P.; Ferrer, J. F.

    1982-01-01

    Plasma of cattle infected with bovine leukemia virus contains a soluble factor that blocks the expression of the viral genome in cultured lymphocytes. The blocking factor is not present in plasma of bovine leukemia virus-free cattle or of cattle infected with common bovine viruses. Blocking of bovine leukemia virus expression by the plasma factor is reversible, and seems to be mediated by a nonimmunoglobulin protein molecule.

  3. Heme protein-induced tubular cytoresistance: expression at the plasma membrane level.

    Science.gov (United States)

    Zager, R A

    1995-05-01

    Following experimental rhabdomyolysis, animals become resistant to heme protein-induced acute renal failure (ARF). The goals of this study were to: (a) ascertain whether this resistance, previously documented only in vivo, is expressed directly at the proximal tubular cell level; (b) determine whether heme proteinuria (vs. other consequences of rhabdomyolysis) is its trigger; and (c) ascertain some of its subcellular determinants. Rats were injected with a borderline toxic dose of glycerol and 24 hours later proximal tubular segments (PTS) were isolated for study. Their vulnerability to diverse forms of injury (FeSO4-induced oxidant stress, hypoxia, Ca2+ ionophore, cytochalasin D, PLA2) was compared to that found in normal PTS. Post-glycerol PTS manifested significant resistance to each insult (decreased lactate dehydrogenase +/- N-acetyl-beta-D-glucosaminidase release). Protection against FeSO4 was virtually complete and it was associated with a 50% decrease in membrane lipid peroxidation. No decrease in hydroxyl radical generation was noted during the FeSO4 challenge (salicylate trap assessment), suggesting a primary increase in membrane resistance to attack. That PLA2 addition caused less deacylation, plasma membrane enzyme (alanine aminopeptidase) release, and LDH leakage from post-glycerol versus normal tubules supported this hypothesis. To test whether cytoresistance was specifically triggered by heme proteins (vs. being a non-specific filtered protein effect, or a result of endotoxin cascade activation), rats were injected with purified myoglobin, non-heme containing filterable proteins, or endotoxin. Only myoglobin induced cytoresistance. In vivo heme oxygenase inhibition (tin-protoporphyrin) did not block the emergence of cytoresistance and it was expressed despite Na,K-ATPase inhibition (ouabain) or cytoskeletal disruption (cytochalasin D). In vivo heat shock failed to protect. In conclusion, (1) rhabdomyolysis induces broad based proximal tubular

  4. Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses

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    Clavel François

    2008-02-01

    Full Text Available Abstract Background Numerous studies have shown that viral quasi-species with genetically diverse envelope proteins (Env replicate simultaneously in patients infected with the human immunodeficiency virus type 1 (HIV-1. Less information is available concerning the extent that envelope sequence diversity translates into a diversity of phenotypic properties, including infectivity and resistance to entry inhibitors. Methods To study these questions, we isolated genetically distinct contemporaneous clonal viral populations from the plasma of 5 HIV-1 infected individuals (n = 70, and evaluated the infectivity of recombinant viruses expressing Env proteins from the clonal viruses in several target cells. The sensitivity to entry inhibitors (enfuvirtide, TAK-799, soluble CD4 and monoclonal antibodies (2G12, 48d, 2F5 was also evaluated for a subset of the recombinant viruses (n = 20. Results Even when comparisons were restricted to viruses with similar tropism, the infectivity for a given target cell of viruses carrying different Env proteins from the same patient varied over an approximately 10-fold range, and differences in their relative ability to infect different target cells were also observed. Variable region haplotypes associated with high and low infectivity could be identified for one patient. In addition, clones carrying unique mutations in V3 often displayed low infectivity. No correlation was observed between viral infectivity and sensitivity to inhibition by any of the six entry inhibitors evaluated, indicating that these properties can be dissociated. Significant inter-patient differences, independent of infectivity, were observed for the sensitivity of Env proteins to several entry inhibitors and their ability to infect different target cells. Conclusion These findings demonstrate the marked functional heterogeneity of HIV-1 Env proteins expressed by contemporaneous circulating viruses, and underscore the advantage of clonal analyses in

  5. Sperm nuclear DNA fragmentation rate is associated with differential protein expression and enriched functions in human seminal plasma.

    Science.gov (United States)

    Intasqui, Paula; Camargo, Mariana; Del Giudice, Paula T; Spaine, Deborah M; Carvalho, Valdemir M; Cardozo, Karina H M; Zylbersztejn, Daniel S; Bertolla, Ricardo P

    2013-10-01

    To analyse the proteomic profile of seminal plasma with the aim of identifying the proteins and post-genomic pathways associated with sperm DNA fragmentation. A cross-sectional study including 89 subjects from a human reproduction service was carried out. All semen samples were assessed for sperm DNA fragmentation using a comet assay. Results from 60 sperm were analysed using Komet 6.0.1 software and the 'Olive tail moment' variable was used to stratify these into low and high sperm DNA fragmentation groups. Seminal plasma proteins from the two groups were pooled and used for proteomic analysis. Quantitative data were used for functional enrichment studies. Seventy-two proteins were identified or quantified in seminal plasma. Of these, nine were differentially expressed in the low group and 21 in the high group. Forty-two proteins were conserved between these groups. Functional enrichment analysis indicated that sperm DNA fragmentation was related to functions such as lipoprotein particle remodelling and regulation, fatty acid binding and immune response. Proteins found exclusively in the low group may be involved in correcting spermatogenesis and/or improving sperm function. Proteins in the high group were associated with increased innate immune response, sperm motility and/or maturation and inhibition of mitochondrial apoptosis. Protein expression and post-genomic pathways of seminal plasma differ according to the rate of sperm DNA integrity. © 2013 The Authors. BJU International © 2013 BJU International.

  6. Differential protein expression in seminal plasma from fertile and infertile males

    Science.gov (United States)

    Cadavid J, Angela P.; Alvarez, Angela; Markert, Udo R.; Maya, Walter Cardona

    2014-01-01

    AIM: The aim of this study was to analyze human seminal plasma proteins in association with male fertility status using the proteomic mass spectrometry technology Surface-Enhanced Laser Desorption Ionization Time-of-Flight (SELDI-TOF-MS). MATERIALS AND METHODS: Semen analysis was performed using conventional methods. Protein profiles of the seminal plasma were obtained by SELDI-TOF mass spectrometry over a strong anion exchanger, ProteinChip® Q10 array. RESULTS AND CONCLUSION: We found statistically significant differences in motility and sperm count between fertile and infertile men. In addition, we observed ten seminal proteins that are significantly up-regulated in the infertile group. In conclusion, comparison of seminal plasma proteome in fertile and infertile men provides new aspects in the physiology of male fertility and might help in identifying novel markers of male infertility. PMID:25395747

  7. Leukocyte-Reduced Platelet-Rich Plasma Alters Protein Expression of Adipose Tissue-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Loibl, Markus; Lang, Siegmund; Hanke, Alexander; Herrmann, Marietta; Huber, Michaela; Brockhoff, Gero; Klein, Silvan; Nerlich, Michael; Angele, Peter; Prantl, Lukas; Gehmert, Sebastian

    2016-08-01

    Application of platelet-rich plasma and stem cells has become important in regenerative medicine. Recent literature supports the use of platelet-rich plasma as a cell culture media supplement to stimulate proliferation of adipose tissue-derived mesenchymal stem cells. The underlying mechanism of proliferation stimulation by platelet-rich plasma has not been investigated so far. Adipose tissue-derived mesenchymal stem cells were cultured in α-minimal essential medium supplemented with platelet-rich plasma or fetal calf serum. Cell proliferation was assessed with cell cycle kinetics using flow cytometric analyses after 48 hours. Differences in proteome expression of the adipose tissue-derived mesenchymal stem cells were analyzed using a reverse-phase protein array to quantify 214 proteins. Complementary Ingenuity Pathways Analysis and gene set enrichment analysis were performed using protein data, and confirmed by Western blot analysis. A higher percentage of adipose tissue-derived mesenchymal stem cells in the S phase in the presence of platelet-rich plasma advocates the proliferation stimulation. Ingenuity Pathways Analysis and gene set enrichment analysis confirm the involvement of the selected proteins in the process of cell growth and proliferation. Ingenuity Pathways Analysis revealed a participation in the top-ranked canonical pathways PI3K/AKT, PTEN, ILK, and IGF-1. Gene set enrichment analysis identified the authors' protein set as being part of significantly regulated protein sets with the focus on cell cycle, metabolism, and the Kyoto Encyclopedia of Genes and Genomes transforming growth factor-β signaling pathway. The present study provides evidence that platelet-rich plasma stimulates proliferation and induces a unique change in the proteomic profile of adipose tissue-derived mesenchymal stem cells. The interpretation of altered expression of regulatory proteins represents a step forward toward achieving good manufacturing practice-compliant criteria

  8. Label-Free Proteome Analysis of Plasma from Patients with Breast Cancer: Stage-Specific Protein Expression

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    Lobo, Marina Duarte Pinto; Moreno, Frederico Bruno Mendes Batista; Souza, Gustavo Henrique Martins Ferreira; Verde, Sara Maria Moreira Lima; Moreira, Renato de Azevedo; Monteiro-Moreira, Ana Cristina de Oliveira

    2017-01-01

    Breast cancer is one of the most commonly diagnosed types of cancer among women. Breast cancer mortality rates remain high probably because its diagnosis is hampered by inaccurate detection methods. Since changes in protein expression as well as modifications in protein glycosylation have been frequently reported in cancer development, the aim of this work was to study the differential expression as well as modifications of glycosylation of proteins from plasma of women with breast cancer at different stages of disease (n = 30) compared to healthy women (n = 10). A proteomics approach was used that depleted albumin and IgG from plasma followed by glycoprotein enrichment using immobilized Moraceae lectin (frutalin)-affinity chromatography and data-independent label-free mass spectrometric analysis. Data are available via ProteomeXchange with identifier PXD003106. As result, 57,016 peptides and 4,175 proteins among all samples were identified. From this, 40 proteins present in unbound (PI—proteins that did not interact with lectin) and bound (PII—proteins that interacted with lectin) fractions were differentially expressed. High levels of apolipoprotein A-II were detected here that were elevated significantly in the early and advanced stages of the disease. Apolipoprotein C-III was detected in both fractions, and its level was increased slightly in the PI fraction of patients with early-stage breast cancer and expressed at higher levels in the PII fraction of patients with early and intermediate stages. Clusterin was present at higher levels in both fractions of patients with early and intermediate stages of breast cancer. Our findings reveal a correlation between alterations in protein glycosylation, lipid metabolism, and the progression of breast cancer. PMID:28210565

  9. Protein Disulfide Isomerase Chaperone ERP-57 Decreases Plasma Membrane Expression of the Human GnRH Receptor

    Science.gov (United States)

    Yánez, Rodrigo Ayala; Conn, P. Michael

    2012-01-01

    Retention of misfolded proteins by the endoplasmic reticulum (ER) is a quality control mechanism involving the participation of endogenous chaperones such as calnexin (CANX) which interact and restrict plasma membrane expression of gonadotropin releasing hormone receptor (GnRHR), a G protein coupled receptor. CANX also interacts with ERP-57, a thiol oxidoreductase chaperone present in the ER. CANX along with ERP-57, promotes the formation of disulfide bond bridges in nascent proteins. The human GnRH receptor (hGnRHR) is stabilized by two disulfide bond bridges (Cys14-Cys200 and Cys114-Cys196), that, when broken, its expression at plasma membrane decreases. To determine if the presence of chaperones CANX and ERP-57 exert an influence over membrane routing and second messenger activation, we assessed the effect of various mutants including those with broken bridges (Cys→Ala) along with the wild type hGnRHR. The effect of chaperones on mutants was insignificant, whereas the overexpression of ERP-57 led to a wild type hGnRHR retention which was further enhanced by cotransfection with CANX cDNA disclosing receptor retention by ERP-57 augmented by CANX, suggesting a quality control mechanism. PMID:20029959

  10. Mesenchymal stromal cell proliferation, gene expression and protein production in human platelet-rich plasma-supplemented media.

    Science.gov (United States)

    Amable, Paola Romina; Teixeira, Marcus Vinicius Telles; Carias, Rosana Bizon Vieira; Granjeiro, José Mauro; Borojevic, Radovan

    2014-01-01

    Platelet-rich plasma (PRP) is increasingly used as a cell culture supplement, in order to reduce the contact of human cells with animal-derived products during in vitro expansion. The effect of supplementation changes on cell growth and protein production is not fully characterized. Human mesenchymal stromal cells from bone marrow, adipose tissue and Wharton's Jelly were isolated and cultured in PRP-supplemented media. Proliferation, in vitro differentiation, expression of cell surface markers, mRNA expression of key genes and protein secretion were quantified. 10% PRP sustained five to tenfold increased cell proliferation as compared to 10% fetal bovine serum. Regarding cell differentiation, PRP reduced adipogenic differentiation and increased calcium deposits in bone marrow and adipose tissue-mesenchymal stromal cells. Wharton's Jelly derived mesenchymal stromal cells secreted higher concentrations of chemokines and growth factors than other mesenchymal stromal cells when cultured in PRP-supplemented media. Bone marrow derived mesenchymal stromal cells secreted higher concentrations of pro-inflammatory and pro-angiogenic proteins. Mesenchymal stromal cells isolated from adipose tissue secreted higher amounts of extracellular matrix components. Mesenchymal stromal cells purified from different tissues have distinct properties regarding differentiation, angiogenic, inflammatory and matrix remodeling potential when cultured in PRP supplemented media. These abilities should be further characterized in order to choose the best protocols for their therapeutic use.

  11. Time course of hepatic gene expression and plasma vitellogenin protein concentrations in estrone-exposed juvenile rainbow trout (Oncorhynchus mykiss).

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    Osachoff, Heather L; Brown, Lorraine L Y; Tirrul, Leena; van Aggelen, Graham C; Brinkman, Fiona S L; Kennedy, Christopher J

    2016-09-01

    Estrone (E1), a natural estrogen hormone found in sewage effluents and surface waters, has known endocrine disrupting effects in fish, thus, it is a contaminant of emerging concern. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to an environmentally-relevant concentration of E1 (24ng/L E1 [0.1nM]) for 7d and then placed in clean water for a 9d recovery period. RNA sequencing showed transcripts from numerous affected biological processes (e.g. immune, metabolic, apoptosis, clotting, and endocrine) were altered by E1 after 4d of treatment. The time course of E1-inducible responses relating to vitellogenesis was examined daily during the two phases of exposure. Hepatic gene expression alterations evaluated by quantitative polymerase chain reaction (QPCR) were found during the treatment period for vitellogenin (VTG), vitelline envelope proteins (VEPs) α, β and γ, and estrogen receptor α1 (ERα1) transcripts. ERα1 was the only transcript induced each day during the treatment phase, thus it was a good indicator of E1 exposure. Gradual increases occurred in VEPβ and VEPγ transcripts, peaking at d7. VTG transcript was only elevated at d4, making it less sensitive than VEPs to this low-level E1 treatment. Inductions of ERα1, VEPα, VEPβ and VEPγ transcripts ceased 1d into the recovery phase. Plasma VTG protein concentrations were not immediately elevated but peaked 7d into the recovery phase. Thus, elevated vitellogenesis-related gene expression and protein production occurred slowly but steadily at this concentration of E1, confirming the sequence of events for transcripts and VTG protein responses to xenoestrogen exposure.

  12. Aggressive periodontitis and chronic arthritis: blood mononuclear cell gene expression and plasma protein levels of cytokines and cytokine inhibitors

    DEFF Research Database (Denmark)

    Sørensen, Lars K; Havemose-Poulsen, Anne; Bendtzen, Klaus

    2009-01-01

    BACKGROUND: Cytokines and cytokine inhibitors have been associated with many immunoinflammatory diseases. In the present study, we examined whether peripheral blood mononuclear cell (PBMC) gene expression mirrors the corresponding plasma levels of clinically important pro- and anti-inflammatory c......BACKGROUND: Cytokines and cytokine inhibitors have been associated with many immunoinflammatory diseases. In the present study, we examined whether peripheral blood mononuclear cell (PBMC) gene expression mirrors the corresponding plasma levels of clinically important pro- and anti...

  13. Oral lichen sclerosus expressing extracellular matrix proteins and IgG4-positive plasma cells.

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    De Aquino Xavier, Flavia Calo; Prates, Alisio Alves; Gurgel, Clarissa Araujo; De Souza, Tulio Geraldo; Andrade, Rodrigo Guimaraes; Goncalves Ramos, Eduardo Antonio; Pedreira Ramalho, Luciana Maria; Dos Santos, Jean Nunes

    2014-09-16

    Lichen sclerosus (LS) is a mucocutaneous disease with uncommon oral involvement. The etiology is not yet well understood, but LS has been associated with autoimmune, genetic, and immunological factors. We report a 47-year-old man with LS that exhibited an asymptomatic white plaque with red patches on the maxillary alveolar mucosa extending to the labial mucosa. He had no other skin disease. Positive immunostaining for tenascin and scarcity of fibronectin suggested extracellular matrix reorganization. Elastin immunostaining indicated a reduction of elastic fibers. Immunoexpression of collagen IV in blood vessels and its absence in the epithelial basement membrane, together with diffuse MMP-9 immunoexpression, suggested altered proteolytic activity. Mast cell staining bordering areas of sclerosis indicated a possible role in the synthesis of collagen. IgG4 positivity in plasma cells suggested a role in the fibrogenesis. This is an unusual presentation of oral LS and we discuss immunohistochemical findings regarding cellular and extracellular matrix components.

  14. Bone morphogenic protein 6: a member of a novel class of prognostic factors expressed by normal and malignant plasma cells inhibiting proliferation and angiogenesis

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    Seckinger, Anja; Meissner, Tobias; Moreaux, Jérôme; Goldschmidt, Hartmut; Fuhler, Gwenny M.; Benner, Axel; Hundemer, Michael; Rème, Thierry; Shaughnessy, John D.; Barlogie, Bart; Bertsch, Uta; Hillengass, Jens; Ho, Anthony D.; Pantesco, Véronique; Jauch, Anna; De Vos, John; Rossi, Jean-François; Möhler, Thomas; Klein, Bernard; Hose, Dirk

    2009-01-01

    Pathogenesis of multiple myeloma is associated with an aberrant expression of pro-proliferative, pro-angiogenic and bone-metabolism modifying factors by malignant plasma cells. Given the frequently long time-span from diagnosis of early-stage plasma cell dyscrasias to overt myeloma and the mostly low proliferation rate of malignant plasma cells, we hypothesize these likewise to express a novel class of inhibitory factors of potential prognostic relevance. Bone morphogenic proteins (BMPs) represent possible candidates as they inhibit proliferation, stimulate bone formation, and have impact on the survival of cancer patients. We assessed expression of BMPs and their receptors by Affymetrix DNA-microarrays (n=779) including CD138-purified primary myeloma cell samples (n=635) of previously untreated patients. BMP6 is the only BMP expressed by malignant and normal plasma cells. Its expression is significantly lower in proliferating myeloma cells, myeloma cell lines, or plasmablasts. BMP6 significantly inhibits proliferation of myeloma cell lines, survival of primary myeloma cells, and in vitro angiogenesis. High BMP6-expression in primary myeloma cell samples delineates significantly superior overall survival for patients undergoing high-dose chemotherapy independent of conventional prognostic factors (ISS-stage, beta-2-microglobulin). PMID:19718049

  15. Growth Arrest-Specific 6 Protein in Patients with Sjogren Syndrome: Determination of the Plasma Level and Expression in the Labial Salivary Gland.

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    Chen-Hung Chen

    Full Text Available Growth arrest-specific protein 6 (Gas6 is a vitamin K-dependent protein expressed by endothelial cells and leukocytes that are involved in cell survival, migration, and proliferation in response to inflammatory processes. The aim of this study was to assess the implications of Gas6 in Sjögren syndrome (SS and its expression in the labial salivary gland.A total of 254 adults, including 159 with primary Sjögren syndrome (pSS, 34 with secondary Sjögren syndrome (sSS, and 61 normal controls, were recruited. Plasma Gas6 concentrations were determined, and Gas6 expressions in labial salivary gland (LSG tissues from controls and pSS and sSS patients were also evaluated. Plasma Gas6 concentrations were significantly lower among patients with pSS than normal controls (13.5 ± 8.6 vs. 19.9 ± 13.4 ng/ml, p < 0.001. There were, however, no significant differences in plasma Gas6 levels between pSS and sSS patients (13.5 ± 8.6 vs. 16.9 ± 11.2 ng/ml, p = 0.068. In multivariate logistic regression analysis, after adjustment for white blood cell count, hemoglobin level, platelet count, lymphocyte count, and C3 and C4 levels, lower plasma Gas6 concentrations were significantly associated with an increased risk of SS. Moreover, by using a semi-quantitative scale to evaluate Gas6 expression in LSG tissues, Gas6 expression was found to be markedly lower in LSG tissues from pSS patients than in tissues from normal controls.Decreased plasma Gas6 concentration and LSG expression were associated with pSS. As such, Gas6 may represent a novel independent risk factor for pSS, with a potential role in salivary gland inflammation and dysfunction.

  16. Post-exercise skeletal muscle glycogen related to plasma cytokines and muscle IL-6 protein content, but not muscle cytokine mRNA expression

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    David Christopher Nieman

    2015-09-01

    Full Text Available Objectives: The purpose of this study was to correlate post-exercise muscle glycogen levels with changes in plasma cytokine, and muscle mRNA cytokine expression and protein content. Methods: Twenty-four male runners (age 36.5±1.8 y, VO2max 60.0±1.5 ml.kg.-1min-1 ran twice (separated by 4 weeks on treadmills to exhaustion at 70% VO2max (average time and distance of 2.24±0.09 h and 24.9±1.1 km. Muscle biopsies from the vastus lateralis and blood samples were collected before and after each run, with IL-6, IL-8, and MCP-1 measured in muscle (mRNA and protein and plasma. Data from the two runs were averaged. Results: Participants experienced a 35.3±4.2% decrease (P<0.001 in skeletal muscle glycogen content (67.5±2.8 to 44.3 ±3.7 mmol/kg wet weight. Muscle mRNA expression for IL-6, IL-8, and MCP-1 increased 7.34±0.90-, 13.9±2.3-, and 4.10±0.60- fold, respectively (all, P<0.001. Skeletal muscle IL-6, IL-8, and MCP-1 protein content increased 35.8±10.6%, 80.6±12.1%, and 105±17.9%, respectively (all P≤0.005. Plasma IL-6, IL-8, and MCP-1 increased 47.1±10.0-, 2.6±0.3-, and 1.6±0.1-fold, respectively (all, P<0.001. Post-exercise muscle glycogen concentrations were negatively correlated with run time to exhaustion (r=-0.70, P<0.001, and changes in muscle IL-6 protein content (r=-0.44, P=0.049, plasma IL-6 (r=-0.72, P<0.001, IL-8 (r=-0.60, P=0.002, and MCP-1 (r=-0.589, P=0.002, but not with changes in muscle IL-8 and MCP-1 protein content, or muscle mRNA expression for IL-6, IL-8, and MCP-1. Conclusions: Prolonged and intensive running increased muscle mRNA expression, muscle protein content, and plasma levels for IL-6, IL-8, and MCP-1, and post-run muscle glycogen levels were most strongly related to plasma cytokine levels.

  17. The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity

    OpenAIRE

    Manoharan, Christine; Wilson, Marieangela C.; Sessions, Richard B; Halestrap, Andrew P.

    2006-01-01

    Monocarboxylate transporters MCT1-MCT4 require basigin (CD147) or embigin (gp70), ancillary proteins with a glutamate residue in their single transmembrane (TM) domain, for plasma membrane (PM) expression and activity. Here we use site-directed mutagenesis and expression in COS cells or Xenopus oocytes to investigate whether this glutamate (Glu218 in basigin) may charge-pair with a positively charged TM-residue of MCT1. Such residues were predicted using a new molecular model of MCT1 based up...

  18. An estrogen-responsive plasma protein expression signature in Atlantic cod (Gadus morhua) revealed by SELDI-TOF MS

    DEFF Research Database (Denmark)

    Nielsen, Mari Mæland; Meyer, Sonnich; Larsen, Bodil Katrine

    2011-01-01

    Compound-specific protein expression signatures( PESs) can be revealed by proteomic techniques. The SELDI-TOF MS approach is advantageous due to its simplicity and high-throughput capacity,however, there are concerns regarding the reproducibility of this method. The aim of this study was to defin...

  19. Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

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    A.R.M. Sabbatini

    2011-02-01

    Full Text Available Histidine-rich glycoprotein (HRG is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD. We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

  20. Photoaffinity Labeling of Plasma Proteins

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    Masaki Otagiri

    2013-11-01

    Full Text Available Photoaffinity labeling is a powerful technique for identifying a target protein. A high degree of labeling specificity can be achieved with this method in comparison to chemical labeling. Human serum albumin (HSA and α1-acid glycoprotein (AGP are two plasma proteins that bind a variety of endogenous and exogenous substances. The ligand binding mechanism of these two proteins is complex. Fatty acids, which are known to be transported in plasma by HSA, cause conformational changes and participate in allosteric ligand binding to HSA. HSA undergoes an N-B transition, a conformational change at alkaline pH, that has been reported to result in increased ligand binding. Attempts have been made to investigate the impact of fatty acids and the N-B transition on ligand binding in HSA using ketoprofen and flunitrazepam as photolabeling agents. Meanwhile, plasma AGP is a mixture of genetic variants of the protein. The photolabeling of AGP with flunitrazepam has been utilized to shed light on the topology of the protein ligand binding site. Furthermore, a review of photoaffinity labeling performed on other major plasma proteins will also be discussed. Using a photoreactive natural ligand as a photolabeling agent to identify target protein in the plasma would reduce non-specific labeling.

  1. Leptin plasma concentrations, leptin gene expression, and protein localization in the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes of the European beaver (Castor fiber).

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    Chojnowska, Katarzyna; Czerwinska, Joanna; Kaminski, Tadeusz; Kaminska, Barbara; Kurzynska, Aleksandra; Bogacka, Iwona

    2017-01-01

    The European beaver (Castor fiber) is the largest seasonal free-living rodent in Eurasia. Since the physiology and endocrine system of this species remains unknown, the present study aimed to determine plasma leptin concentrations and the expression of the leptin gene and protein in the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal (HPG and HPA) axes of beavers during breeding (April), postbreeding (July), and prebreeding (November) seasons. Leptin plasma concentrations did not change in females, whereas in males, leptin plasma concentrations were higher in July than those in April. The presence of leptin mRNA and protein was found in all examined tissues. In females, leptin mRNA expression in the hypothalamus, pituitary, ovaries, and myometrium was markedly higher in July than that in April. In males, leptin mRNA levels varied across the examined tissues of the HPG and HPA. Leptin synthesis increased in the hypothalamus during breeding and postbreeding seasons, but seasonal changes were not observed in the pituitary. In turn, testicular leptin levels were higher during breeding and prebreeding stages. Seasonal differences in the concentrations of leptin mRNA were also observed in the adrenal cortex. In males, leptin mRNA levels were higher in November than those in April or July. In females, leptin synthesis increased in the adrenal cortex during pregnancy relative to other seasons. This is the first ever study to demonstrate seasonal differences in leptin expression in beaver tissues, and our results could suggest that leptin is involved in the regulation of the HPG and HPA axes during various stages of the reproductive cycle in beavers. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Age-related differences in plasma proteins: how plasma proteins change from neonates to adults.

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    Vera Ignjatovic

    Full Text Available The incidence of major diseases such as cardiovascular disease, thrombosis and cancer increases with age and is the major cause of mortality world-wide, with neonates and children somehow protected from such diseases of ageing. We hypothesized that there are major developmental differences in plasma proteins and that these contribute to age-related changes in the incidence of major diseases. We evaluated the human plasma proteome in healthy neonates, children and adults using the 2D-DIGE approach. We demonstrate significant changes in number and abundance of up to 100 protein spots that have marked differences in during the transition of the plasma proteome from neonate and child through to adult. These proteins are known to be involved in numerous physiological processes such as iron transport and homeostasis, immune response, haemostasis and apoptosis, amongst others. Importantly, we determined that the proteins that are differentially expressed with age are not the same proteins that are differentially expressed with gender and that the degree of phosphorylation of plasma proteins also changes with age. Given the multi-functionality of these proteins in human physiology, understanding the differences in the plasma proteome in neonates and children compared to adults will make a major contribution to our understanding of developmental biology in humans.

  3. Analysis of transcriptional regulation and tissue-specific expression of Avicennia marina Plasma Membrane Protein 3 suggests it contributes to Na(+) transport and homoeostasis in A. marina.

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    Chidambaram, Rajalakshmi; Venkataraman, Gayatri; Parida, Ajay

    2015-07-01

    Plasma membrane proteins (PMP3) play a role in cation homoeostasis. The 5' flanking sequence of stress inducible, Avicennia marina PMP3 (AmPMP3prom) was transcriptionally fused to (a) GUS or (b) GFP-AmPMP3 and analyzed in transgenic tobacco. Tissue-histochemical GUS and GFP:AmPMP3 localization are co-incident under basal and stress conditions. AmPMP3prom directed GUS activity is highest in roots. Basal transcription is conferred by a 388bp segment upstream of the translation start site. A 463bp distal enhancer in the AmPMP3prom confers enhanced expression under salinity in all tissues and also responds to increases in salinity. The effect of a central, stem-specific negative regulatory region is suppressed by the distal enhancer. The A. marina rhizosphere encounters dynamic changes in salinity at the inter-tidal interface. The complex, tissue-specific transcriptional responsiveness of AmPMP3 to salinity appears to have evolved in response to these changes. Under salinity, guard cell and phloem-specific expression of GFP:AmPMP3 is highly enhanced. Mesophyll, trichomes, bundle sheath, parenchymatous cortex and xylem parenchyma also show GFP:AmPMP3 expression. Cis-elements conferring stress, root and vascular-specific expression are enriched in the AmPMP3 promoter. Pronounced vascular-specific AmPMP3 expression suggests a role in salinity induced Na(+) transport, storage, and secretion in A. marina.

  4. Human plasma cells express granzyme B.

    Science.gov (United States)

    Xu, Wei; Narayanan, Priya; Kang, Ning; Clayton, Sandra; Ohne, Yoichiro; Shi, Peiqing; Herve, Marie-Cecile; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Gorvel, Jean-Pierre; Oh, Sangkon; Pascual, Virginia; Banchereau, Jacques

    2014-01-01

    While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

  5. Expression of recombinant murine pregnancy-associated plasma protein-A (PAPP-A) and a novel variant (PAPP-Ai) with differential proteolytic activity

    DEFF Research Database (Denmark)

    Søe, Rikke; Overgaard, Michael Toft; Thomsen, Anni R

    2002-01-01

    Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between...

  6. SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats.

    Science.gov (United States)

    Sabino-Silva, Robinson; Alves-Wagner, Ana B T; Burgi, Katia; Okamoto, Maristela M; Alves, Adilson S; Lima, Guilherme A; Freitas, Helayne S; Antunes, Vagner R; Machado, Ubiratan F

    2010-12-01

    Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (∼30%, P acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

  7. Comparative Plasma Protein Profiling of Hemoglobin H Disease

    Directory of Open Access Journals (Sweden)

    Kamonlak Leecharoenkiat

    2014-01-01

    Full Text Available HbH and HbH-constant spring (HbH-CS are the most common forms of α-thalassemia detected in the Thai population. The accumulation of excess β globin chains in these diseases results in increased red cell hemolysis, and patients with HbH-CS normally have a more severe clinical presentation than patients with HbH disease. This study aimed to detect alterations in the expression of plasma proteins of HbH and HbH-CS patients as compared to normal plasma. Platelet poor plasma was separated from HbH and HbH-CS and normal subjects and differential plasma proteins were detected using two-dimensional gel electrophoresis and identified using LC/MS/MS. A total of 14 differentially expressed proteins were detected of which 5 proteins were upregulated and 9 were downregulated. Most of the differentially expressed proteins are liver secreted proteins involved in hemolysis, oxidative stress response, and hemoglobin degradation. Seven proteins were found to be differentially expressed between HbH and HbH-CS. Levels of haptoglobin, a hemoglobin scavenging protein, were significantly increased in HbH patients as compared to HbH-CS patients. The identification of differentially expressed proteins may lead to a better understanding of the biological events underlying the clinical presentation of HbH and HbH-CS patients and can have application as hemolytic markers or severity predictors.

  8. The TNF-alpha system in heart failure and after heart transplantation: plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity

    OpenAIRE

    Riemsdijk-van Overbeeke, Iza; Baan, Carla; Niesters, Bert; Hesse, Cees; Loonen, E.H.M.; Weimar, Willem; Balk, Aggie; Maat, Alex

    1999-01-01

    textabstractBACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To assess the activity of the TNF-alpha system in patients with heart failure and after heart transplantation. METHODS: We measured TNF-alpha mRNA expression of peripheral blood mononuclear ce...

  9. Mating behavior induces changes of expression of Fos protein, plasma testosterone and androgen receptors in the accessory olfactory bulb (AOB) of the male mandarin vole Microtus mandarinus

    OpenAIRE

    Fengqin HE, Fadao TAI

    2009-01-01

    In order to investigate the neuroendocrine mechanism of the mating behavior in the adult male mandarin voles Microtus mandarinus, the radioimmunoassay (RIA) and immunohistochemistry methods were used to investigate the differences in plasma testosterone (T) concentrations and distribution of T immunoreactive neurons (T-IRs), androgen receptor immunoreactive neurons (AR-IRs) and Fos protein immunoreactive neurons (Fos-IRs) in the accessory olfactory bulb (AOB) and the main olfactory bulb (MOB)...

  10. Redox regulation of protein damage in plasma.

    Science.gov (United States)

    Griffiths, Helen R; Dias, Irundika H K; Willetts, Rachel S; Devitt, Andrew

    2014-01-01

    The presence and concentrations of modified proteins circulating in plasma depend on rates of protein synthesis, modification and clearance. In early studies, the proteins most frequently analysed for damage were those which were more abundant in plasma (e.g. albumin and immunoglobulins) which exist at up to 10 orders of magnitude higher concentrations than other plasma proteins e.g. cytokines. However, advances in analytical techniques using mass spectrometry and immuno-affinity purification methods, have facilitated analysis of less abundant, modified proteins and the nature of modifications at specific sites is now being characterised. The damaging reactive species that cause protein modifications in plasma principally arise from reactive oxygen species (ROS) produced by NADPH oxidases (NOX), nitric oxide synthases (NOS) and oxygenase activities; reactive nitrogen species (RNS) from myeloperoxidase (MPO) and NOS activities; and hypochlorous acid from MPO. Secondary damage to proteins may be caused by oxidized lipids and glucose autooxidation. In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites.

  11. 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

    OpenAIRE

    1995-01-01

    To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate co...

  12. Recombinant protein expression in Nicotiana.

    Science.gov (United States)

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  13. Mating behavior induces changes of expression of Fos protein, plasma testosterone and androgen receptors in the accessory olfactory bulb (AOB of the male mandarin vole Microtus mandarinus

    Directory of Open Access Journals (Sweden)

    Fengqin HE, Fadao TAI

    2009-08-01

    Full Text Available In order to investigate the neuroendocrine mechanism of the mating behavior in the adult male mandarin voles Microtus mandarinus, the radioimmunoassay (RIA and immunohistochemistry methods were used to investigate the differences in plasma testosterone (T concentrations and distribution of T immunoreactive neurons (T-IRs, androgen receptor immunoreactive neurons (AR-IRs and Fos protein immunoreactive neurons (Fos-IRs in the accessory olfactory bulb (AOB and the main olfactory bulb (MOB following exposure to clean hard-wood shavings (control group, soiled bedding (exposure group or contact with an estrous female (mating group. Results showed that plasma T concentration was significantly higher in the mating group than that in the exposure group, and both the mating group and the exposure group displayed significantly higher plasma T concentration than the control group. T-IRs, AR-IRs and Fos-IRs were investigated with the immunohistochemistry method in granule cell (GC and mitral cell (MC of the MOB and the AOB in the three groups. There were significantly more T-IRs, AR-IRs and Fos-IRs in MC and GC of the AOB in the mating group than that in the exposure group or the control group. T-IRs, AR-IRs and Fos-IRs did not show significant differences between the exposure group and the control group. Furthermore, obvious differences in MC and GC of the MOB were not found among the three groups. The results confirm that both changes of T and AR in the AOB might be underlying mating behavior in the adult male mandarin voles [Current Zoology 55 (4: 288–295, 2009].

  14. Evaluation of toxicological biomarkers in secreted proteins of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and their expressions in the plasma of rats and incineration workers.

    Science.gov (United States)

    Phark, Sohee; Park, So-Young; Chang, Yoon-Seok; Choi, Seonyoung; Lim, Ji-youn; Kim, Yoonjin; Seo, Jong Bok; Jung, Woon-Won; Sul, Donggeun

    2016-05-01

    Toxicological biomarkers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) were investigated in proteins secreted by HepG2 cells and their expression levels were determined in the plasma of rats exposed to 2,3,7,8-TCDD and in the plasma of incineration workers exposed to dioxins. HepG2 cells were treated with various concentrations of 2,3,7,8-TCDD (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 nM) for 24 or 48 h. MTT and Comet assays were performed to determine cytotoxicities and genotoxicities to select exposure concentrations for the proteomic analysis of proteins secreted by 2,3,7,8-TCDD-treated cells. In the proteomic analysis, dose- and time-dependent toxicological biomarkers were evaluated using two pI ranges (4-7 and 6-9) using a large gel 2-DE system. Fifteen secreted proteins were identified by a nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS and the identities of eight secreted proteins including glyoxalase 1 (GLO 1), homogentisate dioxygenase (HGD), peroxiredoxin 1 (PRX 1), proteasome subunit beta type (PSMB) 5 and 6, UDP-glucose 6-dehydrogenase (UDP-GlcDH), hydroxyacyl-coenzyme A dehydrogenase (HADH) and serotransferrin (STF) were confirmed by western blotting. Of these, PSMB 5 and PRX 1 were also found in the plasma of rats exposed to 2,3,7,8-TCDD, whereas GLO 1, HGD, PSMB 6 and PRX 1 were found in the plasma of incineration workers exposed to dioxins.

  15. Redox regulation of protein damage in plasma

    Directory of Open Access Journals (Sweden)

    Helen R. Griffiths

    2014-01-01

    In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites.

  16. Plasma protein haptoglobin modulates renal iron loading

    DEFF Research Database (Denmark)

    Fagoonee, Sharmila; Gburek, Jakub; Hirsch, Emilio

    2005-01-01

    Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme...

  17. Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate.

    Science.gov (United States)

    Tang, Xiaoyun; Zhao, Yuan Y; Dewald, Jay; Curtis, Jonathan M; Brindley, David N

    2016-04-01

    Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 μg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs.

  18. Proteins of the canine seminal plasma

    Directory of Open Access Journals (Sweden)

    Annice Aquino-Cortez

    2016-05-01

    Full Text Available ABSTRACT: Studies have been performed to identify the proteins present in canine seminal plasma (SP and relate them to sperm quality as well as to discover molecular markers of reproductive tract diseases. There is evidence that heparin-binding proteins, zinc-binding proteins, and lactoferrin as well as the matrix metalloproteinase, superoxide dismutase, catalase, and glutathione peroxidase enzymes are associated with canine sperm quality. Other studies indicate that prolactin and enzymes like arginine esterase, acid phosphatase, and alkaline phosphatase could be successfully used as biomarkers of reproductive disorders. Thus, the present literature review aims to address aspects related to proteins of the canine SP, their influence on fertility, and their importance as biomarkers of reproductive disorders.

  19. YKL-40 tissue expression and plasma levels in patients with ovarian cancer

    DEFF Research Database (Denmark)

    Høgdall, Estrid V S; Ringsholt, Merete; Høgdall, Claus K;

    2009-01-01

    BACKGROUND: YKL-40 (chitinase-3-like-1) is a member of "mammalian chitinase-like proteins". The protein is expressed in many types of cancer cells and the highest plasma YKL-40 levels have been found in patients with metastatic disease, short recurrence/progression-free intervals, and short overall...... survival. The aim of the study was to determine the expression of YKL-40 in tumor tissue and plasma in patients with borderline ovarian tumor or epithelial ovarian cancer (OC), and investigate prognostic value of this marker. METHODS: YKL-40 protein expression was determined by immunohistochemistry...... in tissue arrays from 181 borderline tumors and 473 OC. Plasma YKL-40 was determined by ELISA in preoperative samples from 19 patients with borderline tumor and 76 OC patients. RESULTS: YKL-40 protein expression was found in cancer cells, tumor associated macrophages, neutrophils and mast cells. The tumor...

  20. Niacin increases HDL by reducing hepatic expression and plasma levels of cholesteryl ester transfer protein in APOE*3Leiden.CETP mice

    NARCIS (Netherlands)

    Hoorn, J.W.A. van der; Haan, W. de; Berbée, J.P.P.; Havekes, L.M.; Jukema, J.W.; Rensen, P.C.; Princen, H.M.G.

    2008-01-01

    Objective - Niacin potently decreases plasma triglycerides and LDL-cholesterol. In addition, niacin is the most potent HDL-cholesterol- increasing drug used in the clinic. In the present study, we aimed at elucidation of the mechanism underlying its HDL-raising effect. Methods and Results - InAPOE*3

  1. Seldi-tof MS Profiling of Plasma Proteins in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Shao-Pai Wu

    2006-03-01

    Conclusion: This study clearly demonstrates that the combined technology of SELDI-TOF MS and artificial intelligence is effective in distinguishing protein expression between normal and ovarian cancer plasma. The identified protein peaks may be candidate proteins for early detection of ovarian cancer or evaluation of therapeutic response.

  2. Elevation of plasma phospholipid transfer protein increases the risk of atherosclerosis despite lower apolipoprotein B-containing lipoproteins.

    NARCIS (Netherlands)

    J. Lie (Jessica); M.P.G. de Crom (Rini); T. van Gent (Teus); M.J. van Haperen (Rien); L. Scheek (Leo); F. Sadeghi-Niaraki (Farah); A. van Tol (Arie)

    2004-01-01

    textabstractPlasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoproteins and mediates HDL conversion. PLTP-overexpressing mice have increased atherosclerosis. However, mice do not express cholesteryl ester transfer protein (CETP), which is involved in

  3. Identification of fibrin clot-bound plasma proteins

    NARCIS (Netherlands)

    S. Talens (Simone); F.W.G. Leebeek (Frank); J.A.A. Demmers (Jeroen); D.C. Rijken (Dingeman)

    2012-01-01

    textabstractSeveral proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl2 and aprotinin to citrated platelet-poor plasma and unbound

  4. Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane.

    NARCIS (Netherlands)

    Bosch, R.R.; Bazuine, M.; Wake, M.M.; Span, P.N.; Olthaar, A.J.; Schurmann, A.; Maassen, J.A.; Hermus, A.R.M.M.; Willems, P.H.G.M.; Sweep, C.G.J.

    2003-01-01

    The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rat

  5. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins

    NARCIS (Netherlands)

    Rahimi, Mehran; Ng, E. -P.; Bakhtiari, K.; Vinciguerra, M.; Ahmad, H. Ali; Awala, H.; Mintova, S.; Daghighi, M.; Rostami, F. Bakhshandeh; de Vries, Marcel; Motazacker, M. M.; Peppelenbosch, M. P.; Mahmoudi, M.; Rezaee, F.

    2015-01-01

    The affinity of zeolite nanoparticles (diameter of 8-12 nm) possessing high surface area and high pore volume towards human plasma proteins has been investigated. The protein composition (corona) of zeolite nanoparticles has been shown to be more dependent on the plasma protein concentrations and

  6. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins

    NARCIS (Netherlands)

    Rahimi, Mehran; Ng, E. -P.; Bakhtiari, K.; Vinciguerra, M.; Ahmad, H. Ali; Awala, H.; Mintova, S.; Daghighi, M.; Rostami, F. Bakhshandeh; de Vries, Marcel; Motazacker, M. M.; Peppelenbosch, M. P.; Mahmoudi, M.; Rezaee, F.

    2015-01-01

    The affinity of zeolite nanoparticles (diameter of 8-12 nm) possessing high surface area and high pore volume towards human plasma proteins has been investigated. The protein composition (corona) of zeolite nanoparticles has been shown to be more dependent on the plasma protein concentrations and th

  7. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins

    NARCIS (Netherlands)

    M. Rahimi (Mehran); E.-P. Ng; K. Bakhtiari (Kamran); M. Vinciguerra (Manlio); H.A. Ahmad (H. Ali); H. Awala; S. Mintova; M. Daghighi (Mojtaba); F. Bakhshandeh Rostami; M. de Vries (Marieke); M.M. Motazacker (Mohammad); M.P. Peppelenbosch (Maikel); M. Mahmoudi; F. Rezaee (Farhad)

    2015-01-01

    textabstractThe affinity of zeolite nanoparticles (diameter of 8-12 nm) possessing high surface area and high pore volume towards human plasma proteins has been investigated. The protein composition (corona) of zeolite nanoparticles has been shown to be more dependent on the plasma protein concentra

  8. The 82-plex plasma protein signature that predicts increasing inflammation

    DEFF Research Database (Denmark)

    Tepel, Martin; Beck, Hans C; Tan, Qihua;

    2015-01-01

    transplant recipients and quantified 359 plasma proteins simultaneously using nano-Liquid-Chromatography-Tandem Mass-Spectrometry in individual samples and plasma C-reactive protein on the index day and the next day. Next-day C-reactive protein increased in 59 patients whereas it decreased in 32 patients......The objective of the study was to define the specific plasma protein signature that predicts the increase of the inflammation marker C-reactive protein from index day to next-day using proteome analysis and novel bioinformatics tools. We performed a prospective study of 91 incident kidney....... The prediction model selected and validated 82 plasma proteins which determined increased next-day C-reactive protein (area under receiver-operator-characteristics curve, 0.772; 95% confidence interval, 0.669 to 0.876; P protein signature (P 

  9. Plasma and Plasma Protein Product Transfusion: A Canadian Blood Services Centre for Innovation Symposium.

    Science.gov (United States)

    Zeller, Michelle P; Al-Habsi, Khalid S; Golder, Mia; Walsh, Geraldine M; Sheffield, William P

    2015-07-01

    Plasma obtained via whole blood donation processing or via apheresis technology can either be transfused directly to patients or pooled and fractionated into plasma protein products that are concentrates of 1 or more purified plasma protein. The evidence base supporting clinical efficacy in most of the indications for which plasma is transfused is weak, whereas high-quality evidence supports the efficacy of plasma protein products in at least some of the clinical settings in which they are used. Transfusable plasma utilization remains composed in part of applications that fall outside of clinical practice guidelines. Plasma contains all of the soluble coagulation factors and is frequently transfused in efforts to restore or reinforce patient hemostasis. The biochemical complexities of coagulation have in recent years been rationalized in newer cell-based models that supplement the cascade hypothesis. Efforts to normalize widely used clinical hemostasis screening test values by plasma transfusion are thought to be misplaced, but superior rapid tests have been slow to emerge. The advent of non-vitamin K-dependent oral anticoagulants has brought new challenges to clinical laboratories in plasma testing and to clinicians needing to reverse non-vitamin K-dependent oral anticoagulants urgently. Current plasma-related controversies include prophylactic plasma transfusion before invasive procedures, plasma vs prothrombin complex concentrates for urgent warfarin reversal, and the utility of increased ratios of plasma to red blood cell units transfused in massive transfusion protocols. The first recombinant plasma protein products to reach the clinic were recombinant hemophilia treatment products, and these donor-free equivalents to factors VIII and IX are now being supplemented with novel products whose circulatory half-lives have been increased by chemical modification or genetic fusion. Achieving optimal plasma utilization is an ongoing challenge in the interconnected

  10. Protein expression profiles of human lymph and plasma mapped by 2D-DIGE and 1D SDS–PAGE coupled with nanoLC–ESI–MS/MS bottom-up proteomics

    OpenAIRE

    Clement, Cristina C.; Aphkhazava, David; Nieves, Edward; Callaway, Myrasol; Olszewski, Waldemar; Rotzschke, Olaf; Santambrogio, Laura

    2012-01-01

    In this study a proteomic approach was used to define the protein content of matched samples of afferent prenodal lymph and plasma derived from healthy volunteers. The analysis was performed using two analytical methodologies coupled with nanoliquid chromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEF nanoLC Orbitrap–ESI–MS/MS), and two-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC–ESI–MS/MS). The 253 significantly identified proteins...

  11. Identification of fibrin clot-bound plasma proteins.

    Directory of Open Access Journals (Sweden)

    Simone Talens

    Full Text Available Several proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl(2 and aprotinin to citrated platelet-poor plasma and unbound proteins were washed away with Tris-buffered saline. Non-covalently bound proteins were extracted, separated with 2D gel electrophoresis and visualized with Sypro Ruby. Excised protein spots were analyzed with mass spectrometry. The identity of the proteins was verified by checking the mass of the protein, and, if necessary, by Western blot analysis. Next to established fibrin-binding proteins we identified several novel fibrin clot-bound plasma proteins, including α(2-macroglobulin, carboxypeptidase N, α(1-antitrypsin, haptoglobin, serum amyloid P, and the apolipoproteins A-I, E, J, and A-IV. The latter six proteins are associated with high-density lipoprotein particles. In addition we showed that high-density lipoprotein associated proteins were also present in fibrinogen preparations purified from plasma. Most plasma proteins in a fibrin clot can be classified into three groups according to either blood coagulation, protease inhibition or high-density lipoprotein metabolism. The presence of high-density lipoprotein in clots might point to a role in hemostasis.

  12. Predictable tuning of protein expression in bacteria

    DEFF Research Database (Denmark)

    Bonde, Mads; Pedersen, Margit; Klausen, Michael Schantz

    2016-01-01

    We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expressi...

  13. Complete cDNA sequence of the preproform of human pregnancy-associated plasma protein-A. Evidence for expression in the brain and induction by cAMP

    DEFF Research Database (Denmark)

    Haaning, Jesper; Oxvig, Claus; Overgaard, Michael Toft

    1996-01-01

    A cDNA that encodes the prepropeptide of pregnancy-associated plasma protein-A (preproPAPP-A), a putative metalloproteinase, has been cloned and sequenced. PAPP-A is synthesized in the placenta as a 1627-residue precursor preproprotein with a putative 22-residue signal peptide and a highly basic ...

  14. Coevolution of gene expression among interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  15. Plasma PIVKA proteins in rabbits given warfarin.

    Science.gov (United States)

    Zivelin, A; Rao, L V; Rapaport, S I

    1996-06-01

    The presence of partially carboxylated forms of the vitamin K dependent coagulation factors (PIVKA) was evaluated in the plasma of rabbits treated with warfarin. Excess antigen over activity as measured in rabbit specific assays was taken as evidence for PIVKA. Our data confirm a previous report of the absence of plasma PIVKA prothrombin. In contrast, plasma PIVKA factors VII, IX, and X were demonstrable. A striking excess of plasma factor IX antigen over activity was measured and a large fraction of the factor IX antigen persisted in the plasma after its adsorption with barium citrate.

  16. POLY(N-VINYLPYRROLIDONE)-MODIFIED SURFACES REPEL PLASMA PROTEIN ADSORPTION

    Institute of Scientific and Technical Information of China (English)

    Xiao-li Liu; Zhao-qiang Wu; Dan Li; Hong Chen

    2012-01-01

    The present work aimed to study the interaction between plasma proteins and PVP-modified surfaces under more complex protein conditions.In the competitive adsorption of fibrinogen (Fg) and human serum albumin (HSA),the modified surfaces showed preferential adsorption of HSA.In 100% plasma,the amount of Fg adsorbed onto PVP-modified surfaces was as low as 10 ng/cm2,suggesting the excellent protein resistance properties of the modified surfaces.In addition,immunoblots of proteins eluted from the modified surfaces after plasma contact confirmed that PVP-modified surfaces can repel most plasma proteins,especially proteins that play important roles in the process of blood coagulation.

  17. Comparative changes in plasma protein concentration, hematocrit and plasma volume during exercise, bedrest and + Gz acceleration.

    Science.gov (United States)

    Van Beaumont, W.; Greenleaf, J. E.

    1972-01-01

    Discussion of experiments which indicate that under conditions of a constant red cell volume the proportional changes in hematocrit and plasma volume during exercise are never equal. On the basis of direct measurements and calculated changes of plasma volume it is concluded that during maximal exercise there is a small loss of protein from the plasma. It is clear that changes in content of blood constituents can only be evaluated correctly after determination of changes in plasma volume.

  18. Analytical expressions for radiative opacities of low Z plasmas

    Energy Technology Data Exchange (ETDEWEB)

    Rubiano, J G; Martel, P; Gil, J M; Rodriguez, R; Florido, R; Mendoza, M A [Departamento de Fisica, Universidad de Las Palmas de GC, Las Palmas de GC (Spain); Minguez, E, E-mail: jgarcia@dfis.ulpgc.e [Instituto de Fusion Nuclear (DENIM), Universidad Politecnica de Madrid, Madrid (Spain)

    2010-08-01

    In this work we obtain analytical expressions for the radiative opacity of several low Z plasmas (He, Li, Be, and B) in a wide range of temperatures and densities. These formulas are obtained by fitting the proposed expression to mean opacities data calculated by using the code ABAKO/ RAPCAL. This code computes the radiative properties of plasmas, both in LTE and NLTE conditions, under the detailed-level-accounting approach. It has been successfully validated in the range of interest in previous works.

  19. The proteome response to amyloid protein expression in vivo.

    Directory of Open Access Journals (Sweden)

    Ricardo A Gomes

    Full Text Available Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE. We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

  20. Cold Atmospheric Plasma Manipulation of Proteins in Food Systems

    DEFF Research Database (Denmark)

    Tolouie, Haniye; Hashemi, Maryam; Mohammadifar, Mohammad Amin

    2017-01-01

    Plasma processing has been getting a lot of attention in recent applications as a novel, eco-friendly, and highly efficient approach. Cold plasma has mostly been used to reduce microbial counts in foodstuff and biological materials, as well as in different levels of packaging, particularly in cases...... where there is thermal sensitivity. As it is a very recent application, the impact of cold plasma treatment has been studied on the protein structures of food and pharmaceutical systems, as well as in the packaging industry. Proteins, as a food constituent, play a remarkable role in the techno...... of plasma on the conformation and function of proteins with food origin, especially enzymes and allergens, as well as protein-made packaging films. In enzyme manipulation with plasma, deactivation has been reported to be either partial or complete. In addition, an activity increase has been observed in some...

  1. Prolonged morphine administration alters protein expression in the rat myocardium

    Directory of Open Access Journals (Sweden)

    Drastichova Zdenka

    2011-11-01

    Full Text Available Abstract Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. Results Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. Conclusions The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart.

  2. Coevolution of gene expression among interacting proteins

    OpenAIRE

    2004-01-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically inter...

  3. TRPM4 protein expression in prostate cancer

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Soldini, Davide; Jung, Maria;

    2016-01-01

    BACKGROUND: Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level with bioch......BACKGROUND: Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level.......79-2.62; p = 0.01-0.03 for the two observers) when compared to patients with a lower staining intensity. CONCLUSIONS: TRPM4 protein expression is widely expressed in benign and cancerous prostate tissue, with highest staining intensities found in PCa. Overexpression of TRPM4 in PCa (combination of high...

  4. Glycosylation of hemoglobin and plasma proteins in petrochemical plant workers

    Energy Technology Data Exchange (ETDEWEB)

    Unrug, A.; Tomaszewski, L.

    1985-01-01

    The concentration of glycosylated hemoglobin and (plasma) proteins has been measured in 111 workers of 6 MZRiP departments in Plock and in 54 healthy people. In all subjects the mean concentrations of glycosylated hemoglobin and glycosylated plasma proteins have been in so called wide range of normal values. Significant shifts of glycosylated Hb concentrations have been found in two departments--those of ethylenederivatives and distillation. The concentration of glycosylated plasma proteins has been elevated only in workers of the Catalytic Processes Department.

  5. Proteomic identification of novel differentiation plasma protein markers in hypobaric hypoxia-induced rat model.

    Directory of Open Access Journals (Sweden)

    Yasmin Ahmad

    Full Text Available BACKGROUND: Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia. METHODS: In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h, separated by two-dimensional electrophoresis (2-DE and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF. Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis. RESULTS: Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia. CONCLUSION/SIGNIFICANCE: This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers.

  6. Robust changes in expression of brain-derived neurotrophic factor (BDNF) mRNA and protein across the brain do not translate to detectable changes in BDNF levels in CSF or plasma.

    Science.gov (United States)

    Lanz, Thomas A; Bove, Susan E; Pilsmaker, Catherine D; Mariga, Abigail; Drummond, Elena M; Cadelina, Gregory W; Adamowicz, Wendy O; Swetter, Brentt J; Carmel, Sharon; Dumin, Jo Ann; Kleiman, Robin J

    2012-09-01

    Adult rats were treated acutely with peripheral kainic acid (KA), and changes in brain-derived neurotrophic factor (BDNF) mRNA and protein were tracked over time across multiple brain regions. Despite robust elevation in both mRNA and protein in multiple brain regions, plasma BDNF was unchanged and cerebrospinal fluid (CSF) BDNF levels remained undetectable. Primary neurons were then treated with KA. BDNF was similarly elevated within neurons, but was undetectable in neuronal media. Thus, while deficits in BDNF signaling have been implicated in a number of diseases, these data suggest that extracellular concentrations of BDNF may not be a facile biomarker for changes in neurons.

  7. ABL Tyrosine Kinase Stimulates PUMA Protein Expression

    OpenAIRE

    Oon, Chet K

    2016-01-01

    ABL is an ubiquitously expressed non-receptor tyrosine kinase involved in multiple cellular functions including programmed cell death. Upon DNA damage, ABL has been shown to upregulate PUMA, p53 upregulated modulator of apoptosis, and causes downstream mitochondrial intrinsic apoptotic events. However, the mechanism by which ABL regulates PUMA expression remains unknown. We have shown that ABL does not change PUMA protein subcellular localization through immunofluorescence. Through protein an...

  8. Expression of B-nerve growth factor in rabbit male tract and seminal plasma

    OpenAIRE

    García García, R. M.; Masdeu, M.; A. Sánchez Rodríguez; Bautista, J. M.; Arias Álvarez, María; LORENZO, P. L.; Garcia Rebollar, Pilar

    2015-01-01

    Nerve growth factor (NGF) has been recently identified as an ovulation inductor factor (OIF) in the seminal plasma (SP) (Ratto et al. PNAS 2012; 109:15042-7). The presence of OIF in rabbit has been suggested but this protein has not yet been identified. Our aim was to study the mRNA expression in the rabbit male reproductive tract and to identify the protein β-NGF in the SP.

  9. Relative quantification of several plasma proteins during liver transplantation surgery.

    Science.gov (United States)

    Parviainen, Ville; Joenväärä, Sakari; Tukiainen, Eija; Ilmakunnas, Minna; Isoniemi, Helena; Renkonen, Risto

    2011-01-01

    Plasma proteome is widely used in studying changes occurring in human body during disease or other disturbances. Immunological methods are commonly used in such studies. In recent years, mass spectrometry has gained popularity in high-throughput analysis of plasma proteins. In this study, we tested whether mass spectrometry and iTRAQ-based protein quantification might be used in proteomic analysis of human plasma during liver transplantation surgery to characterize changes in protein abundances occurring during early graft reperfusion. We sampled blood from systemic circulation as well as blood entering and exiting the liver. After immunodepletion of six high-abundant plasma proteins, trypsin digestion, iTRAQ labeling, and cation-exchange fractionation, the peptides were analyzed by reverse phase nano-LC-MS/MS. In total, 72 proteins were identified of which 31 could be quantified in all patient specimens collected. Of these 31 proteins, ten, mostly medium-to-high abundance plasma proteins with a concentration range of 50-2000 mg/L, displayed relative abundance change of more than 10%. The changes in protein abundance observed in this study allow further research on the role of several proteins in ischemia-reperfusion injury during liver transplantation and possibly in other surgery.

  10. Relative Quantification of Several Plasma Proteins during Liver Transplantation Surgery

    Directory of Open Access Journals (Sweden)

    Ville Parviainen

    2011-01-01

    Full Text Available Plasma proteome is widely used in studying changes occurring in human body during disease or other disturbances. Immunological methods are commonly used in such studies. In recent years, mass spectrometry has gained popularity in high-throughput analysis of plasma proteins. In this study, we tested whether mass spectrometry and iTRAQ-based protein quantification might be used in proteomic analysis of human plasma during liver transplantation surgery to characterize changes in protein abundances occurring during early graft reperfusion. We sampled blood from systemic circulation as well as blood entering and exiting the liver. After immunodepletion of six high-abundant plasma proteins, trypsin digestion, iTRAQ labeling, and cation-exchange fractionation, the peptides were analyzed by reverse phase nano-LC-MS/MS. In total, 72 proteins were identified of which 31 could be quantified in all patient specimens collected. Of these 31 proteins, ten, mostly medium-to-high abundance plasma proteins with a concentration range of 50–2000 mg/L, displayed relative abundance change of more than 10%. The changes in protein abundance observed in this study allow further research on the role of several proteins in ischemia-reperfusion injury during liver transplantation and possibly in other surgery.

  11. Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

    Science.gov (United States)

    Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

    2015-11-01

    Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber.

  12. Microdomains of SNARE proteins in the plasma membrane

    NARCIS (Netherlands)

    Bogaart, G. van den; Lang, T.; Jahn, R.

    2013-01-01

    Exocytosis is catalyzed by the engagement of SNARE proteins embedded in the plasma membrane with complementary SNAREs in the membrane of trafficking vesicles undergoing exocytosis. In most cells studied so far, SNAREs are not randomly distributed across the plasma membrane but are clustered and

  13. QSARs for Plasma Protein Binding: Source Data and Predictions

    Data.gov (United States)

    U.S. Environmental Protection Agency — The dataset has all of the information used to create and evaluate 3 independent QSAR models for the fraction of a chemical unbound by plasma protein (Fub) for...

  14. Transport proteins of the plant plasma membrane

    Science.gov (United States)

    Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

  15. Analysis of correlations between protein complex and protein-protein interaction and mRNA expression

    Institute of Scientific and Technical Information of China (English)

    CAI Lun; XUE Hong; LU Hongchao; ZHAO Yi; ZHU Xiaopeng; BU Dongbo; LING Lunjiang; CHEN Runsheng

    2003-01-01

    Protein-protein interaction is a physical interaction of two proteins in living cells. In budding yeast Saccharomyces cerevisiae, large-scale protein-protein interaction data have been obtained through high-throughput yeast two-hybrid systems (Y2H) and protein complex purification techniques based on mass-spectrometry. Here, we collect 11855 interactions between total 2617 proteins. Through seriate genome-wide mRNA expression data, similarity between two genes could be measured. Protein complex data can also be obtained publicly and can be translated to pair relationship that any two proteins can only exist in the same complex or not. Analysis of protein complex data, protein-protein interaction data and mRNA expression data can elucidate correlations between them. The results show that proteins that have interactions or similar expression patterns have a higher possibility to be in the same protein complex than randomized selected proteins, and proteins which have interactions and similar expression patterns are even more possible to exist in the same protein complex. The work indicates that comprehensive integration and analysis of public large-scale bioinformatical data, such as protein complex data, protein-protein interaction data and mRNA expression data, may help to uncover their relationships and common biological information underlying these data. The strategies described here may help to integrate and analyze other functional genomic and proteomic data, such as gene expression profiling, protein-localization mapping and large-scale phenotypic data, both in yeast and in other organisms.

  16. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins.

    Science.gov (United States)

    Rahimi, M; Ng, E-P; Bakhtiari, K; Vinciguerra, M; Ali Ahmad, H; Awala, H; Mintova, S; Daghighi, M; Bakhshandeh Rostami, F; de Vries, M; Motazacker, M M; Peppelenbosch, M P; Mahmoudi, M; Rezaee, F

    2015-11-30

    The affinity of zeolite nanoparticles (diameter of 8-12 nm) possessing high surface area and high pore volume towards human plasma proteins has been investigated. The protein composition (corona) of zeolite nanoparticles has been shown to be more dependent on the plasma protein concentrations and the type of zeolites than zeolite nanoparticles concentration. The number of proteins present in the corona of zeolite nanoparticles at 100% plasma (in vivo state) is less than with 10% plasma exposure. This could be due to a competition between the proteins to occupy the corona of the zeolite nanoparticles. Moreover, a high selective adsorption for apolipoprotein C-III (APOC-III) and fibrinogen on the zeolite nanoparticles at high plasma concentration (100%) was observed. While the zeolite nanoparticles exposed to low plasma concentration (10%) exhibited a high selective adsorption for immunoglobulin gamma (i.e. IGHG1, IGHG2 and IGHG4) proteins. The zeolite nanoparticles can potentially be used for selectively capture of APOC-III in order to reduce the activation of lipoprotein lipase inhibition during hypertriglyceridemia treatment. The zeolite nanoparticles can be adapted to hemophilic patients (hemophilia A (F-VIII deficient) and hemophilia B (F-IX deficient)) with a risk of bleeding, and thus might be potentially used in combination with the existing therapy.

  17. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins

    Science.gov (United States)

    Rahimi, M.; Ng, E.-P.; Bakhtiari, K.; Vinciguerra, M.; Ahmad, H. Ali; Awala, H.; Mintova, S.; Daghighi, M.; Bakhshandeh Rostami, F.; de Vries, M.; Motazacker, M. M.; Peppelenbosch, M. P.; Mahmoudi, M.; Rezaee, F.

    2015-11-01

    The affinity of zeolite nanoparticles (diameter of 8-12 nm) possessing high surface area and high pore volume towards human plasma proteins has been investigated. The protein composition (corona) of zeolite nanoparticles has been shown to be more dependent on the plasma protein concentrations and the type of zeolites than zeolite nanoparticles concentration. The number of proteins present in the corona of zeolite nanoparticles at 100% plasma (in vivo state) is less than with 10% plasma exposure. This could be due to a competition between the proteins to occupy the corona of the zeolite nanoparticles. Moreover, a high selective adsorption for apolipoprotein C-III (APOC-III) and fibrinogen on the zeolite nanoparticles at high plasma concentration (100%) was observed. While the zeolite nanoparticles exposed to low plasma concentration (10%) exhibited a high selective adsorption for immunoglobulin gamma (i.e. IGHG1, IGHG2 and IGHG4) proteins. The zeolite nanoparticles can potentially be used for selectively capture of APOC-III in order to reduce the activation of lipoprotein lipase inhibition during hypertriglyceridemia treatment. The zeolite nanoparticles can be adapted to hemophilic patients (hemophilia A (F-VIII deficient) and hemophilia B (F-IX deficient)) with a risk of bleeding, and thus might be potentially used in combination with the existing therapy.

  18. METHODS OF DETECTING PREGNANCY-ASSOCIATED PLASMA PROTEIN-A2 (PAPP-A2)

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention provides pregnancy associated plasma protein A2 (PAPP-A2), its nucleotide and amino acid sequences, antisense molecules to the nucleotide sequences which encode PAPP-A2, expression vectors for the production of purified PAPP-A2, antibodies capable of binding specifically...

  19. PREGNANCY-ASSOCIATED PLASMA PROTEIN-A2 (PAPP-A2) POLYNUCLEOTIDES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention provides pregnancy associated plasma protein A2 (PAPP-A2), its nucleotide and amino acid sequences antisense molecules to the nucleotide sequences which encode PAPP-A2, expression vectors for the production of purified PAPP-A2, antibodies capable of binding specifically...

  20. METHODS OF DETECTING PREGNANCY-ASSOCIATED PLASMA PROTEIN-A2 (PAPP-A2)

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention provides pregnancy associated plasma protein A2 (PAPP-A2), its nucleotide and amino acid sequences, antisense molecules to the nucleotide sequences which encode PAPP-A2, expression vectors for the production of purified PAPP-A2, antibodies capable of binding specifically...

  1. Differential plasma protein binding to metal oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Zhou J; Mortimer, Gysell; Minchin, Rodney F [School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072 (Australia); Schiller, Tara; Musumeci, Anthony; Martin, Darren, E-mail: r.minchin@uq.edu.a [Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD 4072 (Australia)

    2009-11-11

    Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO{sub 2}, the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO{sub 2} and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.

  2. Interaction of plasma proteins with commercial protein repellent polyvinyl chloride (PVC): a word of caution.

    Science.gov (United States)

    De Somer, F; Van Landschoot, A; Van Nooten, G; Delanghe, J

    2008-07-01

    Protein adsorption onto polymers remains a problem. In recent years, several protein-repellent PVC tubings have been developed. Although several studies report the interaction between plasma coagulation proteins and PVC, few address the interaction with other plasma proteins. Two commercial brands of untreated medical grade PVC tubing, phosphorylcholine-coated PVC tubing, triblock-copolymer (polycaprolactone-polydimethylsiloxane-polycaprolactone)-treated PVC tubing and poly-2-methoxyethylacrylate (PMEA)-coated tubing were exposed for 60 minutes to human plasma. A broad spectrum of plasma proteins was found on all tubing. The adsorbed albumin to total protein ratio is lower than the similar ratio in plasma while alpha1 and alpha2 globulins are over-represented in the protein spectrum. On PMEA tubing, not only alpha globulins, but also beta and gamma globulins, are found in high concentrations in the adsorbed protein. PMEA tubing and uncoated PVC tubing of brand B had a higher amount of protein adsorbed compared against all other tubing (p < 0.05). There were no statistical differences in protein adsorption between the triblock-copolymer-treated tubing, the phosphorylcholine-coated tubing and the uncoated PVC tubing of brand A. The average thickness of the protein layer was 23 nm. Plasma protein adsorption still exists on uncoated and protein-repellent tubing and can initiate a systemic inflammatory reaction.

  3. Protein Adsorption on Various Plasma-Treated Polyethylene Terephthalate Substrates

    Directory of Open Access Journals (Sweden)

    Karin Stana-Kleinschek

    2013-10-01

    Full Text Available Protein adhesion and cell response to plasma-treated polymer surfaces were studied. The polymer polyethylene terephthalate (PET was treated in either an oxygen plasma to make the surface hydrophilic, or a tetrafluoromethane CF4 plasma to make the surface hydrophobic. The plasma source was radiofrequency (RF discharge. The adsorption of albumin and other proteins from a cell-culture medium onto these surfaces was studied using a quartz crystal microbalance (QCM, X-ray photoelectron spectroscopy (XPS and atomic force microscopy (AFM. The cellular response to plasma-treated surfaces was studied as well using an MTT assay and scanning electron microscopy (SEM. The fastest adsorption rate was found on the hydrophilic oxygen plasma-treated sample, and the lowest was found on the pristine untreated sample. Additionally, the amount of adsorbed proteins was higher for the oxygen-plasma-treated surface, and the adsorbed layer was more viscoelastic. In addition, cell adhesion studies support this finding because the best cell adhesion was observed on oxygen-plasma-treated substrates.

  4. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  5. The dynamics of plant plasma membrane proteins: PINs and beyond.

    Science.gov (United States)

    Luschnig, Christian; Vert, Grégory

    2014-08-01

    Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment.

  6. Biotechnology Protein Expression and Purification Facility

    Science.gov (United States)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  7. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane.

    Science.gov (United States)

    Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

    2010-04-01

    Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review.

  8. Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

    Science.gov (United States)

    Koldsø, Heidi; Sansom, Mark S P

    2015-11-25

    The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

  9. Regulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-ARegulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-A

    DEFF Research Database (Denmark)

    Gaidamauskas, Ervinas

    During his PhD studies, Ervinas Gaidamauskas researched the proteins pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2 in vitro. As suggested by its name, PAPP-A plays an important role in pregnancy and fetal development. Additionally, recent studies indicate a newly...

  10. Regulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-ARegulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-A

    DEFF Research Database (Denmark)

    Gaidamauskas, Ervinas

    During his PhD studies, Ervinas Gaidamauskas researched the proteins pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2 in vitro. As suggested by its name, PAPP-A plays an important role in pregnancy and fetal development. Additionally, recent studies indicate a newly...

  11. Hypochlorite-induced oxidation of proteins in plasma

    DEFF Research Database (Denmark)

    Hawkins, C L; Davies, Michael Jonathan

    1999-01-01

    Activated phagocyte cells generate hypochlorite (HOCl) via the release of H2O2 and the enzyme myeloperoxidase. Plasma proteins are major targets for HOCl, although little information is available about the mechanism(s) of oxidation. In this study the reaction of HOCl (at least 50 microM) with dil......Activated phagocyte cells generate hypochlorite (HOCl) via the release of H2O2 and the enzyme myeloperoxidase. Plasma proteins are major targets for HOCl, although little information is available about the mechanism(s) of oxidation. In this study the reaction of HOCl (at least 50 micro......M) with diluted fresh human plasma has been shown to generate material that oxidizes 5-thio-2-nitrobenzoic acid; these oxidants are believed to be chloramines formed from the reaction of HOCl with protein amine groups. Chloramines have also been detected with isolated plasma proteins treated with HOCl. In both....... These results are consistent with protein-derived chloramines, and the radicals derived from them, as contributing agents in HOCl-induced plasma protein oxidation....

  12. Plasma in the near Venus tail: Venus Express observations

    Science.gov (United States)

    Dubinin, E.; Fraenz, M.; Zhang, T. L.; Woch, J.; Wei, Y.; Fedorov, A.; Barabash, S.; Lundin, R.

    2013-12-01

    Although Venus has no global intrinsic magnetic fields, it possesses a long magnetotail of induced origin. The topology of the tail is determined by the interplanetary magnetic field orientation. We present recent plasma and magnetic field observations in the near Venus tail (X≥-3RV) made by the Venus Express spacecraft. We show that ion acceleration in the Venus plasma sheet is produced by the slingshot effect of the draping magnetic field lines, though some features as differential streaming of different ion species point to the existence of other forces. We explain a bell shape of ion spectrograms while the spacecraft crosses the current sheet. The absence of a balance between the lobe magnetic pressure and thermal pressure of plasma in the plasma sheet indicates a dynamic rather than a static equilibrium in the Venus magnetotail. A strong asymmetry of the plasma sheet is controlled by the direction of the motional electric field in the upstream solar wind. In the hemisphere pointed in the direction of the motional electric field, the j×B force accelerates plasma tailward supplying the plasma sheet, while in the opposite hemisphere, the flow pattern occurs less regularly with smaller speeds but higher number densities.

  13. Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1

    Energy Technology Data Exchange (ETDEWEB)

    Sakamoto, Hikaru [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan); Sakata, Keiko; Kusumi, Kensuke [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Kojima, Mikiko; Sakakibara, Hitoshi [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan); Iba, Koh, E-mail: koibascb@kyushu-u.org [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

  14. Localization of porcine seminal plasma (PSP) proteins in the boar reproductive tract and spermatozoa.

    Science.gov (United States)

    Manásková, P; Jonáková, V

    2008-06-01

    Spermadhesins are proteins containing a characteristic CUB domain, originally isolated from seminal plasma and ejaculated spermatozoa in domestic animals. Boar spermadhesins are multifunctional proteins exhibiting ligand-binding abilities with various endogenous ligands present in the male and female reproductive tracts and may play a role in the reproduction process. Porcine spermadhesins (AQN, AWN, PSP protein families) are secreted mainly by the seminal vesicles, but their mRNAs have been found also in the cauda epididymis and prostate. Unlike AQN and AWN spermadhesins, localization of porcine seminal plasma (PSP) proteins in the boar reproductive tract has not been completely resolved. This work has focused on PSP protein expression and localization in the boar reproductive organs and on spermatozoa. Using specific rabbit polyclonal antibodies (anti-PSP I and anti-PSP II), PSP I and PSP II proteins were immunodetected in tissue extracts and in secretory tissues of cauda epididymis, prostate, seminal vesicles and Cowper's glands on the blots and by an indirect immunofluorescence technique, respectively. Moreover, the ability of PSP proteins to bind to epididymal spermatozoa indicated their presence on cauda epididymal and ejaculated spermatozoa. Porcine seminal plasma proteins bind to the sperm surface at ejaculation and may modulate several aspects of sperm activity during reproduction. PSP proteins are produced not only by seminal vesicles and prostate, but also by epididymis. However, their prospective role in sperm epididymal maturation is not clear. Further characterization of seminal plasma protein forms expressed in the individual reproductive organs will help to understand their subsequent role in the reproduction process.

  15. Differentially expressed proteins on postoperative 3

    Directory of Open Access Journals (Sweden)

    Jialili Ainuer

    2011-04-01

    Full Text Available 【Abstract】Objectives: Surgical repair of Achilles tendon (AT rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far. Methods: Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A (immobilization group, n=16 received postoperative cast immobilization; Group B (early motion group, n=16 received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group (Group C. The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing twodimensional polyacrylamide gel electrophoresis (2D-PAGE. PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint (PMF and tandem mass spectrum analysis, using the National Center for Biotechnology Information (NCBI protein database retrieval and then for bioinformatics analysis. Results: A mean of 446.33, 436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were successfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS. We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1

  16. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    Science.gov (United States)

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  17. Designing genes for successful protein expression.

    Science.gov (United States)

    Welch, Mark; Villalobos, Alan; Gustafsson, Claes; Minshull, Jeremy

    2011-01-01

    DNA sequences are now far more readily available in silico than as physical DNA. De novo gene synthesis is an increasingly cost-effective method for building genetic constructs, and effectively removes the constraint of basing constructs on extant sequences. This allows scientists and engineers to experimentally test their hypotheses relating sequence to function. Molecular biologists, and now synthetic biologists, are characterizing and cataloging genetic elements with specific functions, aiming to combine them to perform complex functions. However, the most common purpose of synthetic genes is for the expression of an encoded protein. The huge number of different proteins makes it impossible to characterize and catalog each functional gene. Instead, it is necessary to abstract design principles from experimental data: data that can be generated by making predictions followed by synthesizing sequences to test those predictions. Because of the degeneracy of the genetic code, design of gene sequences to encode proteins is a high-dimensional problem, so there is no single simple formula to guarantee success. Nevertheless, there are several straightforward steps that can be taken to greatly increase the probability that a designed sequence will result in expression of the encoded protein. In this chapter, we discuss gene sequence parameters that are important for protein expression. We also describe algorithms for optimizing these parameters, and troubleshooting procedures that can be helpful when initial attempts fail. Finally, we show how many of these methods can be accomplished using the synthetic biology software tool Gene Designer.

  18. Lowering of plasma phospholipid transfer protein activity by acute hyperglycaemia-induced hyperinsulinaemia in healthy men

    NARCIS (Netherlands)

    vanTol, A; Ligtenberg, JJM; Riemens, SC; vanHaeften, TW; Dullaart, RPF

    1997-01-01

    Human plasma contains two lipid transfer proteins involved in the remodelling of plasma lipoproteins: cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP). CETP mediates the transfer/exchange of cholesterylesters, triglycerides and phospholipids between high-density lip

  19. Streamlined expressed protein ligation using split inteins.

    Science.gov (United States)

    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

  20. Products of DNA, protein and lipid oxidative damage in relation to vitamin C plasma concentration.

    Science.gov (United States)

    Krajcovicová-Kudlácková, M; Dusinská, M; Valachovicová, M; Blazícek, P; Pauková, V

    2006-01-01

    Oxidative stress plays an important role in the pathogenesis of numerous chronic age-related free radical-induced diseases. Improved antioxidant status minimizes oxidative damage to DNA, proteins, lipids and other biomolecules. Diet-derived antioxidants such as vitamin C, vitamin E, carotenoids and related plant pigments are important in antioxidative defense and maintaining health. The results of long-term epidemiological and clinical studies suggest that protective vitamin C plasma concentration for minimum risk of free radical disease is higher than 50 micromol/l. Products of oxidative damage to DNA (DNA strand breaks with oxidized purines and pyrimidines), proteins (carbonyls) and lipids (conjugated dienes of fatty acids, malondialdehyde) were estimated in a group of apparently healthy adult non-smoking population in dependence on different vitamin C plasma concentrations. Under conditions of protective plasma vitamin C concentrations (>50 micromol/l) significantly lower values of DNA, protein and lipid oxidative damage were found in comparison with the vitamin C-deficient group (fruit and vegetable consumption (leading to higher vitamin C intake and higher vitamin C plasma concentrations) on oxidation of DNA, proteins and lipids is also expressed by an inverse significant correlation between plasma vitamin C and products of oxidative damage. The results suggest an important role of higher and frequent consumption of protective food (fruit, vegetables, vegetable oils, nuts, seeds and cereal grains) in prevention of free radical disease.

  1. Plasma concentrations of four pregnancy proteins in complications of pregnancy.

    Science.gov (United States)

    Lin, T M; Halbert, S P; Spellacy, W N; Berne, B H

    1977-08-01

    Toxemia of pregnancy was associated with an elevation of the pregnancy-associated plasma protein (PAPP)-A concentration, as compared to the level in normal pregnancy in the last month of gestation. The other pregnancy proteins measured were not altered in toxemia. In twin pregnancies, the PAPP-A, PAPP-C, and human placental lactogen levels were all increased, particularly PAPP-A. On the other hand, pregnancy zone protein was not affected by twinning. Pregnancy with diabetes showed normal levels of these proteins.

  2. Spectrophotometric and Refractometric Determination of Total Protein in Avian Plasma

    Directory of Open Access Journals (Sweden)

    Rodica Căpriță

    2013-10-01

    Full Text Available The aim of this study was to compare the total protein values obtained in heparin plasma of chickens by a spectrophotometric technique (biuret method, and the values obtained on the same day in the same samples by refractometry. The results obtained by refractometry (average value 2.638±0.153g% were higher than those obtained by the spectrophotometric method (average value 2.441±0.181g%. There was a low correlation (r = 0.6709 between the total protein values, determined with both methods. Protein is the major determinant of plasma refractive index, but glucose contributes too. The refractometric method is not recommended in chickens for the determination of total protein, because avian blood glucose concentration averages about twice than in mammalian blood.

  3. Stereoselective binding of chiral drugs to plasma proteins

    Institute of Scientific and Technical Information of China (English)

    Qi SHEN; Lu WANG; Hui ZHOU; Hui-di JIANG; Lu-shan YU; Su ZENG

    2013-01-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body.The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity,which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles.In this review,the stereoselective binding of chiral drugs to human serum albumin (HSA),α1-acid glycoprotein (AGP)and lipoprotein,three most important proteins in human plasma,are detailed.Furthermore,the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed.Apart from the stereoselectivity of enantiomer-protein binding,enantiomer-enantiomer interactions that may induce allosteric effects are also described.Additionally,the techniques and methods used to determine drug-protein binding parameters are briefly reviewed.

  4. [Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

    Science.gov (United States)

    Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

    2013-02-01

    To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

  5. Engineering genes for predictable protein expression.

    Science.gov (United States)

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  6. Plasma protein carbonyl levels and breast cancer risk.

    Science.gov (United States)

    Rossner, Pavel; Terry, Mary Beth; Gammon, Marilie D; Agrawal, Meenakshi; Zhang, Fang Fang; Ferris, Jennifer S; Teitelbaum, Susan L; Eng, Sybil M; Gaudet, Mia M; Neugut, Alfred I; Santella, Regina M

    2007-01-01

    To study the role of oxidative stress in breast cancer risk, we analysed plasma levels of protein carbonyls in 1050 cases and 1107 controls. We found a statistically significant trend in breast cancer risk in relation to increasing quartiles of plasma protein carbonyl levels (OR = 1.2, 95% CI = 0.9-1.5; OR = 1.5, 95% CI = 1.2-2.0; OR = 1.6, 95% CI = 1.2-2.1, for the 2(nd), 3(rd) and 4(th) quartile relative to the lowest quartile, respectively, P for trend = 0.0001). The increase in risk was similar for younger ( or = 15 grams/day for 4(th) quartile versus lowest quartile OR = 2.3, 95% CI = 1.1-4.7), and hormone replacement therapy use (HRT, OR = 2.6, 95% CI = 1.6-4.4 for 4(th) quartile versus lowest quartile). The multiplicative interaction terms were statistically significant only for physical activity and HRT. The positive association between plasma protein carbonyl levels and breast cancer risk was also observed when the analysis was restricted to women who had not received chemotherapy or radiation therapy prior to blood collection. Among controls, oxidized protein levels significantly increased with cigarette smoking and higher fruit and vegetable consumption, and decreased with alcohol consumption >30 grams per day. Women with higher levels of plasma protein carbonyl and urinary 15F(2t)-isoprostane had an 80% increase in breast cancer risk (OR = 1.8, 95% CI = 1.2-2.6) compared to women with levels below the median for both markers of oxidative stress. In summary, our results suggest that increased plasma protein carbonyl levels may be associated with breast cancer risk.

  7. Use of refractometry for determination of psittacine plasma protein concentration.

    Science.gov (United States)

    Cray, Carolyn; Rodriguez, Marilyn; Arheart, Kristopher L

    2008-12-01

    Previous studies have demonstrated both poor and good correlation of total protein concentrations in various avian species using refractometry and biuret methodologies. The purpose of the current study was to compare these 2 techniques of total protein determination using plasma samples from several psittacine species and to determine the effect of cholesterol and other solutes on refractometry results. Total protein concentration in heparinized plasma samples without visible lipemia was analyzed by refractometry and an automated biuret method on a dry reagent analyzer (Ortho 250). Cholesterol, glucose, and uric acid concentrations were measured using the same analyzer. Results were compared using Deming regression analysis, Bland-Altman bias plots, and Spearman's rank correlation. Correlation coefficients (r) for total protein results by refractometry and biuret methods were 0.49 in African grey parrots (n=28), 0.77 in Amazon parrots (20), 0.57 in cockatiels (20), 0.73 in cockatoos (36), 0.86 in conures (20), and 0.93 in macaws (38) (Prefractometry in Amazon parrots, conures, and macaws (n=25 each, PRefractometry can be used to accurately measure total protein concentration in nonlipemic plasma samples from some psittacine species. Method and species-specific reference intervals should be used in the interpretation of total protein values.

  8. Identification of differentially expressed proteins in vitamin B 12

    Directory of Open Access Journals (Sweden)

    Swati Varshney

    2015-01-01

    Full Text Available Background: Vitamin B 12 (cobalamin is a water-soluble vitamin generally synthesized by microorganisms. Mammals cannot synthesize this vitamin but have evolved processes for absorption, transport and cellular uptake of this vitamin. Only about 30% of vitamin B 12 , which is bound to the protein transcobalamin (TC (Holo-TC [HoloTC] enters into the cell and hence is referred to as the biologically active form of vitamin B 12 . Vitamin B 12 deficiency leads to several complex disorders, including neurological disorders and anemia. We had earlier shown that vitamin B 12 deficiency is associated with coronary artery disease (CAD in Indian population. In the current study, using a proteomics approach we identified proteins that are differentially expressed in the plasma of individuals with low HoloTC levels. Materials and Methods: We used isobaric-tagging method of relative and absolute quantitation to identify proteins that are differently expressed in individuals with low HoloTC levels when compared to those with normal HoloTC level. Results: In two replicate isobaric tags for relative and absolute quantitation experiments several proteins involved in lipid metabolism, blood coagulation, cholesterol metabolic process, and lipoprotein metabolic process were found to be altered in individuals having low HoloTC levels. Conclusions: Our study indicates that low HoloTc levels could be a risk factor in the development of CAD.

  9. NMR Studies of Some Plasma Proteins.

    Science.gov (United States)

    Lawrence, Mark P.

    Available from UMI in association with The British Library. Requires signed TDF. The work reported in this thesis consists of a study of the solution structure of a domain of protein structure found in some of the enzymes involved in blood coagulation. These domains, known as kringles, are of between 78 and 82 residues and contain three conserved disulphide bridges in their primary sequence. The study attempts to elucidate the nature of the lysine-binding site of the fourth kringle of human plasminogen to probe its physiological action, and a theory is developed to explain the overall fold of the protein in terms of its physiological role. The protein structure is found to contain only one small region of secondary structure, an antiparallel beta-sheet of about 8 residues, which provides the support for the binding site. The binding site itself consists of a hydrophobic channel provided by the aromatic residues at positions 61, 63, 71 and 73 in the beta-sheet and a negatively charged site at one end of this channel provided by the aspartic acid residues at positions 54 and 56. The beta-sheet appears to become more tightly defined on binding the kringle with alpha,omega -amino acids which are analogues of lysine and exhibit known anti-fibrinolytic properties. The rest of the solution structure appears to be less clearly defined and relies mainly on the three disulphide bridges and some rather isolated hydrogen bonding for maintenance of the fold. An explanation for this structure with a rigid binding site and a more flexible region for the remainder of the domain is proposed. Shorter studies are reported on the second kringle of bovine prothrombin and the first of human plasminogen which suggest strongly that the kringle fold is conserved.

  10. Plasma protein characteristics of long-term hemodialysis survivors.

    Directory of Open Access Journals (Sweden)

    Yao-Ping Lin

    Full Text Available Hemodialysis (HD patients are under recurrent circulatory stress, and hemodialysis has a high mortality rate. The characteristics of plasma proteomes in patients surviving long-term HD remain obscure, as well as the potential biomarkers in predicting prognoses. This study reports the proteome analyses of patient plasma from non-diabetic long-term HD (LHD, dialysis vintage 14.9±4.1 years, n = 6 and the age/sex/uremic etiology-comparable short-term HD (SHD, dialysis vintage 5.3±2.9 years, n = 6 using 2-DE and mass spectrometry. In addition, a 4-year longitudinal follow-up of 60 non-diabetic HD patients was subsequently conducted to analyze the baseline plasma proteins by ELISA in predicting prognosis. Compared to the SHD, the LHD survivors had increased plasma vitamin D binding proteins (DBP and decreased clusterin, apolipoprotein A-IV, haptoglobin, hemopexin, complement factors B and H, and altered isoforms of α1-antitrypsin and fibrinogen gamma. During the 45.7±15 months for follow-up of the 60 HD patient cases, 16 patients died. Kaplan-Meier analysis demonstrated that HD patients with the lowest tertile of the baseline plasma DBP level have a significantly higher mortality rate. Multivariate Cox regression analysis further indicated that DBP is an independent predictor of mortality. In summary, the altered plasma proteins in LHD implicated accelerated atherosclerosis, defective antioxidative activity, increased inflammation/infection, and organ dysfunction. Furthermore, lower baseline plasma DBP in HD patients is related to mortality. The results suggest that the proteomic approach could help discover the potential biomarker in HD prognoses.

  11. Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758).

    Science.gov (United States)

    Santos, E A A; Sousa, P C; Martins, J A M; Moreira, R A; Monteiro-Moreira, A C O; Moreno, F B M B; Oliveira, M F; Moura, A A; Silva, A R

    2014-06-01

    This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC-MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2±6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7±8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (r=-0.87; P<0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4±1.9 and 8.8±3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-α-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.

  12. Differential regulation of plasma proteins between members of a family with homozygous HbE and HbEβ-thalassemia

    Directory of Open Access Journals (Sweden)

    Suchismita Halder

    2014-09-01

    Full Text Available In this report we’ve compared the plasma protein profiles of 4 individuals in a family. Father and the younger son both are hemoglobin (Hb Eβ-thalassemic {Cod 26 (G-A/IVS 1- 5 (G-C}, but the father never requires transfusion, whereas the younger son requires monthly blood transfusion. Mother and the elder son are HbEE {Cod 26 (G-A/Cod 26 (GA} without any history of transfusion. Proteomic study was done on the plasma fraction of the blood following ammonium sulphate precipitation. Proteins were separated by 2D-gel electrophoresis, expression of proteins compared by densitometry and proteins identified by tandem MALDI mass spectrometry. Proteins responsible in hemolysis, hypercoagulation and hemoglobin scavenging have shown differential regulation, establishing the relation between the differences in the levels of plasma proteins with the progression of the disease phenotype, manifested in the extent of transfusion dependence of the patient.

  13. Plasma protein oxidation and total antioxidant power in premenstrual syndrome

    Institute of Scientific and Technical Information of China (English)

    Eans Tara Tuladhar; Anjali Rao

    2010-01-01

    Objective:To explore whether oxidative stress has any role inpremenstrual syndrome (PMS). Methods: Female volunteers suffering from PMS , in the age group of 20-24 years were compared to their asymptomatic normomennorhoeic counterparts in follicular phase and late luteal phase for ferric reducing antioxidant power of plasma(FRAP), plasma protein thiols(PPT) and protein carbonyls(PPC) levels.Results:There was no significant change in FRAP and PPC levels in controls andPMS groups but PPT decreased significantly in luteal phase ofPMS (P< 0.05) when compared to follicular phase.Conclusions:Estrogen and progesterone, might be responsible for a healthy antioxidant profile inPMS. However, a marked decrease inPPT in luteal phase of PMS group may be due to pro-oxidant nature of estrogen-active in this phase of PMS leading to consumption of the sacrificial antioxidant-protein thiol.

  14. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

    Science.gov (United States)

    Margres, Mark J; Wray, Kenneth P; Seavy, Margaret; McGivern, James J; Herrera, Nathanael D; Rokyta, Darin R

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression

  15. Adsorption of proteins from plasma at polyester non-wovens.

    Science.gov (United States)

    Klomp, A J; Engbers, G H; Mol, J; Terlingen, J G; Feijen, J

    1999-07-01

    Polyester non-wovens in filters for the removal of leukocytes from platelet concentrates (PCs) must be platelet compatible. In PC filtration, the adsorption of proteins at the plasma-non-woven interface can be of great importance with respect to the yield of platelets. Unmodified and radio frequency glow discharge (RFGD) treated poly(ethylene terephthalate) non-woven (NW-PET) and two commercial surface-modified non-wovens were contacted with human plasma. Protein desorption by sodium dodecyl sulphate (SDS) was evaluated by X-ray photoelectron spectroscopy (XPS). The desorbed proteins were characterized by gel electrophoresis and immunoblotting. Compared to the commercial surface-modified non-wovens, unmodified and RFGD-treated NW-PETs adsorbed a relatively high amount of protein. Significantly more protein was removed from the hydrophobic NW-PET by SDS than from the hydrophilic RFGD-treated non-wovens. RFGD treatment of NW-PET reduces the reversibility of protein adsorption. Less albumin and fibrinogen were removed from the RFGD-treated non-wovens than from NW-PET. In addition, a large amount of histidine-rich glycoprotein was removed from RFGD-treated non-wovens, but not from NW-PET. The different behaviour of RFGFD-treated non-wovens towards protein adsorption is probably caused by differences in the chemical reactivity of the non-woven surfaces.

  16. Disproportional changes in hematocrit, plasma volume, and proteins during exercise and bed rest.

    Science.gov (United States)

    Van Beaumont, W.; Greenleaf, J. E.; Juhos, L.

    1972-01-01

    The interrelationships between the changes in plasma volume, hematocrit, and plasma proteins during muscular exercise and bed rest were investigated. Proportionally, the changes in hematocrit are always smaller than the changes in plasma volume. For this reason changes in the concentration of blood constituents can only be quantitated on the basis of plasma volume changes. During short periods of intensive exercise, there was a small loss of plasma proteins. With prolonged submaximal exercise there was a net gain in plasma protein, which contributes to stabilization of the vascular volume. Prolonged bed rest induced hypoproteinemia; this loss of plasma protein probably plays an important role in recumbency hypovolemia.

  17. Fine tuning of IRF-4 expression by SWAP-70 controls the initiation of plasma cell development.

    Science.gov (United States)

    Chopin, Michaël; Chacón-Martínez, Carlos Andrés; Jessberger, Rolf

    2011-10-01

    The generation of plasma cells (PCs) is key for proper humoral immune responses. The transcription factors IRF-4 and BLIMP-1 (B-lymphocyte induce maturation protein-1) control PC commitment, but the underlying regulatory mechanisms are incompletely understood. Here we have identified SWAP-70 as being critically involved in Toll-like receptor (TLR)-triggered PC differentiation. Upon activation through various TLRs, Swap-70(-/-) B cells were activated and proliferated normally. However, expression of BLIMP-1 was markedly reduced and PC differentiation was impaired. Four hours of LPS stimulation were sufficient to drive PC differentiation, and SWAP-70 was required during this initial period. Swap-70(-/-) B cells pre-activated in vitro failed to efficiently differentiate into PCs upon adoptive transfer into recipient mice. Re-introduction of SWAP-70 into Swap-70(-/-) B cells rescued their development into PCs, and SWAP-70 over-expression in wild-type (WT) B cells increased PC generation. In the absence of SWAP-70, IRF-4 protein levels were reduced and the IRF-4(high) B220(+) CD138(-) compartment, including PC precursors, was strongly diminished. Ectopic expression of SWAP-70 increases IRF-4 protein levels and PC differentiation in WT and Swap-70(-/-) B cells, and IRF-4 over-expression in Swap-70(-/-) B cells elevates PC differentiation to WT levels. Thus, in a dose-dependent manner, SWAP-70 controls IRF-4 protein expression and thereby regulates the initiation of PC differentiation.

  18. Radioimmunoassay for pregnancy-associated plasma protein A

    Energy Technology Data Exchange (ETDEWEB)

    Sinosich, M.J. (Royal North Shore Hospital, Sidney, New South Wales, Australia); Teisner, B.; Folkerson, J.; Saunders, D.M.; Grudzinskas, J.G.

    1982-01-01

    A specific and highly sensitive radioimmunoassay for determination of pregnancy-associated plasma protein A in human serum is described. The minimum detection limit for this protein was 2.9 ..mu..g/L. The within- and between-assay coefficients of variation were 4.0 and 4.5%, respectively. The circulating protein was detected within 32 days of conception in eight normal pregnancies and within 21 days in a twin pregnancy. Circulating concentrations in the mother at term were consistently higher (10-fold) than in matched amniotic fluid; none was detected in the umbilical circulation. This protein was also detected in the circulation of patients with hydatidiform mole. This assay will permit investigations into the clinical evaluation of measurements of the protein during early pregnancy and trophoblastic disease.

  19. Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein

    DEFF Research Database (Denmark)

    Nielsen, Marianne Jensby; Petersen, Steen Vang; Jacobsen, Christian

    2006-01-01

    Haptoglobin-related protein (Hpr) is a primate-specific plasma protein associated with apolipoprotein L-I (apoL-I)-containing high-density lipoprotein (HDL) particles shown to be a part of the innate immune defense. Despite the assumption hitherto that Hpr does not bind to hemoglobin, the present...

  20. Plasma membrane microdomains regulate turnover of transport proteins in yeast

    Science.gov (United States)

    Grossmann, Guido; Malinsky, Jan; Stahlschmidt, Wiebke; Loibl, Martin; Weig-Meckl, Ina; Frommer, Wolf B.; Opekarová, Miroslava; Tanner, Widmar

    2008-01-01

    In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins. PMID:19064668

  1. Do plasma proteins distinguish between liposomes of varying charge density?

    KAUST Repository

    Capriotti, Anna Laura

    2012-03-01

    Cationic liposomes (CLs) are one of the most employed nonviral nanovector systems in gene therapy. However, their transfection efficiency is strongly affected by interactions with plasma components, that lead to the formation of a "protein corona" onto CL surface. The interactions between nanoparticles entering the body and biomolecules have an essential role for their biodistribution. Because the knowledge of proteins adsorbed onto vector surface could be useful in the screening of new, more efficient and more biocompatible liposomal formulations, the behavior of three CLs with different membrane charge densities was investigated. The proteins of the three coronas were identified by nano-liquid chromatography-tandem mass spectrometry, and quantified with label-free spectral counting strategy. Fibrinogen displayed higher association with CLs with high membrane charge density, while apolipoproteins and C4b-binding protein with CLs with low membrane charge density. These results are discussed in terms of the different lipid compositions of CLs and may have a deep biological impact for in vivo applications. Surface charge of nanoparticles is emerging as a relevant factor determining the corona composition after interaction with plasma proteins. Remarkably, it is also shown that the charge of the protein corona formed around CLs is strongly related to their membrane charge density. © 2012 Elsevier B.V.

  2. Dietary zinc depletion and repletion affects plasma proteins: an analysis of the plasma proteome.

    Science.gov (United States)

    Grider, Arthur; Wickwire, Kathie; Ho, Emily; Chung, Carolyn S; King, Janet

    2013-02-01

    Zinc (Zn) deficiency is a problem world-wide. Current methods for assessing Zn status are limited to measuring plasma or serum Zn within populations suspected of deficiency. Despite the high prevalence of Zn deficiency in the human population there are no methods currently available for sensitively assessing Zn status among individuals. The purpose of this research was to utilize a proteomic approach using two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify protein biomarkers that were sensitive to changes in dietary Zn levels in humans. Proteomic analysis was performed in human plasma samples (n = 6) obtained from healthy adult male subjects that completed a dietary Zn depletion/repletion protocol, current dietary zinc intake has a greater effect on fractional zinc absorption than does longer term zinc consumption in healthy adult men. Chung et al. (Am J Clin Nutr 87 (5):1224-1229, 2008). After a 13 day Zn acclimatization period where subjects consumed a Zn-adequate diet, the male subjects consumed a marginal Zn-depleted diet for 42 days followed by consumption of a Zn-repleted diet for 28 days. The samples at baseline, end of depletion and end of repletion were pre-fractionated through immuno-affinity columns to remove 14 highly abundant proteins, and each fraction separated by 2DE. Following staining by colloidal Coomassie blue and densitometric analysis, three proteins were identified by mass spectrometry as affected by changes in dietary Zn. Fibrin β and chain E, fragment double D were observed in the plasma protein fraction that remained bound to the immunoaffinity column. An unnamed protein that was related to immunoglobulins was observed in the immunodepleted plasma fraction. Fibrin β increased two-fold following the Zn depletion period and decreased to baseline values following the Zn repletion period; this protein may serve as a viable biomarker for Zn status in the future.

  3. Expression of Contractile Protein Isoforms in Microgravity

    Science.gov (United States)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  4. Increased capillary permeability for plasma proteins in oral contraceptive users.

    Science.gov (United States)

    Tollan, A; Kvenild, K; Strand, H; Oian, P; Maltau, J M

    1992-05-01

    The transcapillary fluid balance was examined in eleven women before administration of a monophasic oral contraceptive (desogestrel 0.15 mg, ethinylestradiol 0.03 mg), and after three and six months of use. The interstitial colloid osmotic pressure was measured by the "wick" method, and the interstitial hydrostatic pressure by the "wick-in-needle" method in subcutaneous tissue on thorax and leg. During the six-month observation period, the following changes were observed: Plasma colloid osmotic pressure decreased (mean 1.8 mmHg, p = 0.047), as well as serum albumin (mean 5.1 g/l, p = 0.0006), total protein concentration (mean 2.8 g/l, p = 0.0006), hemoglobin (mean 0.5 g/dl, p = 0.014) and hematocrit (mean 1.8%, p = 0.047). Blood pressure and body weight remained unchanged, but foot volume showed a significant increase. The colloid osmotic pressure gradient (plasma-interstitium) was significantly reduced. The results indicate an increase in plasma volume in addition to an increased capillary permeability to plasma proteins during oral contraceptive use. We suggest that the observed changes in transcapillary fluid balance is caused by the estrogen component of the oral contraceptive pill.

  5. A Protein Extract from Chicken Reduces Plasma Homocysteine in Rats

    Directory of Open Access Journals (Sweden)

    Vegard Lysne

    2015-06-01

    Full Text Available The present study aimed to evaluate effects of a water-soluble protein fraction of chicken (CP, with a low methionine/glycine ratio, on plasma homocysteine and metabolites related to homocysteine metabolism. Male Wistar rats were fed either a control diet with 20% w/w casein as the protein source, or an experimental diet where 6, 14 or 20% w/w of the casein was replaced with the same amount of CP for four weeks. Rats fed CP had reduced plasma total homocysteine level and markedly increased levels of the choline pathway metabolites betaine, dimethylglycine, sarcosine, glycine and serine, as well as the transsulfuration pathway metabolites cystathionine and cysteine. Hepatic mRNA level of enzymes involved in homocysteine remethylation, methionine synthase and betaine-homocysteine S-methyltransferase, were unchanged, whereas cystathionine gamma-lyase of the transsulfuration pathway was increased in the CP treated rats. Plasma concentrations of vitamin B2, folate, cobalamin, and the B-6 catabolite pyridoxic acid were increased in the 20% CP-treated rats. In conclusion, the CP diet was associated with lower plasma homocysteine concentration and higher levels of serine, choline oxidation and transsulfuration metabolites compared to a casein diet. The status of related B-vitamins was also affected by CP.

  6. Spectrophotometric and Refractometric Determination of Total Protein in Avian Plasma

    OpenAIRE

    2013-01-01

    The aim of this study was to compare the total protein values obtained in heparin plasma of chickens by a spectrophotometric technique (biuret method), and the values obtained on the same day in the same samples by refractometry. The results obtained by refractometry (average value 2.638±0.153g%) were higher than those obtained by the spectrophotometric method (average value 2.441±0.181g%). There was a low correlation (r = 0.6709) between the total protein values, determined with both methods...

  7. Effects of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis

    DEFF Research Database (Denmark)

    Larsen, Mogens; Røntved, Christine Maria; Theil, Peter Kappel

    2017-01-01

    enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[13C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [125I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [13C...

  8. 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.

  9. Cancer associated proteins in blood plasma: Determining normal variation.

    Science.gov (United States)

    Stenemo, Markus; Teleman, Johan; Sjöström, Martin; Grubb, Gabriel; Malmström, Erik; Malmström, Johan; Niméus, Emma

    2016-07-01

    Protein biomarkers have the potential to improve diagnosis, stratification of patients into treatment cohorts, follow disease progression and treatment response. One distinct group of potential biomarkers comprises proteins which have been linked to cancer, known as cancer associated proteins (CAPs). We determined the normal variation of 86 CAPs in 72 individual plasma samples collected from ten individuals using SRM mass spectrometry. Samples were collected weekly during 5 weeks from ten volunteers and over one day at nine fixed time points from three volunteers. We determined the degree of the normal variation depending on interpersonal variation, variation due to time of day, and variation over weeks and observed that the variation dependent on the time of day appeared to be the most important. Subdivision of the proteins resulted in two predominant protein groups containing 21 proteins with relatively high variation in all three factors (day, week and individual), and 22 proteins with relatively low variation in all factors. We present a strategy for prioritizing biomarker candidates for future studies based on stratification over their normal variation and have made all data publicly available. Our findings can be used to improve selection of biomarker candidates in future studies and to determine which proteins are most suitable depending on study design.

  10. Protein expression of sensory and motor nerves: Two-dimensional gel electrophoresis and mass spectrometry.

    Science.gov (United States)

    Ren, Zhiwu; Wang, Yu; Peng, Jiang; Zhang, Li; Xu, Wenjing; Liang, Xiangdang; Zhao, Qing; Lu, Shibi

    2012-02-15

    The present study utilized samples from bilateral motor branches of the femoral nerve, as well as saphenous nerves, ventral roots, and dorsal roots of the spinal cord, to detect differential protein expression using two-dimensional gel electrophoresis and nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry techniques. A mass spectrum was identified using the Mascot search. Results revealed differential expression of 11 proteins, including transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an unnamed protein product (gi|55628), glyceraldehyde-3-phosphate dehydrogenase-like protein, lactoylglutathione lyase, adenylate kinase isozyme 1, two unnamed proteins products (gi|55628 and gi|1334163), and poly(rC)-binding protein 1 in motor and sensory nerves. Results suggested that these proteins played roles in specific nerve regeneration following peripheral nerve injury and served as specific markers for motor and sensory nerves.

  11. Protein expression of sensory and motor nerves Two-dimensional gel electrophoresis and mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    Zhiwu Ren; Yu Wang; Jiang Peng; Li Zhang; Wenjing Xu; Xiangdang Liang; Qing Zhao; Shibi Lu

    2012-01-01

    The present study utilized samples from bilateral motor branches of the femoral nerve, as well as saphenous nerves, ventral roots, and dorsal roots of the spinal cord, to detect differential protein expression using two-dimensional gel electrophoresis and nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry techniques. A mass spectrum was identified using the Mascot search. Results revealed differential expression of 11 proteins, including transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an unnamed protein product (gi|55628), glyceraldehyde-3-phosphate dehydrogenase-like protein, lactoylglutathione lyase, adenylate kinase isozyme 1, two unnamed proteins products (gi|55628 and gi|1334163), and poly(rC)-binding protein 1 in motor and sensory nerves. Results suggested that these proteins played roles in specific nerve regeneration following peripheral nerve injury and served as specific markers for motor and sensory nerves.

  12. Seminal plasma proteomic and gene expression and ngf location and receptors (TRK1 and NGFR) in rabbit genital system

    OpenAIRE

    JÃsy Maria Arruda de Alencar

    2015-01-01

    The aim this study were (i) to map and identify proteins in seminal plasma of New Zealand white rabbits strain, using the techniques of two-dimensional electrophoresis coupled to mass spectrometry and to estimate associations between these proteins with sperm parameters and (ii) characterize expression of the nerve growth factor polypeptide beta ( -NGF) and its cognate neurotrophic receptor tyrosine kinase type 1 (NTRK1), and nerve growth factor receptor (NGFR) at gonads and sex glands in adu...

  13. Specific alterations in plasma proteins during depressed, manic, and euthymic states of bipolar disorder

    Energy Technology Data Exchange (ETDEWEB)

    Song, Y.R. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Wu, B. [Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Yang, Y.T.; Chen, J. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Zhang, L.J.; Zhang, Z.W. [Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Shi, H.Y. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Huang, C.L.; Pan, J.X. [Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Xie, P. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China)

    2015-09-08

    Bipolar disorder (BD) is a common psychiatric mood disorder affecting more than 1-2% of the general population of different European countries. Unfortunately, there is no objective laboratory-based test to aid BD diagnosis or monitor its progression, and little is known about the molecular basis of BD. Here, we performed a comparative proteomic study to identify differentially expressed plasma proteins in various BD mood states (depressed BD, manic BD, and euthymic BD) relative to healthy controls. A total of 10 euthymic BD, 20 depressed BD, 15 manic BD, and 20 demographically matched healthy control subjects were recruited. Seven high-abundance proteins were immunodepleted in plasma samples from the 4 experimental groups, which were then subjected to proteome-wide expression profiling by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry. Proteomic results were validated by immunoblotting and bioinformatically analyzed using MetaCore. From a total of 32 proteins identified with 1.5-fold changes in expression compared with healthy controls, 16 proteins were perturbed in BD independent of mood state, while 16 proteins were specifically associated with particular BD mood states. Two mood-independent differential proteins, apolipoprotein (Apo) A1 and Apo L1, suggest that BD pathophysiology may be associated with early perturbations in lipid metabolism. Moreover, down-regulation of one mood-dependent protein, carbonic anhydrase 1 (CA-1), suggests it may be involved in the pathophysiology of depressive episodes in BD. Thus, BD pathophysiology may be associated with early perturbations in lipid metabolism that are independent of mood state, while CA-1 may be involved in the pathophysiology of depressive episodes.

  14. Engineering Escherichia coli for Functional Expression of Membrane Proteins

    NARCIS (Netherlands)

    Ho, Franz Y; Poolman, Bert

    2015-01-01

    A major bottleneck in the characterization of membrane proteins is low yield of functional protein in recombinant expression. Microorganisms are widely used for recombinant protein production, because of ease of cultivation and high protein yield. However, the target proteins do not always obtain th

  15. QSAR Models for the Prediction of Plasma Protein Binding

    Directory of Open Access Journals (Sweden)

    Zeshan Amin

    2013-02-01

    Full Text Available Introduction: The prediction of plasma protein binding (ppb is of paramount importance in the pharmacokinetics characterization of drugs, as it causes significant changes in volume of distribution, clearance and drug half life. This study utilized Quantitative Structure – Activity Relationships (QSAR for the prediction of plasma protein binding. Methods: Protein binding values for 794 compounds were collated from literature. The data was partitioned into a training set of 662 compounds and an external validation set of 132 compounds. Physicochemical and molecular descriptors were calculated for each compound using ACD labs/logD, MOE (Chemical Computing Group and Symyx QSAR software packages. Several data mining tools were employed for the construction of models. These included stepwise regression analysis, Classification and Regression Trees (CART, Boosted trees and Random Forest. Results: Several predictive models were identified; however, one model in particular produced significantly superior prediction accuracy for the external validation set as measured using mean absolute error and correlation coefficient. The selected model was a boosted regression tree model which had the mean absolute error for training set of 13.25 and for validation set of 14.96. Conclusion: Plasma protein binding can be modeled using simple regression trees or multiple linear regressions with reasonable model accuracies. These interpretable models were able to identify the governing molecular factors for a high ppb that included hydrophobicity, van der Waals surface area parameters, and aromaticity. On the other hand, the more complicated ensemble method of boosted regression trees produced the most accurate ppb estimations for the external validation set.

  16. Interactions between plasma proteins and naturally occurring polyphenols.

    Science.gov (United States)

    Li, Min; Hagerman, Ann E

    2013-05-01

    The plant natural products known as polyphenols are found at micronutrient levels in fruits, vegetables, and plant-based beverages such as wine, tea, coffee and cocoa. Consumption of a fruit- and vegetable-rich diet, the "Mediterranean diet", has been epidemiologically related to health benefits especially for chronic diseases including diabetes, cardiovascular disease, and Alzheimer's disease. The abundance of polyphenols in plant-rich diets, and the potent bioactivities of polyphenols, provide indirect evidence for a role for polyphenols in maintaining good health. However, molecular mechanisms for therapeutic or preventative activity have not been demonstrated in vivo. We summarize the chemical classes of natural polyphenols, their bioactivities and bioavailability and metabolism. Because many polyphenols bind protein, we focus on the potential of protein binding to mediate the health-related effects of polyphenols. We discuss interactions with plasma proteins as the first target organ past the digestive tract for these orally-ingested compounds.

  17. Tissue expression and plasma levels of adrenomedullin in renal cancer patients

    DEFF Research Database (Denmark)

    Michelsen, Jens; Thiesson, Helle; Walter, Steen;

    2006-01-01

    The peptide AM (adrenomedullin) is stimulated by hypoxia through HIF-1 (hypoxia-inducible factor-1). The majority of human CC-RCCs (clear cell renal cell carcinomas) display mutations in the tumour suppressor protein von Hippel-Lindau, which leads to constitutively elevated HIF-1. We hypothesized......RNA and peptide expression in tissue and AM plasma concentration were determined. HIF-1alpha was localized in tissue by immunohistochemistry. AM mRNA was elevated in CC-RCC compared with adjacent renal cortex (6-fold, n=18; P

  18. Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Jeffery Constance J

    2010-11-01

    Full Text Available Abstract Background Transmembrane proteins (TM proteins make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY. All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%. In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical

  19. A simple and straightforward expression for curling probe electron density diagnosis in reactive plasmas

    Science.gov (United States)

    Arshadi, Ali; Brinkmann, Ralf Peter; Hotta, Masaya; Nakamura, Keiji

    2017-04-01

    Active plasma resonance spectroscopy (APRS) refers to the family of plasma diagnostic methods which utilize the ability of plasmas to resonate at frequencies close to the plasma frequency. APRS operates by exciting the plasma with a weak RF signal by means of a small electric probe. The response of the plasma is recorded by a network analyzer (NA). A mathematical model is applied to derive characteristics like the electron density and the electron temperature. The curling probe is a promising realization of APRS. The curling probe is well-qualified for the local measurement of the electron density in reactive plasmas. This spiral probe resonates in plasma at a larger density dependent frequency than the plasma frequency. This manuscript represents a simple and straightforward expression relating this resonance frequency to the electron density of the plasma. A good agreement is observed between the proposed expression and the results obtained from previous studies and numerical simulations.

  20. Krill protein hydrolysate reduces plasma triacylglycerol level with concurrent increase in plasma bile acid level and hepatic fatty acid catabolism in high-fat fed mice

    Directory of Open Access Journals (Sweden)

    Marie S. Ramsvik

    2013-11-01

    Full Text Available Background: Krill powder, consisting of both lipids and proteins, has been reported to modulate hepatic lipid catabolism in animals. Fish protein hydrolysate diets have also been reported to affect lipid metabolism and to elevate bile acid (BA level in plasma. BA interacts with a number of nuclear receptors and thus affects a variety of signaling pathways, including very low density lipoprotein (VLDL secretion. The aim of the present study was to investigate whether a krill protein hydrolysate (KPH could affect lipid and BA metabolism in mice. Method: C57BL/6 mice were fed a high-fat (21%, w/w diet containing 20% crude protein (w/w as casein (control group or KPH for 6 weeks. Lipids and fatty acid composition were measured from plasma, enzyme activity and gene expression were analyzed from liver samples, and BA was measured from plasma. Results: The effect of dietary treatment with KPH resulted in reduced levels of plasma triacylglycerols (TAG and non-esterified fatty acids (NEFAs. The KPH treated mice had also a marked increased plasma BA concentration. The increased plasma BA level was associated with induction of genes related to membrane canalicular exporter proteins (Abcc2, Abcb4 and to BA exporters to blood (Abcc3 and Abcc4. Of note, we observed a 2-fold increased nuclear farnesoid X receptor (Fxr mRNA levels in the liver of mice fed KPH. We also observed increased activity of the nuclear peroxiosme proliferator-activated receptor alpha (PPARα target gene carnitine plamitoyltransferase 2 (CPT-2. Conclusion: The KPH diet showed to influence lipid and BA metabolism in high-fat fed mice. Moreover, increased mitochondrial fatty acid oxidation and elevation of BA concentration may regulate the plasma level of TAGs and NEFAs.

  1. Simultaneous Mars Express / MGS observations of plasma near Mars

    Science.gov (United States)

    Brain, D.; Luhmann, J.; Halekas, J.; Frahm, R.; Winningham, D.; Barabash, S.

    2006-12-01

    Since late 2003, Mars Express (MEX) and Mars Global Surveyor (MGS) have been making complementary in situ measurements (in terms of both instrument and orbit) of the Martian plasma environment. Study of MGS and MEX data in tandem provides an opportunity to mitigate the shortcomings of each dataset and increase our overall understanding of the Martian solar wind interaction and atmospheric escape. Close passes of spacecraft (conjunctions) are one particularly powerful means of increasing the utility of measurements, as evidenced by the Cluster mission at Earth. At Mars, conjunctions might be used to obtain more complete simultaneous and/or co-located plasma measurements, which can be used to study a variety of phenomena, including measurements of auroral-like particle acceleration near crustal fields and the three-dimensional motion and shape of plasma boundaries. We will present an analysis of approximately forty conjunctions (instances with instantaneous spacecraft separation smaller than 400 km) of MEX and MGS identified between January 2004 and February 2006. The closest pass was ~40~km, near the South Pole. Conjunctions occur both at mid-latitudes (when the surface-projected orbit tracks of the two spacecraft nearly overlap), and at the poles. We will present comparisons of MEX Analyzer of Space Plasmas and Energetic Atoms (ASPERA-3) data with MGS Magnetometer and Electron Reflectometer (MAG/ER) data for these events. Our case studies include intercomparison of MEX and MGS electron data, the addition of MGS magnetic field and MEX ion data, and the inclusion of solar wind proxy information to establish context. In addition to these close conjunctions, we will present the preliminary results of a search for times when MEX and MGS pass through the same region of space separated by a delay (for time evolution of plasma populations in certain regions), and times when they occupy the same flux tube (for spatial evolution of particle distributions). Continued study of

  2. Changes in total plasma content of electrolytes and proteins with maximal exercise.

    Science.gov (United States)

    Van Beaumont, W.; Strand, J. C.; Petrofsky, J. S.; Hipskind, S. G.; Greenleaf, J. E.

    1973-01-01

    To determine to what extent the increases in concentration of plasma proteins and electrolytes with short maximal work were a result of hemoconcentration, the changes in plasma volume and total content of the plasma constituents were simultaneously evaluated. The results obtained from six human subjects indicated that in comparison to preexercise values there was a net decrease in total content of plasma protein, sodium, and chloride in the first 2 min of the postexercise period, due primarily to a significant loss (13-15%) of plasma fluid. The total plasma potassium content was increased immediately after exercise but was significantly below the preexercise plasma content after 2 min of recovery.

  3. Expression and significance of heat shock protein 70 and glucose-regulated protein 94 in human esophageal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ping Wang; Guo-Zhen Liu; Ai-Li Song; Rui-Fen Chen; Hai-Yan Li; Yu Liu

    2005-01-01

    AIM: To investigate the expression and significance of heat shock protein 70 (HSP70) and glucose-regulated protein 94 (grp94) in human esophageal carcinoma and adjacent normal tissues.METHODS: The expression of HSP70 and grp94 in 78human esophageal cancer and adjacent normal tissues was studied by immunohistochemistry and pathology photograph analysis.RESULTS: Both esophageal cancer and adjacent normal tissues could express HSP70 and grp94. Of the 78 cases of esophageal carcinoma, 95.0%(72/78) showed positive HSP70, mainly stained in nuclei, while grp94 was mainly stained in cell plasma, and the positive rate was 71.8%(56/78).There was a significant difference in the expression of HSP70 and grp94 between esophageal cancer and adjacent normal tissues (P<0.01). Compared with adjacent normal tissues, there was a significant difference between differential types and HSP70 expression (P<0.01).CONCLUSION: HSP70 and grp94 express differently in cell plasma and nuclei. The expression intensity of HSP70is related to the differentiation of esophageal carcinoma.

  4. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    Institute of Scientific and Technical Information of China (English)

    GAO Lei; LI Xia; GUO Zheng; ZHU MingZhu; LI YanHui; RAO ShaoQi

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interaction data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automatically selects the most appropriate functional classes as specific as possible during the learning process, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to "biology process" by three measures particularly designed for functional classes organized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  5. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interac-tion data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automati-cally selects the most appropriate functional classes as specific as possible during the learning proc-ess, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to “biology process” by three measures particularly designed for functional classes organ-ized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  6. Effects of ADMA upon gene expression: an insight into the pathophysiological significance of raised plasma ADMA.

    Directory of Open Access Journals (Sweden)

    Caroline L Smith

    2005-10-01

    Full Text Available BACKGROUND: Asymmetric dimethylarginine (ADMA is a naturally occurring inhibitor of nitric oxide synthesis that accumulates in a wide range of diseases associated with endothelial dysfunction and enhanced atherosclerosis. Clinical studies implicate plasma ADMA as a major novel cardiovascular risk factor, but the mechanisms by which low concentrations of ADMA produce adverse effects on the cardiovascular system are unclear. METHODS AND FINDINGS: We treated human coronary artery endothelial cells with pathophysiological concentrations of ADMA and assessed the effects on gene expression using U133A GeneChips (Affymetrix. Changes in several genes, including bone morphogenetic protein 2 inducible kinase (BMP2K, SMA-related protein 5 (Smad5, bone morphogenetic protein receptor 1A, and protein arginine methyltransferase 3 (PRMT3; also known as HRMT1L3, were confirmed by Northern blotting, quantitative PCR, and in some instances Western blotting analysis to detect changes in protein expression. To determine whether these changes also occurred in vivo, tissue from gene deletion mice with raised ADMA levels was examined. More than 50 genes were significantly altered in endothelial cells after treatment with pathophysiological concentrations of ADMA (2 microM. We detected specific patterns of changes that identify pathways involved in processes relevant to cardiovascular risk and pulmonary hypertension. Changes in BMP2K and PRMT3 were confirmed at mRNA and protein levels, in vitro and in vivo. CONCLUSION: Pathophysiological concentrations of ADMA are sufficient to elicit significant changes in coronary artery endothelial cell gene expression. Changes in bone morphogenetic protein signalling, and in enzymes involved in arginine methylation, may be particularly relevant to understanding the pathophysiological significance of raised ADMA levels. This study identifies the mechanisms by which increased ADMA may contribute to common cardiovascular diseases and

  7. Effects of ADMA upon gene expression: an insight into the pathophysiological significance of raised plasma ADMA.

    Directory of Open Access Journals (Sweden)

    Caroline L Smith

    2005-10-01

    Full Text Available Asymmetric dimethylarginine (ADMA is a naturally occurring inhibitor of nitric oxide synthesis that accumulates in a wide range of diseases associated with endothelial dysfunction and enhanced atherosclerosis. Clinical studies implicate plasma ADMA as a major novel cardiovascular risk factor, but the mechanisms by which low concentrations of ADMA produce adverse effects on the cardiovascular system are unclear.We treated human coronary artery endothelial cells with pathophysiological concentrations of ADMA and assessed the effects on gene expression using U133A GeneChips (Affymetrix. Changes in several genes, including bone morphogenetic protein 2 inducible kinase (BMP2K, SMA-related protein 5 (Smad5, bone morphogenetic protein receptor 1A, and protein arginine methyltransferase 3 (PRMT3; also known as HRMT1L3, were confirmed by Northern blotting, quantitative PCR, and in some instances Western blotting analysis to detect changes in protein expression. To determine whether these changes also occurred in vivo, tissue from gene deletion mice with raised ADMA levels was examined. More than 50 genes were significantly altered in endothelial cells after treatment with pathophysiological concentrations of ADMA (2 microM. We detected specific patterns of changes that identify pathways involved in processes relevant to cardiovascular risk and pulmonary hypertension. Changes in BMP2K and PRMT3 were confirmed at mRNA and protein levels, in vitro and in vivo.Pathophysiological concentrations of ADMA are sufficient to elicit significant changes in coronary artery endothelial cell gene expression. Changes in bone morphogenetic protein signalling, and in enzymes involved in arginine methylation, may be particularly relevant to understanding the pathophysiological significance of raised ADMA levels. This study identifies the mechanisms by which increased ADMA may contribute to common cardiovascular diseases and thereby indicates possible targets for therapies.

  8. Forced KLF4 expression increases the generation of mature plasma cells and uncovers a network linked with plasma cell stage.

    Science.gov (United States)

    Schoenhals, Matthieu; Jourdan, Michel; Seckinger, Anja; Pantesco, Véronique; Hose, Dirk; Kassambara, Alboukadel; Moreaux, Jérôme; Klein, Bernard

    2016-07-17

    A role of the transcription factor Krüppel-like factor 4 (KLF4) in the generation of mature plasma cells (PC) is unknown. Indeed, KLF4 is critical in controlling the differentiation of various cell linages, particularly monocytes and epithelial cells. KLF4 is expressed at low levels in pro-B cells and its expression increases as they mature into pre-B cells, resting naïve B cells and memory B cells. We show here that KLF4 is expressed in human bone marrow plasma cells and its function was studied using an in vitro model of differentiation of memory B cells into long lived plasma cells. KLF4 is rapidly lost when memory B cells differentiate into highly cell cycling plasmablasts, poorly cycling early plasma cells and then quiescent long-lived plasma cells. A forced expression of KLF4 in plasmablasts enhances the yield of their differentiation into early plasma cell and long lived plasma cells, by inhibiting apoptosis and upregulating previously unknown plasma cell pathways.

  9. 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

  10. Resistin in dairy cows: plasma concentrations during early lactation, expression and potential role in adipose tissue.

    Directory of Open Access Journals (Sweden)

    Maxime Reverchon

    Full Text Available Resistin is an adipokine that has been implicated in energy metabolism regulation in rodents but has been little studied in dairy cows. We determined plasma resistin concentrations in early lactation in dairy cows and investigated the levels of resistin mRNA and protein in adipose tissue and the phosphorylation of several components of insulin signaling pathways one week post partum (1 WPP and at five months of gestation (5 MG. We detected resistin in mature bovine adipocytes and investigated the effect of recombinant bovine resistin on lipolysis in bovine adipose tissue explants. ELISA showed that plasma resistin concentration was low before calving, subsequently increasing and reaching a peak at 1 WPP, decreasing steadily thereafter to reach pre-calving levels at 6 WPP. Plasma resistin concentration was significantly positively correlated with plasma non esterified fatty acid (NEFA levels and negatively with milk yield, dry matter intake and energy balance between WPP1 to WPP22. We showed, by quantitative RT-PCR and western blotting, that resistin mRNA and protein levels in adipose tissue were higher at WPP1 than at 5 MG. The level of phosphorylation of several early and downstream insulin signaling components (IRβ, IRS-1, IRS-2, Akt, MAPK ERK1/2, P70S6K and S6 in adipose tissue was also lower at 1 WPP than at 5 MG. Finally, we showed that recombinant bovine resistin increased the release of glycerol and mRNA levels for ATGL (adipose triglyceride lipase and HSL (hormone-sensitive lipase in adipose tissue explants. Overall, resistin levels were high in the plasma and adipose tissue and were positively correlated with NEFA levels after calving. Resistin is expressed in bovine mature adipocytes and promotes lipid mobilization in adipose explants in vitro.

  11. Proteomic identification of plasma protein tyrosine phosphatase alpha and fibronectin associated with liver fluke, Opisthorchis viverrini, infection.

    Directory of Open Access Journals (Sweden)

    Jarinya Khoontawad

    Full Text Available Opisthorchiasis caused by Opisthorchis viverrini induces periductal fibrosis via host immune/inflammatory responses. Plasma protein alteration during host-parasite interaction-mediated inflammation may provide potential diagnostic and/or prognostic biomarkers. To search for target protein changes in O. viverrini-infected hamsters, a 1-D PAGE gel band was trypsin-digested and analyzed by a LC-MS/MS-based proteomics approach in the plasma profile of infected hamsters, and applied to humans. Sixty seven proteins were selected for further analysis based on at least two unique tryptic peptides with protein ID score >10 and increased expression at least two times across time points. These proteins have not been previously identified in O. viverrini-associated infection. Among those, proteins involved in structural (19%, immune response (13%, cell cycle (10% and transcription (10% were highly expressed. Western blots revealed an expression level of protein tyrosine phosphatase alpha (PTPα which reached a peak at 1 month and subsequently tended to decrease. Fibronectin significantly increased at 1 month and tended to increase with time, supporting proteomic analysis. PTPα was expressed in the cytoplasm of inflammatory cells, while fibronectin was observed mainly in the cytoplasm of fibroblasts and the extracellular matrix at periductal fibrosis areas. In addition, these protein levels significantly increased in the plasma of O. viverrini-infected patients compared to healthy individuals, and significantly decreased at 2-months post-treatment, indicating their potential as disease markers. In conclusion, our results suggest that plasma PTPα and fibronectin may be associated with opisthorchiasis and the hamster model provides the basis for development of novel diagnostic markers in the future.

  12. Mapping of 79 loci for 83 plasma protein biomarkers in cardiovascular disease

    DEFF Research Database (Denmark)

    Folkersen, Lasse Westergaard; Fauman, Eric; Sabater-Lleal, Maria

    2017-01-01

    Recent advances in highly multiplexed immunoassays have allowed systematic large-scale measurement of hundreds of plasma proteins in large cohort studies. In combination with genotyping, such studies offer the prospect to 1) identify mechanisms involved with regulation of protein expression...... identified 79 genome-wide significant (pmining, manual curation, and network-based methods incorporating information on expression quantitative trait loci (eQTL), we...... propose plausible causal mechanisms for 25 trans-acting loci, including a potential post-translational regulation of stem cell factor by matrix metalloproteinase 9 and receptor-ligand pairs such as RANK-RANK ligand. Using public GWA study data, we further evaluate all 79 loci for their causal effect...

  13. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  14. Strain engineering for improved expression of recombinant proteins in bacteria.

    Science.gov (United States)

    Makino, Tomohiro; Skretas, Georgios; Georgiou, George

    2011-05-14

    Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance recombinant protein expression have been developed. These methodologies now enable the generation of optimized E. coli expression strains in a manner analogous to metabolic engineering for the synthesis of low-molecular-weight compounds. In this review, we provide an overview of strain engineering approaches useful for enhancing the expression of hard-to-produce proteins, including heterologous membrane proteins.

  15. Serum Copper and Plasma Protein Status in Normal Pregnancy

    Directory of Open Access Journals (Sweden)

    Nushrat Noor, Nasim Jahan, Nayma Sultana

    2012-12-01

    Full Text Available AbstractBackground: Gradual alteration of serum copper and some plasma protein levels may occur with advancement of pregnancy, which is associated with increased maternal and infant morbidity and mortality.Objective: To observe serum copper and plasma protein levels in normal pregnant women of different trimesters in order to find out their nutritional status.Methods: This cross sectional study was carried out in the Department of Physiology, Sir Salimullah Medical College (SSMC, Dhaka, between 1st January 2010 and December 2010. Ninety normal pregnant women of different trimesters with age 20-30 years were included in the study group. They were selected from Out Patient Department of Obstetrics and Gynaecology, SSMC. Age matched 30 non-pregnant women were taken as control. Serum copper level was measured by Spectrophotometric method, serum total protein and albumin levels were estimated by standard method. Statistical analysis was done by one way ANOVA, Bonferroni and Pearson’s correlation coefficient test as applicable.Results: Serum Cu levels were significantly higher in all trimesters of pregnant women compared to control. Again, this value was significantly higher in 3rd trimester than that of in 1st and 2nd trimester and also in 2nd trimester than that of in 1st trimester. In addition, mean serum total protein level was significantly lower in 3rd trimester than control but no statistically significant difference was observed among different trimesters. Again, mean serum albumin level was significantly lower in 2nd and 3rd trimester than 1st trimester and control. In addition, serum Cu concentration showed significant positive correlation with different trimesters of gestation.Conclusion: This study reveals that hypercupremia along with hypoproteinemia occur in pregnant women from 1st to 3rd trimester of gestation. This gradual alteration of micro and macronutrients become more profound with advancement of pregnancy.

  16. Differential Protein Expression in the Hemolymph of Bithynia siamensis goniomphalos Infected with Opisthorchis viverrini

    Science.gov (United States)

    Suwannatrai, Kulwadee; Suwannatrai, Apiporn; Tabsripair, Pairat; Welbat, Jariya Umka; Tangkawattana, Sirikachorn; Cantacessi, Cinzia; Mulvenna, Jason; Tesana, Smarn; Loukas, Alex

    2016-01-01

    Bithynia siamensis goniomphalos is a freshwater snail that serves as the first intermediate host of the human liver fluke Opisthorchis viverrini. This parasite is a major public health problem in different countries throughout the Greater Mekong sub-region (Thailand, southern Vietnam, Lao PDR and Cambodia). Chronic O. viverrini infection also results in a gradual increase of fibrotic tissues in the biliary tract that are associated with hepatobiliary diseases and contribute to cholangiocarcinoma (a fatal type of bile duct cancer). Infectivity of the parasite in the snail host is strongly correlated with destruction of helminths by the snail’s innate immune system, composed of cellular (hemocyte) and humoral (plasma) defense factors. To better understand this important host-parasite interface we applied sequential window acquisition of all theoretical spectra mass spectrometry (SWATH-MS) to identify and quantify the proteins from the hemolymph of B. siamensis goniomphalos experimentally infected with O. viverrini and compare them to non-infected snails (control group). A total of 362 and 242 proteins were identified in the hemocytes and plasma, respectively. Of these, 145 and 117 proteins exhibited significant differences in expression upon fluke infection in hemocytes and plasma, respectively. Among the proteins with significantly different expression patterns, we found proteins related to immune response (up-regulated in both hemocyte and plasma of infected snails) and proteins belonging to the structural and motor group (mostly down-regulated in hemocytes but up-regulated in plasma of infected snails). The proteins identified and quantified in this work will provide important information for the understanding of the factors involved in snail defense against O. viverrini and might facilitate the development of new strategies to control O. viverrini infection in endemic areas. PMID:27893749

  17. Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

    Science.gov (United States)

    Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

    2007-08-17

    Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

  18. Strain engineering for improved expression of recombinant proteins in bacteria

    OpenAIRE

    Skretas Georgios; Makino Tomohiro; Georgiou George

    2011-01-01

    Abstract Protein expression in Escherichia coli represents the most facile approach for the preparation of non-glycosylated proteins for analytical and preparative purposes. So far, the optimization of recombinant expression has largely remained a matter of trial and error and has relied upon varying parameters, such as expression vector, media composition, growth temperature and chaperone co-expression. Recently several new approaches for the genome-scale engineering of E. coli to enhance re...

  19. Calreticulin: Roles in Cell-Surface Protein Expression

    Directory of Open Access Journals (Sweden)

    Yue Jiang

    2014-09-01

    Full Text Available In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

  20. Protein secretion is required for pregnancy-associated plasma protein-A to promote lung cancer growth in vivo.

    Directory of Open Access Journals (Sweden)

    Hong Pan

    Full Text Available Pregnancy-associated plasma protein-A (PAPPA has been reported to regulate the activity of insulin-like growth factor (IGF signal pathway through proteolytic degradation of IGF binding proteins (IGFBPs thereby increasing the local concentration of free IGFs available to receptors. In this study we found that PAPPA is secreted from two out of seven lung cancer cell lines examined. None of immortalized normal bronchial epithelial cells (HBE tested secrets PAPPA. There is no correlation between expression level and secretion of PAPPA in these cells. A cell line over-expressing PAPPA accompanied with secretion shows no notable changes in proliferation under cell culture conditions in vitro, but displays significantly augmentation of tumor growth in vivo in a xenograft model. In contrast, a cell line over-expressing PAPPA without secretion exhibits reduction of tumor growth both in vitro and in vivo. Down-regulation of PAPPA expression and secretion by RNAi knockdown decreases tumor growth after implanted in vivo. The tumor promoting activity of PAPPA appears to be mediated mainly through augmentation of the IGF signaling pathway as indicated by notable increases in downstream Akt kinase phosphorylation in tumor samples. Our results indicate that PAPPA secretion may play an important role in lung cancer growth and progression.

  1. Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis▿

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-01-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs. PMID:19251885

  2. Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-05-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

  3. Expression and structural analysis of membrane proteins

    OpenAIRE

    Eifler, Nora

    2006-01-01

    1.1 Membrane Proteins Between one quarter and one third of all genes in eukaryotic and prokaryotic organisms code for integral membrane proteins (IMPs) (Essen, 2002). These proteins are essential parts of biological membranes and confer various functions, such as energy conversion, transport, biosynthesis of lipids, signal transduction, or cell recognition. The enormous economical potential of membrane proteins is highlighted by the family of G-protein-coupled receptors (GPC...

  4. ADAM10 regulates transcription factor expression required for plasma cell function.

    Directory of Open Access Journals (Sweden)

    Natalia S Chaimowitz

    Full Text Available A disintegrin and metalloprotease 10 (ADAM10 is a key regulator of cellular processes by shedding extracellular domains of transmembrane proteins. We have previously demonstrated that deletion of B cell expressed ADAM10 results in changes in lymphoid tissue architecture and impaired germinal center (GC formation. In this study, mice were generated in which ADAM10 is deleted in B cells following class switch recombination (ADAM10(Δ/ΔIgG1-cre(+/- mice. Despite normal GC formation, antibody responses were impaired in ADAM10(Δ/ΔIgG1-cre(+/- mice, implicating ADAM10 in post-GC and extrafollicular B cell terminal differentiation. Surprisingly, plasma cell (PC numbers were normal in ADAM10(Δ/ΔIgG1-cre(+/- mice when compared to controls. However, PCs isolated from ADAM10(Δ/ΔIgG1-cre(+/- mice exhibited decreased expression of transcription factors important for PC function: Prdm1, Xbp1 and Irf4. Bcl6 is a GC transcriptional repressor that inhibits the PC transcriptional program and thus must be downregulated for PC differentiation to occur. Bcl6 expression was increased in PCs isolated from ADAM10(Δ/ΔIgG1-cre(+/- mice at both the mRNA and protein level. These results demonstrate that ADAM10 is required for proper transcription factor expression in PCs and thus, for normal PC function.

  5. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Kokoro [Fujifilm Corporation, Analysis Technology Center (Japan); Kojima, Chojiro, E-mail: kojima@protein.osaka-u.ac.j [Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences (Japan)

    2010-11-15

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in {sup 1}H-{sup 15}N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  6. Analysis of the plasma proteome in COPD: Novel low abundance proteins reflect the severity of lung remodeling.

    Science.gov (United States)

    Merali, Salim; Barrero, Carlos A; Bowler, Russell P; Chen, Diane Er; Criner, Gerard; Braverman, Alan; Litwin, Samuel; Yeung, Anthony; Kelsen, Steven G

    2014-04-01

    The search for COPD biomarkers has largely employed a targeted approach that focuses on plasma proteins involved in the systemic inflammatory response and in lung injury and repair. This proof of concept study was designed to test the idea that an open, unbiased, in-depth proteomics approach could identify novel, low abundance plasma proteins i.e., ng/mL concentration, which could serve as potential biomarkers. Differentially expressed proteins were identified in a discovery group with severe COPD (FEV1 <45% predicted; n = 10). Subjects with normal lung function matched for age, sex, ethnicity and smoking history served as controls (n = 10). Pooled plasma from each group was exhaustively immunodepleted of abundant proteins, d separated by 1-D gel electrophoresis and extensively fractionated prior to LC-tandem mass spectroscopy (GeLC-MS). Thirty one differentially expressed proteins were identified in the discovery group including markers of lung defense against oxidant stress, alveolar macrophage activation, and lung tissue injury and repair. Four of the 31 proteins (i.e., GRP78, soluble CD163, IL1AP and MSPT9) were measured in a separate verification group of 80 subjects with varying COPD severity by immunoassay. All 4 were significantly altered in COPD and 2 (GRP78 and soluble CD163) correlated with both FEV1 and the extent of emphysema. In-depth, plasma proteomic analysis identified a group of novel, differentially expressed, low abundance proteins that reflect known pathogenic mechanisms and the severity of lung remodeling in COPD. These proteins may also prove useful as COPD biomarkers.

  7. Effect of Addition of Concentrated Proteins and Seminal Plasma Low Molecular Weight Proteins in Freezing and Thawing of Equine Semen

    Directory of Open Access Journals (Sweden)

    Bruno Fagundes

    2011-07-01

    Full Text Available Difficulties in obtaining equine frozen semen with potential fertility are recognized. This study was designed to investigate the effect of seminal plasma on frozen/thawing of eight stallion semen from different breed using the following treatments: Seminal plasma with ten-fold concentrated proteins with molecular weight above 10 kDa on frozen extender; Part of seminal plasma with proteins under 10 kDa on frozen extender; Conventional freezing, using whole seminal plasma on frozen extender. Using the parameter of 30% of seminal motility post-thawing as index of good freezability, it was verified an increased percentage of stallions that presented good freezability when semen was frozen with seminal plasma containing ten-fold concentrated proteins with molecular weight above 10 kDa on frozen extender. These results, suggested the use of seminal plasma concentrated proteins from own stallion to freezing/thawing semen.

  8. Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

    Science.gov (United States)

    Li, Lunyi; Shi, Xiaoshan; Guo, Xingdong; Li, Hua; Xu, Chenqi

    2014-03-01

    Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms.

  9. Cloning and expression of special F protein from human liver

    Institute of Scientific and Technical Information of China (English)

    Shu-Ye Liu; Xin-Da Yu; Chun-Juan Song; Wei Lu; Jian-Dong Zhang; Xin-Rong Shi; Ying Duan; Ju Zhang

    2007-01-01

    AIM:To clone human liver special F protein and to express it in a prokaryotic system.METHODS:Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this,cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein's cDNA was subcloned into the expression vector pET-15b and transformed into E coli BL21 (DEB) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein.RESULTS:The cDNA clone of human liver special F protein (1134bp) was successfully produced,with the cDNA sequence being published in Gene-bank:DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted.CONCLUSION:F protein expresses cDNA clone in a proKaryotic system,which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein.

  10. The Analysis of Sialylation, N-Glycan Branching, and Expression of O-Glycans in Seminal Plasma of Infertile Men

    Directory of Open Access Journals (Sweden)

    Ewa M. Kratz

    2015-01-01

    Full Text Available Carbohydrates are known to mediate some events involved in successful fertilization. Although some studies on the glycosylation of seminal plasma proteins are available, the total glycan profile was rarely analyzed as a feature influencing fertilization potential. In this work we aimed to compare some glycosylation traits in seminal plasma glycoproteins of fertile and infertile men. The following findings emerge from our studies: (1 in human seminal plasma the presence and alterations of O-linked glycans were observed; (2 the expression of SNA-reactive sialic acid significantly differs between asthenozoospermia and both normozoospermic (fertile and infertile groups; (3 the expression of PHA-L-reactive highly branched N-glycans was significantly lower in oligozoospermic patients than in both normozoospermic groups. Indication of the appropriate lectins that would enable the possibly precise determination of the glycan profile seems to be a good supplement to mass spectrum analysis. Extension of the lectin panel is useful for the further research.

  11. Boost protein expression through co-expression of LEA-like peptide in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Shinya Ikeno

    Full Text Available The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently.

  12. Intake of Mung Bean Protein Isolate Reduces Plasma Triglyceride Level in Rats

    Directory of Open Access Journals (Sweden)

    Nobuhiko Tachibana

    2013-09-01

    Full Text Available ABSTRACTBackground: Mung bean is well known as a starch source, but the physiological effects of mung bean protein have received little attention. In this study, we isolated mung bean protein from de-starched mung bean solutions, and investigated its influence on lipid metabolism. Objective: The aim of this study is to clarify the influence of the lipid metabolism by consumption of mung bean protein isolate (MPIMethods: Diets containing either mung bean protein isolate (MPI or casein were fed to normal rats for 28 days.Results: Both groups ate the same amount of food, but the plasma triglyceride level, relative liver weight and liver lipid contents (cholesterol and triglyceride pool in the MPI group were significantly lower than in the casein group. In the MPI group, the expression of sterol regulatory-element binding factor 1 (SREBF1 mRNA in the liver was significantly different when compared with the casein group. The significantly lower levels of insulin and free fatty acids in the MPI-fed rats may be due to the regulation of genes related to lipid metabolism in the liver.Conclusions: These results suggest that MPI may improve the plasma lipid profile by normalizing insulin sensitivity.Keywords: mung bean, Vigna radiata L., 8S globulin, triglyceride, β-conglycinin, 7S globulin, insulin sensitivity, SREBF1

  13. Increased plasma retinol binding protein 4 levels in patients with inflammatory cardiomyopathy.

    Science.gov (United States)

    Bobbert, Peter; Weithäuser, Alice; Andres, Janin; Bobbert, Thomas; Kühl, Uwe; Schultheiss, Heinz Peter; Rauch, Ursula; Skurk, Carsten

    2009-12-01

    Chronic heart failure (CHF) is associated with a higher risk for diabetes mellitus. Retinol binding protein 4 (RBP 4) is an adipose tissue-derived protein with pro-diabetogenic effects. A complete understanding of the association of CHF and insulin resistance remains elusive. The purpose of this study was to examine the relationship between CHF and diabetes mellitus. Plasma levels of RBP 4, insulin, and interleukins (IL) 2, 8, and 10, were assessed in patients with dilated cardiomyopathy (DCM, n = 53), dilated inflammatory cardiomyopathy (DCMi, n = 54), and controls (n = 20). In addition, a possible mechanism of RBP 4 regulation was examined in adipocytes in vitro. Plasma levels of RBP 4 and insulin were measured by a specific ELISA. Interleukin concentrations were obtained by multiplex ELISA. Cell culture with 3T3-L1 adipocytes was performed to measure RBP 4 mRNA expression after stimulation with IL-8. RBP 4 levels were significantly increased in patients with DCMi (52.95 +/- 20.42 microg/mL) compared with DCM (35.54 +/- 23.08 microg/mL) and the control group (27.3 +/- 18.51 microg/mL). RBP 4 was positively correlated with IL-8 (r=0.416, P < 0.05) in human plasma in patients with DCMi. Moreover, increased insulin resistance was observed in patients with DCMi compared with the control and DCM groups. In vitro, IL-8 induced a significant upregulation of RBP 4 mRNA expression in adipocytes. Elevated RBP 4 plasma concentrations, induced by IL-8, might be one mechanism leading to a higher incidence of diabetes in patients with DCMi.

  14. Retinol status and expression of retinol-related proteins in methionine-choline deficient rats.

    Science.gov (United States)

    Miyazaki, Hiroshi; Takitani, Kimitaka; Koh, Maki; Inoue, Akiko; Kishi, Kanta; Tamai, Hiroshi

    2014-01-01

    Retinol and its derivative, retinoic acid, have pleiotropic functions including vision, immunity, hematopoiesis, reproduction, cell differentiation/growth, and development. Non-alcoholic fatty liver disease (NAFLD) is one of the most common diseases in developed countries and encompasses a broad spectrum of forms, ranging from steatosis to steatohepatitis, which develops further to cirrhosis. Retinol status has an important role in liver homeostasis. The purpose of this study was to evaluate the retinol status and expression of retinol-related proteins, including enzymes and binding proteins, in methionine-choline deficient (MCD) rats as a model of NAFLD. We examined retinol levels in the plasma and liver and gene expression for β-carotene 15,15'-monooxygenase (BCMO), lecithIn: retinol acyltransferase (LRAT), aldehyde dehydrogenase 1A1 (ALDH1A1), ALDH1A2, and cellular retinol binding protein (CRBP)-I in MCD rats. The plasma retinol levels in MCD rats were lower than those in the controls, whereas hepatic retinol levels in MCD rats were higher. BCMO expression in the intestine and liver in MCD rats was lower, whereas that in the testes and the kidneys was higher than in control rats. Expression of LRAT, CRBP-I, ALDH1A1, and ALDH1A2 in the liver of MCD rats was also higher. Altered expression of retinol-related proteins may affect retinol status in NAFLD.

  15. Plasma from hemorrhaged mice activates CREB and increases cytokine expression in lung mononuclear cells through a xanthine oxidase-dependent mechanism.

    Science.gov (United States)

    Shenkar, R; Abraham, E

    1996-02-01

    Hemorrhage rapidly increases plasma xanthine oxidase levels as well as the expression of proinflammatory and immunoregulatory cytokines in the lungs. To determine the role of circulating xanthine oxidase (XO), as well as other plasma factors, in affecting pulmonary cytokine expression, we conducted studies in which plasma from hemorrhaged mice was transferred into unhemorrhaged recipient mice. Administration of posthemorrhage plasma to recipient mice increased the levels of mRNA for interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta 1 (TGF-beta 1) in lung mononuclear cells. No enhancement of mRNA levels for these cytokines was found in the lungs of mice given allopurinol-treated posthemorrhage plasma or fed a tungsten-enriched, XO-depleting diet prior to transfer of posthemorrhage plasma. Among the nuclear transcriptional regulatory factors examined, only the cyclic AMP response-element binding protein (CREB) was activated in nuclear extracts from lung mononuclear cells of mice that were given posthemorrhage plasma. No activation of nuclear factor-kappa B (NF-kappa B), nuclear factor interleukin 6 (NF-IL6), activating protein-1 (AP-1), or serum protein-1 (SP-1) was found. These results suggest that the mechanism for hemorrhage-induced increases in pulmonary cytokine expression is by activation of the enhancer CREB through a tissue XO-dependent pathway initiated by plasma-borne mediators.

  16. Expression of potein complexes using multiple E. coli protein co-expression systems: a benchmarking study

    NARCIS (Netherlands)

    Busso, D.; Peleg, Y.; Folkers, G.E.; Celie, P.H.N.

    2011-01-01

    Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for opti

  17. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    Science.gov (United States)

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  18. Caenorhabditis elegans paraoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins

    Science.gov (United States)

    Chen, Yushu; Bharill, Shashank; Altun, Zeynep; O’Hagan, Robert; Coblitz, Brian; Isacoff, Ehud Y.; Chalfie, Martin

    2016-01-01

    Caenorhabditis elegans senses gentle touch via a mechanotransduction channel formed from the DEG/ENaC proteins MEC-4 and MEC-10. An additional protein, the paraoxonase-like protein MEC-6, is essential for transduction, and previous work suggested that MEC-6 was part of the transduction complex. We found that MEC-6 and a similar protein, POML-1, reside primarily in the endoplasmic reticulum and do not colocalize with MEC-4 on the plasma membrane in vivo. As with MEC-6, POML-1 is needed for touch sensitivity, the neurodegeneration caused by the mec-4(d) mutation, and the expression and distribution of MEC-4 in vivo. Both proteins are likely needed for the proper folding or assembly of MEC-4 channels in vivo as measured by FRET. MEC-6 detectably increases the rate of MEC-4 accumulation on the Xenopus oocyte plasma membrane. These results suggest that MEC-6 and POML-1 interact with MEC-4 to facilitate expression and localization of MEC-4 on the cell surface. Thus MEC-6 and POML-1 act more like chaperones for MEC-4 than channel components. PMID:26941331

  19. Expression of Hsp27 and Hsp70 in lymphocytes and plasma in healthy workers and coal miners with lung cancer.

    Science.gov (United States)

    Wang, Haijiao; Xing, Jingcai; Wang, Feng; Han, Wenhui; Ren, Houmao; Wu, Tangchun; Chen, Weihong

    2010-08-01

    In coal mines, main occupational hazard is coal-mine dust, which can cause health problem including coal workers' pneumoconiosis and lung cancer. Some heat shock proteins (Hsps) have been reported as an acute response to a wide variety of stressful stimuli. Whether Hsps protect against chronic environmental coal-mine dust over years is unknown. It is also interesting to know that whether the expression of Hsp27 and Hsp70 proteins as a marker for exposure is associated risk of lung cancer among coal miners. We investigated the association between levels of Hsp27 and Hsp70 expression in lymphocytes and plasma and levels of coal-mine dust exposure in workplace or risk of lung cancer in 42 cancer-free non-coal miners, 99 cancer-free coal miners and 51 coal miners with lung cancer in Taiyuan city in China. The results showed that plasma Hsp27 levels were increased in coal miners compared to non-coal miners (P<0.01). Except high cumulative coal-mine dust exposure (OR=13.62, 95%CI=6.05-30.69) and amount of smoking higher than 24 pack-year (OR=2.72, 95% CI=1.37-5.42), the elevated levels of plasma Hsp70 (OR=13.00, 95% CI=5.14-32.91) and plasma Hsp27 (OR=2.97, 95% CI=1.40-6.32) and decreased expression of Hsp70 in lymphocytes (OR=2.36, 95% CI=1.05-5.31) were associated with increased risk of lung cancer. These findings suggest that plasma Hsp27 may be a potential marker for coal-mine dust exposure. And the expression of Hsp27 and Hsp70 levels in plasma and lymphocytes may be used as biomarkers for lung cancer induced by occupational coal-mine dust exposure.

  20. Identification of calcium-binding proteins associated with the human sperm plasma membrane

    National Research Council Canada - National Science Library

    Naaby-Hansen, Soren; Diekman, Alan; Shetty, Jagathpala; Flickinger, Charles J; Westbrook, Anne; Herr, John C

    2010-01-01

    The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation...

  1. GLUT4 protein expression in obese and lean 12-month-old rats: insights from different types of data analysis

    Directory of Open Access Journals (Sweden)

    P.M. Seraphim

    2001-10-01

    Full Text Available GLUT4 protein expression in white adipose tissue (WAT and skeletal muscle (SM was investigated in 2-month-old, 12-month-old spontaneously obese or 12-month-old calorie-restricted lean Wistar rats, by considering different parameters of analysis, such as tissue and body weight, and total protein yield of the tissue. In WAT, a ~70% decrease was observed in plasma membrane and microsomal GLUT4 protein, expressed as µg protein or g tissue, in both 12-month-old obese and 12-month-old lean rats compared to 2-month-old rats. However, when plasma membrane and microsomal GLUT4 tissue contents were expressed as g body weight, they were the same. In SM, GLUT4 protein content, expressed as µg protein, was similar in 2-month-old and 12-month-old obese rats, whereas it was reduced in 12-month-old obese rats, when expressed as g tissue or g body weight, which may play an important role in insulin resistance. Weight loss did not change the SM GLUT4 content. These results show that altered insulin sensitivity is accompanied by modulation of GLUT4 protein expression. However, the true role of WAT and SM GLUT4 contents in whole-body or tissue insulin sensitivity should be determined considering not only GLUT4 protein expression, but also the strong morphostructural changes in these tissues, which require different types of data analysis.

  2. Aberrant Glycosylation of Plasma Proteins in Severe Preeclampsia Promotes Monocyte Adhesion

    Science.gov (United States)

    Kazanjian, Avedis A.; Tinnemore, Deborah; Gafken, Philip R.; Ogata, Yuko; Napolitano, Peter G.; Stallings, Jonathan D.; Ippolito, Danielle L.

    2014-01-01

    Glycosylation of plasma proteins increases during pregnancy. Our objectives were to investigate an anti-inflammatory role of these proteins in normal pregnancies and determine whether aberrant protein glycosylation promotes monocyte adhesion in preeclampsia. Plasma was prospectively collected from nonpregnant controls and nulliparous patients in all 3 trimesters. Patients were divided into cohorts based on the applicable postpartum diagnosis. U937 monocytes were preconditioned with enzymatically deglycosylated plasma, and monocyte adhesion to endothelial cell monolayers was quantified by spectrophotometry. Plasma from nonpregnant controls, first trimester normotensives, and first trimester patients with mild preeclampsia inhibited monocyte–endothelial cell adhesion (P < .05), but plasma from first trimester patients with severe preeclampsia and second and third trimester normotensives did not. Deglycosylating plasma proteins significantly increased adhesion in all the cohorts. These results support a role of plasma glycoprotein interaction in monocyte–endothelial cell adhesion and could suggest a novel therapeutic target for severe preeclampsia. PMID:23757314

  3. A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker

    Directory of Open Access Journals (Sweden)

    Mueller-Roeber Bernd

    2010-05-01

    Full Text Available Abstract Background Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL. Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols

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

    Science.gov (United States)

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

    2015-02-01

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

  5. Expression of Helicobacter pylori Hsp60 protein and its immunogenicity

    Institute of Scientific and Technical Information of China (English)

    Yang Bai; Liang-Ren Li; Ji-De Wang; Ye Chen; Jian-Feng Jin; Zhao-Shan Zhang; Dian-Yuan Zhou; Ya-Li Zhang

    2003-01-01

    AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity.METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+), which was transformed into BL21 (DE3) E. coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments.RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2 % of the total bacterial protein,and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself.CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.

  6. Evaluation of somatic embryos of alfalfa for recombinant protein expression.

    Science.gov (United States)

    Fu, Guohua; Grbic, Vojislava; Ma, Shengwu; Tian, Lining

    2015-02-01

    Somatic embryos of alfalfa can accumulate higher levels of recombinant proteins comparing to vegetative organs. Somatic embryos may be explored as a new system for new protein production for plants. Plants have been explored via genetic engineering as an inexpensive system for recombinant protein production. However, protein expression levels in vegetative tissues have been low, which limits the commercial utilization of plant expression systems. Somatic embryos resemble zygotic embryos in many aspects and may accumulate higher levels of proteins as true seed. In this study, somatic embryo of alfalfa (Medicago sativa L.) was investigated for the expression of recombinant proteins. Three heterologous genes, including the standard scientific reporter uid that codes for β-glucuronidase and two genes of interest: ctb coding for cholera toxin B subunit (CTB), and hIL-13 coding for human interleukin 13, were independently introduced into alfalfa via Agrobacterium-mediated transformation. Somatic embryos were subsequently induced from transgenic plants carrying these genes. Somatic embryos accumulated approximately twofold more recombinant proteins than vegetative organs including roots, stems, and leaves. The recombinant proteins of CTB and hIL-13 accumulated up to 0.15 and 0.18 % of total soluble protein in alfalfa somatic embryos, respectively. The recombinant proteins expressed in somatic embryos also exhibited biological activities. As somatic embryos can be induced in many plant species and their production can be scaled up via different avenues, somatic embryos may be developed as an efficient expression system for recombinant protein production.

  7. Maltose-Binding Protein (MBP, a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    Directory of Open Access Journals (Sweden)

    Raphael Reuten

    Full Text Available Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST, SlyD, and serum albumin (ser alb tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome, which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  8. Quantitative analysis of plasma proteins in whole blood-derived fresh frozen plasma prepared with three pathogen reduction technologies.

    Science.gov (United States)

    Larrea, Luis; Ortiz-de-Salazar, María-Isabel; Martínez, Patricia; Roig, Roberto

    2015-06-01

    Several plasma pathogen reduction technologies (PRT) are currently available. We evaluated three plasma PRT processes: Cerus Amotosalen (AM), Terumo BCT riboflavin (RB) and Macopharma methylene blue (MB). RB treatment resulted in the shortest overall processing time and in the smallest volume loss (1%) and MB treatment in the largest volume loss (8%). MB treatment retained the highest concentrations of factors II, VII, X, IX, Protein C, and Antithrombin and the AM products of factor V and XI. Each PRT process evaluated offered distinct advantages such as procedural simplicity and volume retention (RB) and overall plasma protein retention (MB).

  9. Differential expression of SLAMS and other modulatory molecules by human plasma cells during normal maturation.

    Science.gov (United States)

    Rodríguez-Bayona, Beatriz; Ramos-Amaya, Ana; Brieva, José A

    2011-01-30

    Plasma cells (PCs) are specialized in antibody (Ab) production and they are, therefore, responsible for maintaining humoral immune responses. The human PC compartment is heterogeneous. PCs from inductive secondary lymphoid organs and from peripheral blood (PB) show less capability for prolonged survival and Ab production than bone marrow (BM) PCs, a pool consisting of fully mature cells. The HLDA9 workshop has allowed the use of labeled-monoclonal Abs (moAbs) recognizing a variety of recently identified lymphocyte modulatory surface receptors. In this study, flow cytometry analysis has been used to define the presence of these receptors on human PCs obtained from human tonsil (as an example of inductive organ), from PB and from BM. It was found that human PCs commonly expressed SLAMF1 (CD150), SLAMF2 (CD48), SLAMF3 (CD229), SLAMF6 (CD352) and SLAMF7 (CD319), but not SLAMF4 (CD244). In addition, PCs distinctively showed a low level of SLAMF5 (CD84) and a very high level of SLAMF7 expression in comparison with earlier stages of B cell maturation. All PC subsets exhibited a similar pattern of expression of SLAMF receptors suggesting a stage-dependent role for these proteins. In addition, most circulating PCs clearly expressed TNFRSF14 (CD270), BTLA (CD272), B7-1 (CD80) and B7-2 (CD86), and a substantial fraction of them were also positive for TNFRSF18 (CD357), FCRL1 (CD307a) and LAIR-1 (CD305). In contrast, tonsil and BM PCs only exhibited partial expression of TNFRSF14 and B7-2, a pattern of molecular expression similar to that detected on germinal center (GC) B cells. Present results indicate that human PCs exhibit a common pattern of SLAMF proteins, but differ in the rest of the receptors examined; this difference might be associated with their distinctive homing and functional requirements. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Plant Antifreeze Proteins and Their Expression Regulatory Mechanism

    Institute of Scientific and Technical Information of China (English)

    Lin Yuan-zhen; Lin Shan-zhi; Zhang Zhi-yi; Zhang Wei; Liu Wen-feng

    2005-01-01

    Low temperature is one of the major limiting environmental factors which constitutes the growth, development,productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identification and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.

  11. An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis

    DEFF Research Database (Denmark)

    Khan, Junaid A.; Wang, Qi; Sjölund, Richard D.

    2007-01-01

    that have a similar overall domain structure of an amino-terminal signal peptide, plastocyanin-like copper-binding domain, proline/serine-rich domain, and carboxy-terminal hydrophobic domain. The amino- and carboxy-terminal domains of the 21.5-kD sieve element-specific ENOD are posttranslationally cleaved...... from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both...... floral and vegetative tissues, the sieve element-specific ENOD is expressed only within the phloem. Members of the ENOD subfamily of the cupredoxin superfamily do not appear to bind copper and have unknown functions. Phenotypic analysis of homozygous T-DNA insertion mutants for the gene At3g20570 shows...

  12. Effects of immunosuppressive treatment on protein expression in rat kidney

    Directory of Open Access Journals (Sweden)

    Kędzierska K

    2014-09-01

    Full Text Available Karolina Kędzierska,1 Katarzyna Sporniak-Tutak,2 Krzysztof Sindrewicz,2 Joanna Bober,3 Leszek Domański,1 Mirosław Parafiniuk,4 Elżbieta Urasińska,5 Andrzej Ciechanowicz,6 Maciej Domański,1 Tomasz Smektała,2 Marek Masiuk,5 Wiesław Skrzypczak,6 Małgorzata Ożgo,6 Joanna Kabat-Koperska,1 Kazimierz Ciechanowski1 1Department of Nephrology, Transplantology, and Internal Medicine, 2Department of Dental Surgery, 3Department of Medical Chemistry, 4Department of Forensic Medicine, 5Department of Pathomorphology, Pomeranian Medical University, 6Department of Physiology, Cytobiology, and Proteomics, West Pomeranian University of Technology, Szczecin, Poland Abstract: The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents' toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins' synthesis. Very slight differences

  13. Binding patterns of seminal plasma plasma proteins on bovine epididymal and ejaculated sperm membrane

    Directory of Open Access Journals (Sweden)

    C.E.A. Souza

    2011-06-01

    Full Text Available The present study was designed to investigate the topographical distribution of seminal plasma (SP proteins on epididymal and ejaculated bovine sperm. Using immunocytochemistry and confocal microscopy the binding patterns of bovine SP proteins BSP-A3, albumin, transferrin, prostaglandin D-synthase (PGDS and nucleobindin in ejaculated and cauda epididymal sperm from adult bulls were evaluated. Experiments were performed using sperm from 5 males. Data showed a positive signal, only detected for anti-PGDS, in the acrosomal cap of epididymal and ejaculated sperm. In ejaculated sperm, a very weak signal for nucleobindin 2 in the midpiece and equatorial regions was detected, using the anti-rat nucleobindin. BSP-A3 was detected on all sperm regions studied, with a more evidenced signal in acrosome and midpiece. However, no binding was detected for albumin or transferrin in neither epididymal nor ejaculated sperm. In conclusion, PGDS, BSP-A3 and nucleobindin interact directly with bovine sperm, with specific topographic distribution. These findings may add to the knowledge of how these proteins modulate sperm functions, thus providing fundamental support for studies designed to evaluate how they influence sperm functions.

  14. The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

    Science.gov (United States)

    Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

    2011-10-01

    Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.

  15. Characterization of auxin-binding proteins from zucchini plasma membrane

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  16. A STUDY ON CYCLOOXYGENASE -2 PROTEIN EXPRESSION IN ESOPHAGEAL CAICONOGENESIS

    Institute of Scientific and Technical Information of China (English)

    王立峰; 张伟; 王吾如; 王洪平; 韩双廷; 曲平; 刘义; 李茉; 刘伯齐; 林培中

    2001-01-01

    To investigate cyclooxygenase- 2(Cox-2) protein expression in esophageal cancer and precancerous lesions. Methods: One hundred twenty biopsy specimens from esophageal carcinoma and 113 from patients with esophageal premalingnant lesions, 27 from individuals with normal esophageal mucosa and 3 from Barrett's esophagus were examined for Cox-2 protein expression by immunohistochemistry. Results: Cox-2 protein was not observed in normal esophageal squamous and glandular epithelium, hyperplasia from mild to severe dysplasia lesions and carcinoma in situ. Positive Cox-2 protein expression was found in 4 of 60 specimens of invasive squamous-cell carcinomas, 21 of 30 specimens of esophageal adenocarcinomas and in 3 of 3 Barret's esophageal tissues. Conclusion: The Cox-2 protein expression may be associated with the development of the esophageal adenocarcinomas but not esophageal squamous-cell carcinomas.

  17. Insulin influenced expression of myelin proteins in diabetic peripheral neuropathy.

    Science.gov (United States)

    Rachana, Kuruvanthe S; Manu, Mallahalli S; Advirao, Gopal M

    2016-08-26

    Diabetic peripheral neuropathy (DPN) is one of the downstream complications of diabetes. This complication is caused by the deficiency of insulin action and subsequent hyperglycemia, but the details of their pathogenesis remain unclear. Hence, it is of critical importance to understand how such hormonal variation affects the expression of myelin proteins such as myelin basic protein (MBP) and myelin associated glycoprotein (MAG) in the peripheral nerve. An earlier report from our lab has demonstrated the expression of insulin receptors (IR) in Schwann cells (SCs) of sciatic nerve. To assess the neurotrophic role of insulin in diabetic neuropathy, we studied the expression of these myelin proteins under control, DPN and insulin treated DPN subjects at developmental stages. Further, the expression of these myelin proteins was correlated with the expression of insulin receptor. Expression of myelin proteins was significantly reduced in the diabetic model compared to normal, and upregulated in insulin treated diabetic rats. Similarly, an in vitro study was also carried out in SCs grown at high glucose and insulin treated conditions. The expression pattern of myelin proteins in SCs was comparable to that of in vivo samples. In addition, quantitative study of myelin genes by real time PCR has also showed the significant expression pattern change in the insulin treated and non-treated DPN subjects. Taken together, these results corroborate the critical importance of insulin as a neurotrophic factor in demyelinized neurons in diabetic neuropathy.

  18. PTK6/BRK is expressed in the normal mammary gland and activated at the plasma membrane in breast tumors.

    Science.gov (United States)

    Peng, Maoyu; Emmadi, Rajyasree; Wang, Zebin; Wiley, Elizabeth L; Gann, Peter H; Khan, Seema A; Banerji, Nilanjana; McDonald, William; Asztalos, Szilard; Pham, Thao N D; Tonetti, Debra A; Tyner, Angela L

    2014-08-15

    Protein Tyrosine kinase 6 (PTK6/BRK) is overexpressed in the majority of human breast tumors and breast tumor cell lines. It is also expressed in normal epithelial linings of the gastrointestinal tract, skin, and prostate. To date, expression of PTK6 has not been extensively examined in the normal human mammary gland. We detected PTK6 mRNA and protein expression in the immortalized normal MCF-10A human mammary gland epithelial cell line, and examined PTK6 expression and activation in a normal human breast tissue microarray, as well as in human breast tumors. Phosphorylation of tyrosine residue 342 in the PTK6 activation loop corresponds with its activation. Similar to findings in the prostate, we detect nuclear and cytoplasmic PTK6 in normal mammary gland epithelial cells, but no phosphorylation of tyrosine residue 342. However, in human breast tumors, striking PTK6 expression and phosphorylation of tyrosine 342 is observed at the plasma membrane. PTK6 is expressed in the normal human mammary gland, but does not appear to be active and may have kinase-independent functions that are distinct from its cancer promoting activities at the membrane. Understanding consequences of PTK6 activation at the plasma membrane may have implications for developing novel targeted therapies against this kinase.

  19. Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

    Energy Technology Data Exchange (ETDEWEB)

    Witt, P.L.; Bownds, M.D.

    1987-03-24

    Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger.

  20. Inhibition of platelet (/sup 3/H)- imipramine binding by human plasma protein fractions

    Energy Technology Data Exchange (ETDEWEB)

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity (/sup 3/H)-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha/sub 1/ acid glycoprotein, high density and low density lipoprotein, IgG and ..cap alpha../sub 1/-antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of (/sup 3/H)-imipramine binding site.

  1. Expression and characterization of plasma membrane aquaporins in stomatal complexes of Zea mays.

    Science.gov (United States)

    Heinen, Robert B; Bienert, Gerd Patrick; Cohen, David; Chevalier, Adrien S; Uehlein, Norbert; Hachez, Charles; Kaldenhoff, Ralf; Le Thiec, Didier; Chaumont, François

    2014-10-01

    Stomata, the microscopic pores on the surface of the aerial parts of plants, are bordered by two specialized cells, known as guard cells, which control the stomatal aperture according to endogenous and environmental signals. Like most movements occurring in plants, the opening and closing of stomata are based on hydraulic forces. During opening, the activation of plasma membrane and tonoplast transporters results in solute accumulation in the guard cells. To re-establish the perturbed osmotic equilibrium, water follows the solutes into the cells, leading to their swelling. Numerous studies have contributed to the understanding of the mechanism and regulation of stomatal movements. However, despite the importance of transmembrane water flow during this process, only a few studies have provided evidence for the involvement of water channels, called aquaporins. Here, we microdissected Zea mays stomatal complexes and showed that members of the aquaporin plasma membrane intrinsic protein (PIP) subfamily are expressed in these complexes and that their mRNA expression generally follows a diurnal pattern. The substrate specificity of two of the expressed ZmPIPs, ZmPIP1;5 and ZmPIP1;6, was investigated by heterologous expression in Xenopus oocytes and yeast cells. Our data show that both isoforms facilitate transmembrane water diffusion in the presence of the ZmPIP2;1 isoform. In addition, both display CO2 permeability comparable to that of the CO2 diffusion facilitator NtAQP1. These data indicate that ZmPIPs may have various physiological roles in stomatal complexes.

  2. Clinical significance of PHPT1 protein expression in lung cancer

    Institute of Scientific and Technical Information of China (English)

    XU An-jian; XIA Xiang-hou; DU Song-tao; GU Jun-chao

    2010-01-01

    Background in our previous studies, we found the expression of 14-kD phosphohistidine phosphatase (PHPT1) was associated with lung cancer cells migration and invasion, and PHPT1 mRNA expression level in lung cancer tissues clinically correlated with lymph node metastasis. in the present study, we aimed to further investigate the expression of PHPT1 protein in lung cancer.Methods Expression of PHPT1 protein in tissue samples from 146 lung cancers and 30 normal tissues adjacent to lung cancers was assessed using immunohistochemical method. Fisher's exact test was used to analyze expression patterns of PHPT1 protein in these tissue types. Meanwhile, we studied the correlation between expression of PHPT1 protein and clinicopathological features in lung cancer.Results Significantly higher expression levels of PHPT1 protein were found in lung cancer samples (53.42%) than in normal tissues adjacent to lung cancer (23.33%) (P=0.003). Fisher's exact test showed that lung cancer stage positively correlated with expression of PHPT1 protein (P=0.02), and lung cancer samples with lymph node metastasis showed higher PHPT1 protein expression (P=0.016) than the samples without lymph node metastasis.Conclusions The results of this study agree with findings from our previous study of PHPT1 mRNA expression in lung cancer tissues, and strongly suggest that PHPT1 protein is closely associated with the carcinogenesis and metastasis of lung cancer. Thus, therapy targeting PHPT1 (inhibition or silencing) could be potentially benefited for lung cancer patients.

  3. E-cadherin expression and its clinical significance in 41 cases of spinal plasma cell myeloma

    Institute of Scientific and Technical Information of China (English)

    Wang Liangzhe; Liu Huimin; He Jin; Li Yuli; Xu Yi; Chen Bing

    2009-01-01

    Objective: To investigate the expression level of E-cadherin in spinal plasma cell myeloma and its relationship with the clinicopathological indexes of spinal plasma cell myeloma. Methods: The expression of E-cadherin was detected in 41 cases of spinal plasma cell myeloma and 14 cases of simple bone cysts by using S-P (streptavidin-perosidase) immunohistochemical staining. The relationship between E-cadherin expression and the clinicopathological features was analyzed. Results: In spinal plasma cell myelomas, the positive expression rate of E-cadherin (46.34 %) was higher than that in simple bone cyst tissues (all negative) (P0.05), but had relationship with the classification of light chain. The expression rate in lambda chain subgroup was lower than that in the others (P<0.05). We also found the E-cadherin negative patients had a higher overall survival rate than those who had strong E-cadherin expression (P<0.05). Conclusion: E-cadherin expressed higher in spinal plasma cell myelomas than in simple bone cyst tissues and its expression had relationship with the light chain classification in spinal plasma cell myeloma. The patients with no stain of E-cadherin may have a poor prognosis. Detecting the type of light chain associated with expression of E-cadherin might be helpful in evaluating the outcome of spinal PCM patients.

  4. Blood plasma proteins and protein fractions in roe deer Capreolus capreolus L.

    Directory of Open Access Journals (Sweden)

    Dorota CYGAN-SZCZEGIELNIAK

    2015-09-01

    Full Text Available The aim of the research was to investigate some selected biochemical blood parameters in roe deer (Capreolus capreolus L.. The experiment covered 15 from 2 to 3-year-old bucks from Kuyavian-Pomeranian Voivodeship. The animals were shot by individual hunters on the shooting grounds during the hunting season of 2008/2009 (in the accordance with the Journal of Laws No 48. The material for the research was blood plasma obtained after centrifuging full, nonhemolyzed blood. The blood was collected from the zygomatic vein directly to the test tubes with EDTA and transported in cooling conditions to the laboratory. After transporting the samples of blood to a certified analytical laboratory, the following elements of the obtained blood plasma were examined: ceruloplasmin . using turbidimetric method; transferrin . using immunoturbimetric method; troponin- using a third generation assay on an Elecsys; total protein, albumin, globulin . using spectrophotometric method and total iron . using colorimetric method. The results were statistically analyzed, i.e. the correlation between the parameters was measured by means of Pearsonfs correlation coefficient. The analysis of the results revealed a number of statistically significant relations between the parameters under the investigation, especially among the compounds directly responsible for metabolism of iron and copper. A statistically important positive correlation was observed between ceruloplasmin and ferritin (r = 0.563; P.0.05 and a negative one between transferrin and troponin (r = -0.609; P.0.05. Moreover, the content of transferrin . an iron-binding protein . was 0.17 g/l, while the concentration of iron was 58 ƒĘmol/l. The content of ceruloplasmin . a protein responsible for metabolism of copper . was very low (0.036 g/l. The level of proteins in the blood plasma of the animals under the research was approximately 72 g/l, with the share of albumins about 46%. The albumin-globulin ratio was 0.86.

  5. Stable protein expression in mammalian cells using baculoviruses.

    Science.gov (United States)

    Lackner, Andreas; Kreidl, Emanuel; Peter-Vörösmarty, Barbara; Spiegl-Kreinecker, Sabine; Berger, Walter; Grusch, Michael

    2012-01-01

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been widely used in biotechnology for protein expression in insect cells. Baculoviruses use arthropods as their natural hosts and are unable to replicate in mammalian cells. However, AcMNPV is able to enter many mammalian cell types and can be used for transgene expression if engineered to contain suitable expression cassettes. In this chapter, we describe the construction and application of a recombinant baculovirus containing a bicistronic expression cassette that can be used for stable protein expression in mammalian cells. As an example, the generation of glioblastoma and hepatocellular carcinoma cell lines stably expressing green fluorescent protein after puromycin selection is shown.

  6. Plasma levels of soluble endothelial cell protein C receptor in patients with Wegener's granulomatosis

    NARCIS (Netherlands)

    Boomsma, MM; Stearns-Kurosawa, DJ; Stegeman, CA; Raschi, E; Meroni, PL; Kurosawa, S; Tervaert, JWC

    Elevated soluble thrombomodulin (sTM) levels are an accepted marker of endothelial damage. The physiological significance of plasma endothelial protein C receptor (sEPCR) levels is not known. To assess the relevance of this plasma protein in Wegener's granulomatosis (WG), sEPCR levels were measured

  7. Interaction between La(III) and proteins on the plasma membrane of horseradish

    Science.gov (United States)

    Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

    2012-06-01

    Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

  8. Estimation of protein content in the plasma of young chickens by a refractometric method.

    Science.gov (United States)

    Morgan, G W; Thaxton, P; Edens, F W

    1975-07-01

    This study was conducted to evaluate a refractometric method for determination of protein content of chicken plasma. Comparison of the results obtained with the refractometric and the Lowry methods indicated that refractometry, when used with due caution in a typical laboratory situation, provided a simple, fast, inexpensive and valid method for determining the protein content of plasma from young chickens.

  9. Chemometrics of differentially expressed proteins from colorectal cancer patients

    Institute of Scientific and Technical Information of China (English)

    Lay-Chin Yeoh; Saravanan Dharmaraj; Boon-Hui Gooi; Manjit Singh; Lay-Harn Gam

    2011-01-01

    AIM: To evaluate the usefulness of differentially expressed proteins from colorectal cancer (CRC) tissues for differentiating cancer and normal tissues. METHODS: A Proteomic approach was used to identify the differentially expressed proteins between CRC and normal tissues. The proteins were extracted using Tris buffer and thiourea lysis buffer (TLB) for extraction of aqueous soluble and membrane-associated proteins, respectively. Chemometrics, namely principal component analysis (PCA) and linear discriminant analysis (LDA), were used to assess the usefulness of these proteins for identifying the cancerous state of tissues. RESULTS: Differentially expressed proteins identified were 37 aqueous soluble proteins in Tris extracts and 24 membrane-associated proteins in TLB extracts. Based on the protein spots intensity on 2D-gel images, PCA by applying an eigenvalue > 1 was successfully used to reduce the number of principal components (PCs) into 12 and seven PCs for Tris and TLB extracts, respectively, and subsequently six PCs, respectively from both the extracts were used for LDA. The LDA classification for Tris extract showed 82.7% of original samples were correctly classified, whereas 82.7% were correctly classified for the cross-validated samples. The LDA for TLB extract showed that 78.8% of original samples and 71.2% of the cross-validated samples were correctly classified. CONCLUSION: The classification of CRC tissues by PCA and LDA provided a promising distinction between normal and cancer types. These methods can possibly be used for identification of potential biomarkers among the differentially expressed proteins identified.

  10. Plasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways

    Institute of Scientific and Technical Information of China (English)

    Mary; Louisa; Holton; Michael; Emerson; Ludwig; Neyses; Angel; L; Armesilla

    2010-01-01

    Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular freecalcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulindependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.

  11. Relationship between Sustained Reductions in Plasma Lipid and Lipoprotein Concentrations with Apheresis and Plasma Levels and mRNA Expression of PTX3 and Plasma Levels of hsCRP in Patients with HyperLp(alipoproteinemia

    Directory of Open Access Journals (Sweden)

    Claudia Stefanutti

    2016-01-01

    Full Text Available The effect of lipoprotein apheresis (Direct Adsorption of Lipids, DALI (LA on plasma levels of pentraxin 3 (PTX3, an inflammatory marker that reflects coronary plaque vulnerability, and expression of PTX3 mRNA was evaluated in patients with hyperLp(alipoproteinemia and angiographically defined atherosclerosis/coronary artery disease. Eleven patients, aged 55±9.3 years (mean ± SD, were enrolled in the study. PTX3 soluble protein levels in plasma were unchanged by 2 sessions of LA; however, a downregulation of mRNA expression for PTX3 was observed, starting with the first session of LA (p<0.001. The observed reduction was progressively increased in the interval between the first and second LA sessions to achieve a maximum decrease by the end of the second session. A statistically significantly greater treatment-effect correlation was observed in patients undergoing weekly treatments, compared with those undergoing treatment every 15 days. A progressive reduction in plasma levels of C-reactive protein was also seen from the first session of LA, with a statistically significant linear correlation for treatment-effect in the change in plasma levels of this established inflammatory marker (R2=0.99; p<0.001. Our findings suggest that LA has anti-inflammatory and endothelium protective effects beyond its well-established efficacy in lowering apoB100-containing lipoproteins.

  12. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells

    Directory of Open Access Journals (Sweden)

    Gay Bernard

    2011-09-01

    Full Text Available Abstract Background Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. Results We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. Conclusion These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.

  13. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells.

    Science.gov (United States)

    Clerc, Isabelle; Laverdure, Sylvain; Torresilla, Cynthia; Landry, Sébastien; Borel, Sophie; Vargas, Amandine; Arpin-André, Charlotte; Gay, Bernard; Briant, Laurence; Gross, Antoine; Barbeau, Benoît; Mesnard, Jean-Michel

    2011-09-19

    Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.

  14. Expression of a Carrot Antifreeze Protein Gene in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Ma Xinyu; Shen Xin; Lu Cunfu

    2003-01-01

    The recombinant expression vectorpET43. lb-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol. L-1 IPTG (isopropyl-β-D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein.The analysis of product solubility revealed that pET43. 1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.

  15. A Phospholipid-Protein Complex from Krill with Antioxidative and Immunomodulating Properties Reduced Plasma Triacylglycerol and Hepatic Lipogenesis in Rats

    Directory of Open Access Journals (Sweden)

    Marie S. Ramsvik

    2015-07-01

    Full Text Available Dietary intake of marine omega-3 polyunsaturated fatty acids (n-3 PUFAs can change the plasma profile from atherogenic to cardioprotective. In addition, there is growing evidence that proteins of marine origin may have health benefits. We investigated a phospholipid-protein complex (PPC from krill that is hypothesized to influence lipid metabolism, inflammation, and redox status. Male Wistar rats were fed a control diet (2% soy oil, 8% lard, 20% casein, or diets where corresponding amounts of casein and lard were replaced with PPC at 3%, 6%, or 11% (wt %, for four weeks. Dietary supplementation with PPC resulted in significantly lower levels of plasma triacylglycerols in the 11% PPC-fed group, probably due to reduced hepatic lipogenesis. Plasma cholesterol levels were also reduced at the highest dose of PPC. In addition, the plasma and liver content of n-3 PUFAs increased while n-6 PUFAs decreased. This was associated with increased total antioxidant capacity in plasma and increased liver gene expression of mitochondrial superoxide dismutase (Sod2. Finally, a reduced plasma level of the inflammatory mediator interleukin-2 (IL-2 was detected in the PPC-fed animals. The present data show that PPC has lipid-lowering effects in rats, and may modulate risk factors related to cardiovascular disease progression.

  16. Effects of Salvianolic Acid B on Protein Expression in Human Umbilical Vein Endothelial Cells

    Science.gov (United States)

    Chang, Tsong-Min; Shi, Guey-Yueh; Wu, Hua-Lin; Wu, Chieh-Hsi; Su, Yan-Di; Wang, Hui-Lin; Wen, Hsin-Yun; Huang, Huey-Chun

    2011-01-01

    Salvianolic acid B (Sal B), a pure water-soluble compound extracted from Radix Salviae miltiorrhizae, has been reported to possess potential cardioprotective efficacy. To identify proteins or pathways by which Sal B might exert its protective activities on the cardiovascular system, two-dimensional gel electrophoresis-based comparative proteomics was performed, and proteins altered in their expression level after Sal B treatment were identified by MALDI-TOF MS/MS. Human umbilical vein endothelial cells (HUVECs) were incubated at Sal B concentrations that can be reached in human plasma by pharmacological intervention. Results indicated that caldesmon, an actin-stabilizing protein, was downregulated in Sal B-exposed HUVECs. Proteins that showed increased expression levels upon Sal B treatment were vimentin, T-complex protein 1, protein disulfide isomerase, tropomyosin alpha, heat shock protein beta-1, UBX domain-containing protein 1, alpha enolase, and peroxiredoxin-2. Additionally, Sal B leads to increased phosphorylation of nucleophosmin in a dose-dependent manner and promotes proliferation of HUVECs. We found that Sal B exhibited a coordinated regulation of enzymes and proteins involved in cytoskeletal reorganization, oxidative stress, and cell growth. Our investigation would provide understanding to the endothelium protection information of Sal B. PMID:21423689

  17. Effects of Salvianolic Acid B on Protein Expression in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Tsong-Min Chang

    2011-01-01

    Full Text Available Salvianolic acid B (Sal B, a pure water-soluble compound extracted from Radix Salviae miltiorrhizae, has been reported to possess potential cardioprotective efficacy. To identify proteins or pathways by which Sal B might exert its protective activities on the cardiovascular system, two-dimensional gel electrophoresis-based comparative proteomics was performed, and proteins altered in their expression level after Sal B treatment were identified by MALDI-TOF MS/MS. Human umbilical vein endothelial cells (HUVECs were incubated at Sal B concentrations that can be reached in human plasma by pharmacological intervention. Results indicated that caldesmon, an actin-stabilizing protein, was downregulated in Sal B-exposed HUVECs. Proteins that showed increased expression levels upon Sal B treatment were vimentin, T-complex protein 1, protein disulfide isomerase, tropomyosin alpha, heat shock protein beta-1, UBX domain-containing protein 1, alpha enolase, and peroxiredoxin-2. Additionally, Sal B leads to increased phosphorylation of nucleophosmin in a dose-dependent manner and promotes proliferation of HUVECs. We found that Sal B exhibited a coordinated regulation of enzymes and proteins involved in cytoskeletal reorganization, oxidative stress, and cell growth. Our investigation would provide understanding to the endothelium protection information of Sal B.

  18. Protein alterations induced by long-term agonist treatment of HEK293 cells expressing thyrotropin-releasing hormone receptor and G11alpha protein.

    Science.gov (United States)

    Drastichova, Zdenka; Bourova, Lenka; Hejnova, Lucie; Jedelsky, Petr; Svoboda, Petr; Novotny, Jiri

    2010-01-01

    This study aimed to determine whether sustained stimulation with thyrotropin-releasing hormone (TRH), a peptide with important physiological functions, can possibly affect expression of plasma membrane proteins in HEK293 cells expressing high levels of TRH receptor and G(11)alpha protein. Our previous experiments using silver-stained two-dimensional polyacrylamide gel electrophoretograms did not reveal any significant changes in an overall composition of membrane microdomain proteins after long-term treatment with TRH of these cells (Matousek et al. 2005 Cell Biochem Biophys 42: 21-40). Here we used a purified plasma membrane fraction prepared by Percoll gradient centrifugation and proteins resolved by 2D electrophoresis were stained with SYPRO Ruby gel stain. The high enrichment in plasma membrane proteins of this preparation was confirmed by a multifold increase in the number of TRH receptors and agonist stimulated G-protein activity, compared to postnuclear supernatant. By a combination of these approaches we were able to determine a number of clearly discernible protein changes in the plasma membrane-enriched fraction isolated from cells treated with TRH (1 x 10(-5) M, 16 h): 4 proteins disappeared, the level of 18 proteins decreased and the level of 39 proteins increased. Our concomitant immunochemical determinations also indicated a clear down-regulation of G(q/11)alpha proteins in preparations from hormone-treated cells. In parallel, we observed decrease in caspase 3 and alterations in some other apoptotic marker proteins, which were in line with the presumed antiapoptotic effect of TRH.

  19. Plasma membrane proteins Yro2 and Mrh1 are required for acetic acid tolerance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Takabatake, Akiko; Kawazoe, Nozomi; Izawa, Shingo

    2015-03-01

    Yro2 and its paralogous protein Mrh1 of Saccharomyces cerevisiae have seven predicted transmembrane domains and predominantly localize to the plasma membrane. Their physiological functions and regulation of gene expression have not yet been elucidated in detail. We herein demonstrated that MRH1 was constitutively expressed, whereas the expression of YRO2 was induced by acetic acid stress and entering the stationary phase. Fluorescence microscopic analysis revealed that Mrh1 and Yro2 were distributed as small foci in the plasma membrane under acetic acid stress conditions. The null mutants of these genes (mrh1∆, yro2∆, and mrh1∆yro2∆) showed delayed growth and a decrease in the productivity of ethanol in the presence of acetic acid, indicating that Yro2 and Mrh1 are involved in tolerance to acetic acid stress.

  20. Analysis of interferon gamma protein expression in zebrafish (Danio rerio).

    Science.gov (United States)

    Yoon, Sohye; Alnabulsi, Ayham; Wang, Ting Yu; Lee, Po Tsang; Chen, Tzong-Yueh; Bird, Steve; Zou, Jun; Secombes, Christopher J

    2016-10-01

    IFN-γ is a major effector cytokine, produced to induce type I immune responses. It has been cloned in several fish species including zebrafish, however to date few studies have looked at IFN-γ protein expression and bioactivity in fish. Hence, the current study focused on developing a monoclonal antibody (moAb) against zfIFN-γ. We show that the zfIFN-γ moAb specifically recognises E. coli produced recombinant IFN-γ protein and zfIFN-γ produced in transfected HEK293 cells, by Western blot analysis. Next we analysed the production of the native protein following expression induced by PHA stimulation of leukocytes in vitro or antigen re-stimulation in vivo. We show the IFN-γ protein is produced as a dimer, and that a good correlation exists between transcript expression levels and protein levels.

  1. Heterologous Protein Expression by Lactococcus lactis

    NARCIS (Netherlands)

    Villatoro-Hernández, J.; Kuipers, O.P.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.

    2012-01-01

    This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a m

  2. Immunohistochemical expression of latent membrane protein 1 ...

    African Journals Online (AJOL)

    EB

    2013-09-03

    Sep 3, 2013 ... Department of Pathology, Ibn Rochd University Medical Center, Casablanca, ... NPC is a characteristic tumor displaying epidemiological, genetic and regional distribution properties that ... expression and histological type, age and sex distributions was ..... according to geographical location and we could.

  3. Multiplexed LC-MS/MS analysis of horse plasma proteins to study doping in sport.

    Science.gov (United States)

    Barton, Chris; Beck, Paul; Kay, Richard; Teale, Phil; Roberts, Jane

    2009-06-01

    The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC-MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race-horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis-driven discovery of doping biomarker candidates.

  4. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    Science.gov (United States)

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression.

  5. Performance benchmarking of four cell-free protein expression systems.

    Science.gov (United States)

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  6. Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

    OpenAIRE

    Josic, Djuro; Breen, Lucas; Clifton, James; Gajdosik, Martina Srajer; Gaso-Sokac, Dajana; Rucevic, Marijana; Müller, Egbert

    2012-01-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in se...

  7. Impact of elevated plasma serotonin on global gene expression of murine megakaryocytes.

    Directory of Open Access Journals (Sweden)

    Charles P Mercado

    Full Text Available BACKGROUND: Serotonin (5-HT is a biogenic amine that also acts as a mitogen and a developmental signal early in rodent embryogenesis. Genetic and pharmacological disruption of 5-HT signaling causes various diseases and disorders via mediating central nervous system, cardiovascular system, and serious abnormalities on a growing embryo. Today, neither the effective modulators on 5-HT signaling pathways nor the genes affected by 5-HT signal are well known yet. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to identify the genes altered by 5-HT signaling pathways, we analyzed the global gene expression via the Illumina array platform using the mouse WG-6 v2.0 Expression BeadChip containing 45,281 probe sets representing 30,854 genes in megakaryocytes isolated from mice infused with 5-HT or saline. We identified 723 differentially expressed genes of which 706 were induced and 17 were repressed by elevated plasma 5-HT. CONCLUSIONS/SIGNIFICANCE: Hierarchical gene clustering analysis was utilized to represent relations between groups and clusters. Using gene ontology mining tools and canonical pathway analyses, we identified multiple biological pathways that are regulated by 5-HT: (i cytoskeletal remodeling, (ii G-protein signaling, (iii vesicular transport, and (iv apoptosis and survival. Our data encompass the first extensive genome-wide based profiling in the progenitors of platelets in response to 5-HT elevation in vivo.

  8. Small-scale expression of proteins in E. coli.

    Science.gov (United States)

    Zerbs, Sarah; Giuliani, Sarah; Collart, Frank

    2014-01-01

    Proteins participate in virtually every cellular activity, and a knowledge of protein function is essential for an understanding of biological systems. However, protein diversity necessitates the application of an array of in vivo and in vitro approaches for characterization of the functional and biochemical properties of proteins. Methods that enable production of proteins for in vitro studies are critical for determination of the molecular, kinetic, and thermodynamic properties of these molecules. Ideally, proteins could be purified from the original source; however, the native host is often unsuitable for a number of reasons. Consequently, systems for heterologous protein production are commonly used to produce large amounts of protein. Heterologous expression hosts are chosen using a number of criteria, including genetic tractability, advantageous production or processing characteristics (secretion or posttranslational modifications), or economy of time and growth requirements. The subcloning process also provides an opportunity to introduce purification tags, epitope tags, fusions, truncations, and mutations into the coding sequence that may be useful in downstream purification or characterization applications. Bacterial systems for heterologous protein expression have advantages in ease of use, cost, short generation times, and scalability. These expression systems have been widely used by high-throughput protein production projects and often represent an initial experiment for any expression target. Escherichia coli has been studied for many years as a model bacterial organism and is one of the most popular hosts for heterologous protein expression (Terpe, 2006). Its protein production capabilities have been intensively studied, and the ease of genetic manipulation in this organism has led to the development of strains engineered exclusively for use in protein expression. These resources are widely available from commercial sources and public repositories

  9. Cardiac C-type natriuretic peptide gene expression and plasma concentrations in neonatal piglets

    DEFF Research Database (Denmark)

    Hansen, Lasse; Smith, Julie; Goetze, Jens P

    2014-01-01

    in plasma and cardiac tissue extracts were quantified by a porcine-specific radioimmunoassay. RESULTS: Cardiac CNP mRNA contents (n=24) were low compared to sites of known expression, where porcine seminal vesicle CNP mRNA contents were 200-fold higher. In addition, plasma proCNP concentrations...

  10. Genome-wide screens for expressed hypothetical proteins

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Durhuus, Jon Ambæk; Rasmussen, Lene Juel

    2012-01-01

    A hypothetical protein (HP) is defined as a protein that is predicted to be expressed from an open reading frame, but for which there is no experimental evidence of translation. HPs constitute a substantial fraction of proteomes of human as well as of other organisms. With the general belief...

  11. Improved means and methods for expressing recombinant proteins

    NARCIS (Netherlands)

    Poolman, Berend; Martinez Linares, Daniel; Gul, Nadia

    2014-01-01

    The invention relates to the field of genetic engineering and the production of recombinant proteins in microbial host cells. Provided is a method for enhanced expression of a recombinant protein of interest in a microbial host cell, comprising providing a microbial host cell wherein the function of

  12. Patterns of fluorescent protein expression in Scleractinian corals.

    Science.gov (United States)

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  13. Biofield-effect protein-sensor: Plasma functionalization of polyaniline, protein immobilization, and sensing mechanism

    Science.gov (United States)

    Cho, Chae-Ryong; Lee, Hyun-Uk; Ahn, Kyun; Jeong, Se-Young; Choi, Jun-Hee; Kim, Jinwoo; Cho, Jiung

    2014-06-01

    We report the fabrication of a biofield-effect protein-sensor (BioFEP) based on atmospheric-pressure plasma (AP) treatment of a conducting polyaniline (PANI) film. Successive H2 and O2 AP (OHAP) treatment generated dominant hydrophilic -OH and O=CO- functional groups on the PANI film surface, which served as strong binding sites to immobilize bovine serum albumin (BSA) protein molecules. The output current changes of the BioFEP as a function of BSA concentration were obtained. The resistance of the OHAP surface could be sensitively increased from 2.5 × 108 Ω to 2.0 × 1012 Ω with increasing BSA concentrations in the range of 0.025-4 μg/ml. The results suggest that the method is a simple and cost-effective tool to determine the concentration of BSA by measuring electrical resistance.

  14. Effect of anticoagulants and glucose on refractometric estimation of protein in canine and rabbit plasma.

    Science.gov (United States)

    Dubin, S; Hunt, P

    1978-10-01

    The effect of ethylenediaminetetraacetate (EDTA) compounds on the refractometric estimation of plasma protein concentration was attributed largely to osmotic fluid shifts, as reflected in changes in hematocrit, and to addition of total solids to the plasma. With H4EDTA, these two mechanisms were additive and caused increased plasma protein readings of significant magnitude even at recommended (1--2 mg/ml) anticoagulant concentrations. For the potassium and sodium salts, the two mechanisms were partly compensatory, which ameliorated the effect at 1--2 mg/ml concentration. At higher concentrations, such as might occur if a blood collecting tube were incompletely filled, all of the EDTA compounds caused technically significant over-estimation of plasma protein. When dextrose (d-glucose) was added in-vitro to canine blood, in amounts analogous to clinical hyperglycemia, the effect upon plasma protein estimation was minimal.

  15. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  16. Binary gene induction and protein expression in individual cells

    Directory of Open Access Journals (Sweden)

    Conolly Rory B

    2006-04-01

    Full Text Available Abstract Background Eukaryotic gene transcription is believed to occur in either a binary or a graded fashion. With binary induction, a transcription activator (TA regulates the probability with which a gene template is switched from the inactive to the active state without affecting the rate at which RNA molecules are produced from the template. With graded, also called rheostat-like, induction the gene template has continuously varying levels of transcriptional activity, and the TA regulates the rate of RNA production. Support for each of these two mechanisms arises primarily from experimental studies measuring reporter proteins in individual cells, rather than from direct measurement of induction events at the gene template. Methods and results In this paper, using a computational model of stochastic gene expression, we have studied the biological and experimental conditions under which a binary induction mode operating at the gene template can give rise to differentially expressed "phenotypes" (i.e., binary, hybrid or graded at the protein level. We have also investigated whether the choice of reporter genes plays a significant role in determining the observed protein expression patterns in individual cells, given the diverse properties of commonly-used reporter genes. Our simulation confirmed early findings that the lifetimes of active/inactive promoters and half-lives of downstream mRNA/protein products are important determinants of various protein expression patterns, but showed that the induction time and the sensitivity with which the expressed genes are detected are also important experimental variables. Using parameter conditions representative of reporter genes including green fluorescence protein (GFP and β-galactosidase, we also demonstrated that graded gene expression is more likely to be observed with GFP, a longer-lived protein with low detection sensitivity. Conclusion The choice of reporter genes may determine whether protein

  17. Prolonged morphine administration alters protein expression in the rat myocardium

    OpenAIRE

    Drastichova Zdenka; Skrabalova Jitka; Neckar Jan; Kolar Frantisek; Novotny Jiri

    2011-01-01

    Abstract Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Protein...

  18. Vacuolar Sorting Receptor (VSR) Proteins Reach the Plasma Membrane in Germinating Pollen Tubes

    Institute of Scientific and Technical Information of China (English)

    Hao Wang; Xiao-Hong Zhuang; Stefan Hillmer; David G. Robinson; Li-Wen Jiang

    2011-01-01

    Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants.Confocal immunofluorescent and immunogold Electron Microscope (EM) studies have localized VSRs to PVCs or multivesicular bodies (MVBs) and trans-Golgi network (TGN) in various plant cell types,including suspension culture cells,root cells,developing and germinating seeds.Here,we provide evidence that VSRs reach plasma membrane (PM) in growing pollen tubes.Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that,in addition to the previously demonstrated PVC/MVB localization,VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution.Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes.Using a high-speed Spinning Disc Confocal Microscope,the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR.In contrast,the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.

  19. Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

    Science.gov (United States)

    Pople, J E; Moore, A E; Talbot, D C S; Barrett, K E; Jones, D A; Lim, F L

    2014-10-01

    Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker

  20. A high confidence, manually validated human blood plasma protein reference set

    DEFF Research Database (Denmark)

    Schenk, Susann; Schoenhals, Gary J; de Souza, Gustavo

    2008-01-01

    sources, including the HUPO PPP dataset. CONCLUSION: Superior instrumentation combined with rigorous validation criteria gave rise to a set of 697 plasma proteins in which we have very high confidence, demonstrated by an exceptionally low false peptide identification rate of 0.29%.......BACKGROUND: The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list...

  1. The clinical expression of hereditary protein C and protein S deficiency: : a relation to clinical thrombotic risk-factors and to levels of protein C and protein S

    NARCIS (Netherlands)

    Henkens, C. M. A.; van der Meer, J.; Hillege, J. L.; Bom, V. J. J.; Halie, M. R.; van der Schaaf, W.

    We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were

  2. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    Directory of Open Access Journals (Sweden)

    Rydzak Thomas

    2012-09-01

    Full Text Available Abstract Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative

  3. Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

    Science.gov (United States)

    Bedbrook, Claire N; Kato, Mihoko; Ravindra Kumar, Sripriya; Lakshmanan, Anupama; Nath, Ravi D; Sun, Fei; Sternberg, Paul W; Arnold, Frances H; Gradinaru, Viviana

    2015-08-20

    Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.

  4. Expression and purification of splicing proteins from mammalian cells.

    Science.gov (United States)

    Allemand, Eric; Hastings, Michelle L

    2014-01-01

    Pre-mRNA splicing is a complex process that is carried out by a large ribonucleoprotein enzyme, termed the spliceosome, which comprises up to 200 proteins. Despite this complexity, the role of individual spliceosomal proteins in the splicing reaction has been successfully investigated using cell-free assays. In many cases, the splicing factor of interest must be expressed and purified in order to study its function in vitro. Posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination of splicing factors are important for activity. Thus, their purification from mammalian cells presents numerous advantages. Here, we describe a method for expression and purification of splicing proteins from mammalian cells.

  5. Isolation and Identification of Concanavalin A Binding Glycoproteins from Human Seminal Plasma: A Step Towards Identification of Male Infertility Marker Proteins

    Directory of Open Access Journals (Sweden)

    Anil Kumar Tomar

    2011-01-01

    Full Text Available Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma.

  6. Isolation and Identification of Concanavalin A Binding Glycoproteins from Human Seminal Plasma: A Step Towards Identification of Male Infertility Marker Proteins

    Science.gov (United States)

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Raj, Isha; Singh, Sarman; Singh, Tej P.; Yadav, Savita

    2011-01-01

    Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma. PMID:22182811

  7. Protein expression analysis of inflammation-related colon carcinogenesis

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    Yasui Yumiko

    2009-01-01

    Full Text Available Background: Chronic inflammation is a risk factor for colorectal cancer (CRC development. The aim of this study was to determine the differences in protein expression between CRC and the surrounding nontumorous colonic tissues in the mice that received azoxymethane (AOM and dextran sodium sulfate (DSS using a proteomic analysis. Materials and Methods: Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight, followed by 2% (w/v DSS in their drinking water for seven days, starting one week after the AOM injection. Colonic adenocarcinoma developed after 20 weeks and a proteomics analysis based on two-dimensional gel electrophoresis and ultraflex TOF/TOF mass spectrometry was conducted in the cancerous and nontumorous tissue specimens. Results: The proteomic analysis revealed 21 differentially expressed proteins in the cancerous tissues in comparison to the nontumorous tissues. There were five markedly increased proteins (beta-tropomyosin, tropomyosin 1 alpha isoform b, S100 calcium binding protein A9, and an unknown protein and 16 markedly decreased proteins (Car1 proteins, selenium-binding protein 1, HMG-CoA synthase, thioredoxin 1, 1 Cys peroxiredoxin protein 2, Fcgbp protein, Cytochrome c oxidase, subunit Va, ETHE1 protein, and 7 unknown proteins. Conclusions: There were 21 differentially expressed proteins in the cancerous tissues of the mice that received AOM and DSS. Their functions include metabolism, the antioxidant system, oxidative stress, mucin production, and inflammation. These findings may provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies to treat carcinogenesis in the inflamed colon.

  8. Microfluidic chips for protein differential expression profiling.

    Science.gov (United States)

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  9. Variation in Protein Intake Induces Variation in Spider Silk Expression

    Science.gov (United States)

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  10. Variation in protein intake induces variation in spider silk expression.

    Directory of Open Access Journals (Sweden)

    Sean J Blamires

    Full Text Available BACKGROUND: It is energetically expensive to synthesize certain amino acids. The proteins (spidroins of spider major ampullate (MA silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. METHODOLOGY/PRINCIPAL FINDINGS: We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. CONCLUSIONS: Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact

  11. Soluble Proteins Form Film by the Treatment of Low Temperature Plasma

    Science.gov (United States)

    Ikehara, Sanae; Sakakita, Hajime; Ishikawa, Kenji; Akimoto, Yoshihiro; Nakanishi, Hayao; Shimizu, Nobuyuki; Hori, Masaru; Ikehara, Yuzuru

    2015-09-01

    It has been pointed out that low temperature plasma in atmosphere was feasible to use for hemostasis without heat injury. Indeed, earlier studies demonstrated that low temperature plasma played an important role to stimulate platelets to aggregate and turned on the proteolytic activities of coagulation factors, resulting in the acceleration of the natural blood coagulation process. On the other hands, our developed equips could immediately form clots upon the contact with plasma flair, while the histological appearance was different from natural coagulation. Based on these findings in formed clots, we sought to determine if plasma flair supplied by our devices was capable of forming film using a series of soluble proteins Following plasma treatment, films were formed from bovine serum albumin, and the other plasma proteins at physiological concentration. Analysis of trans-electron microscope demonstrated that plasma treatment generated small protein particles and made them fuse to be larger aggregations The combined results demonstrated that plasma are capable of aggregating soluble proteins and that platelets and coagulation factors are not necessary for plasma induced blood coagulation. Supported in part by Grants-in-Aid for Scientific Research on Priority Area (21590454, 24590498, and 24108006 to Y. I.).

  12. Cholesteryl ester transfer protein alters liver and plasma triglyceride metabolism through two liver networks in female mice.

    Science.gov (United States)

    Palmisano, Brian T; Le, Thao D; Zhu, Lin; Lee, Yoon Kwang; Stafford, John M

    2016-08-01

    Elevated plasma TGs increase risk of cardiovascular disease in women. Estrogen treatment raises plasma TGs in women, but molecular mechanisms remain poorly understood. Here we explore the role of cholesteryl ester transfer protein (CETP) in the regulation of TG metabolism in female mice, which naturally lack CETP. In transgenic CETP females, acute estrogen treatment raised plasma TGs 50%, increased TG production, and increased expression of genes involved in VLDL synthesis, but not in nontransgenic littermate females. In CETP females, estrogen enhanced expression of small heterodimer partner (SHP), a nuclear receptor regulating VLDL production. Deletion of liver SHP prevented increases in TG production and expression of genes involved in VLDL synthesis in CETP mice with estrogen treatment. We also examined whether CETP expression had effects on TG metabolism independent of estrogen treatment. CETP increased liver β-oxidation and reduced liver TG content by 60%. Liver estrogen receptor α (ERα) was required for CETP expression to enhance β-oxidation and reduce liver TG content. Thus, CETP alters at least two networks governing TG metabolism, one involving SHP to increase VLDL-TG production in response to estrogen, and another involving ERα to enhance β-oxidation and lower liver TG content. These findings demonstrate a novel role for CETP in estrogen-mediated increases in TG production and a broader role for CETP in TG metabolism. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

  13. Differential Protein Expression in Congenital and Acquired Cholesteatomas.

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    Seung-Ho Shin

    Full Text Available Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5-3, plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5-3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5-3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins

  14. Advanced oxidation protein products in plasma: stability during storage and correlation with other clinical characteristics.

    Science.gov (United States)

    Matteucci, E; Biasci, E; Giampietro, O

    2001-12-01

    Proteins are susceptible to free radical damage. We measured advanced oxidation protein products (AOPP) in the plasma of 56 hospitalised patients. Concentrations of AOPP were expressed as chloramine-T equivalents by measuring absorbance in acidic conditions at 340 nm in the presence of potassium iodide. We also determined erythrocyte sedimentation rate (ESR), circulating urea, creatinine, glucose, uric acid, electrolytes, lipids, total proteins and fractions and fibrinogen. Twenty-four samples were processed both immediately and after 7, 15, 30, 90, 180 and 438 days of storage at both at -20 degrees C and -80 degrees C (aliquots were frozen and thawed only once) to evaluate AOPP stability. The remaining 32 samples were also processed for thiobarbituric-acid-reactive substances (TBARS). Mean AOPP concentration in all 56 patients was 48.3+/-37.2 microM. Mean basal concentration of AOPP in the 24 plasma samples (55.0+/-47.1 microM) showed no significant change at each intermediate determination, yet significantly increased after 438 days of storage both at -80 degrees C (96.6+/-83.2, p<0.01) and, markedly, at -20 degrees C (171.3+/-94.6, p<0.001). TBARS concentration was 1.59+/-0.65 micromol/l. Multiple regression analysis evidenced that AOPP concentration was positively correlated (multiple r=0.62, p<0.001) with serum urea and triglycerides, but negatively correlated with patient age (indeed, serum albumin and total proteins decreased with increasing age, r=0.3, p<0.05). TBARS concentration was associated with ESR and serum glucose (multiple r=0.73, p<0.001), yet positively with AOPP (r=0.39, simple p<0.05). We conclude that AOPP remain stable during sample storage both at -20 degrees C and -80 degrees C for 6 months. Renal failure and hypertriglyceridemia probably enhance the in vivo process of AOPP formation. Oxidative damage as measured by TBARS may be increased because of exposure to hyperglycemia causing nonenzymatic glycation of plasma proteins.

  15. Hypochlorite-induced damage to plasma and proteins: formation of nitrogen-centred radicals and their role in protein oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Hawkins, C.L.; Davies, M.J. [Heart Research Institute, Camperdown, NSW (Australia)

    1998-12-31

    The respiratory burst of activated phagocyte cells results in the generation of hypochlorite (HOCl) via the release of the hydrogen peroxide and the enzyme myeloperoxidase. Little information is available about the mechanisms and intermediates involved in these reactions. Electron paramagnetic resonance (EPR) spectroscopy with spin trapping has been employed to identify radicals formed in fresh human plasma and isolated proteins and peptides on treatment with HOCI. Reaction of plasma with HOCI in the presence of a spin trap gives broad, anisotropic radical adducts consistent with the formation of large, slowly-tumbling, protein-derived radicals. The identity of the plasma-derived radical adducts was investigated further by the incubation of the pre-formed adducts with the non-specific proteolytic enzyme pronase. This treatment gave sharper, signals consistent with the release of more mobile, low-molecular-weight spin adducts from the initial protein-derived adducts. The hyperfine couplings of these sharper signals are characteristic of the formation of nitrogen-centred radical adducts. Similar or identical species are observed on treatment with isolated human serum albumin, suggesting that this is a major site of HOCI-induced oxidation. Reaction of HOCI-treated plasma or isolated proteins/peptides with excess methionine eliminates radical formation, consistent with lysine-derived chloramines (via homolysis or heterolysis of N-CI bonds) being the radical source. The effect of HOCI on the structural integrity of the plasma proteins was investigated by SDS-PAGE. It was demonstrated that incubation of HOCI-treated plasma or proteins, after removal of excess oxidant, resulted in a time- and HOCI-dependent fragmentation of the proteins. No evidence was obtained for the presence of either discrete fragments or aggregated material. This suggests that the reaction of HOCI with plasma proteins results in the formation of a large number of random fragments. Treatment with

  16. Enzyme hydrolysis of plasma proteins in a CSTR ultrafiltration reactor: Performances and modeling.

    Science.gov (United States)

    Bressollier, P; Petit, J M; Julien, R

    1988-05-01

    By investigating the effects of four operating variables-volume (V), Ultrafiltration flux (J), enzyme concentration (E), and substrate concentration (S)-on capacity (K) and conversion rate (epsilon) of a hollow fiber CSTR, the performances of the CSTR and the kinetic constants of the reaction were determined. A model which takes into account the course of fractional conversion (X) according to the modified space-time parameter, tau (integrated form of V, J, S, and E), was devised by employing the relationship to integrate the equation for the reaction rate of the CSTR and the expression of the modified space time. Correlation of this model and the experimentally obtained results demonstrates that the characteristics for an ultrafiltration membrane reactor for enzymatic hydrolysis by alcalase of plasma proteins are close to those of an ideal CSTR. Optimal scaling up, however, remains dependent on the compromise which may be obtained between capacity and the conversion rate.

  17. Changes of pregnancy-associated plasma protein-A in patients with acute coronary syndrome

    Institute of Scientific and Technical Information of China (English)

    LIU Jin-lai; ZHANG Hui; XIE Xu-jing; CHEN Lin; ZHAO Chang-lin

    2005-01-01

    @@ The term vulnerable patient has been proposed to define subjects susceptible to an acute coronary syndrome (ACS) or sudden cardiac death based on plaque characteristics, blood abnorma-lities, or myocardial vulnerability.1 It will be important in the future to identify both vulnerable patients and vulnerable plaques. Atherosclerotic arteries obtained at autopsy from patients who died suddenly of cardiac causes indicate that pregnancy-associated plasma protein-A (PAPP-A) was abundantly expressed in plaque cells and in the extracellular matrix of ruptured and eroded unstable plaques, but not in stable plaques.2 Here we examined circulating PAPP-A levels in patients with ACS in order to evaluate its potential use in identifying vulnerable patients.

  18. Correlation of plasma FL expression with bone marrow irradiation dose.

    Directory of Open Access Journals (Sweden)

    Mary Sproull

    Full Text Available PURPOSE: Ablative bone marrow irradiation is an integral part of hematopoietic stem cell transplantation. These treatment regimens are based on classically held models of radiation dose and the bone marrow response. Flt-3 ligand (FL has been suggested as a marker of hematopoiesis and bone marrow status but the kinetics of its response to bone marrow irradiation has yet to be fully characterized. In the current study, we examine plasma FL response to total body and partial body irradiation in mice and its relationship with irradiation dose, time of collection and pattern of bone marrow exposure. MATERIALS/METHODS: C57BL6 mice received a single whole body or partial body irradiation dose of 1-8 Gy. Plasma was collected by mandibular or cardiac puncture at 24, 48 and 72 hr post-irradiation as well as 1-3 weeks post-irradiation. FL levels were determined via ELISA assay and used to generate two models: a linear regression model and a gated values model correlating plasma FL levels with radiation dose. RESULTS: At all doses between 1-8 Gy, plasma FL levels were greater than control and the level of FL increased proportionally to the total body irradiation dose. Differences in FL levels were statistically significant at each dose and at all time points. Partial body irradiation of the trunk areas, encompassing the bulk of the hematopoietically active bone marrow, resulted in significantly increased FL levels over control but irradiation of only the head or extremities did not. FL levels were used to generate a dose prediction model for total body irradiation. In a blinded study, the model differentiated mice into dose received cohorts of 1, 4 or 8 Gy based on plasma FL levels at 24 or 72 hrs post-irradiation. CONCLUSION: Our findings indicate that plasma FL levels might be used as a marker of hematopoietically active bone marrow and radiation exposure in mice.

  19. ZBTB32 is an early repressor of the CIITA and MHC class II gene expression during B cell differentiation to plasma cells.

    Science.gov (United States)

    Yoon, Hye Suk; Scharer, Christopher D; Majumder, Parimal; Davis, Carl W; Butler, Royce; Zinzow-Kramer, Wendy; Skountzou, Ioanna; Koutsonanos, Dimitrios G; Ahmed, Rafi; Boss, Jeremy M

    2012-09-01

    CIITA and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly, but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when B lymphocyte-induced maturation protein-1 (Blimp-1), the regulator of plasma cell fate and suppressor of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. Short hairpin RNA depletion of ZBTB32 in a plasma cell line resulted in re-expression of CIITA and I-A. Compared with conditional Blimp-1 knockout and wild-type B cells, B cells from ZBTB32/ROG-knockout mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and a potential regulatory partner of Blimp-1 in repressing gene expression.

  20. Identification of Differentially Expressed Serum Proteins in Infectious Purpura Fulminans

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    Ting He

    2014-01-01

    Full Text Available Purpura fulminans (PF is a life-threatening hemorrhagic condition. Because of the rarity and randomness of the disease, no improvement in treatment has been made for a long time. In this study, we assessed the serum proteome response to PF by comparing serum proteins between healthy controls and PF patient. Liquid chromatography with tandem mass spectrometry (LC-MS/MS approach was used after depleting 6 abundant proteins of serum. In total, 262 proteins were confidently identified with 2 unique peptides, and 38 proteins were identified significantly up- (≥2 or downregulated (≤0.5 based on spectral counting ratios (SpCPF/N. In the 38 proteins with significant abundance changes, 11 proteins were previously known to be associated with burn or sepsis response, but 27 potentially novel proteins may be specifically associated with PF process. Two differentially expressed proteins, alpha-1-antitrypsin (SERPINA1 and alpha-2 antiplasmin (SERPINF2, were validated by Western blot. This is the first study where PF patient and healthy controls are compared in a proteomic study to elucidate proteins involved in the response to PF. This study provides an initial basis for future studies of PF, and the differentially expressed proteins might provide new therapeutic targets to decrease the mortality of PF.

  1. Meal composition and plasma amino acid ratios: Effect of various proteins or carbohydrates, and of various protein concentrations

    Science.gov (United States)

    Yokogoshi, Hidehiko; Wurtman, Richard J.

    1986-01-01

    The effects of meals containing various proteins and carbohydrates, and of those containing various proportions of protein (0 percent to 20 percent of a meal, by weight) or of carbohydrate (0 percent to 75 percent), on plasma levels of certain large neutral amino acids (LNAA) in rats previously fasted for 19 hours were examined. Also the plasma tryptophan ratios (the ratio of the plasma trytophan concentration to the summed concentrations of the other large neutral amino acids) and other plasma amino acid ratios were calculated. (The plasma tryptophan ratio has been shown to determine brain tryptophan levels and, thereby, to affect the synthesis and release of the neurotransmitter serotonin). A meal containing 70 percent to 75 percent of an insulin-secreting carbohydrate (dextrose or dextrin) increased plasma insulin levels and the tryptophan ratio; those containing 0 percent or 25 percent carbohydrate failed to do so. Addition of as little as 5 percent casein to a 70 percent carbohydrate meal fully blocked the increase in the plasma tryptophan ratio without affecting the secretion of insulin - probably by contributing much larger quantities of the other LNAA than of tryptophan to the blood. Dietary proteins differed in their ability to suppress the carbohydrate-induced rise in the plasma tryptophan ratio. Addition of 10 percent casein, peanut meal, or gelatin fully blocked this increase, but lactalbumin failed to do so, and egg white did so only partially. (Consumption of the 10 percent gelatin meal also produced a major reduction in the plasma tyrosine ratio, and may thereby have affected brain tyrosine levels and catecholamine synthesis.) These observations suggest that serotonin-releasing neurons in brains of fasted rats are capable of distinguishing (by their metabolic effects) between meals poor in protein but rich in carbohydrates that elicit insulin secretion, and all other meals. The changes in brain serotonin caused by carbohydrate-rich, protein

  2. Meal composition and plasma amino acid ratios: Effect of various proteins or carbohydrates, and of various protein concentrations

    Science.gov (United States)

    Yokogoshi, Hidehiko; Wurtman, Richard J.

    1986-01-01

    The effects of meals containing various proteins and carbohydrates, and of those containing various proportions of protein (0 percent to 20 percent of a meal, by weight) or of carbohydrate (0 percent to 75 percent), on plasma levels of certain large neutral amino acids (LNAA) in rats previously fasted for 19 hours were examined. Also the plasma tryptophan ratios (the ratio of the plasma trytophan concentration to the summed concentrations of the other large neutral amino acids) and other plasma amino acid ratios were calculated. (The plasma tryptophan ratio has been shown to determine brain tryptophan levels and, thereby, to affect the synthesis and release of the neurotransmitter serotonin). A meal containing 70 percent to 75 percent of an insulin-secreting carbohydrate (dextrose or dextrin) increased plasma insulin levels and the tryptophan ratio; those containing 0 percent or 25 percent carbohydrate failed to do so. Addition of as little as 5 percent casein to a 70 percent carbohydrate meal fully blocked the increase in the plasma tryptophan ratio without affecting the secretion of insulin - probably by contributing much larger quantities of the other LNAA than of tryptophan to the blood. Dietary proteins differed in their ability to suppress the carbohydrate-induced rise in the plasma tryptophan ratio. Addition of 10 percent casein, peanut meal, or gelatin fully blocked this increase, but lactalbumin failed to do so, and egg white did so only partially. (Consumption of the 10 percent gelatin meal also produced a major reduction in the plasma tyrosine ratio, and may thereby have affected brain tyrosine levels and catecholamine synthesis.) These observations suggest that serotonin-releasing neurons in brains of fasted rats are capable of distinguishing (by their metabolic effects) between meals poor in protein but rich in carbohydrates that elicit insulin secretion, and all other meals. The changes in brain serotonin caused by carbohydrate-rich, protein

  3. Translational Modulation of Proteins Expressed from Bicistronic Vectors

    Directory of Open Access Journals (Sweden)

    Prasun J. Mishra

    2009-11-01

    Full Text Available Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment.

  4. Depletion of abundant plasma proteins by poly(N-isopropylacrylamide-acrylic acid) hydrogel particles

    DEFF Research Database (Denmark)

    Such-Sanmartín, Gerard; Ventura-Espejo, Estela; Jensen, Ole N

    2014-01-01

    at higher efficiency than low abundance proteins, which are enriched in the supernatants, whereas (2) hydrogel particles incubated with high concentrations of plasma capture and irreversibly trap abundant proteins. During the elution step, irreversibly trapped proteins remain captured while low abundance...... (SRM) liquid chromatography (LC)-MS/MS. This novel use of hydrogel particles opens new perspectives for biomarker analysis based on mass spectrometry....

  5. Differential Expression of Non-Shelterin Genes Associated with High Telomerase Levels and Telomere Shortening in Plasma Cell Disorders

    Science.gov (United States)

    Panero, Julieta; Stella, Flavia; Schutz, Natalia; Fantl, Dorotea Beatriz; Slavutsky, Irma

    2015-01-01

    Telomerase, shelterin proteins and various interacting factors, named non-shelterin proteins, are involved in the regulation of telomere length (TL). Altered expression of any of these telomere-associated genes can lead to telomere dysfunction, causing genomic instability and disease development. In this study, we investigated the expression profile of a set of non-shelterin genes involved in essential processes such as replication (RPA1), DNA damage repair pathways (MRE11-RAD50-NBS1) and stabilization of telomerase complex (DKC1), in 35 patients with monoclonal gammopathy of undetermined significance (MGUS) and 40 cases with multiple myeloma (MM). Results were correlated with hTERT expression, TL and clinical parameters. Overall, a significant increase in DKC1, RAD50, MRE11, NBS1 and RPA1 expression along with an upregulation of hTERT in MM compared with MGUS was observed (p≤0.032). Interestingly, in both entities high mRNA levels of non-shelterin genes were associated with short TLs and increased hTERT expression. Significant differences were observed for DKC1 in MM (p ≤0.026), suggesting an important role for this gene in the maintenance of short telomeres by telomerase in myeloma plasma cells. With regard to clinical associations, we observed a significant increase in DKC1, RAD50, MRE11 and RPA1 expression in MM cases with high bone marrow infiltration (p≤0.03) and a tendency towards cases with advanced ISS stage, providing the first evidence of non-shelterin genes associated to risk factors in MM. Taken together, our findings bring new insights into the intricate mechanisms by which telomere-associated proteins collaborate in the maintenance of plasma cells immortalization and suggest a role for the upregulation of these genes in the progression of the disease. PMID:26366868

  6. Differential Expression of Non-Shelterin Genes Associated with High Telomerase Levels and Telomere Shortening in Plasma Cell Disorders.

    Directory of Open Access Journals (Sweden)

    Julieta Panero

    Full Text Available Telomerase, shelterin proteins and various interacting factors, named non-shelterin proteins, are involved in the regulation of telomere length (TL. Altered expression of any of these telomere-associated genes can lead to telomere dysfunction, causing genomic instability and disease development. In this study, we investigated the expression profile of a set of non-shelterin genes involved in essential processes such as replication (RPA1, DNA damage repair pathways (MRE11-RAD50-NBS1 and stabilization of telomerase complex (DKC1, in 35 patients with monoclonal gammopathy of undetermined significance (MGUS and 40 cases with multiple myeloma (MM. Results were correlated with hTERT expression, TL and clinical parameters. Overall, a significant increase in DKC1, RAD50, MRE11, NBS1 and RPA1 expression along with an upregulation of hTERT in MM compared with MGUS was observed (p≤0.032. Interestingly, in both entities high mRNA levels of non-shelterin genes were associated with short TLs and increased hTERT expression. Significant differences were observed for DKC1 in MM (p ≤0.026, suggesting an important role for this gene in the maintenance of short telomeres by telomerase in myeloma plasma cells. With regard to clinical associations, we observed a significant increase in DKC1, RAD50, MRE11 and RPA1 expression in MM cases with high bone marrow infiltration (p≤0.03 and a tendency towards cases with advanced ISS stage, providing the first evidence of non-shelterin genes associated to risk factors in MM. Taken together, our findings bring new insights into the intricate mechanisms by which telomere-associated proteins collaborate in the maintenance of plasma cells immortalization and suggest a role for the upregulation of these genes in the progression of the disease.

  7. Construction, Expression and Purification of SUMO1-GST Fusion Protein

    Institute of Scientific and Technical Information of China (English)

    QIAO Xiao-fang; FANG Xue-dong; LIU Jun

    2011-01-01

    Sumoylation is an important protein modification discovered recently. SUMO(small ubiquitin-related modifier) pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein,SUMO, ligated to the target protein. The purification of SUMO proteins is a key step to reveal their function. The purpose of this study was to construct the recombinant SUMO1 gene cloned to a pGEX-4T-1 vector to express and purify the SUMO1-GST fusion protein in Escherichia coli. First, the full length DNA sequence of SUMO1 gene was amplified by PCR and was ligated to pMD18-T vector. Then the SUMO1 gene was subcloned to pGEX-4T-1 prokaryotic expression vector between BamHI and XhoI sites, and transformed in Escherichia coli DH5a cells. The right colonies were identified by restrictive enzyme digestion and sequencing. The correct rebombinant plasmid of pGEX-4T-1-SUMO1 was transformed in Escherichia coli BL21 cells and then induced by IPTG(isopropyl-β-D-lthiogalacto-pyranoside) to express the SUMO1-GST fusion protein. The highly purified SUMOl-GST(glutathione S-transferase) fusion protein was obtained by affinity chromatography. Finally, the properties of SUMO1-GST fusion protein were confirmed by Coomassie brilliant blue strain and Western blot analysis. The recombinant plasmid of pGEX-4T-1-SUMO 1 was successfully constructed, and SUMO1-GST fusion proteins were successfully expressed.

  8. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    Science.gov (United States)

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

  9. Identification of altered plasma proteins by proteomic study in valvular heart diseases and the potential clinical significance.

    Directory of Open Access Journals (Sweden)

    Ge Gao

    Full Text Available BACKGROUND: Little is known about genetic basis and proteomics in valvular heart disease (VHD including rheumatic (RVD and degenerative (DVD valvular disease. The present proteomic study examined the hypothesis that certain proteins may be associated with the pathological changes in the plasma of VHD patients. METHODS AND RESULTS: Differential protein analysis in the plasma identified 18 differentially expressed protein spots and 14 corresponding proteins or polypeptides by two-dimensional electrophoresis and mass spectrometry in 120 subjects. Two up-regulated (complement C4A and carbonic anhydrase 1 and three down-regulated proteins (serotransferrin, alpha-1-antichymotrypsin, and vitronectin were validated by ELISA in enlarging samples. The plasma levels (n = 40 for each of complement C4A in RVD (715.8±35.6 vs. 594.7±28.2 ng/ml, P = 0.009 and carbonic anhydrase 1 (237.70±15.7 vs. 184.7±10.8 U/L, P = 0.007 in DVD patients were significantly higher and that of serotransferrin (2.36±0.20 vs. 2.93±0.16 mg/ml, P = 0.025 and alpha-1-antichymotrypsin (370.0±13.7 vs. 413.0±11.6 µg/ml, P = 0.019 in RVD patients were significantly lower than those in controls. The plasma vitronectin level in both RVD (281.3±11.0 vs. 323.2±10.0 µg/ml, P = 0.006 and DVD (283.6±11.4 vs. 323.2±10.0 µg/ml, P = 0.011 was significantly lower than those in normal controls. CONCLUSIONS: We have for the first time identified alterations of 14 differential proteins or polypeptides in the plasma of patients with various VHD. The elevation of plasma complement C4A in RVD and carbonic anhydrase 1 in DVD and the decrease of serotransferrin and alpha-1-antichymotrypsin in RVD patients may be useful biomarkers for these valvular diseases. The decreased plasma level of vitronectin - a protein related to the formation of valvular structure - in both RVD and DVD patients might indicate the possible genetic deficiency in these patients.

  10. Plasma cytokine expression after lower-limb compression in rats

    Directory of Open Access Journals (Sweden)

    Mauricio Wanderley Moral Sgarbi

    2015-02-01

    Full Text Available OBJECTIVES: Muscle injury due to crushing (muscle compression injury is associated with systemic manifestations known as crush syndrome. A systemic inflammatory reaction may also be triggered by isolated muscle injury. The aim of this study was to investigate the plasma levels of interleukins (IL 1, 6 and 10 and tumor necrosis factor alpha (TNF-α, which are markers for possible systemic inflammatory reactions, after isolated muscle injury resulting from lower-limb compression in rats.METHODS: Male Wistar rats were subjected to 1 h of compression of their lower limbs by means of a rubber band. The plasma levels of IL 1, 6 and 10 and TNF-α were measured 1, 2 and 4 h after the rats were released from compression.RESULTS: The plasma levels of IL 10 decreased in relation to those of the other groups, with a statistically significant difference (p < 0.05. The method used did not detect the presence of IL 1, IL 6 or TNF-α.CONCLUSION: Our results demonstrated that the changes in plasma levels of IL 10 that were found may have been a sign of the presence of circulating interleukins in this model of lower-limb compression in rats.

  11. Expression of genes encoding extracellular matrix proteins: a macroarray study.

    Science.gov (United States)

    Futyma, Konrad; Miotła, Paweł; Różyńska, Krystyna; Zdunek, Małgorzata; Semczuk, Andrzej; Rechberger, Tomasz; Wojcierowski, Jacek

    2014-12-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

  12. Mycorrhizal fungi influence on silver uptake and membrane protein gene expression following silver nanoparticle exposure

    Science.gov (United States)

    Noori, Azam; White, Jason C.; Newman, Lee A.

    2017-02-01

    The rapid growth of nanotechnology and the high demand for nanomaterial use have greatly increased the risk of particle release into the environment. Understanding nanomaterial interactions with crop species and their associated microorganisms is critical to food safety and security. In the current study, tomato was inoculated with mycorrhizal fungi and subsequently exposed to 12, 24, or 36 mg/kg of 2- or 15-nm silver nanoparticles (Ag-NPs). Mycorrhizal (M) and non-mycorrhizal (NM) tomatoes exposed to 36 mg/kg of 2-nm Ag-NPs accumulated 1300 and 1600 μg/g silver in their tissues, respectively. Mycorrhizal plants accumulated 14% less silver compared to non-mycorrhizal plants. To begin to understand the mechanisms by which plants accumulate NPs, the expression of two aquaporin channel genes, the plasma membrane intrinsic protein (PIP) and the tonoplast membrane intrinsic protein (TIP), and one potassium channel (KC) gene were studied. In non-mycorrhizal plants, the expression of KC, PIP, and TIP was eight, five, and nine times higher than the control, respectively. These expressions for mycorrhizal plants were 5.8, 3.5, and 2 times higher than controls, respectively. The expression of KC and PIP, which are located on the plasma membrane, was 3.5 and 2.5, respectively, times higher than TIP, which is located on the tonoplast. PIP expression was significantly higher in NM tomatoes exposed to 12 mg/kg of 2-nm Ag-NPs compared to M plants. These results show that mycorrhizal colonization decreases Ag accumulation in NP-exposed plants and also moderates changes in expression level of membrane transport proteins.

  13. Expression of SKP2 Protein in Lung Carcinoma

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jun; YANG Chun-lu; ZHANG Huan; DING Wei-zhong; LIU Zhi-ping; LIU Jing-yi

    2008-01-01

    Objective:To study the expressive characteristics of SKP2 protein in lung carcinoma and its implication for prognosis.Methods:The expression of SKP2 protein was detected in 89 non small cell lung carcinoma,13 small cell lung carcinoma,10 lung benign lesion tissues by Tissue Chip and Immunohistochemistry technology.Results:The positive rate of SKP2 protein staining was(23.52±13.57)% in non small cell lung carcinoma and (53.85+12.26)% in small cell lung carcinoma,which were significantly higher than(2.91±1.27)% in lung benign lesion tissues.It was highest in small cell lung carcinoma and lowest in lung benign lesion tissues,with a significant difference between them(P=0.000).The expressive level of SKP2 protein in lung carcinoma tissues was closely related to cell differentiation,lymph node metastasis and pathological types,but not to age,sex,smoking history,tumor site and size,and TNM staging.The survival analysis revealed that the 5-year survival rate of lung carcinoma patients was lower in SKP2 protein positive expression group than that in negative expression group(P1=0.003/0.002;r=-0.275,P2=0.005).Conclusion:The positive expression of SKP2 protein is higher in lung carcinoma than in lung benign lesion tissues.in particular,much higher in small cell lung carcinoma.In lung carcinoma,its expressive level was closely related to cell differentiation,lymph node metastasis and pathological types.Moreover,it may be an independent factor to prognosis of patients with lung carcinoma.

  14. EXPRESSION AND SIGNIFICANCE OF ERK PROTEIN IN HUMAN BREAST CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    张秀梅; 李柏林; 宋敏; 宋继谒

    2004-01-01

    Objective: To investigate the expression of ERK and p-ERK protein in human breast cancer and their corresponding tissue, to assess the significance of ERK signal pathway in tumorigenesis and progression of breast carcinoma. Methods: 40 breast cancer cases were used in S-P immunohistochemistry technique and Western Blot study. Results: The expression of ERK1, ERK2, and p- ERK protein levels increased remarkably in breast cancer tissues in comparison to normal tissues (P<0.01). The expression was upregulated by 1.32-, 1.53-and 4.27-fold, respectively. The overexpressions of ERK1, ERK2, and p- ERK proteins were obviously correlated with clinical stage of breast cancer. Protein levels of ERK and p-ERK were higher in stage III patients than in stage I and stage II patients (P<0.05). These proteins were strongly related with axillary lymph node metastasis of breast cancer, but not correlated with histopathological type and status of ER and PR of breast cancer. Expression of ERK1, and ERK2, protein showed a positive linear correlation. Conclusion: ERK signal transduction pathway is a key factor during human breast tumorigenesis and breast cancer progression.

  15. Expression of Extracellular Superoxide Dismutase Protein in Diabetes

    Directory of Open Access Journals (Sweden)

    Chul Han Kim

    2013-09-01

    Full Text Available Background Diabetes is characterized by chronic hyperglycemia, which can increase reactiveoxygen species (ROS production by the mitochondrial electron transport chain. The formationof ROS induces oxidative stress and activates oxidative damage-inducing genes in cells. Noresearch has been published on oxidative damage-related extracellular superoxide dismutase(EC-SOD protein levels in human diabetic skin. We investigated the expression of EC-SOD indiabetic skin compared with normal skin tissue in vivo.Methods The expression of EC-SOD protein was evaluated by western blotting in 6 diabeticskin tissue samples and 6 normal skin samples. Immunohistochemical staining was also carriedout to confirm the EC-SOD expression level in the 6 diabetic skin tissue samples.Results The western blotting showed significantly lower EC-SOD protein expression in thediabetic skin tissue than in the normal tissue. Immunohistochemical examination of EC-SODprotein expression supported the western blotting analysis.Conclusions Diabetic skin tissues express a relatively small amount of EC-SOD protein andmay not be protected against oxidative stress. We believe that EC-SOD is related to the alteredmetabolic state in diabetic skin, which elevates ROS production.

  16. HIP2: An online database of human plasma proteins from healthy individuals

    Directory of Open Access Journals (Sweden)

    Shen Changyu

    2008-04-01

    Full Text Available Abstract Background With the introduction of increasingly powerful mass spectrometry (MS techniques for clinical research, several recent large-scale MS proteomics studies have sought to characterize the entire human plasma proteome with a general objective for identifying thousands of proteins leaked from tissues in the circulating blood. Understanding the basic constituents, diversity, and variability of the human plasma proteome is essential to the development of sensitive molecular diagnosis and treatment monitoring solutions for future biomedical applications. Biomedical researchers today, however, do not have an integrated online resource in which they can search for plasma proteins collected from different mass spectrometry platforms, experimental protocols, and search software for healthy individuals. The lack of such a resource for comparisons has made it difficult to interpret proteomics profile changes in patients' plasma and to design protein biomarker discovery experiments. Description To aid future protein biomarker studies of disease and health from human plasma, we developed an online database, HIP2 (Healthy Human Individual's Integrated Plasma Proteome. The current version contains 12,787 protein entries linked to 86,831 peptide entries identified using different MS platforms. Conclusion This web-based database will be useful to biomedical researchers involved in biomarker discovery research. This database has been developed to be the comprehensive collection of healthy human plasma proteins, and has protein data captured in a relational database schema built to contain mappings of supporting peptide evidence from several high-quality and high-throughput mass-spectrometry (MS experimental data sets. Users can search for plasma protein/peptide annotations, peptide/protein alignments, and experimental/sample conditions with options for filter-based retrieval to achieve greater analytical power for discovery and validation.

  17. Raf-1 kinase inhibitory protein expression in thyroid carcinomas.

    Science.gov (United States)

    Kim, Hyun-Soo; Kim, Gou Young; Lim, Sung-Jig; Kim, Youn Wha

    2010-12-01

    Raf-1 kinase inhibitory protein (RKIP) has been implicated in several fundamental signal transduction pathways that control cellular growth, differentiation, apoptosis and migration. RKIP is reduced in a variety of human carcinomas, but RKIP expression in thyroid carcinomas has not been analyzed at the protein level. In this study, we examined the immunohistochemical expression of RKIP in various subtypes of thyroid carcinoma. Immunostaining for RKIP was performed on 104 cases of primary thyroid carcinoma (40 papillary, 29 follicular, 11 medullary, 11 poorly differentiated, and 13 anaplastic carcinomas) and 26 cases of nodal metastatic tumor (17 papillary, 4 medullary, and 5 anaplastic carcinomas). Normal thyroid tissue and all cases of follicular, papillary, and medullary carcinomas showed uniform, strong cytoplasmic immunoreactivity for RKIP. With the exception of one case, poorly differentiated carcinomas also revealed strong RKIP expression. In contrast, RKIP expression was completely absent in all anaplastic carcinomas. The transition zone from the differentiated carcinoma component (strong RKIP expression) to the anaplastic carcinoma component (no RKIP expression) demonstrated a completely opposite pattern of RKIP immunoreactivity. This reduction of RKIP expression in anaplastic carcinoma was statistically significant (P carcinomas showed uniform, strong cytoplasmic RKIP immunoreactivity, in contrast, in metastatic anaplastic carcinomas, RKIP expression was completely absent. RKIP expression is significantly reduced in anaplastic thyroid carcinoma as compared to other subtypes of thyroid carcinoma. Further studies are necessary to elucidate the precise mechanism of RKIP action in anaplastic thyroid carcinoma.

  18. Expression and biochemical characterization of recombinant human epididymis protein 4.

    Science.gov (United States)

    Hua, Ling; Liu, Yunhui; Zhen, Shuai; Wan, Deyou; Cao, Jiyue; Gao, Xin

    2014-10-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system.

  19. Purification of Pregnancy-associated Plasma Protein-A and Preparation of Its Antibodies

    Institute of Scientific and Technical Information of China (English)

    CHENG; Bin-yan; LI; Zi-ying; YUAN; Zhi-gang; ZHANG; Xue-feng; LIU; Yi-bing

    2013-01-01

    Pregnancy-associated plasma protein-A(PAPP-A)is isolated from the plasma of pregnant women.It is producted by the syntrophoblast tissue of the placenta and decidual cells.PAPP-A belongs to macromolecular glycoprotein.As a sensitive serum marker,the decreased PAPP-A levels during the first

  20. Detection of boar sperm plasma membrane protein using Rhodamine 640; implications for cryobiology and physiology

    Science.gov (United States)

    Rhodamine 640 (R640) was used to detect changes in boar sperm plasma membrane protein (PMP) during cryopreservation; a poorly understood phenomenon. The protocol was adapted for boar sperm so that semen samples (n = 17) could be analyzed for PMP (R640 positive) and plasma membrane integrity (PMI; Y...

  1. Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

    DEFF Research Database (Denmark)

    Schindler, J.; Ye, J. Y.; Jensen, Ole Nørregaard

    2013-01-01

    Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane...

  2. Early Diagnosis of Intestinal Ischemia Using Urinary and Plasma Fatty Acid Binding Proteins

    NARCIS (Netherlands)

    Thuijls, Geertje; van Wijck, Kim; Grootjans, Joep; Derikx, Joep P. M.; van Bijnen, Annemarie A.; Heineman, Erik; Dejong, Cornelis H. C.; Buurman, Wim A.; Poeze, Martijn

    2011-01-01

    Objective: This study aims at improving diagnosis of intestinal ischemia, by measuring plasma and urinary fatty acid binding protein (FABP) levels. Methods: Fifty consecutive patients suspected of intestinal ischemia were included and blood and urine were sampled at time of suspicion. Plasma and uri

  3. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

    DEFF Research Database (Denmark)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen;

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membran...

  4. A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito.

    Science.gov (United States)

    Pradel, Gabriele; Hayton, Karen; Aravind, L; Iyer, Lakshminarayan M; Abrahamsen, Mitchell S; Bonawitz, Annemarie; Mejia, Cesar; Templeton, Thomas J

    2004-06-07

    The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage-specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane-associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

  5. Automation of plasma protein binding assay using rapid equilibrium dialysis device and Tecan workstation.

    Science.gov (United States)

    Ye, Zhengqi; Zetterberg, Craig; Gao, Hong

    2017-03-14

    Binding of drug molecules to plasma proteins is an important parameter in assessing drug ADME properties. Plasma protein binding (PPB) assays are routinely performed during drug discovery and development. A fully automated PPB assay was developed using rapid equilibrium dialysis (RED) device and Tecan workstation coupled to an automated incubator. The PPB assay was carried out in unsealed RED plates which allowed the assay to be fully automated. The plasma pH was maintained at 7.4 during the 6-h dialysis under 2% CO2 condition. The samples were extracted with acetonitrile and analyzed by liquid chromatography tandem mass spectrometry. The percent bound results of 10 commercial drugs in plasma protein binding were very similar between the automated and manual assays, and were comparable to literature values. The automated assay increases laboratory productivity and is applicable to high-throughput screening of drug protein binding in drug discovery.

  6. Proteomic profiling of human plasma exosomes identifies PPARgamma as an exosome-associated protein.

    Science.gov (United States)

    Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W; Shen, Rong-Fong; Daniels, Mathew P; Levine, Stewart J

    2009-01-16

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-gamma (PPARgamma), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARgamma as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  7. Expression of Structural Protein E2 of Hepatitis C Virus

    Institute of Scientific and Technical Information of China (English)

    GUO Tai-lin; YE Lin-bo; MAO Can-quan

    2005-01-01

    The E2 glycoprotein is one of the structural components of the hepatitis C virus (HCV) virion. It elicits production of neutralising antibodies against the virus, and is involved in viral morphogenesis. The protein is considered as a major candidate for anti- HCV vaccine. Despitethis, little is known about this protein. Previous studies have focused on the and functional analysis of the glycosylated forms. This report describes expression of the E2 (recE2) in different forms and in different expression systems in Escherichia coli cells and in mammalian cells in order to obtain enough protein efficiently in vitro, in addition we also analysed the usage of rare codons in the genes of E2 and CORE. All results have shown that great efforts should be made to improve the expression efficiency of E2 in bacteria or mammalian cells.

  8. Expression and Purification of SARS Coronavirus Membrane Protein

    Institute of Scientific and Technical Information of China (English)

    戴五星; 雷明军; 吴少庭; 陈智浩; 梁靓; 潘晖榕; 秦莉; 高士同; 袁仕善; 张仁利

    2004-01-01

    To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The re combinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropylβ-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0. 992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.

  9. Expression of protein-coding genes embedded in ribosomal DNA

    DEFF Research Database (Denmark)

    Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik

    2007-01-01

    Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns...... that encode reverse transcriptase-like genes, and group I introns and archaeal introns that encode homing endonuclease genes (HEGs). Although rDNA-embedded protein genes are widespread in nuclei, organelles and bacteria, there is surprisingly little information available on how these genes are expressed....... Exceptions include a handful of HEGs from group I introns. Recent studies have revealed unusual and essential roles of group I and group I-like ribozymes in the endogenous expression of HEGs. Here we discuss general aspects of rDNA-embedded protein genes and focus on HEG expression from group I introns...

  10. Using ion exchange chromatography to purify a recombinantly expressed protein.

    Science.gov (United States)

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.

  11. The expression and significance of p53 protein and Ki-67 protein in pterygium

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    Ljubojević Vesna

    2016-01-01

    Full Text Available Background/Aim. Pterygium is considered to be a degenerative disease of the conjunctiva, however, the presence of tumor markers in pterygium reinforces the hypothesis that this lesion is similar to tumor. Inactivation of p53 function removes an obstacle to increased proliferation. Factors affecting the prevalence of p53 expression in pterygium deserve investigation. The aim of the study was to investigate the expression of p53 and Ki-67 proteins in pterygium and normal conjunctiva, the effects of gender and age on p53 expression, and the relationship between the expression of p53 and Ki-67 proteins. Methods. A total of 34 samples of pterygium and 34 samples of the normal conjunctiva were analyzed. The samples were studied by immunohistochemistry using antibodies against p53 and Ki-67. Results. Totally 15 (44% samples of pterygia were p53 positive. Correlations between the expression of p53 protein and sex, and age were not established. The number of Ki-67 positive cells in pterygium (9.74% was significantly higher than the number of Ki-67 positive cells in the normal conjunctiva (1.74%, (p = 0.001. Between the expression of p53 protein and Ki-67 protein in pterygium there was a significant positive correlation (p = 0.000. Conclusion. The prevalence of p53 positive samples of pterygium was 44%. The influence of sex and age on p53 protein expression in pterygium was not found. The increased proliferative acivity was present in the epithelium of pterygium. The expression of Ki-67 protein is associated with the expression of p53 protein in pterygium. The findings of our study support the thesis of pterygium as tissue growth disorder.

  12. Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification.

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    Jayde A Gawthorne

    Full Text Available In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an "effector" protein normally produced by enterohemorrhagic E. coli strains and "injected" into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.

  13. Plasma proteins production and excretion in diabetic nephropathy in ...

    African Journals Online (AJOL)

    Journal of African Association of Physiological Sciences ... Subjects, materials, and methods: Plasma albumin, and fibrinogen ... Results: A direct relationship was found between albuminuria and albumin concentration (r=0.59, p<0.05).

  14. Ubiquitination regulates the plasma membrane expression of renal UT-A urea transporters.

    Science.gov (United States)

    Stewart, Gavin S; O'Brien, Jennifer H; Smith, Craig P

    2008-07-01

    The renal UT-A urea transporters UT-A1, UT-A2, and UT-A3 are known to play an important role in the urinary concentrating mechanism. The control of the cellular localization of UT-A transporters is therefore vital to overall renal function. In the present study, we have investigated the effect of ubiquitination on UT-A plasma membrane expression in Madin-Darby canine kidney (MDCK) cell lines expressing each of the three renal UT-A transporters. Inhibition of the ubiquitin-proteasome pathway caused an increase in basal transepithelial urea flux across MDCK-rat (r)UT-A1 and MDCK-mouse (m)UT-A2 monolayers (P UT-A transporter expression in the plasma membrane (P UT-A3 expression in the plasma membrane (P UT-A urea transporters, but that this is not the mechanism primarily used by vasopressin to produce its physiological effects.

  15. SPINK 1 Protein Expression and Prostate Cancer Progression

    Science.gov (United States)

    Flavin, Richard; Pettersson, Andreas; Hendrickson, Whitney K.; Fiorentino, Michelangelo; Finn, Stephen; Kunz, Lauren; Judson, Gregory L.; Lis, Rosina; Bailey, Dyane; Fiore, Christopher; Nuttall, Elizabeth; Martin, Neil E.; Stack, Edward; Penney, Kathryn L.; Rider, Jennifer R.; Sinnott, Jennifer; Sweeney, Christopher; Sesso, Howard D.; Fall, Katja; Giovannucci, Edward; Kantoff, Philip; Stampfer, Meir; Loda, Massimo; Mucci, Lorelei A.

    2014-01-01

    Purpose SPINK1 over-expression has been described in prostate cancer and is linked with poor prognosis in many cancers. The objective of this study was to characterize the association between SPINK1 over-expression and prostate cancer specific survival. Experimental Design The study included 879 participants in the US Physicians’ Health Study and Health Professionals Follow–Up Study, diagnosed with prostate cancer (1983 – 2004) and treated by radical prostatectomy. Protein tumor expression of SPINK1 was evaluated by immunohistochemistry on tumor tissue microarrays. Results 74/879 (8%) prostate cancer tumors were SPINK1 positive. Immunohistochemical data was available for PTEN, p-Akt, pS6, stathmin, androgen receptor (AR) and ERG (as a measure of the TMPRSS2:ERG translocation). Compared to SPINK1 negative tumors, SPINK1 positive tumors showed higher PTEN and stathmin expression, and lower expression of AR (p<0.01). SPINK1 over-expression was seen in 47 of 427 (11%) ERG negative samples and in 19 of 427 (4%) ERG positive cases (p=0.0003). We found no significant associations between SPINK1 status and Gleason grade or tumor stage. There was no association between SPINK1 expression and biochemical recurrence (p=0.56). Moreover, there was no association between SPINK1 expression and prostate cancer mortality (there were 75 lethal cases of prostate cancer during a mean of 13.5 years follow-up [HR 0.71 (95% confidence interval 0.29–1.76)]). Conclusions Our results suggest that SPINK1 protein expression may not be a predictor of recurrence or lethal prostate cancer amongst men treated by radical prostatectomy. SPINK1 and ERG protein expression do not appear to be entirely mutually exclusive, as some previous studies have suggested. PMID:24687926

  16. Expression and export: recombinant protein production systems for Aspergillus.

    Science.gov (United States)

    Fleissner, André; Dersch, Petra

    2010-07-01

    Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.

  17. Decreased Expression of GPER1 Gene and Protein in Goiter

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    Raquel Weber

    2015-01-01

    Full Text Available Goiter is more common in women, suggesting that estrogen could be involved in its physiopathology. The presence of classical estrogen receptors (ERα and ERβ has been described in thyroid tissue, suggesting a direct effect of estrogen on the gland. A nonclassic estrogen receptor, the G-protein-coupled estrogen receptor (GPER1, has been described recently in several tissues. However, in goiter, the presence of this receptor has not been studied yet. We investigated GPER1 gene and protein expressions in normal thyroid and goiter using reverse transcription quantitative polymerase chain reaction (RT-qPCR and Western blot, respectively. In normal thyroid (n=16 and goiter (n=19, GPER1 gene was expressed in all samples, while GPER1 protein was expressed in all samples of normal thyroid (n=15 but in only 72% of goiter samples (n=13. When comparing GPER1 gene and protein levels in both conditions, gene expression and protein levels were higher in normal thyroid than in goiter, suggesting a role of this receptor in this condition. Further studies are needed to elucidate the role of GPER1 in normal thyroid and goiter.

  18. p53 and MDM2 protein expression in actinic cheilitis.

    Science.gov (United States)

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  19. p53 and MDM2 protein expression in actinic cheilitis

    Directory of Open Access Journals (Sweden)

    Maria da Conceição Andrade de Freitas

    2008-12-01

    Full Text Available Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976 parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  20. Interfacial polymerization for colorimetric labeling of protein expression in cells.

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    Jacob L Lilly

    Full Text Available Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  1. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Science.gov (United States)

    Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  2. Altered micro-ribonucleic acid expression profiles of extracellular microvesicles in the seminal plasma of patients with oligoasthenozoospermia.

    Science.gov (United States)

    Abu-Halima, Masood; Ludwig, Nicole; Hart, Martin; Leidinger, Petra; Backes, Christina; Keller, Andreas; Hammadeh, Mohamad; Meese, Eckart

    2016-10-01

    To determine whether microRNA (miRNA) expression profile is different in extracellular microvesicles collected from seminal plasma of men with oligoasthenozoospermia, to gain further insight into molecular mechanisms underlying male infertility. Microarray with quantitative real-time polymerase chain reaction validation and Western blot analysis confirmation. University research and clinical institutes. A total of 24 men, including 12 oligoasthenozoospermic subfertile men and 12 normozoospermic men. None. Statistically significant altered miRNA expression profiles in oligoasthenozoospermic subfertile men compared with normozoospermic fertile men. Extracellular microvesicles including exosomes were isolated from seminal plasma by ultracentrifugation. Presence of exosome-specific proteins was confirmed by Western blotting. In the extracellular microvesicles, we analyzed 1,205 miRNAs by microarray and identified 36 miRNAs with altered expression levels in oligoasthenozoospermic compared with normozoospermic fertile men. Seven miRNAs were overexpressed and 29 miRNAs were underexpressed in oligoasthenozoospermic men. Using quantitative real-time polymerase chain reaction as an independent method, we confirmed the significantly higher expression levels of miR-765 and miR-1275 and the significantly lower expression level of miR-15a in oligoasthenozoospermic subfertile men as compared with the normozoospermic men. We identified altered expression levels of miRNAs in extracellular microvesicles from seminal plasma as part of the molecular events in the male genital tract. These miRNAs may help to understand the molecular mechanisms underlying male infertility. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. Biochemical and cytogenetic studies of Poecilia from eastern México. I. Comparative microelectrophoresis of plasma proteins of seven species

    OpenAIRE

    Balsano, J. S.; Rasch, Ellen M.

    2016-01-01

    Over 2000 fisch plasmas from six species of Poecilia were collected from 33 populations in eastern Mexico and one from western Mexico. These plasmas were electrophoretically separated in 7.5% polyacrylamide gel which was stained for specific enzymes or total protein. Identiflcations of albumin band mobilities were verified by mixing isoaliquots of test plasmas with plasmas of known standards and by comparing test plasmas with plasmas from F1 hybrid progreny of known parentage.In the latipinna...

  4. Recombinant protein expression by targeting pre-selected chromosomal loci

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    Krömer Wolfgang

    2009-12-01

    Full Text Available Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs were targeted by Flp recombinase mediated cassette exchange (RMCE. The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context

  5. hTERT protein expression is independent of clinicopathological parameters and c-Myc protein expression in human breast cancer

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    Meligonis G

    2005-01-01

    Full Text Available Abstract Background Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. The hTERT (human telomerase reverse transcriptase subunit seems to be the rate-limiting determinant of telomerase and knowledge of factors controlling hTERT transcription may be useful in therapeutic strategies. The hTERT promoter contains binding sites for c-Myc and there is some experimental and in vitro evidence that c-Myc may increase hTERT expression. We previously reported no correlation between c-Myc mRNA expression and hTERT mRNA or telomerase activity in human breast cancer. This study aims to examine the correlation between hTERT expression as determined by immunohistochemistry and c-Myc expression, lymph node status, and tumour size and grade in human breast cancer. Materials and methods The immunohistochemical expression of hTERT and c-Myc was investigated in 38 malignant breast tumours. The expression of hTERT was then correlated with the lymph node status, c-Myc expression and other clinicopathological parameters of the tumours. Results hTERT expression was positive in 27 (71% of the 38 tumours. 15 (79% of 19 node positive tumours were hTERT positive compared with 11 (63% of 19 node negative tumours. The expression was higher in node positive tumours but this failed to reach statistical significance (p = 0.388. There was no significant association with tumour size, tumour grade or c-Myc expression. However, hTERT expression correlated positively with patients' age (correlation coefficient = 0.415, p = 0.0097. Conclusion hTERT protein expression is independent of lymph node status, tumour size and grade and c-Myc protein expression in human breast cancer

  6. Spectrophotometric determination of total proteins in blood plasma: a comparative study among dye-binding methods

    OpenAIRE

    Dimas Augusto Morozin Zaia; Fábio Rangel Marques; Cássia Thaïs Bussamra Vieira Zaia

    2005-01-01

    A comparative study between the biuret method (standard method for total proteins) and spectrophotometric methods using dyes (Bradford, 3',3",5',5"-tetrabromophenolphthalein ethyl ester-TBPEE, and erythrosin-B) was carried out for the determination of total proteins in blood plasma from rats. Bradford method showed the highest sensitivity for proteins and biuret method showed the lowest. For all the methods, the absorbance for different proteins (BSA, casein, and egg albumin) was measured and...

  7. Voltage-dependent anion channels (VDACs, porin) expressed in the plasma membrane regulate the differentiation and function of human osteoclasts.

    Science.gov (United States)

    Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki

    2013-01-01

    Fewer molecules have been identified on human than murine osteoclasts, the former differing from murine osteoclasts in many ways. We show that voltage-dependent anion channels (VDACs, porin) are expressed in the plasma membrane of human osteoclasts. A search for novel proteins expressed in the plasma membrane of human osteoclasts identified VDAC. Anti-VDAC antibodies inhibited human osteoclastogenesis in vitro. VDAC expression was detected in membranes by immunoelectron microscopy and immunocytochemical double staining. The VDAC protein functions as a Cl(-) channel. VDACs regulate bone resorption, which show using Osteologic™ plates. The epitope of the antibody lay within a 10-amino acid sequence in the VDAC. The findings suggest that the VDAC is, at least partly, a novel Cl(-) channel regulating the differentiation and function of human osteoclasts. VDACs may play a crucial role in acidifying the resorption lacunae between osteoclasts and bone. Inhibitors of VDACs could be used to treat diseases involving increased resorption, such as osteoporosis, rheumatoid arthritis, and Paget's disease. © 2012 International Federation for Cell Biology.

  8. Expression profile of heat shock protein 70 in indigenous Huainan partridge chicken exposed to low temperature

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    Xing Yong Chen

    2014-04-01

    Full Text Available It is clear that heat shock protein 70 (HSP70 is responsible for stressful conditions. However, the expression level and profile of HSP70 during cold stress are still unknown. In this study, the expression profile of HSP70 in the heart, liver, muscle and spleen of Huainan partridge chicken exposed to low temperature was investigated. HSP70 expression was showed tissue-dependent with highest expression in muscle, followed by liver and heart; conversely, there was no evidence of changes in spleen, where there were two expression peaks during cold stress, before 3 and after 72 h, respectively. The plasma creatine kinase (CK activity exhibited a significant increase (P<0.01 after 1 h of cold stress exposure, and then decreased till to the lowest level after 72 h of cold stress exposure. On the other hand, nitric oxide content arose and reached the peak level (P<0.01 after 3 h of cold stress exposure, and then suddenly decreased to the original level with the duration of exposure time. In conclusion, mRNA expression of HSP70 turned out to be tissueand time-dependent in muscle, liver and heart in broilers under cold stress exposure. The distinct expression of HSP70 suggested that highenergy supply and balance of CK activity might be responsible for the HSP70 high expression.

  9. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    Science.gov (United States)

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  10. Nanoparticle size matters in the formation of plasma protein coronas on Fe3O4 nanoparticles.

    Science.gov (United States)

    Hu, Zhengyan; Zhang, Hongyan; Zhang, Yi; Wu, Ren'an; Zou, Hanfa

    2014-09-01

    When nanoparticles (NPs) enter into biological systems, proteins would interact with NPs to form the protein corona that can critically impact the biological identity of the nanomaterial. Owing to their fundamental scientific interest and potential applications, Fe3O4 NPs of different sizes have been developed for applications in cell separation and protein separation and as contrast agents in magnetic resonance imaging (MRI), etc. Here, we investigated whether nanoparticle size affects the formation of protein coronas around Fe3O4 NPs. Both the identification and quantification results demonstrated that particle size does play an important role in the formation of plasma protein coronas on Fe3O4 NPs; it not only influenced the protein composition of the formed plasma protein corona but also affected the abundances of the plasma proteins within the coronas. Understanding the different binding profiles of human plasma proteins on Fe3O4 NPs of different sizes would facilitate the exploration of the bio-distributions and biological fates of Fe3O4 NPs in biological systems.

  11. Protein expression on Cr resistant microorganism using electrophoresis method

    Directory of Open Access Journals (Sweden)

    SAJIDAN

    2009-01-01

    Full Text Available Fatmawati U, Suranto, Sajidan. 2009. Protein expression on Cr resistant microorganism using electrophoresis method. Nusantara Bioscience 1: 31-37. Hexavalent chromium (Cr(VI is known as toxic heavy metals, so the need is reduced to Cr(III is much less toxicity. Pseudomonas aeruginosa, Pseudomonas putida, Klebsiella pneumoniae, Pantoea sp. and Saccharomyces cerevisiae are resistant Cr(VI microorganism and have ability to reduce Cr(VI. The aim of this research is to know ability of microorganism to reduce Cr(VI and to know protein band pattern between Cr(VI resistant microorganism and non resistant microorganism which inoculated on LB broth. SDS-PAGE was used to indentify protein expression. While, Cr(VI concentration was identified by 1.5 diphenylcarbazide method. The quantitative data was analyzed by two factorial ANOVA that continued with DMRT at 1% level test. The qualitative data i.e. protein expression analyzed by relative mobility (Rf. The results showed that the ability of microorganisms to reduce Cr(VI at initial concentration of 0.5 ppm, 1 ppm, 5 ppm and 10 ppm may vary, the average percentage of the ability of each microorganism in reducing Cr(VI is P. putida (65% > S. cerevisiae (64.45% >. P. aeruginosa (60.73% > Pantoea sp. (50.22% > K. pneumoniae (47.82% > without microorganisms (34.25%. The adding microorganisms have significantly influenced toward reduction of Cr(VI. The SDS-PAGE shows that protein expression between resistant and not resistant microorganisms are no different, but resistant microorganisms have more protein (protein band is thicker.

  12. The role of cholesteryl ester transfer protein and phospholipid transfer protein in the remodeling of plasma high-density lipoproteins.

    Science.gov (United States)

    Lagrost, L

    1997-08-01

    Recent studies demonstrated that alterations in the size distribution of high-density lipoproteins (HDLs) constitute reliable markers for the risk of coronary artery disease. These observations suggested that the determination of the size distribution of HDL subpopulations by using polyacrylamide gradient gel electrophoresis might constitute an effective tool in clinical practice for the detection of patients with elevated risk. During the last decade, concordant observations revealed that all the HDL subpopulations are metabolically interrelated, and their relative abundances are dependent on the activity of several plasma factors, among them the cholesteryl ester transfer protein (CETP) and the phospholipid transfer protein (PLTP). As reviewed in the present article, although both CETP and PLTP can promote the size redistribution or conversion of HDL, the two plasma lipid transfer proteins can alter differently the plasma HDL distribution profile through distinct mechanisms. (Trends Cardiovasc Med 1997;7:218-224). © 1997, Elsevier Science Inc.

  13. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    Science.gov (United States)

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  14. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    Science.gov (United States)

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  15. Interaction between Fibrinogen and Insulin-Like Growth Factor-Binding Protein-1 in Human Plasma under Physiological Conditions.

    Science.gov (United States)

    Gligorijević, N; Nedić, O

    2016-02-01

    Fibrinogen is a plasma glycoprotein and one of the principle participants in blood coagulation. It interacts with many proteins during formation of a blood clot, including insulin-like growth factors (IGFs) and their binding proteins (IGFBP). Fibrinogen complexes were found as minor fractions in fibrinogen preparations independently of the coagulation process, and their presence influences the kinetics of polymerization. The idea of this work was to investigate whether fibrinogen in human plasma interacts with IGFBPs independently of the tissue injury or coagulation process. The results have shown that fibrinogen forms complexes with IGFBP-1 under physiological conditions. Several experimental approaches have confirmed that complexes are co-isolated with fibrinogen from plasma, they are relatively stable, and they appear as a general feature of human plasma. Several other experiments excluded the possibility that alpha-2 macroglobulin/IGFBP-1 complexes or IGFBP-1 oligomers contributed to IGFBP-1 immunoreactivity. The role of fibrinogen/IGFBP-1 complexes is still unknown. Further investigation in individuals expressing both impaired glucose control and coagulopathy could contribute to identification and understanding of their possible physiological role.

  16. Platelet adhesion onto wettability gradient surfaces in the absence and presence of plasma proteins.

    Science.gov (United States)

    Lee, J H; Lee, H B

    1998-08-01

    A wettability gradient was prepared on lowdensity polyethylene (PE) sheets by treating them in air with a corona from a knife-type electrode the power of which increased gradually along the sample length. The PE surfaces oxidized gradually with the increasing corona power and a wettability gradient was created on the surfaces, as evidenced by the measurement of water contact angles, Fourier transform infrared spectroscopy in the attenuated total reflectance mode, and electron spectroscopy for chemical analysis. The wettability gradient surfaces prepared were used to investigate the adhesion behavior of platelets in the absence and presence of plasma proteins in terms of the surface hydrophilicity/hydrophobicity of polymeric materials. The platelets adhered to the wettability gradient surfaces along the sample length were counted and examined by scanning electron microscopy (SEM). It was observed that the platelet adhesion in the absence of plasma proteins increased gradually as the surface wettability increased along the sample length. The platelets adhered to the hydrophilic positions of the gradient surface also were more activated (possessed more pseudo pods as examined by SEM) than on the more hydrophobic ones. However, platelet adhesion in the presence of plasma proteins decreased gradually with the increasing surface wettability; the platelets adhered to the surface also were more activated on the hydrophobic positions of the gradient surface. This result is closely related to plasma protein adsorption on the surface. Plasma protein adsorption on the wettability gradient surface increased with the increasing surface wettability. More plasma protein adsorption on the hydrophilic positions of the gradient surface caused less platelet adhesion, probably due to platelet adhesion inhibiting proteins, such as high-molecular-weight kininogen, which preferably adsorbs onto the surface by the so-called Vroman effect. It seems that both the presence of plasma proteins

  17. Breast Cancer Resistance Protein Expression and 5-Fluorouracil Resistance

    Institute of Scientific and Technical Information of China (English)

    JIAN-HUI YUAN; ZHI-XIONG ZHUANG; JIN-QUAN CHENG; LONG-YUAN JIANG; WEI-DONG JI; LIANG-FENG GUO; JIAN-JUN LIU; XING-YUN XU; JING-SONG HE; XIAN-MING WANG

    2008-01-01

    To filtrate breast cancer resistance protein (BCRP)-mediated resistant agents and to investigate clinical relationship between BCRP expression and drug resistance. Methods MTT assay was performed to filtrate BCRP-mediated resistant agents with BCRP expression cell model and to detect chemosensitivity of breast cancer tissue specimens to these agents. A high performance liquid chromatography (HPLC) assay was established, and was used to measure the relative dose of intracellular retention resistant agents. RT-PCR and immununohistochemistry (IHC) were employed to investigate the BCRP expression in breast cancer tissue specimens. Results MTT assay showed that the expression of BCRP increased with the increasing resistance of 5-fluorouracil (5-Fu) (P=0.8124, P<0.01). Condusion Resistance to 5-Fu can be mediated by BCRP. Clinical chemotherapy for breast cancer patients can be optimized based on BCRP-positive expression.

  18. Regenerating human muscle fibres express GLUT3 protein

    DEFF Research Database (Denmark)

    Gaster, M; Beck-Nielsen, H; Schrøder, H D

    2002-01-01

    The presence of the GLUT3 glucose transporter protein in human muscle cells is a matter of debate. The present study was designed to establish whether GLUT3 is expressed in mature human skeletal muscle fibres and, if so, whether its expression changes under different conditions, such as metabolic...... stress (obesity, obese non-insulin-dependent diabetes mellitus), hypertrophy (training), de- and reinnervation (amyotrophic lateral sclerosis) or regeneration (polymyositis). We used an immunohistochemical approach to detect and localise GLUT3. GLUT3 immunoreactivity was not detectable in adult skeletal...... muscle fibres, nor did metabolic stress, training or de- and re-innervation induce GLUT3 expression, while a few GLUT3 expressing fibres were seen in some cases of polymyositis. In contrast, GLUT4 was expressed in all investigated muscle fibres. GLUT3 immunoreactivity was found in perineural...

  19. Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: II. Intrinsic Protein Fluorescence.

    Science.gov (United States)

    Caldwell, C R

    1987-07-01

    The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32 degrees C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30 degrees C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32 degrees C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14 degrees C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32 degrees C which could account for the complex temperature dependence of the barley root plasma membrane ATPase.

  20. Rice Hypersensitive Induced Reaction Protein 1 (OsHIR1 associates with plasma membrane and triggers hypersensitive cell death

    Directory of Open Access Journals (Sweden)

    Sun Sai-Ming

    2010-12-01

    Full Text Available Abstract Background In plants, HIR (Hypersensitive Induced Reaction proteins, members of the PID (Proliferation, Ion and Death superfamily, have been shown to play a part in the development of spontaneous hypersensitive response lesions in leaves, in reaction to pathogen attacks. The levels of HIR proteins were shown to correlate with localized host cell deaths and defense responses in maize and barley. However, not much was known about the HIR proteins in rice. Since rice is an important cereal crop consumed by more than 50% of the populations in Asia and Africa, it is crucial to understand the mechanisms of disease responses in this plant. We previously identified the rice HIR1 (OsHIR1 as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1. Here we show that OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1. Result Through electron microscopy studies using wild type rice plants, OsHIR1 was found to mainly localize to the plasma membrane, with a minor portion localized to the tonoplast. Moreover, the plasma membrane localization of OsHIR1 was enhanced in transgenic rice plants overexpressing its interacting protein partner, OsLRR1. Co-localization of OsHIR1 and OsLRR1 to the plasma membrane was confirmed by double-labeling electron microscopy. Pathogen inoculation studies using transgenic Arabidopsis thaliana expressing either OsHIR1 or OsLRR1 showed that both transgenic lines exhibited increased resistance toward the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. However, OsHIR1 transgenic plants produced more extensive spontaneous hypersensitive response lesions and contained lower titers of the invading pathogen, when compared to OsLRR1 transgenic plants. Conclusion The OsHIR1 protein is mainly localized to the plasma membrane, and its subcellular localization in that compartment is enhanced by OsLRR1. The expression of OsHIR1 may sensitize the plant

  1. Functional Expression of Aquaporin-2 Tagged with Photoconvertible Fluorescent Protein in mpkCCD Cells

    Directory of Open Access Journals (Sweden)

    Kay-Pong Yip

    2015-05-01

    Full Text Available Background: Vasopressin induced trafficking of aquaporin-2 (AQP2 containing vesicles has been studied in kidney cell lines using conventional fluorescent proteins as tags. However, trafficking of fluorescent tagged AQP2, which resembles the vectorial translocation of native AQP2 from cytoplasm to apical membrane has not been demonstrated at real time. Using a photoconvertible fluorescent protein tag on AQP2 might allow the simultaneous tracking of two separate populations of AQP2 vesicle after subcellular local photoconversion. Methods: A spacer was used to link a photoconvertible fluorescent protein (mEos2 to the amino-terminus of AQP2. The DNA constructs were expressed in mpkCCD cells. The trafficking of chimeric protein was visualized with high speed confocal microscopy in 4 dimensions. Results: Chimeric AQP2 expressed in mpkCCD cell conferred osmotic water permeability to the cells. Subcellular photoconversion with a 405 nm laser pulse converted green chimeras to red chimeras locally. Forskolin stimulation triggered chimeric AQP2 to translocate from acidic organelles to apical plasma membrane. By serendipity, the rate of apical accumulation was found to increase when mEos2 was tagged to the carboxyl-terminus in at least one of the AQP2 molecules within the tetramer. Conclusion: Functional photoconvertible chimeric AQP2 was successfully expressed in mpkCCD cells, in which forskolin induced apical trafficking and accumulation of chimeric AQP2. The proof-of-concept to monitor two populations of AQP2 vesicle simultaneously was demonstrated.

  2. Direct protein introduction into plant cells using a multi-gas plasma jet.

    Science.gov (United States)

    Yanagawa, Yuki; Kawano, Hiroaki; Kobayashi, Tomohiro; Miyahara, Hidekazu; Okino, Akitoshi; Mitsuhara, Ichiro

    2017-01-01

    Protein introduction into cells is more difficult in plants than in mammalian cells, although it was reported that protein introduction was successful in shoot apical meristem and leaves only together with a cell-penetrating peptide. In this study, we tried to introduce superfolder green fluorescent protein (sGFP)-fused to adenylate cyclase as a reporter protein without a cell-penetrating peptide into the cells of tobacco leaves by treatment with atmospheric non-thermal plasmas. For this purpose, CO2 or N2 plasma was generated using a multi-gas plasma jet. Confocal microscopy indicated that sGFP signals were observed inside of leaf cells after treatment with CO2 or N2 plasma without substantial damage. In addition, the amount of cyclic adenosine monophosphate (cAMP) formed by the catalytic enzyme adenylate cyclase, which requires cellular calmodulin for its activity, was significantly increased in leaves treated with CO2 or N2 plasma, also indicating the introduction of sGFP-fused adenylate cyclase into the cells. These results suggested that treatment with CO2 or N2 plasma could be a useful technique for protein introduction into plant tissues.

  3. Secreted Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

    Science.gov (United States)

    Feyertag, Felix; Berninsone, Patricia M; Alvarez-Ponce, David

    2017-03-01

    The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E-R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein-protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E-R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E-R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E-R anticorrelation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    Directory of Open Access Journals (Sweden)

    Nishizaki Takashi

    2006-07-01

    Full Text Available Abstract Background Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. Methods The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. Results The Bcl-2 protein expression was found to be decreased in 105 (42% cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089 associated with a worse disease free survival (DFS, while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. Conclusion The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer.

  5. A large-scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand-binding domains of taste receptor.

    Science.gov (United States)

    Yamashita, Atsuko; Nango, Eriko; Ashikawa, Yuji

    2017-08-19

    Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo- or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co-expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large-scale expression of hetero-multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high-expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large-scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes. © 2017 The Protein Society.

  6. Expression of Prokaryotic Integral Membrane Proteins in E. coli.

    Science.gov (United States)

    Love, James D

    2017-01-01

    Production of prokaryotic membrane proteins for structural and functional studies in E. coli can be parallelized and miniaturized. All stages from cloning, expression, purification to detergent selection can be investigated using high-throughput techniques to rapidly and economically find tractable targets.

  7. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Directory of Open Access Journals (Sweden)

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  8. Heterologous expression of membrane proteins: choosing the appropriate host.

    Directory of Open Access Journals (Sweden)

    Florent Bernaudat

    Full Text Available BACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals, functions (transporters, receptors, enzymes and topologies (between 0 and 13 transmembrane segments. The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.

  9. Heterologous expression of membrane proteins: choosing the appropriate host.

    Science.gov (United States)

    Bernaudat, Florent; Frelet-Barrand, Annie; Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. © 2011 Bernaudat et al.

  10. CD27-triggering on primary plasma cell leukaemia cells has anti-apoptotic effects involving mitogen activated protein kinases

    NARCIS (Netherlands)

    Guikema, JEJ; Vellenga, E; Abdulahad, WH; Hovenga, S; Bos, NA

    2004-01-01

    Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multiple myeloma (MM) and is characterized by a poor prognosis. In a previous study we demonstrated that PCL plasma cells display a high expression of CD27, in contrast to MM plasma cells. The present study was

  11. Plasma Levels of Monocyte Chemotactic Protein-1 Are Associated with Clinical Features and Angiogenesis in Patients with Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Toni Valković

    2016-01-01

    Full Text Available The aim of this pilot study was to determine the plasma levels of monocyte chemotactic protein-1 (MCP-1 and possible associations with angiogenesis and the main clinical features of untreated patients with multiple myeloma (MM. ELISA was used to determine plasma MCP-1 levels in 45 newly diagnosed MM patients and 24 healthy controls. The blood vessels were highlighted by immunohistochemical staining, and computer-assisted image analysis was used for more objective and accurate determination of two parameters of angiogenesis: microvessel density (MVD and total vascular area (TVA. The plasma levels of MCP-1 were compared to these parameters and the presence of anemia, renal dysfunction, and bone lesions. A significant positive correlation was found between plasma MCP-1 concentrations and TVA (p=0.02. The MCP-1 levels were significantly higher in MM patients with evident bone lesions (p=0.01, renal dysfunction (p=0.02, or anemia (p=0.04. Therefore, our preliminary results found a positive association between plasma MCP-1 levels, angiogenesis (expressed as TVA, and clinical features in patients with MM. However, additional prospective studies with a respectable number of patients should be performed to authenticate these results and establish MCP-1 as a possible target of active treatment.

  12. THE CLINICAL EXPRESSION OF HEREDITARY PROTEIN-C AND PROTEIN-S DEFICIENCY - A RELATION TO CLINICAL THROMBOTIC RISK-FACTORS AND TO LEVELS OF PROTEIN-C AND PROTEIN-S

    NARCIS (Netherlands)

    HENKENS, CMA; VANDERMEER, J; HILLEGE, JL; BOM, VJJ; HALIE, MR; van der Schaaf, W

    1993-01-01

    We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were

  13. Differential expression of miRNAs in the seminal plasma and serum of testicular cancer patients.

    Science.gov (United States)

    Pelloni, Marianna; Coltrinari, Giulia; Paoli, Donatella; Pallotti, Francesco; Lombardo, Francesco; Lenzi, Andrea; Gandini, Loredana

    2016-10-28

    Various microRNAs from the miR-371-3 and miR-302a-d clusters have recently been proposed as markers for testicular germ cell tumours. Upregulation of these miRNAs has been found in both the tissue and serum of testicular cancer patients, but they have never been studied in human seminal plasma. The aim of this study was, therefore, to assess the differences in the expression of miR-371-3 and miR-302a-d between the seminal plasma and serum of testicular cancer patients, and to identify new potential testicular cancer markers in seminal plasma. We investigated the serum and seminal plasma of 28 pre-orchiectomy patients subsequently diagnosed with testicular cancer, the seminal plasma of another 20 patients 30 days post-orchiectomy and a control group consisting of 28 cancer-free subjects attending our centre for an andrological check-up. Serum microRNA expression was analysed using RT-qPCR. TaqMan Array Card 3.0 platform was used for microRNA profiling in the seminal plasma of cancer patients. Results for both miR-371-3 and the miR-302 cluster in the serum of testicular cancer patients were in line with literature reports, while miR-371and miR-372 expression in seminal plasma showed the opposite trend to serum. On array analysis, 37 miRNAs were differentially expressed in the seminal plasma of cancer patients, and the upregulated miR-142 and the downregulated miR-34b were validated using RT-qPCR. Our study investigated the expression of miRNAs in the seminal plasma of patients with testicular cancer for the first time. Unlike in serum, miR-371-3 cannot be considered as markers in seminal plasma, whereas miR-142 levels in seminal plasma may be a potential marker for testicular cancer.

  14. Plasma and saliva miR-21 expression in colorectal cancer patients.

    Science.gov (United States)

    Sazanov, A A; Kiselyova, E V; Zakharenko, A A; Romanov, M N; Zaraysky, M I

    2017-05-01

    MicroRNA-21 (miR-21) expression was quantified by real-time qRT-PCR in peripheral blood and saliva samples obtained from patients diagnosed with colorectal cancer (CRC) of varying degrees of malignancy and healthy volunteers. All patients had adenocarcinoma located in the distal colon at different stages. Significant differences were detected between the control group and the total experimental group of CRC patients (plasma, P = 0.0001; saliva, P = 5e-12). MiR-21 expression was also significantly different in certain subgroups of patients with CRC disease stages II-IV as compared to the control group. No correlation of miR-21 expression was found with regard to gender and age of patents. Also, there were no significant individual correlations and linear regression of miR-21 expression in the plasma and saliva. The estimated diagnostic sensitivity and specificity of miR-21 expression were respectively 65 and 85% in the plasma, and 97 and 91% in the saliva. Our data suggest that miR-21 in both the saliva and plasma could be a proper biomarker for CRC screening, although the saliva miR-21 expression test looks preferable due to its higher sensitivity, specificity, and technical simplicity.

  15. Optimization of translation profiles enhances protein expression and solubility.

    Science.gov (United States)

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  16. Optimization of translation profiles enhances protein expression and solubility.

    Directory of Open Access Journals (Sweden)

    Anne-Katrin Hess

    Full Text Available mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  17. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    Science.gov (United States)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  18. A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma

    DEFF Research Database (Denmark)

    Havelund, Jesper F.; Wojdyla, Katarzyna; Davies, Michael J.

    2017-01-01

    Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues...... in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine...

  19. The E4 protein; structure, function and patterns of expression

    Energy Technology Data Exchange (ETDEWEB)

    Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  20. A method for studies on interactions between a gold-based drug and plasma proteins based on capillary electrophoresis with inductively coupled plasma mass spectrometry detection

    DEFF Research Database (Denmark)

    Nguyen, Tam T T N; Østergaard, Jesper; Gammelgaard, Bente

    2015-01-01

    An analytical method based on capillary electrophoresis (CE) and inductively coupled plasma mass spectrometry (ICP-MS) detection was developed for studies on the interaction of gold-containing drugs and plasma proteins using auranofin as example. A detection limit of 18 ng/mL of auranofin...... was the major auranofin-interacting protein in plasma. The CE-ICP-MS method is proposed as a novel approach for kinetic studies of the interactions between gold-based drugs and plasma proteins. Graphical Abstract Development of a CE-ICP-MS based method allows for studies on interaction of the gold containing...

  1. A comprehensive analysis of the Streptococcus pyogenes and human plasma protein interaction network.

    Science.gov (United States)

    Sjöholm, Kristoffer; Karlsson, Christofer; Linder, Adam; Malmström, Johan

    2014-07-01

    Streptococcus pyogenes is a major human bacterial pathogen responsible for severe and invasive disease associated with high mortality rates. The bacterium interacts with several human blood plasma proteins and clarifying these interactions and their biological consequences will help to explain the progression from mild to severe infections. In this study, we used a combination of mass spectrometry (MS) based techniques to comprehensively quantify the components of the S. pyogenes-plasma protein interaction network. From an initial list of 181 interacting human plasma proteins defined using liquid chromatography (LC)-MS/MS analysis we further subdivided the interacting protein list using selected reaction monitoring (SRM) depending on the level of enrichment and protein concentration on the bacterial surface. The combination of MS methods revealed several previously characterized interactions between the S. pyogenes surface and human plasma along with many more, so far uncharacterised, possible plasma protein interactions with S. pyogenes. In follow-up experiments, the combination of MS techniques was applied to study differences in protein binding to a S. pyogenes wild type strain and an isogenic mutant lacking several important virulence factors, and a unique pair of invasive and non-invasive S. pyogenes isolates from the same patient. Comparing the plasma protein-binding properties of the wild type and the mutant and the invasive and non-invasive S. pyogenes bacteria revealed considerable differences, underlining the significance of these protein interactions. The results also demonstrate the power of the developed mass spectrometry method to investigate host-microbial relationships with a large proteomics depth and high quantitative accuracy.

  2. Transient expression and cellular localization of recombinant proteins in cultured insect cells

    Science.gov (United States)

    Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

  3. Inhibition of hepatitis C virus protein expression by RNA interference.

    Science.gov (United States)

    Sen, Adrish; Steele, Robert; Ghosh, Asish K; Basu, Arnab; Ray, Ranjit; Ray, Ratna B

    2003-10-01

    Hepatitis C virus (HCV) is a serious human pathogen and an estimated 170 million people are infected worldwide. Current therapeutic regimens have shown limited efficacy against selected genotypes of the virus. The phenomenon of RNA interference can be used to selectively block homologous genes post-transcriptionally, and has revolutionized approaches to study gene function. In this report, we have demonstrated that small interfering RNAs (siRNAs) targeted against NS5A of HCV genotype 1a specifically inhibit NS5A RNA and protein expression in a human hepatoma (HepG2) cell line. Expression of endogenous alpha-actin and the ds-RNA activated serine/threonine kinase-PKR were unaltered, demonstrating that the inhibitory effect observed from siRNA was specific to the HCV NS5A protein. We next examined whether siRNA directed against NS5A could inhibit core protein expression, the first gene product synthesized in virus infected cells due to its localization at the 5' end of the HCV polyprotein. For this purpose, a full-length cDNA clone from HCV (H77, genotype 1a) was used, and results indicated that the introduction of NS5A targeted siRNA resulted in an inhibition of NS5A and core protein expression. Moreover, we observed that this siRNA effectively inhibited NS5A mediated activation of the IL-8 promoter. Taken together, our results demonstrated that siRNA was effective in inhibiting HCV protein expression, and may have therapeutic potential to limit HCV replication in chronically infected patients.

  4. Isolation and characterization of gelatin-binding proteins from goat seminal plasma

    Directory of Open Access Journals (Sweden)

    Lazure Claude

    2003-04-01

    Full Text Available Abstract A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation and destabilization (capacitation process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80°C was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins. Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in

  5. Function of plasma membrane microdomain-associated proteins during legume nodulation.

    Science.gov (United States)

    Qiao, Zhenzhen; Libault, Marc

    2017-08-17

    Plasma membrane microdomains are plasma membrane sub-compartments enriched in sphingolipids and sterols, and composed by a specific set of proteins. They are involved in recognizing signal molecules, transducing these signals, and controlling endocytosis and exocytosis processes. In a recent study, applying biochemical and microscopic methods, we characterized the soybean GmFWL1 protein, a major regulator of soybean nodulation, as a new membrane microdomain-associated protein. Interestingly, upon rhizobia inoculation of the soybean root system, GmFWL1 and one of its interacting partners, GmFLOT2/4, both translocate to the root hair cell tip, the primary site of interaction and infection between soybean and Rhizobium. The role of GmFWL1 as a plasma membrane microdomain-associated protein is also supported by immunoprecipitation assays performed on soybean nodules, which revealed 178 GmFWL1 protein partners including a large number of microdomain-associated proteins such as GmFLOT2/4. In this addendum, we provide additional information about the identity of the soybean proteins repetitively identified as GmFWL1 protein partners. Their function is discussed especially in regard to plant-microbe interactions and microbial symbiosis. This addendum will provide new insights in the role of plasma membrane microdomains in regulating legume nodulation.

  6. Evaluation of the refractometric method for the determination of total protein in avian plasma or serum.

    Science.gov (United States)

    Lumeij, J T; de Bruijne, J J

    1985-07-01

    Serum total protein concentrations in pigeon blood determined with the biuret method (TPB-se) were compared with total protein concentrations in plasma (TPR-pl) and serum (TPR-se) obtained by estimation from refractive index. The refractometric method consistently yielded higher values (Prefractometric method for determination of TP in pigeon blood is not recommended.

  7. Expression and Characterization of Human Heart Type Fatty Acid Binding Protein in Pichia Pastoris

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    H-FABP is regarded as a tissue-specific protein existing only in myocardial cells. It is released from the cardiac tissue and gets into the plasma when a heart attack occurs; the myocardial infarction is a good case in point. As a result, the detection of H-FABP will be an early and important biomarker for the disease concerned. The objective of the study is to prepare the recombinant H-FABP by aeukaryotic expression system, pichia, to produce the protein mimicking natural H-FABP, as an immunogen for the production of the specific antibody. A gene fragment encoding H-FABP was cloned in the expressing vector pPICZα, after sequencing. The recombinant plasmid was transformed into the competent cells of the X-33 strain by means of electroporation. The expression of the target peptide indueed by methanol was screened by means of Western blotting, with the available MAb( Clone 6B6 ). Highly expressive engineer strains were obtained. The production of recombinant H-FABP under induction was about 0.7 g/L, with an Mr of 14.5 kDa and recognized by a commercially available MAb (Clone 6B6). The recombinant vector was successfully constructed. Following this, H-FABP was expressed in X-33, and it would become the source of the preparation of specific antibodies, to develop diagnostic kits.

  8. Grape seed extract inhibits VEGF expression via reducing HIF-1alpha protein expression.

    Science.gov (United States)

    Lu, Jianming; Zhang, Keqiang; Chen, Shiuan; Wen, Wei

    2009-04-01

    Grape seed extract (GSE) is a widely consumed dietary supplement that has antitumor activity. Here, we have investigated the inhibitory effect of GSE on the expression of vascular endothelial growth factor (VEGF) and the mechanism underlying this action. We found that GSE inhibited VEGF messenger RNA (mRNA) and protein expression in U251 human glioma cells and MDA-MB-231 human breast cancer cells. GSE inhibited transcriptional activation of the VEGF gene through reducing protein but not mRNA expression of hypoxia-inducible factor (HIF) 1alpha. The inhibitory effect of GSE on HIF-1alpha expression was mainly through inhibiting HIF-1alpha protein synthesis rather than promoting protein degradation. Consistent with this result, GSE-suppressed phosphorylation of several important components involved in HIF-1alpha protein synthesis, such as Akt, S6 kinase and S6 protein. Furthermore, in the MDA-MB-231 tumor, we found that GSE treatment inhibited the expression of VEGF and HIF-1alpha and the phosphorylation of S6 kinase without altering the subcellular localization of HIF-1alpha, correlating with reduced vessel density and tumor size. Depletion of polyphenol with polyvinylpyrrolidone abolished the inhibitory activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the inhibitory activity. Taken together, our results indicate that GSE inhibits VEGF expression by reducing HIF-1alpha protein synthesis through blocking Akt activation. This finding provides new insight into the mechanisms of anticancer activity of GSE and reveals a novel molecular mechanism underlying the antiangiogenic action of GSE.

  9. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    Directory of Open Access Journals (Sweden)

    Jeremy A. Kroemer

    2015-01-01

    Full Text Available Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification.

  10. Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors.

    Science.gov (United States)

    Pizzamiglio, Sara; De Bortoli, Maida; Taverna, Elena; Signore, Michele; Veneroni, Silvia; Cho, William Chi-Shing; Orlandi, Rosaria; Verderio, Paolo; Bongarzone, Italia

    2017-02-14

    We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.

  11. Morphine Withdrawal Modifies Prion Protein Expression in Rat Hippocampus

    Science.gov (United States)

    Mattei, Vincenzo; Martellucci, Stefano; Santilli, Francesca; Manganelli, Valeria; Garofalo, Tina; Candelise, Niccolò; Caruso, Alessandra; Sorice, Maurizio; Scaccianoce, Sergio

    2017-01-01

    The hippocampus is a vulnerable brain structure susceptible to damage during aging and chronic stress. Repeated exposure to opioids may alter the brain so that it functions normally when the drugs are present, thus, a prolonged withdrawal might lead to homeostatic changes headed for the restoration of the physiological state. Abuse of morphine may lead to Reacting Oxygen Species-induced neurodegeneration and apoptosis. It has been proposed that during morphine withdrawal, stress responses might be responsible, at least in part, for long-term changes of hippocampal plasticity. Since prion protein is involved in both, Reacting Oxygen Species mediated stress responses and synaptic plasticity, in this work we investigate the effect of opiate withdrawal in rats after morphine treatment. We hypothesize that stressful stimuli induced by opiate withdrawal, and the subsequent long-term homeostatic changes in hippocampal plasticity, might modulate the Prion protein expression. Our results indicate that abstinence from the opiate induced a time-dependent and region-specific modification in Prion protein content, indeed during morphine withdrawal a selective unbalance of hippocampal Prion Protein is observable. Moreover, Prion protein overexpression in hippocampal tissue seems to generate a dimeric structure of Prion protein and α-cleavage at the hydrophobic domain. Stress factors or toxic insults can induce cytosolic dimerization of Prion Protein through the hydrophobic domain, which in turn, it stimulates the α-cleavage and the production of neuroprotective Prion protein fragments. We speculate that this might be the mechanism by which stressful stimuli induced by opiate withdrawal and the subsequent long-term homeostatic changes in hippocampal plasticity, modulate the expression and the dynamics of Prion protein. PMID:28081197

  12. Growth, testis size, spermatogenesis, semen parameters and seminal plasma and sperm membrane protein profile during the reproductive development of male goats supplemented with de-oiled castor cake.

    Science.gov (United States)

    Oliveira, C H A; Silva, A M; Silva, L M; van Tilburg, M F; Fernandes, C C L; Velho, A L M C; Moura, A A; Moreno, F B M B; Monteiro-Moreira, A C O; Moreira, R A; Lima, I M T; Rondina, D

    2015-06-01

    The present study was conducted to evaluate the effect of de-oiled castor cake on reproductive traits of crossbreed goats. Fourteen males were grouped into two lots (n = 7/group), as described: group without de-oiled castor cake (WCC) and group fed with de-oiled castor cake (CC). Goats received two diets containing a mixture of Bermudagrass hay and concentrates with the same energy (73% total digestive nutrients) and protein content (15% crude protein) during 150 days, corresponding to ages from 40 (puberty) to 60 weeks. Blood plasma concentrations of urea, albumin, lactate dehydrogenase, creatinine, alanine aminotransferase and testosterone were determined. We also evaluated scrotal circumference, sperm parameters, quantitative aspects of spermatogenesis and daily sperm production (DSP), as well as the proteome of seminal plasma and sperm membrane. Seminal fluid and sperm proteins were analyzed by 2D SDS-PAGE and mass spectrometry. After 150 days of castor cake feeding, animals had no changes in the biochemical composition of blood plasma, suggesting the absence of intoxication by ingestion of ricin. There were no alterations in dry mater intake, weight gain, testis size, peripheral concentrations of testosterone, sperm concentration, motility and morphology. Sertoli and germ cell populations in the testis and DSP were not affected either. However, there were significant variations in the expression of five seminal plasma proteins and four sperm membrane proteins. In conclusion, the replacement of soybean meal by castor cake (with ricin concentrations of 50mg/kg) did not interfere with the growth and core reproductive development of male goats. However, the diet with ricin altered the expression of certain seminal plasma and sperm membrane proteins, which play roles in sperm function and fertilization. Lower expression of these proteins may impair the ricin-fed animals to perform as high-fertility sires.

  13. Novel biomarkers of nasopharyngeal carcinoma metastasis risk identified by reverse phase protein array based tumor profiling with consideration of plasma Epstein-Barr virus DNA load.

    Science.gov (United States)

    Xu, Tao; Su, Bojin; Huang, Peiyu; Wei, Weihong; Deng, Yanming; Sehgal, Vasudha; Wang, Donghui; Jiang, Jun; Zhang, Guoyi; Li, Anfei; Yang, Huiling; Claret, Francois X

    2017-05-01

    In patients with Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC), intertumor heterogeneity causes interpatient heterogeneity in the risk of distant metastasis. We aimed to identify novel biomarkers of metastasis risk using reverse phase protein array (RPPA) profiling of NPC patients at risk for metastasis and considering plasma EBV DNA load. A total of 98 patients with NPC with and without metastasis after treatment, matched with respect to clinical parameters, are enrolled. Total protein expression is measured by RPPA, and protein functions are analyzed by pathway bioinformatics. The RPPA analysis revealed a profile of 70 proteins that are differentially expressed in metastatic and nonmetastatic tumors. Plasma EBV DNA load after treatment correlated with protein expression level better than plasma EBV DNA load before treatment did. The biomarkers of NPC metastasis identified by proteomics regulate signaling pathways involved in cell cycle progression, apoptosis, and epithelial-mesenchymal transition. The authors identified 26 biomarkers associated with 5-year distant failure-free survival in univariate analysis; five biomarkers remained significant in multivariate analysis. A comprehensive RPPA profiling study is warranted to identify novel metastasis-related biomarkers and further examine the activation state of signaling proteins to improve estimation of metastasis risk for patients with EBV-associated NPC. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Increased cholesterol efflux from cultured fibroblasts to plasma from hypertriglyceridemic type 2 diabetic patients: roles of pre beta-HDL, phospholipid transfer protein and cholesterol esterification.

    Science.gov (United States)

    de Vries, R; Groen, A K; Perton, F G; Dallinga-Thie, G M; van Wijland, M J A; Dikkeschei, L D; Wolffenbuttel, B H R; van Tol, A; Dullaart, R P F

    2008-02-01

    We tested whether hypertriglyceridemia associated with type 2 diabetes mellitus is accompanied by alterations in pre beta-HDL, which are considered to be initial acceptors of cell-derived cholesterol, and by changes in the ability of plasma to promote cellular cholesterol efflux. In 28 hypertriglyceridemic and 56 normotriglyceridemic type 2 diabetic patients, and in 56 control subjects, we determined plasma lipids, HDL cholesterol and phospholipids, plasma pre beta-HDL and pre beta-HDL formation, phospholipid transfer protein (PLTP) activity, plasma cholesterol esterification (EST) and cholesteryl ester transfer (CET) and the ability of plasma to stimulate cholesterol efflux out of cultured human fibroblasts. HDL cholesterol and HDL phospholipids were lower, whereas plasma PLTP activity, EST and CET were higher in hypertriglyceridemic diabetic patients than in the other groups. Pre beta-HDL levels and pre beta-HDL formation were unaltered, although the relative amount of pre beta-HDL (expressed as % of total plasma apo A-I) was increased in hypertriglyeridemic diabetic patients. Cellular cholesterol efflux to plasma from hypertriglyceridemic diabetic patients was increased compared to efflux to normotriglyceridemic diabetic and control plasma, but efflux to normotriglyceridemic diabetic and control plasma did not differ. Multiple linear regression analysis demonstrated that cellular cholesterol efflux to plasma was positively and independently related to pre beta-HDL formation, PLTP activity and EST (multiple r=0.48), but not to the diabetic state. In conclusion, cholesterol efflux from fibroblasts to normotriglyceridemic diabetic plasma is unchanged. Efflux to hypertriglyceridemic diabetic plasma is enhanced, in association with increased plasma PLTP activity and cholesterol esterification. Unaltered pre beta-HDL formation in diabetic hypertriglyceridemia, despite low apo A-I, could contribute to maintenance of cholesterol efflux.

  15. PEG-albumin plasma expansion increases expression of MCP-1 evidencing increased circulatory wall shear stress: an experimental study.

    Directory of Open Access Journals (Sweden)

    C Makena Hightower

    Full Text Available Treatment of blood loss with plasma expanders lowers blood viscosity, increasing cardiac output. However, increased flow velocity by conventional plasma expanders does not compensate for decreased viscosity in maintaining vessel wall shear stress (WSS, decreasing endothelial nitric oxide (NO production. A new type of plasma expander using polyethylene glycol conjugate albumin (PEG-Alb causes supra-perfusion when used in extreme hemodilution and is effective in treating hemorrhagic shock, although it is minimally viscogenic. An acute 40% hemodilution/exchange-transfusion protocol was used to compare 4% PEG-Alb to Ringer's lactate, Dextran 70 kDa and 6% Hetastarch (670 kDa in unanesthetized CD-1 mice. Serum cytokine analysis showed that PEG-Alb elevates monocyte chemotactic protein-1 (MCP-1, a member of a small inducible gene family, as well as expression of MIP-1α, and MIP-2. MCP-1 is specific to increased WSS. Given the direct link between increased WSS and production of NO, the beneficial resuscitation effects due to PEG-Alb plasma expansion appear to be due to increased WSS through increased perfusion and blood flow rather than blood viscosity.

  16. Gene expression responses of HeLa cells to chemical species generated by an atmospheric plasma flow

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Mayo, E-mail: yokoyama@plasma.ifs.tohoku.ac.jp [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Johkura, Kohei, E-mail: kohei@shinshu-u.ac.jp [Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan); Sato, Takehiko, E-mail: sato@ifs.tohoku.ac.jp [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan)

    2014-08-08

    Highlights: • Response of HeLa cells to a plasma-irradiated medium was revealed by DNA microarray. • Gene expression pattern was basically different from that in a H{sub 2}O{sub 2}-added medium. • Prominently up-/down-regulated genes were partly shared by the two media. • Gene ontology analysis showed both similar and different responses in the two media. • Candidate genes involved in response to ROS were detected in each medium. - Abstract: Plasma irradiation generates many factors able to affect the cellular condition, and this feature has been studied for its application in the field of medicine. We previously reported that hydrogen peroxide (H{sub 2}O{sub 2}) was the major cause of HeLa cell death among the chemical species generated by high level irradiation of a culture medium by atmospheric plasma. To assess the effect of plasma-induced factors on the response of live cells, HeLa cells were exposed to a medium irradiated by a non-lethal plasma flow level, and their gene expression was broadly analyzed by DNA microarray in comparison with that in a corresponding concentration of 51 μM H{sub 2}O{sub 2}. As a result, though the cell viability was sufficiently maintained at more than 90% in both cases, the plasma-medium had a greater impact on it than the H{sub 2}O{sub 2}-medium. Hierarchical clustering analysis revealed fundamentally different cellular responses between these two media. A larger population of genes was upregulated in the plasma-medium, whereas genes were downregulated in the H{sub 2}O{sub 2}-medium. However, a part of the genes that showed prominent differential expression was shared by them, including an immediate early gene ID2. In gene ontology analysis of upregulated genes, the plasma-medium showed more diverse ontologies than the H{sub 2}O{sub 2}-medium, whereas ontologies such as “response to stimulus” were common, and several genes corresponded to “response to reactive oxygen species.” Genes of AP-1 proteins, e.g., JUN

  17. Pathogen inactivation in fresh frozen plasma using riboflavin and ultraviolet light: Effects on plasma proteins and coagulation factor VIII

    Directory of Open Access Journals (Sweden)

    Stanojković Zoran

    2011-01-01

    Full Text Available Background/Aim. Riboflavin (vitamin B2 activated by ultraviolet (UV light, produces active oxygen which damages cell membrane and prevents replication of the carrier of diseases (viruses, bacteria, protozoa in all blood products. The aim of this study was to establish the influence of the process of photo inactivation in pathogens using riboflavin and UV rays on the concentration of coagulation factor VIII:C (FVIII:C and proteins in plasma that were treated before freezing. Methods. The examination included 20 units of plasma, separated from whole blood donated by voluntary blood donors around 6 hours from the moment of collection. The units were pooled and separated in to two groups: one consisted of 10 control units and the other of 10 experimental units. Experimental units of the plasma were treated by riboflavin (35 mL and UV rays (6.24 J/mL, 265-370 nm on Mirasol aparature (Caridian BCT Biotechnologies, USA in approximate duration of 6 minutes. Furthermore, 35 mL of saline solution was added to the control plasma. One sample for examining was taken from the control plasma (KG and two residual were taken from experimental plasma after the addition of riboflavin either before (EG1 or post illumination (EG2. Results. Comparing the mean values of FVIII:C (% we noticed statistically significantly higher level in the EG1 group than in the EG2 group (65.00 ± 4.52 vs 63.20 ± 4.73; t = 4.323, p = 0.002, while between the KG and experimental groups (EG1 and EG2 there was no statistically significant difference in the concentration of FVIII:C. There was a statistically significant decrease of albumin concentration (g/L in the EG2 group comparing to the KG (33.35 ± 0.94 vs 31.94 ± 0.84; t = 3.534, p = 0.002, but there was no mentioned difference in albumin concentration between the KG and the EG1, so as between the EG1 and the EG2. Conclusion. Plasma inactivated by riboflavin and UV rays (Mirasol PRT sistem, Caridian BCT, USA keeps all the

  18. Type 2 diabetes mellitus is associated with differential effects on plasma cholesteryl ester transfer protein and phospholipid transfer protein activities and concentrations

    NARCIS (Netherlands)

    Dullaart, RPF; De Vries, R; Scheek, L; Borggreve, SE; Van Gent, T; Dallinga-Thie, GM; Ito, M; Nagano, M; Sluiter, WJ; Hattori, H; Van Tol, A

    2004-01-01

    Background: Human plasma contains two lipid transfer proteins, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), which are crucial in reverse cholesterol transport. Methods: Plasma CETP and PLTP activity levels and concentrations in 16 type 2 diabetic patients and 1

  19. Expression and significance of P53 protein and MDM-2 protein in human gliomas

    Institute of Scientific and Technical Information of China (English)

    WANG An-liu; LIU Zhao-xia; LI Guang; ZHANG Li-wei

    2011-01-01

    Background P53 is one of the most studied tumor suppressors in the cancer research, and over 50% of human tumors carry P53 mutations. MDM-2 is amplified and/or overexpressed in a variety of human tumors of diverse tissue origin. The aim of this study was to examine the expression of P53 protein and MDM-2 protein in gliomas, and to investigate the relationship between the expression of the two proteins and the histopathological grades of glioma. The relationship between MDM-2 protein expression and P53 protein expression was also analyzed.Methods The expression of P53 protein and MDM-2 protein was immunohistochemically detected using monoclonal antibodies in 242 paraffin embedded tissues, including 30 normal brain tissues from patients with craniocerebral injury and 212 tissues from patients with primary glioma (grade Ⅰ-Ⅱ group: 5 cases of grade Ⅰ, 119 cases of grade Ⅱ; and grade Ⅲ-Ⅳ group: 53 cases of grade Ⅲ, and 35 cases of grade Ⅳ).Results The P53 positive rate was significantly higher in the glioma groups than in the control group (P <0.0001). The P53 positive rate was significantly higher in glioma tissues of grade Ⅲ-V than in glioma tissues of grade Ⅰ-Ⅱ group (P=0.001). The MDM-2 positive rate was significantly higher in glioma groups than in the control group (P <0.0001).There was no significant difference in the MDM-2 positive rate between the two glioma groups (P=0.936). The expression of P53 protein was not related to expression of MDM-2 protein (P=0.069)Conclusions Overexpression of P53 protein might be related to the occurrence and progression of glioma.Overexpression of MDM-2 protein may play an important role in glioma tumorigenesis, but may not be involved in glioma progression. The overexpression of MDM-2 protein was an early event in malignant transformation of glioma. MDM-2 may be a key player in glioma in its own right.

  20. The Rab11-FIP1/RCP gene codes for multiple protein transcripts related to the plasma membrane recycling system.

    Science.gov (United States)

    Jin, Min; Goldenring, James R

    2006-06-01

    Rab11a is a member of the Rab11 small GTPase family, and plays an important role in plasma membrane recycling. Rab11-Family Interacting Protein 1 (Rab11-FIP1) binds to Rab11 through a carboxyl-terminal amphipathic alpha helix. We have identified eight alternatively spliced Rab11-FIP1 gene transcripts from human chromosome 8. Among them, Rab11-FIP1A-D have carboxyl terminal Rab11 binding domains, while Rab11-FIP1E-H do not contain the Rab11 binding domain. While Rab11-FIP1B and F gene transcripts are ubiquitous, other Rab11-FIP1 transcripts demonstrate more limited patterns of expression in human tissue cDNAs. EGFP-Rab11-FIP1A-D proteins over-expressed in HeLa cells targeted to Rab11a-containing membranes, while EGFP-Rab11-FIP1E/F and H proteins did not localize with recycling system membranes. However, transferrin trafficking was not significantly altered in HeLa cells over-expressing expressing any of the EGFP-Rab11-FIP1 proteins. Rabbit polyclonal antibodies specific for Rab11-FIP1B and Rab11-FIP1C/RCP demonstrated that Rab11-FIP1B and Rab11-FIP1C/RCP are expressed endogenously. Strikingly, endogenous staining for Rab11-FIP1C/RCP only partially co-localized with EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B, and EGFP-Rab11a in the perinuclear region, indicating that Rab11-FIP1C/RCP resides in a differentiable subcellular compartment within the plasma membrane recycling system compared with Rab11-FIP1A and Rab11-FIP1B. These data suggest that Rab11-FIP1 proteins may play coordinated roles in regulating plasma membrane recycling with regional specificity within the Rab11a-containing recycling system.

  1. In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy

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    Saubaméa Bruno

    2010-03-01

    Full Text Available Abstract Background Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs. CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. Methods Protein expression in whole spheroids (150 μm in diameter from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. Results We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9+ cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively. Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to

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

    Science.gov (United States)

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

    2015-10-14

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

  3. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A1

    Science.gov (United States)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen; Folch-Puy, Emma; Foronjy, Robert; Jalili, Roxana; Jendresen, Christian Bille; Kimura, Masashi; Kraft, Edward; Lindemose, Søren; Lu, Jin; McLain, Teri; Nutt, Leta; Ramon-Garcia, Santiago; Smith, Joseph; Spivak, Aaron; Wang, Michael L.; Zanic, Marija; Lin, Sue-Hwa

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic-bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers. PMID:18765283

  4. A highly stable nonbiofouling surface with well-packed grafted zwitterionic polysulfobetaine for plasma protein repulsion.

    Science.gov (United States)

    Chang, Yung; Liao, Shih-Chieh; Higuchi, Akon; Ruaan, Ruoh-Chyu; Chu, Chih-Wei; Chen, Wen-Yih

    2008-05-20

    An ideal nonbiofouling surface for biomedical applications requires both high-efficient antifouling characteristics in relation to biological components and long-term material stability from biological systems. In this study we demonstrate the performance and stability of an antifouling surface with grafted zwitterionic sulfobetaine methacrylate (SBMA). The SBMA was grafted from a bromide-covered gold surface via surface-initiated atom transfer radical polymerization to form well-packed polymer brushes. Plasma protein adsorption on poly(sulfobetaine methacrylate) (polySBMA) grafted surfaces was measured with a surface plasmon resonance sensor. It is revealed that an excellent stable nonbiofouling surface with grafted polySBMA can be performed with a cycling test of the adsorption of three model proteins in a wide range of various salt types, buffer compositions, solution pH levels, and temperatures. This work also demonstrates the adsorption of plasma proteins and the adhesion of platelets from human blood plasma on the polySBMA grafted surface. It was found that the polySBMA grafted surface effectively reduces the plasma protein adsorption from platelet-poor plasma solution to a level superior to that of adsorption on a surface terminated with tetra(ethylene glycol). The adhesion and activation of platelets from platelet-rich plasma solution were not observed on the polySBMA grafted surface. This work further concludes that a surface with good hemocompatibility can be achieved by the well-packed surface-grafted polySBMA brushes.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-05-01

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

  6. Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

    Science.gov (United States)

    Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

    2015-10-20

    Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

  7. The Role of Bromodomain Proteins in Regulating Gene Expression

    Directory of Open Access Journals (Sweden)

    Michael F. Duffy

    2012-05-01

    Full Text Available Histone modifications are important in regulating gene expression in eukaryotes. Of the numerous histone modifications which have been identified, acetylation is one of the best characterised and is generally associated with active genes. Histone acetylation can directly affect chromatin structure by neutralising charges on the histone tail, and can also function as a binding site for proteins which can directly or indirectly regulate transcription. Bromodomains specifically bind to acetylated lysine residues on histone tails, and bromodomain proteins play an important role in anchoring the complexes of which they are a part to acetylated chromatin. Bromodomain proteins are involved in a diverse range of functions, such as acetylating histones, remodeling chromatin, and recruiting other factors necessary for transcription. These proteins thus play a critical role in the regulation of transcription.

  8. Suppression of adenylyl cyclase-mediated cAMP production by plasma membrane associated cytoskeletal protein 4.1G.

    Science.gov (United States)

    Goto, Toshihiro; Chiba, Ayano; Sukegawa, Jun; Yanagisawa, Teruyuki; Saito, Masaki; Nakahata, Norimichi

    2013-03-01

    It has been shown lately that activity of G protein-coupled receptors (GPCRs) is regulated by an array of proteins binding to carboxy (C)-terminus of GPCRs. Proteins of 4.1 family are subsets of subcortical cytoskeletal proteins and are known to stabilize cellular structures and proteins at the plasma membrane. One of the 4.1 family proteins, 4.1G has been shown to interact with the C-terminus of GPCRs and regulate intracellular distribution of the receptors, including parathyroid hormone (PTH)/PTH-related protein receptor (PTHR). PTHR is coupled to trimeric G proteins G(s) and G(q), which activate the adenylyl cyclase/cyclic AMP (cAMP) pathway and phospholipase C pathway, respectively. During the course of investigation of the role of 4.1G on adenylyl cyclase/cAMP signaling pathway, we found that 4.1G suppressed forskolin-induced cAMP production in cells. The cAMP accumulation induced by forskolin was decreased in HEK293 cells overexpressing 4.1G or increased in 4.1G-knockdown cells. Furthermore, PTH -(1-34)-stimulated cAMP production was also suppressed in the presence of exogenously expressed 4.1G despite its activity to increase the distribution of PTHR to the cell surface. In cells overexpressing FERM domain-deleted 4.1G, a mutant form of the protein deficient in plasma membrane distribution, neither forskolin-induced nor PTH -(1-34)-stimulated cAMP production was not altered. The suppression of the forskolin-induced cAMP production was observed even in membrane preparations of 4.1G-overexpressing cells. In 4.1G-knockdown HEK293 cells, plasma membrane distribution of adenylyl cyclase 6, one of the major subtypes of the enzyme in the cells, showed a slight decrease, in spite of the increased production of cAMP in those cells when stimulated by forskolin. Also, cytochalasin D treatment did not cause any influence on forskolin-induced cAMP production in HEK293 cells. These data indicate that plasma membrane-associated 4.1G regulates GPCR-mediated G(s) signaling

  9. Semen quality and concentration of soluble proteins in the seminal plasma of Alpine bucks Semen quality and concentration of soluble proteins in the seminal plasma of Alpine bucks

    Directory of Open Access Journals (Sweden)

    Simone Eliza Facione Guimarães

    2010-06-01

    Full Text Available It was aimed to study the in vitro seminal quality analyzed by complementary tests and to compare them with physical, morphological and biochemical aspects of male goat semen of the Alpine breed. This experiment took place at the Federal University of Viçosa, situated at 20º45’ S latitude and 42º51’ W longitude, Southwest of Brazil. It was done during the summer months of January and February, and three adult male goats of the Alpine breed were used in intensive conditions. The semen was collected by artificial vagina method. In all semen samples (45 ejaculates, after the physical and morphological analysis, the hiposmotic test was done. In 24 ejaculates, it were done thermo-resistance test, and in 21 ejaculates it were determined the concentration of total soluble proteins in seminal plasma. The male goats presented difference in the semen physical and morphological aspects, in the hiposmotic test and thermo-resistance test, but they did not presented difference in total soluble proteins concentration in seminal plasma. Results of the slow thermo-resistance test and hiposmotic test were positively correlated (r = 0.60. It was concluded, according to our results, that the concentration of total soluble proteins in seminal plasma can not be used as a parameter to predict the seminal quality of Alpine bucks.It was aimed to study the in vitro seminal quality analyzed by complementary tests and to compare them with physical, morphological and biochemical aspects of male goat semen of the Alpine breed. This experiment took place at the Federal University of Viçosa, situated at 20º45’ S latitude and 42º51’ W longitude, Southwest of Brazil. It was done during the summer months of January and February, and three adult male goats of the Alpine breed were used in intensive conditions. The semen was collected by artificial vagina method. In all semen samples (45 ejaculates, after the physical and morphological analysis, the hiposmotic test

  10. Effect of whey protein on plasma amino acids in diabetic mice.

    Science.gov (United States)

    Han, Ting; Cai, Donglian; Geng, Shanshan; Wang, Ying; Zhen, Hui; Wu, Peiying

    2013-12-01

    The aim of this study was to investigate the effect of whey protein on plasma amino acid levels in a mouse model of type II diabetes, using high-performance liquid chromatography (HPLC). The composition and content of amino acids in the whey proteins were analyzed using HPLC. Type I and type II diabetic mouse models were prepared using streptozotocin (STZ) and normal mice were used as a control. The ICR mice in each group were then randomly divided into four subgroups, to which 0, 10, 20 and 40% whey protein, respectively, was administered for four weeks. Changes in the plasma amino acid levels were observed in each group. The proportions of leucine, isoleucine and valine in the whey proteins were 14.40, 5.93 and 5.32% of the total amino acids, respectively, that is, the branched-chain amino acid content was 25.65%. The levels of branched-chain amino acids increased in the plasma of the normal and model mice following the administration of whey proteins by gavage and the amino acid levels increased as the concentration of the administered protein increased. In addition, the branched-chain amino acid levels in the blood of the model mice were higher than those in the normal mice. The levels of plasma amino acids in diabetic mice increased following gavage with whey protein, which is rich in branched-chain amino acids.

  11. A rapid and simple assay for growth hormone-binding protein activity in human plasma.

    Science.gov (United States)

    Baumann, G; Shaw, M A; Amburn, K

    1988-12-01

    The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subjects and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins.

  12. A pilot study of muscle plasma protein changes after exercise

    DEFF Research Database (Denmark)

    Dahlqvist, Julia R; Voss, Line G; Lauridsen, Thomas

    2014-01-01

    INTRODUCTION: Creatine kinase (CK) and myoglobin (Mb) do not possess all good qualities as biomarkers of skeletal muscle damage. We investigated the utility of troponin I (TnI) and telethonin (Tcap) as markers and examined their temporal profiles after skeletal muscle damage. METHODS: Plasma...... profiles were measured before and after exercise in 3 groups: subjects affected by either Becker muscular dystrophy or McArdle disease, and healthy subjects. RESULTS: Mb and TnI appeared early in the blood, and the increase of TnI was only observed in patients with muscle disease. The CK increase was more...... delayed in plasma. Tcap was not detectable at any time. CONCLUSIONS: Our results suggest that TnI is a marker of more severe damage signifying sarcomeric damage, and it could therefore be an important supplement to CK and Mb in clinical practice. Tcap is not useful as a marker for skeletal muscle damage....

  13. Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

    Science.gov (United States)

    Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

    2012-01-01

    Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization.

  14. Bacteria and protozoa differentially modulate the expression of Rab proteins.

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    Elsa Seixas

    Full Text Available Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endocytic pathway. Intracellular pathogens can interfere with the function of these proteins in order to subvert host immune responses. However, it is unknown if this subversion can be achieved through the modulation of Rab gene expression. We compared the expression level of 23 distinct Rab GTPases in mouse macrophages after infection with the protozoan Plasmodium berghei, and the bacteria Escherichia coli and Salmonella enterica. We found that P. berghei induces an increase in the expression of a different set of Rab genes than E. coli and S. enterica, which behaved similarly. Strikingly, when one of the Rab proteins whose expression was increased by P. berghei, namely Rab14, was silenced, we observed a significant increase in the phagocytosis of P. berghei, whereas Rab14 overexpression led to a decrease in phagocytosis. This suggests that the parasite might induce the increase of Rab14 expression for its own advantage. Similarly, when Rab9a, whose expression was increased by E. coli and S. enterica, was silenced, we observed an increase in the phagocytosis of both bacterial species, whereas Rab9a overexpression caused a reduction in phagocytosis. This further suggests that the modulation of Rab gene expression could represent a mechanism of immune evasion. Thus, our study analyzes the modulation of Rab gene expression induced by bacteria and protozoa and suggests that this modulation could be necessary for the success of microbial infection.

  15. New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

    Science.gov (United States)

    Alshaikh, N A; Rosing, J; Thomassen, M C L G D; Castoldi, E; Simioni, P; Hackeng, T M

    2017-02-17

    Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation.

  16. Heat Shock Protein 90 (Hsp90 Expression and Breast Cancer

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    Christos A. Papadimitriou

    2012-09-01

    Full Text Available Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc. are currently under evaluation.

  17. Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma)

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Luckenbach, J Adam; Madsen, Steffen S;

    2008-01-01

    (-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control...... levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter...... hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation...

  18. Transgenic expression of green fluorescent protein in mouse oxytocin neurones.

    Science.gov (United States)

    Young, W S; Iacangelo, A; Luo, X Z; King, C; Duncan, K; Ginns, E I

    1999-12-01

    Routine targeting of neurones for expression of exogenous genes would facilitate our ability to manipulate their internal milieu or functions, providing insight into physiology of neurones. The magnocellular neurones of the paraventricular and supraoptic nuclei of the hypothalamus have been the objects of limited success by this approach. Here we report on the placement of the enhanced green fluorescent protein (eGFP) coding sequence at various locations within an oxytocin transgene. Placement within the first exon yielded little to no expression, whereas placement in the third exon (as an in-frame fusion with the carboxyl terminus of the oxytocin preprohormone) resulted in cell-specific expression of eGFP in oxytocin neurones. Furthermore, placement of the eGFP sequence downstream of a picornavirus internal ribosomal entry site (IRES), also in the third exon, allowed expression of the eGFP as a separate protein. Other coding sequences should now be amenable to expression within oxytocin neurones to study their physiology.

  19. Plasma proteins as biomarkers of the aging process.

    Science.gov (United States)

    Vranckx, R; Savu, L; Lambert, N; de Conchard, G V; Grosse, R; Mourey, M S; Corman, B

    1995-02-01

    This study was designed to characterize the rat serum proteins as biomarkers of the normal aging process. Crossed immunoelectrophoresis or electroimmunodiffusion quantitation of proteins was performed in rats aged 6, 12, 24, and 30 mo. Selection of healthy animals was based on confrontation of crossed immunoelectrophoresis patterns with those of experimentally inflamed young adults and with individual anatomopathological data. Convergence of inflammatory patterns and severe histological lesions was the exclusion criterion. Senescence-induced decrease was demonstrated for eight proteins [negative senescence reactants (SRs-)] and increase for six proteins [positive SRs (SRs+)]. Most SRs belonged to the class of proteins responsive to acute inflammation [acute phase reactants (APRs)]. One SR+, the thyroxine-binding globulin, a high-affinity thyroid hormone binder, emerged as a particularly reliable senescence biomarker, showing the highest aging-related variation (8-fold increase from 6 to 30 mo) and not belonging to the APR class. Chronic treatment with perindopril, an angiotensin I-converting enzyme inhibitor used in heart and renal disease therapy, significantly enhanced thyroxine-binding capacity, possibly by preventing age-related alterations of serum lipids. Serum protein patterns prove valuable both as indexes for selecting aging animals free from superimposed pathologies and as parameters of senescence-induced changes in protein biosynthesis.

  20. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    Science.gov (United States)

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

  1. Heat-shock protein expression in canine corneal wound healing.

    Science.gov (United States)

    Peterson, Cornelia W M; Carter, Renee T; Bentley, Ellison; Murphy, Christopher J; Chandler, Heather L

    2016-05-01

    Heat-shock proteins, particularly the 70-kDa member (Hsp70), have been implicated in facilitating wound healing in multiple tissues. Expression and localization of three HSPs were assessed in normal and wounded canine corneas to elucidate a role in epithelial healing. Paraffin-embedded normal corneas, acute and repeatedly abraded corneas, and keratectomies of spontaneous chronic corneal epithelial defects (SCCEDs) were subjected to routine immunohistochemistry for Hsp27, 47, and 70 expression. Ex vivo corneal defects were created and treated with anti-HSPs or IgG controls, and wound healing was monitored. Primary cultures of canine corneal stromal fibroblasts and corneal epithelial cells were treated with exogenous Hsp70, and an artificial wound was created in vitro to monitor restoration of the monolayer. Normal canine corneas exhibited constitutive expression of all HSPs evaluated. Inducible expression was demonstrated in acutely wounded tissues, and expression in the chronically abraded corneas was relocalized. All HSP expression was below the limits of detection in the epithelium of SCCED samples. Inhibition of HSPs in culture resulted in delayed wound healing when compared to controls. Hsp70-treated fibroblasts demonstrated significantly (P healing response, and suppressed expression may contribute to the pathophysiology of nonhealing defects. © 2015 American College of Veterinary Ophthalmologists.

  2. BMP-7 PROTEIN EXPRESSION IS DOWNREGULATED IN HUMAN DIABETIC NEPHROPATHY.

    Science.gov (United States)

    Ivanac-Janković, Renata; Ćorić, Marijana; Furić-Čunko, Vesna; Lovičić, Vesna; Bašić-Jukić, Nikolina; Kes, Petar

    2015-06-01

    Bone morphogenetic protein-7 (BMP-7) is expressed in all parts of the normal kidney parenchyma, being highest in the epithelium of proximal tubules. It protects kidney against acute and chronic injury, inflammation and fibrosis. Diabetic nephropathy is the leading cause of chronic kidney disease, and is characterized by decreased expression of BMP-7. The aim of our study was to analyze whether the expression of BMP-7 is significantly changed in advanced stages of human diabetic nephropathy. Immunohistochemical analysis of the expression of BMP-7 was performed on archival material of 30 patients that underwent renal biopsy and had confirmed diagnosis of diabetic nephropathy. Results showed that BMP-7 was differently expressed in the cytoplasm of epithelial cells of proximal tubules and podocytes among all stages of diabetic nephropathy. At early stages of diabetic nephropathy, BMP-7 was strongly positive in proximal tubules and podocytes, while low expression was recorded in the majority of samples at advanced stages. In conclusion, increased expression of BMP-7 at initial stages of diabetic nephropathy with subsequent decrease at advanced stage highlights the role of BMP-7 in the protection of kidney structure and function. Further investigations should be focused on disturbances of BMP-7 receptors and signaling pathways in patients with diabetic nephropathy.

  3. Whey Protein Delays Gastric Emptying and Suppresses Plasma Fatty Acids and Their Metabolites Compared to Casein, Gluten, and Fish Protein

    DEFF Research Database (Denmark)

    Stanstrup, Jan; Schou, Simon S; Holmer-Jensen, Jens

    2014-01-01

    Whey protein has been demonstrated to improve fasting lipid and insulin response in overweight and obese individuals. To establish new hypotheses for this effect and to investigate the impact of stomach emptying, we compared plasma profiles after intake of whey isolate (WI), casein, gluten (GLU...

  4. The Effect of Post-Resistance Exercise Amino Acids on Plasma MCP-1 and CCR2 Expression

    Directory of Open Access Journals (Sweden)

    Adam J. Wells

    2016-07-01

    Full Text Available The recruitment and infiltration of classical monocytes into damaged muscle is critical for optimal tissue remodeling. This study examined the effects of an amino acid supplement on classical monocyte recruitment following an acute bout of lower body resistance exercise. Ten resistance-trained men (24.7 ± 3.4 years; 90.1 ± 11.3 kg; 176.0 ± 4.9 cm ingested supplement (SUPP or placebo (PL immediately post-exercise in a randomized, cross-over design. Blood samples were obtained at baseline (BL, immediately (IP, 30-min (30P, 1-h (1H, 2-h (2H, and 5-h (5H post-exercise to assess plasma concentrations of monocyte chemoattractant protein 1 (MCP-1, myoglobin, cortisol and insulin concentrations; and expressions of C-C chemokine receptor-2 (CCR2, and macrophage-1 antigen (CD11b on classical monocytes. Magnitude-based inferences were used to provide inferences on the true effects of SUPP compared to PL. Changes in myoglobin, cortisol, and insulin concentrations were similar between treatments. Compared to PL, plasma MCP-1 was “very likely greater” (98.1% likelihood effect in SUPP at 2H. CCR2 expression was “likely greater” at IP (84.9% likelihood effect, “likely greater” at 1H (87.7% likelihood effect, “very likely greater” at 2H (97.0% likelihood effect, and “likely greater” at 5H (90.1% likelihood effect in SUPP, compared to PL. Ingestion of SUPP did not influence CD11b expression. Ingestion of an amino acid supplement immediately post-exercise appears to help maintain plasma MCP-1 concentrations and augment CCR2 expression in resistance trained men.

  5. 1-溴丙烷对雄性大鼠血浆和脑组织中NSE、MBP 蛋白表达影响%Effect of 1-bromopropane inhalation on the expression of neuron specific enolase and myelin basic protein in plasma and brain tissue of male rats

    Institute of Scientific and Technical Information of China (English)

    李宏玲; 刘浩中; 宋向荣; 陈晓燕; 谢植伟; 赵娜; 蔡婷峰; 梁旻炜; 王海兰

    2016-01-01

    Objective To determine the effects of 1-bromopropane (1-BP) subacute inhalation on the expression of neuron specific enolase (NSE) and myelin basic protein (MBP) in plasma and brain tissue in male rats.Methods Specific pathogen free adult male Wistar rats were randomly divided into 4 groups with 12 rats in each group:the control group , the low-, medium-and high-dose groups with 1-BP exposure levels at 0, 1 250, 2 500 and 5 000 mg/m3 , respectively.The rats were given continuous dynamic inhalation of 1-BP for 6 hours per day, 5 days per week, for continuous 4 weeks.The rats were sacrificed at the end of exposure , 9 rats from each group were randomly chosen and the blood from abdominal aorta was collected and the plasma was isolated .The plasma levels of NSE and MBP were measured by enzyme-linked immunosorbent assay.The whole brain, pallium, cerebellum and brainstem were isolated for detection of organ coefficient . The rest of 3 rats in each group were processed for histopathologic examination and the expressions of NSE and MBP were evaluated by immunohistochemistry .Results The organ coefficients of whole brain , pallium, cerebellum and brainstem in the high-dose group were higher than those in the control group [ ( 0.754 ±0.056 )% vs ( 0.663 ±0.035 )%, ( 0.382 ± 0.037)%vs (0.339 ±0.021)%, (0.115 ±0.008)% vs (0.098 ±0.006)% and (0.213 ±0.018)% vs (0.183 ± 0.014)%, respectively, P0.05].Histopathological examination showed that a few necrotic nerve cells were observed in hippocampus of rats in high-dose group.The expressions of NSE and MBP in brain tissue of rats in control , low-and medium-dose groups showed no significant difference .The down-regulated expression of NSE and MBP were only observed in cells of hippocampus of rats in the high -dose group.Conclusion The 1-BP induced neural toxicity was reflected in the function of central nervous system rather than in the structural morphology.The plasma NSE might be one of the effect biomarkers for

  6. Protein retention on plasma-treated hierarchical nanoscale gold-silver platform

    Science.gov (United States)

    Fang, Jinghua; Levchenko, Igor; Mai-Prochnow, Anne; Keidar, Michael; Cvelbar, Uros; Filipic, Gregor; Han, Zhao Jun; Ostrikov, Kostya (Ken)

    2015-08-01

    Dense arrays of gold-supported silver nanowires of about 100 nm in diameter grown directly in the channels of nanoporous aluminium oxide membrane were fabricated and tested as a novel platform for the immobilization and retention of BSA proteins in the microbial-protective environments. Additional treatment of the silver nanowires using low-temperature plasmas in the inductively-coupled plasma reactor and an atmospheric-pressure plasma jet have demonstrated that the morphology of the nanowire array can be controlled and the amount of the retained protein may be increased due to the plasma effect. A combination of the neutral gold sublayer with the antimicrobial properties of silver nanowires could significantly enhance the efficiency of the platforms used in various biotechnological processes.

  7. Rational use of plasma protein and tissue binding data in drug design.

    Science.gov (United States)

    Liu, Xingrong; Wright, Matthew; Hop, Cornelis E C A

    2014-10-23

    It is a commonly accepted assumption that only unbound drug molecules are available to interact with their targets. Therefore, one of the objectives in drug design is to optimize the compound structure to increase in vivo unbound drug concentration. In this review, theoretical analyses and experimental observations are presented to illustrate that low plasma protein binding does not necessarily lead to high in vivo unbound plasma concentration. Similarly, low brain tissue binding does not lead to high in vivo unbound brain tissue concentration. Instead, low intrinsic clearance leads to high in vivo unbound plasma concentration, and low efflux transport activity at the blood-brain barrier leads to high unbound brain concentration. Plasma protein and brain tissue binding are very important parameters in understanding pharmacokinetics, pharmacodynamics, and toxicities of drugs, but these parameters should not be targeted for optimization in drug design.

  8. Protein retention on plasma-treated hierarchical nanoscale gold-silver platform

    Science.gov (United States)

    Fang, Jinghua; Levchenko, Igor; Mai-Prochnow, Anne; Keidar, Michael; Cvelbar, Uros; Filipic, Gregor; Han, Zhao Jun; Ostrikov, Kostya (Ken)

    2015-01-01

    Dense arrays of gold-supported silver nanowires of about 100 nm in diameter grown directly in the channels of nanoporous aluminium oxide membrane were fabricated and tested as a novel platform for the immobilization and retention of BSA proteins in the microbial-protective environments. Additional treatment of the silver nanowires using low-temperature plasmas in the inductively-coupled plasma reactor and an atmospheric-pressure plasma jet have demonstrated that the morphology of the nanowire array can be controlled and the amount of the retained protein may be increased due to the plasma effect. A combination of the neutral gold sublayer with the antimicrobial properties of silver nanowires could significantly enhance the efficiency of the platforms used in various biotechnological processes. PMID:26307515

  9. A new essential protein discovery method based on the integration of protein-protein interaction and gene expression data

    Directory of Open Access Journals (Sweden)

    Li Min

    2012-03-01

    Full Text Available Abstract Background Identification of essential proteins is always a challenging task since it requires experimental approaches that are time-consuming and laborious. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which have produced unprecedented opportunities for detecting proteins' essentialities from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. However, the network topology-based centrality measures are very sensitive to the robustness of network. Therefore, a new robust essential protein discovery method would be of great value. Results In this paper, we propose a new centrality measure, named PeC, based on the integration of protein-protein interaction and gene expression data. The performance of PeC is validated based on the protein-protein interaction network of Saccharomyces cerevisiae. The experimental results show that the predicted precision of PeC clearly exceeds that of the other fifteen previously proposed centrality measures: Degree Centrality (DC, Betweenness Centrality (BC, Closeness Centrality (CC, Subgraph Centrality (SC, Eigenvector Centrality (EC, Information Centrality (IC, Bottle Neck (BN, Density of Maximum Neighborhood Component (DMNC, Local Average Connectivity-based method (LAC, Sum of ECC (SoECC, Range-Limited Centrality (RL, L-index (LI, Leader Rank (LR, Normalized α-Centrality (NC, and Moduland-Centrality (MC. Especially, the improvement of PeC over the classic centrality measures (BC, CC, SC, EC, and BN is more than 50% when predicting no more than 500 proteins. Conclusions We demonstrate that the integration of protein-protein interaction network and gene expression data can help improve the precision of predicting essential proteins. The new centrality measure, PeC, is an effective essential protein discovery method.

  10. Generation of transgenic dogs that conditionally express green fluorescent protein.

    Science.gov (United States)

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  11. Transcriptional Modulation of Heat-Shock Protein Gene Expression

    Directory of Open Access Journals (Sweden)

    Anastasis Stephanou

    2011-01-01

    Full Text Available Heat-shock proteins (Hsps are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex signaling pathways regulating Hsp expression may lead to novel therapeutic targets.

  12. Transcriptional modulation of heat-shock protein gene expression.

    Science.gov (United States)

    Stephanou, Anastasis; Latchman, David S

    2011-01-01

    Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex signaling pathways regulating Hsp expression may lead to novel therapeutic targets.

  13. Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics.

    Science.gov (United States)

    Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

    2016-01-06

    The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics.

  14. Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography

    Directory of Open Access Journals (Sweden)

    Geisler Markus

    1998-01-01

    Full Text Available In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993 et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 &mgr;M and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

  15. Calorie restriction reduces pinealectomy-induced insulin resistance by improving GLUT4 gene expression and its translocation to the plasma membrane.

    Science.gov (United States)

    Zanquetta, Melissa M; Seraphim, Patricia M; Sumida, Doris H; Cipolla-Neto, Jose; Machado, Ubiratan F

    2003-10-01

    The present study aimed to investigate insulin sensitivity and GLUT4 expression protein in pinealectomized rats, as well as to determining the effects of melatonin and calorie restriction on the changes induced by pinealectomy. Wistar rats were pinealectomized (Pinx) or sham operated (Sham), and studied 30 days later. Melatonin replacement treatment (50 g/100 g body weight) was continued for 30 days after pinealectomy. Calorie restriction was performed by offering 60% of the standard food intake. In vivo insulin sensitivity was evaluated using the glucose disappearance constant (kITT) during an insulin tolerance test, and GLUT4 mRNA and protein were assessed by Northern and Western blotting, respectively. The in vitro effect of melatonin on GLUT4 protein content in plasma membrane was investigated in adipocytes isolated from intact rats. Compared with Sham rats, Pinx rats showed decreased kITT (40%), GLUT4 expression in white adipose tissue (WAT, approximately 70%), and unchanged GLUT4 expression in skeletal muscle. Melatonin treatment in Pinx rats restored the kITT and GLUT4 protein to control values. No in vitro effects of melatonin (10-9 m) upon GLUT4 protein were observed. Calorie restriction of Pinx rats increased their kITT value ( approximately 40%), total GLUT4 protein content ( approximately 240%) and its translocation to the plasma membrane ( approximately 80%) in WAT. The results show that pinealectomy, for lack of melatonin, decreased insulin sensitivity as well as GLUT4 gene expression. Calorie restriction improved insulin sensitivity in Pinx rats, and this was related to increased GLUT4 gene expression and insulin-induced GLUT4 translocation to the plasma membrane in WAT.

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

    Directory of Open Access Journals (Sweden)

    David L. Springer

    2004-01-01

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

  17. Prognostic value of plasma C-reactive protein in the evaluation of paraquat poisoning patients

    Institute of Scientific and Technical Information of China (English)

    Zong NingΔ; Yu-Long BaiΔ; Hua Lu; Kang-Lin Mo

    2015-01-01

    Objective:To investigate the prognostic value of plasma C-reactive protein (CRP) level in patients with paraquat poisoning. Methods: This study included 162 patients with paraquat poisoning. The data of plasma paraquat,CRP level and arterial blood gas were analyzed. Cox regression analysis was applied to evaluate the risk factors of prognosis. Receiver operating characteristics curve analysis and area under curve were used to calculate the predictive power of significant variable. Differences in patient survival were determined using the Kaplan-Meier method and a log-rank test. Results:PlasmaCRP level was significantly increased in non-survival patients compared with survival patients (P Conclusions: These results suggest that plasmaCRP level is distinct increased in patients with paraquat poisoning, and the plasmaCRP level may be useful for the prediction of prognosis in paraquat poisoning.

  18. Expression patterns of protein kinase D 3 during mouse development

    Directory of Open Access Journals (Sweden)

    Lutz Sylke

    2008-04-01

    Full Text Available Abstract Background The PKD family of serine/threonine kinases comprises a single member in Drosophila (dPKD, two isoforms in C. elegans (DKF-1 and 2 and three members, PKD1, PKD2 and PKD3 in mammals. PKD1 and PKD2 have been the focus of most studies up to date, which implicate these enzymes in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, immune responses, apoptosis and cell proliferation. Concerning PKD3, a role in the formation of vesicular transport carriers at the trans-Golgi network (TGN and in basal glucose transport has been inferred from in vitro studies. So far, however, the physiological functions of the kinase during development remain unknown. Results We have examined the expression pattern of PKD3 during the development of mouse embryos by immunohistochemistry. Using a PKD3 specific antibody we demonstrate that the kinase is differentially expressed during organogenesis. In the developing heart a strong PKD3 expression is constantly detected from E10 to E16.5. From E12.5 on PKD3 is increasingly expressed in neuronal as well as in the supporting connective tissue and in skeletal muscles. Conclusion The data presented support an important role for PKD3 during development of these tissues.

  19. Polygenic Overlap Between C-Reactive Protein, Plasma Lipids, and Alzheimer Disease.

    Science.gov (United States)

    Desikan, Rahul S; Schork, Andrew J; Wang, Yunpeng; Thompson, Wesley K; Dehghan, Abbas; Ridker, Paul M; Chasman, Daniel I; McEvoy, Linda K; Holland, Dominic; Chen, Chi-Hua; Karow, David S; Brewer, James B; Hess, Christopher P; Williams, Julie; Sims, Rebecca; O'Donovan, Michael C; Choi, Seung Hoan; Bis, Joshua C; Ikram, M Arfan; Gudnason, Vilmundur; DeStefano, Anita L; van der Lee, Sven J; Psaty, Bruce M; van Duijn, Cornelia M; Launer, Lenore; Seshadri, Sudha; Pericak-Vance, Margaret A; Mayeux, Richard; Haines, Jonathan L; Farrer, Lindsay A; Hardy, John; Ulstein, Ingun Dina; Aarsland, Dag; Fladby, Tormod; White, Linda R; Sando, Sigrid B; Rongve, Arvid; Witoelar, Aree; Djurovic, Srdjan; Hyman, Bradley T; Snaedal, Jon; Steinberg, Stacy; Stefansson, Hreinn; Stefansson, Kari; Schellenberg, Gerard D; Andreassen, Ole A; Dale, Anders M

    2015-06-09

    Epidemiological findings suggest a relationship between Alzheimer disease (AD), inflammation, and dyslipidemia, although the nature of this relationship is not well understood. We investigated whether this phenotypic association arises from a shared genetic basis. Using summary statistics (P values and odds ratios) from genome-wide association studies of >200 000 individuals, we investigated overlap in single-nucleotide polymorphisms associated with clinically diagnosed AD and C-reactive protein (CRP), triglycerides, and high- and low-density lipoprotein levels. We found up to 50-fold enrichment of AD single-nucleotide polymorphisms for different levels of association with C-reactive protein, low-density lipoprotein, high-density lipoprotein, and triglyceride single-nucleotide polymorphisms using a false discovery rate threshold <0.05. By conditioning on polymorphisms associated with the 4 phenotypes, we identified 55 loci associated with increased AD risk. We then conducted a meta-analysis of these 55 variants across 4 independent AD cohorts (total: n=29 054 AD cases and 114 824 healthy controls) and discovered 2 genome-wide significant variants on chromosome 4 (rs13113697; closest gene, HS3ST1; odds ratio=1.07; 95% confidence interval=1.05-1.11; P=2.86×10(-8)) and chromosome 10 (rs7920721; closest gene, ECHDC3; odds ratio=1.07; 95% confidence interval=1.04-1.11; P=3.38×10(-8)). We also found that gene expression of HS3ST1 and ECHDC3 was altered in AD brains compared with control brains. We demonstrate genetic overlap between AD, C-reactive protein, and plasma lipids. By conditioning on the genetic association with the cardiovascular phenotypes, we identify novel AD susceptibility loci, including 2 genome-wide significant variants conferring increased risk for AD. © 2015 American Heart Association, Inc.

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

    Science.gov (United States)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2006-01-01

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

  2. Recombinant Dragline Silk-Like Proteins-Expression and Purification.

    Science.gov (United States)

    Gaines, William A; Marcotte, William R

    2011-03-01

    Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.

  3. Disposable bioreactors for inoculum production and protein expression.

    Science.gov (United States)

    Eibl, Regine; Löffelholz, Christian; Eibl, Dieter

    2014-01-01

    Disposable bioreactors have been increasingly implemented over the past ten years. This relates to both R & D and commercial manufacture, in particular, in animal cell-based processes. Among the numerous disposable bioreactors which are available today, wave-mixed bag bioreactors and stirred bioreactors are predominant. Whereas wave-mixed bag bioreactors represent the system of choice for inoculum production, stirred systems are often preferred for protein expression. For this reason, the authors present protocols instructing the reader how to use the wave-mixed BIOSTAT CultiBag RM 20 L for inoculum production and the stirred UniVessel SU 2 L for recombinant protein production at benchtop scale. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing the human placental secreted alkaline phosphatase (SEAP).

  4. Cloning and expression of SLC1OA4,a putative organic anion transport protein

    Institute of Scientific and Technical Information of China (English)

    Patrick L Splinter; Konstantinos N Lazaridis; Paul A Dawson; Nicholas F LaRusso

    2006-01-01

    AIM:To determine if novel bile acid transporters may be expressed in human tissues.METHODS:SLC10A1 (NTCP) was used as a probe to search the NCBI database for homology to previously uncharacterized ESTs. The homology search identified an EST (termed SLC10A4) that shares sequence identity with SLC10A1 and SLC10A2 (ASBT). We performed Northern blot analysis and RT-PCR to determine the tissue distribution of SLC10A4. SLC10A4 was cloned in frame with an epitope tag and overexpressed in CHO cells to determine cellular localization and functional analysis of bile acid uptake.RESULTS:Northern analysis revealed that SLC 10A4 mRNA is ubiquitously expressed fn human tissues with the highest levels of mRNA expression in brain,placenta, and liver. In SLC10A4-transfected CHO cells,immunoblotting analysis and immunofluorescence staining demonstrated a 49-kDa protein that is expressed at the plasma membrane and intracellular compartments.Functional analysis of SLC10A4 showed no significant taurocholate uptake in the presence of sodium when compared to untransfected CHO cells.CONCLUSION:To date, we have shown that this protein has no capacity to transport taurocholate relative to SLC1041; however, given its ubiquitous tissue distribution, it may play a more active role in transporting other endogenous organic anions.

  5. Clinical Significance and Expression of PAF and TNF-alpha in Seminal Plasma of Leukocytospermic Patients

    Directory of Open Access Journals (Sweden)

    Chaodong Liu

    2012-01-01

    Full Text Available Objective. Discuss the changes and roles of PAF in the reproductive tract infection by observing the expression of platelet activating factor (PAF and tumor necrosis factor α (TNF-α in seminal plasma of patients with leukocytospermia. Methods. The seminal plasma was obtained from 22 cases of leukocytospermia and 15 cases of normal males; the peroxidase dyeing method was adopted for seminal plasma white blood count; the ELISA was adopted to test PAF and TNF-α concentration in seminal plasma. Result. PAF concentration ( ng/mL of leukocytospermia group was significantly lower than the normal group ( ng/mL, while TNF-α ( ng/mL was significantly higher than that of normal group ( ng/mL. There was negative correlation between PAF and TNF-α , (, ; the same situation existed in PAF and WBC (, ; but TNF-α was positively correlated to WBC (, . Conclusion. (1 Low expression of PAF and high expression of TNF-α in leukocytospermia affect the sperm motility, which is one of the reasons that leads to infertility. (2 Lower expression of PAF has its particularity during the reproductive tract infection.

  6. Cardiac C-type natriuretic peptide gene expression and plasma concentrations in neonatal piglets

    DEFF Research Database (Denmark)

    Hansen, Lasse H; Smith, Julie; Goetze, Jens Peter

    2014-01-01

    and cardiac tissue extracts were quantified by a porcine-specific radioimmunoassay. RESULTS: Cardiac CNP mRNA contents (n=24) were low compared to sites of known expression, where porcine seminal vesicle CNP mRNA contents were 200-fold higher. In addition, plasma proCNP concentrations in the newborn piglets...

  7. Age-related variations of protein carbonyls in human saliva and plasma: is saliva protein carbonyls an alternative biomarker of aging?

    Science.gov (United States)

    Wang, Zhihui; Wang, Yanyi; Liu, Hongchen; Che, Yuwei; Xu, Yingying; E, Lingling

    2015-06-01

    Free radical hypothesis which is one of the most acknowledged aging theories was developed into oxidative stress hypothesis. Protein carbonylation is by far one of the most widely used markers of protein oxidation. We studied the role of age and gender in protein carbonyl content of saliva and plasma among 273 Chinese healthy subjects (137 females and 136 males aged between 20 and 79) and discussed the correlation between protein carbonyl content of saliva and plasma. Protein carbonyl content of saliva and plasma were, respectively, 2.391 ± 0.639 and 0.838 ± 0.274 nmol/mg. Variations of saliva and plasma different age groups all reached significant differences in both male and female (all p saliva and plasma protein carbonyls were found to be significantly correlated with age (r = 0.6582 and r = 0.5176, all p saliva and plasma protein carbonyl levels (all p > 0.05). Saliva and plasma protein carbonyls were positively related (r = 0.4405, p saliva and plasma protein carbonyls/ferric reducing ability of plasma (FRAP) ratios were proved to be significantly correlated with age (r = 0.7796 and r = 0.6938, all p saliva protein carbonyls/FRAP ratio and plasma protein carbonyls/FRAP ratio were also correlated (r = 0.5573, p saliva protein carbonyls seem to be an alternative biomarker of aging while the mechanisms of protein carbonylation and oxidative stress and the relationship between saliva protein carbonyls and diseases need to be further investigated.

  8. Splice Isoforms of Phosducin-like Protein Control the Expression of Heterotrimeric G Proteins*

    Science.gov (United States)

    Gao, Xueli; Sinha, Satyabrata; Belcastro, Marycharmain; Woodard, Catherine; Ramamurthy, Visvanathan; Stoilov, Peter; Sokolov, Maxim

    2013-01-01

    Heterotrimeric G proteins play an essential role in cellular signaling; however, the mechanism regulating their synthesis and assembly remains poorly understood. A line of evidence indicates that the posttranslational processing of G protein β subunits begins inside the protein-folding chamber of the chaperonin containing t-complex protein 1. This process is facilitated by the ubiquitously expressed phosducin-like protein (PhLP), which is thought to act as a CCT co-factor. Here we demonstrate that alternative splicing of the PhLP gene gives rise to a transcript encoding a truncated, short protein (PhLPs) that is broadly expressed in human tissues but absent in mice. Seeking to elucidate the function of PhLPs, we expressed this protein in the rod photoreceptors of mice and found that this manipulation caused a dramatic translational and posttranslational suppression of rod heterotrimeric G proteins. The investigation of the underlying mechanism revealed that PhLPs disrupts the folding of Gβ and the assembly of Gβ and Gγ subunits, events normally assisted by PhLP, by forming a stable and apparently inactive tertiary complex with CCT preloaded with nascent Gβ. As a result, the cellular levels of Gβ and Gγ, which depends on Gβ for stability, decline. In addition, PhLPs evokes a profound and rather specific down-regulation of the Gα transcript, leading to a complete disappearance of the protein. This study provides the first evidence of a generic mechanism, whereby the splicing of the PhLP gene could potentially and efficiently regulate the cellular levels of heterotrimeric G proteins. PMID:23888055

  9. Relationship between the plasma levels of neurodegenerative proteins and motor subtypes of Parkinson's disease.

    Science.gov (United States)

    Ding, Jian; Zhang, Jiejin; Wang, Xixi; Zhang, Li; Jiang, Siming; Yuan, Yongsheng; Li, Junyi; Zhu, Lin; Zhang, Kezhong

    2017-03-01

    The aim of our study is to examine the plasma levels of the four kinds of neurodegenerative proteins in plasma: α-syn, T-tau, P-tau181, and Aβ-42 in Parkinson's disease (PD) and to evaluate the relationship between their plasma levels and PD motor subtypes. 84 patients with PD were enrolled in our study, and finally, 73 of them were classified into the tremor-dominant subtype (TD) and the postural instability gait difficulty subtype (PIGD). Their motor performance was evaluated by a series of clinical assessments: Freezing of Gait Questionnaire (FOGQ), Timed Up and Go (TUGs), Tinetti balance, and Tinetti gait. Plasma levels of these proteins were measured by enzyme-linked immunosorbent assay (ELISA). The plasma level of α-syn was significantly higher in PD patients when compared to controls (p = 0.004), and significantly higher in the PIGD group when compared to the TD group (p = 0.03). While the plasma level of Aβ-42 was significantly lower in PD patients than in controls (p = 0.002), and significantly lower in the PIGD group than in the TD group (p = 0.05). In PD patients, the plasma level of α-syn (r = -0.355, p score, even after performing multiple linear regression (p = 0.002). While the plasma level of Aβ-42 (r = -0.261, p score and remained correlate when performed multiple linear regression (p = 0.005). The patients with PIGD subtype are characterized with a lower level of plasma Aβ-42 and a higher plasma level of α-syn, which may be used as biomarkers for diagnosis and progression of the subtypes of PD.

  10. Pregnancy-associated plasma protein-A and the vulnerable plaque

    DEFF Research Database (Denmark)

    Jespersen, Camilla H B; Vestergaard, Kirstine R; Schou, Morten

    2014-01-01

    For more than a decade, pregnancy-associated plasma protein-A (PAPP-A) has been examined for its relation to acute coronary syndrome (ACS) and the vulnerable plaque. This review summarizes the current knowledge of plasma PAPP-A in relation to nonpregnant individuals focusing on patients with ACS,......, discusses its use as a possible biomarker for diagnosis and prognosis in ACS, briefly describes the challenges in different assay technologies and describes the effect of heparin administration on PAPP-A concentrations in plasma....

  11. Micro patterning of cell and protein non-adhesive plasma polymerized coatings for biochip applications

    DEFF Research Database (Denmark)

    Bouaidat, Salim; Berendsen, C.; Thomsen, P.;

    2004-01-01

    Micro scale patterning of bioactive surfaces is desirable for numerous biochip applications. Polyethyleneoxide-like (PEO-like) coating with non-fouling functionality has been deposited using low frequency AC plasma polymerization. The non-fouling properties of the coating were tested with human...... cells ( HeLa) and fluorescence labeled proteins (isothiocyanate-labeled bovine serum albumin, i.e. FITC-BSA). The PEO-like coatings were fabricated by plasma polymerization of 12-crown-4 (ppCrown) with plasma polymerized hexene (ppHexene) as adhesion layer. The coatings were micro patterned using...

  12. Association between plasma metabolites and gene expression profiles in five porcine endocrine tissues

    Directory of Open Access Journals (Sweden)

    Bassols Anna

    2011-07-01

    Full Text Available Abstract Background Endocrine tissues play a fundamental role in maintaining homeostasis of plasma metabolites such as non-esterified fatty acids and glucose, the levels of which reflect the energy balance or the health status of animals. However, the relationship between the transcriptome of endocrine tissues and plasma metabolites has been poorly studied. Methods We determined the blood levels of 12 plasma metabolites in 27 pigs belonging to five breeds, each breed consisting of both females and males. The transcriptome of five endocrine tissues i.e. hypothalamus, adenohypophysis, thyroid gland, gonads and backfat tissues from 16 out of the 27 pigs was also determined. Sex and breed effects on the 12 plasma metabolites were investigated and associations between genes expressed in the five endocrine tissues and the 12 plasma metabolites measured were analyzed. A probeset was defined as a quantitative trait transcript (QTT when its association with a particular metabolic trait achieved a nominal P value Results A larger than expected number of QTT was found for non-esterified fatty acids and alanine aminotransferase in at least two tissues. The associations were highly tissue-specific. The QTT within the tissues were divided into co-expression network modules enriched for genes in Kyoto Encyclopedia of Genes and Genomes or gene ontology categories that are related to the physiological functions of the corresponding tissues. We also explored a multi-tissue co-expression network using QTT for non-esterified fatty acids from the five tissues and found that a module, enriched in hypothalamus QTT, was positioned at the centre of the entire multi-tissue network. Conclusions These results emphasize the relationships between endocrine tissues and plasma metabolites in terms of gene expression. Highly tissue-specific association patterns suggest that candidate genes or gene pathways should be investigated in the context of specific tissues.

  13. Transcriptional Modulation of Heat-Shock Protein Gene Expression

    OpenAIRE

    Anastasis Stephanou; Latchman, David S.

    2011-01-01

    Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex sig...

  14. Transcriptional modulation of heat-shock protein gene expression.

    OpenAIRE

    A. Stephanou; Latchman, D S

    2011-01-01

    Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex sig...

  15. Valproic acid: in vitro plasma protein binding and interaction with phenytoin.

    Science.gov (United States)

    Cramer, J A; Mattson, R H

    1979-01-01

    Because valproic acid (VPA) is highly bound to plasma protein, several variables affecting binding will significantly alter the quantity of free drug which is pharmacologically active. Therefore, total VPA plasma concentrations do not reflect the therapeutic strength of the drug in tissue. We have performed equilibrium dialysis and ultrafiltration studies of VPA binding to plasma protein. The converging data in these in vitro studies indicate a clinically significant alteration in the percent of free VPA when total drug concentration exceeds 80 micrograms/ml. Saturation of drug binding sites probably occurs in this range. At 20--60 micrograms/ml VPA there is 5% free drug, with a significant increase to 8% free at 80 micrograms/ml; free drug increases to over 20% at 145 micrograms/ml total VPA. Human plasma, which is low in albumin, has twice the quantity of free VPA as normal plasma (10 versus 5% free). The clinical evidence of interaction between VPA and phenytoin is confirmed in vitro by the increase in the free fraction of both drugs. VPA binding decreases by 3--6%, while phenytoin binding decreases 5--6% as both drugs reach high plasma concentrations. When appropriate, laboratory reports should be available defining concentration of free drug in plasma for optimal interpretation of drug concetrations relative to clinical effects.

  16. Effects of plant proteins on postprandial, free plasma amino acid concentrations in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Larsen, Bodil Katrine; Dalsgaard, Anne Johanne Tang; Pedersen, Per Bovbjerg

    2012-01-01

    proteins from wheat, peas, field beans, sunflower and soybean. Blood samples were obtained from the caudal vein of 7 fish in each dietary treatment group prior to feeding, as well as: 2, 4, 6, 8, 12, 24, 48 and 72 h after feeding (sampling 7 new fish at each time point), and plasma amino acid......Postprandial patterns in plasma free amino acid concentrations were investigated in juvenile rainbow trout (Oncorhynchus mykiss) fed either a fish meal based diet (FM) or a diet (VEG) where 59% of fish meal protein (corresponding to 46% of total dietary protein) was replaced by a matrix of plant...... concentrations were subsequently measured by HPLC. Nutrient digestibility and ammonia excretion of the two experimental diets were measured in a parallel experiment using a modified Guelph setup. Results showed that the appearance of most amino acids (essential and non-essential) in the plasma was delayed...

  17. Lithium an emerging contaminant: bioavailability, effects on protein expression, and homeostasis disruption in short-term exposure of rainbow trout.

    Science.gov (United States)

    Tkatcheva, Victoria; Poirier, David; Chong-Kit, Richard; Furdui, Vasile I; Burr, Christopher; Leger, Ray; Parmar, Jaspal; Switzer, Teresa; Maedler, Stefanie; Reiner, Eric J; Sherry, James P; Simmons, Denina B D

    2015-04-01

    Worldwide production of lithium (Li) has increased dramatically during the past decade, driven by the demand for high charge density batteries. Information about Li in the aquatic environment is limited. The present study was designed to explore the effects of Li in rainbow trout (Oncorhynchus mykiss). Juvenile trout were exposed to a nominal concentration of 1.0mg Li/L in three separate exposures. Major ion concentrations were measured in brain and plasma by ion chromatography. Plasma proteins and fatty acids were measured by HPLC-MS/MS. Lithium accumulated in the brain and plasma. Arachidonic acid was elevated in plasma after 48h. Elevated concentrations of Li in brain were associated with depressed concentrations of sodium, magnesium, potassium and ammonium relative to the control. In plasma, sodium and calcium were also depressed. Several changes occurred to plasma proteins corresponding to Li exposure: inhibition of prostaglandin synthase (Ptgs2), increased expression of copper transporting ATP synthases, and Na(+)/K(+) ATPase. To our knowledge, ours is the first study to demonstrate elevated Li concentrations in fish brain, with associated effects on ion regulation. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

    Science.gov (United States)

    Allen, Robert S.; Tilbrook, Kimberley; Warden, Andrew C.; Campbell, Peter C.; Rolland, Vivien; Singh, Surinder P.; Wood, Craig C.

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia. PMID:28316608

  19. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

    Science.gov (United States)

    Allen, Robert S; Tilbrook, Kimberley; Warden, Andrew C; Campbell, Peter C; Rolland, Vivien; Singh, Surinder P; Wood, Craig C

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

  20. Hypoxia decreases podocyte expression of slit diaphragm proteins

    Directory of Open Access Journals (Sweden)

    Lu H

    2012-07-01

    Full Text Available Hong Lu,1 Gaurav Kapur,1 Tej K Mattoo,1 William D Lyman1,21Carman and Ann Adams Department of Pediatrics, 2Children's Research Center of Michigan, Children's Hospital of Michigan, Detroit, MI, USABackground: Chronic hypoxia contributes to progressive tubulointerstitial injury and, consequently, renal failure. However, the effect of hypoxia on glomerular podocytes, which are integral to the slit diaphragm complex and responsible for selectivity of the glomerular filtration barrier, has not been completely determined. Methods: Conditionally immortalized mouse podocyte cells were exposed to hypoxic (1% O2 or normoxic (room air conditions for 24, 48, or 72 hours, after which cell viability was determined by MTT assay. Cells were stained with podocin and phalloidin to determine podocin and intracellular actin distribution. Expression of synaptopodin, CD2-associated protein (CD2AP, NcK, transforming growth factor-β1 (TGF-β1, hypoxia-inducible factor (HIF-1α were evaluated by real-time polymerase chain reaction.Results: Podocytes exposed to hypoxia had significantly reduced viability at 48 (87% and 72 hours (66%. There was disarrangement of intracellular filament actin by phalloidin staining, a 30% weaker fluorescence intensity by podocin staining, significantly reduced expression of synaptopodin (12%, CD2AP (42%, NcK (38%, and increased expression of TGF-β1 and P-ERK after hypoxia treatment.Conclusion: Podocyte exposure to hypoxia leads to reduced viability and SD protein expression, which may explain persistent and/or increasing proteinuria in patients with progressive renal failure. Increased expression of TGF-β1 and P-ERK is associated with apoptosis and fibrosis, which could be the link between hypoxia and glomerular injury.Keywords: podocytes, hypoxia, slit-diaphragm proteins

  1. Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes

    Science.gov (United States)

    Caldwell, Charles R.

    1987-01-01

    The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32°C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30°C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32°C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14°C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32°C which could account for the complex temperature dependence of the barley root plasma membrane ATPase. PMID:16665545

  2. Differential expression of ribosomal proteins in myelodysplastic syndromes.

    Science.gov (United States)

    Rinker, Elizabeth B; Dueber, Julie C; Qualtieri, Julianne; Tedesco, Jason; Erdogan, Begum; Bosompem, Amma; Kim, Annette S

    2016-02-01

    Aberrations of ribosomal biogenesis have been implicated in several congenital bone marrow failure syndromes, such as Diamond-Blackfan anaemia, Shwachman-Diamond syndrome and Dyskeratosis Congenita. Recent studies have identified haploinsufficiency of RPS14 in the acquired bone marrow disease isolated 5q minus syndrome, a subtype of myelodysplastic syndromes (MDS). However, the expression of various proteins comprising the ribosomal subunits and other proteins enzymatically involved in the synthesis of the ribosome has not been explored in non-5q minus MDS. Furthermore, differences in the effects of these expression alterations among myeloid, erythroid and megakaryocyte lineages have not been well elucidated. We examined the expression of several proteins related to ribosomal biogenesis in bone marrow biopsy specimens from patients with MDS (5q minus patients excluded) and controls with no known myeloid disease. Specifically, we found that there is overexpression of RPS24, DKC1 and SBDS in MDS. This overexpression is in contrast to the haploinsufficiency identified in the congenital bone marrow failure syndromes and in acquired 5q minus MDS. Potential mechanisms for these differences and aetiology for these findings in MDS are discussed.

  3. Expression of DNA-dependent protein kinase in human granulocytes

    Institute of Scientific and Technical Information of China (English)

    Annahita SALLMYR; Anna MILLER; Aida GABDOULKHAKOVA; Valentina SAFRONOVA; Gunnel HENRIKSSON; Anders BREDBERG

    2004-01-01

    Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNAPK in P MN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration.In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.

  4. Altered retinol status and expression of retinol-related proteins in streptozotocin-induced type 1 diabetic model rats

    OpenAIRE

    Takitani, Kimitaka; Inoue, Keisuke; Koh, Maki; Miyazaki, Hiroshi; Inoue, Akiko; Kishi, Kanta; Tamai, Hiroshi

    2015-01-01

    Diabetes is a metabolic disorder characterized by chronic hyperglycemia. Advanced diabetes is associated with severe complications and impaired nutritional status. Here, we assessed the expression of retinol-associated proteins, including β-carotene 15,15'-monooxygenase (BCMO), lecithin:retinol acyltransferase (LRAT), aldehyde dehydrogenase (ALDH), and cytochrome P450 26A1 (CYP26A1), and measured retinol levels in the plasma and liver of streptozotocin (STZ)-induced type 1 diabetic model rats...

  5. Expression of P16 protein and Bcl-2 protein in malignant eyelid tumors

    Institute of Scientific and Technical Information of China (English)

    牛膺筠; 周占宇; 刘夫玲; 王红云

    2002-01-01

    Objective To investigate the relationship between P16 gene (the tumor suppressor gene) and the bcl-2 gene (the apoptosis inhibitor gene) and the incidence and development of malignant eyelid tumors. Methods The streptavidin-biotin-peroxidase complex immunohistochemistry method was used to study the expression of P16 gene and the bcl-2 gene in 96 cases of malignant eyelid tumors. Results Among the 96 cases, there were 40 basal cell carcinomas (BCCs), 33 squamous carcinomas and 23 sebaceous carcinoma, with P16 protein positive (nuclear staining) rates 70%, 54.6% and 56.5%, respectively. The P16 positive rate was negatively correlated with the degree of tumor histological differentiation, and the rate difference between the high differentiated carcinomas was significant (P<0.05). Positive Bcl-2 protein expression was detected in the cytoplasm. All 40 BCC cases were Bcl-2 positive, and nearly all of the tumor cells showed positive cytoplasmic expression, while in the 33 squamous cell carcinoma cases only one showed positive focal reaction, and the staining in the other 32 cases was relatively faint. None of the 23 sebaceous carcinomas expressed Bcl-2. Conclusions The expression of the P16 protein was related to the occurrence and degree of differentiation of malignant eyelid tumors. The overexpression of the Bcl-2 protein suggests that suppression of apoptosis might play a role in the tumorigenesis of BCC.

  6. The interleukin-6 receptor alpha-chain (CD126) is expressed by neoplastic but not normal plasma cells.

    Science.gov (United States)

    Rawstron, A C; Fenton, J A; Ashcroft, J; English, A; Jones, R A; Richards, S J; Pratt, G; Owen, R; Davies, F E; Child, J A; Jack, A S; Morgan, G

    2000-12-01

    Interleukin-6 (IL-6) is reported to be central to the pathogenesis of myeloma, inducing proliferation and inhibiting apoptosis in neoplastic plasma cells. Therefore, abrogating IL-6 signaling is of therapeutic interest, particularly with the development of humanized anti-IL-6 receptor (IL-6R) antibodies. The use of such antibodies clinically requires an understanding of IL-6R expression on neoplastic cells, particularly in the cycling fraction. IL-6R expression levels were determined on plasma cells from patients with myeloma (n = 93) and with monoclonal gammopathy of undetermined significance (MGUS) or plasmacytoma (n = 66) and compared with the levels found on normal plasma cells (n = 11). In addition, 4-color flow cytometry was used to assess the differential expression by stage of differentiation and cell cycle status of the neoplastic plasma cells. IL-6R alpha chain (CD126) was not detectable in normal plasma cells, but was expressed in approximately 90% of patients with myeloma. In all groups, the expression levels showed a normal distribution. In patients with MGUS or plasmacytoma, neoplastic plasma cells expressed significantly higher levels of CD126 compared with phenotypically normal plasma cells from the same marrow. VLA-5(-) "immature" plasma cells showed the highest levels of CD126 expression, but "mature" VLA-5(+) myeloma plasma cells also overexpressed CD126 when compared with normal subjects. This study demonstrates that CD126 expression is restricted to neoplastic plasma cells, with little or no detectable expression by normal cells. Stromal cells in the bone marrow microenvironment do not induce the overexpression because neoplastic cells express higher levels of CD126 than normal plasma cells from the same bone marrow in individuals with MGUS. (Blood. 2000;96:3880-3886)

  7. Influence of dietary fish proteins on plasma and liver cholesterol concentrations in rats.

    Science.gov (United States)

    Zhang, X; Beynen, A C

    1993-05-01

    The effects of amount and type of dietary fish proteins on plasma and liver cholesterol concentrations were evaluated in female rats. The isonitrogenous diets used contained 10 g cholesterol/kg and were carefully balanced for residual fat, cholesterol, Ca, Mg and P in the protein preparations. Cod meal, soya-bean protein or casein was incorporated into the diets as the only source of dietary protein at three levels: either 24, 48 or 72 g N/kg diet. Extra protein was added to the diet at the expense of the glucose component. In a second experiment soya-bean protein, casein, cod meal, whiting meal or plaice meal was added to the diet at a level of 24 g N/kg. When compared with casein, cod meal and soya-bean protein decreased plasma and liver cholesterol concentrations. A further cholesterol-lowering effect was achieved by increasing the proportion of either soya-bean protein or cod meal in the diet. Substitution of casein for glucose did not influence plasma and liver cholesterol concentrations. Plaice meal in the diet produced lower group mean plasma cholesterol concentrations than did whiting meal. In rats fed on the diet containing plaice meal, liver cholesterol concentrations were significantly lower than those in their counterparts fed on either cod meal or whiting meal. The present study demonstrates that different fish proteins in the diet have different effects on cholesterol metabolism and that the cholesterol-influencing properties of cod meal can be enhanced by the incorporation of higher proportions of this protein in the diet.

  8. Differential Gene Expression and Protein Phosphorylation as Factors Regulating the State of the Arabidopsis SNX1 Protein Complexes in Response to Environmental Stimuli

    Science.gov (United States)

    Brumbarova, Tzvetina; Ivanov, Rumen

    2016-01-01

    Endosomal recycling of plasma membrane proteins contributes significantly to the regulation of cellular transport and signaling processes. Members of the Arabidopsis (Arabidopsis thaliana) SORTING NEXIN (SNX) protein family were shown to mediate the endosomal retrieval of transporter proteins in response to external challenges. Our aim is to understand the possible ways through which external stimuli influence the activity of SNX1 in the root. Several proteins are known to contribute to the function of SNX1 through direct protein–protein interaction. We, therefore, compiled a list of all Arabidopsis proteins known to physically interact with SNX1 and employed available gene expression and proteomic data for a comprehensive analysis of the transcriptional and post-transcriptional regulation of this interactome. The genes encoding SNX1-interaction partners showed distinct expression patterns with some, like FAB1A, being uniformly expressed, while others, like MC9 and BLOS1, were expressed in specific root zones and cell types. Under stress conditions known to induce SNX1-dependent responses, two genes encoding SNX1-interacting proteins, MC9 and NHX6, showed major gene-expression variations. We could also observe zone-specific transcriptional changes of SNX1 under iron deficiency, which are consistent with the described role of the SNX1 protein. This suggests that the composition of potential SNX1-containing protein complexes in roots is cell-specific and may be readjusted in response to external stimuli. On the level of post-transcriptional modifications, we observed stress-dependent changes in the phosphorylation status of SNX1, FAB1A, and CLASP. Interestingly, the phosphorylation events affecting SNX1 interactors occur in a pattern which is largely complementary to transcriptional regulation. Our analysis shows that transcriptional and post-transcriptional regulation play distinct roles in SNX1-mediated endosomal recycling under external stress. PMID:27725825

  9. Efficient expression and purification of biologically active human cystatin proteins.

    Science.gov (United States)

    Chauhan, Sakshi; Tomar, Raghuvir S

    2016-02-01

    Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.

  10. The E4 protein; structure, function and patterns of expression.

    Science.gov (United States)

    Doorbar, John

    2013-10-01

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1^E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein's flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1^E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1^E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1^E4, these kinases regulate one of

  11. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    Science.gov (United States)

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

  12. Expression of parathyroid hormone-related protein in ameloblastomas.

    Science.gov (United States)

    Ohtsuru, Mitsunobu

    2005-12-01

    Parathyroid hormone-related protein (PTHrP) was first discovered as a causative protein for hypercalcemia, which is often seen in the malignant tumor. PTHrP binds to the parathyroid hormone 1 receptor (PTH1R) for signal transduction. PTHrP-PTH1R interactions were associated with bone resorption. The present study, therefore, sought to clarify the expression of PTHrP, parathyroid hormone (PTH) and PTH1R in ameloblastoma, using RT-PCR (N = 8), immunohistochemistry (N = 23) and ELISA (N = 11) techniques. PTHrP and B-actin mRNA were detected in the all samples. Expression of PTHrP was also seen in all of the 23 cases in ameloblastoma by immunohistochemistry. There was a significant difference in PTHrP concentration by ELISA between typical unicystic type and solid type including unicystic type 3 (p = 0.0427). Only one exhibited the weak expression of PTH1R mRNA. PTH1R was observed on osteoblasts in bone around the tumor but no expression was observed on ameloblastoma cells in tumor parenchyma by immunohistochemistry. PTH was not detected in ameloblastoma by RT-PCR, immunohistochemistory as well as ELISA. In addition, hypercalcemia and increase of serum PTHrP level was observed in one case of 8 ameloblastomas. It was suggested that PTHrP level may be associated with local bone infiltration and hypercalcemia in ameloblastoma.

  13. Myocardin-related transcription factor regulates Nox4 protein expression

    DEFF Research Database (Denmark)

    Rozycki, Matthew; Bialik, Janne Folke; Speight, Pam

    2016-01-01

    TGFβ-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator...... translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application...... of contact uncoupling and TGFβ. Nox4 knockdown abrogates epithelial-myofibroblast transition-associated reactive oxygen species production. Laser capture microdissection reveals increased Nox4 expression in the tubular epithelium also during obstructive nephropathy. MRTF down-regulation/inhibition suppresses...

  14. Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström macroglobulinemia.

    Science.gov (United States)

    Gaudette, B T; Dwivedi, B; Chitta, K S; Poulain, S; Powell, D; Vertino, P; Leleu, X; Lonial, S; Chanan-Khan, A A; Kowalski, J; Boise, L H

    2016-01-28

    Waldenström macroglobulinemia (WM) is a proliferative disorder of IgM-secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels, which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for the inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing.

  15. Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.

    Science.gov (United States)

    Santner, Aaron A; Croy, Carrie H; Vasanwala, Farha H; Uversky, Vladimir N; Van, Ya-Yue J; Dunker, A Keith

    2012-09-18

    Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have a low level of sequence complexity and a high net charge but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently used fusions such as maltose-binding protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physiochemical properties, they did not perturb the structure, conformational stability, or function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically disordered fusions and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.

  16. Complement inhibitory proteins expression in placentas of thrombophilic women Complement inhibitory proteins expression in placentas of thrombophilic women

    Directory of Open Access Journals (Sweden)

    Przemysław Krzysztof Wirstlein

    2012-10-01

    Full Text Available Factors controlling complement activation appear to exert a protective effect on pregnancy. This is
    particularly important in women with thrombophilia. The aim of this study was to determine the transcript and
    protein levels of complement decay-accelerating factor (DAF and membrane cofactor protein (MCP in the
    placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry staining
    of inhibitors of the complement cascade, DAF and MCP proteins, in the placentas of thrombophilic women.
    Placentas were collected from eight women with inherited thrombophilia and ten with acquired thrombophilia.
    The levels of DAF and MCP transcripts were evaluated by qPCR, the protein level was evaluated by Western
    blot. We observed a higher transcript (p < 0.05 and protein (p < 0.001 levels of DAF and MCP in the placentas
    of thrombophilic women than in the control group. DAF and MCP were localized on villous syncytiotrophoblast
    membranes, but the assessment of staining in all groups did not differ. The observed higher expression level of
    proteins that control activation of complement control proteins is only seemingly contradictory to the changes
    observed for example in the antiphospholipid syndrome. However, given the hitherto known biochemical changes
    associated with thrombophilia, a mechanism in which increased expression of DAF and MCP in the placentas is
    an effect of proinflammatory cytokines, which accompanies thrombophilia, is probable.Factors controlling complement activation appear to exert a protective effect on pregnancy. This is
    particularly important in women with thrombophilia. The aim of this study was to determine the transcript and
    protein levels of complement decay-accelerating factor (DAF and membrane cofactor protein (MCP in the
    placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry

  17. Total plasma protein in very preterm babies: prognostic value and comparison with illness severity scores.

    Directory of Open Access Journals (Sweden)

    Silvia Iacobelli

    Full Text Available OBJECTIVE: We aimed to investigate the predictive value for severe adverse outcome of plasma protein measurements on day one of life in very preterm infants and to compare total plasma protein levels with the validated illness severity scores CRIB, CRIB-II, SNAP-II and SNAPPE-II, regarding their predictive ability for severe adverse outcome. METHODS: We analyzed a cohort of infants born at 24-31 weeks gestation, admitted to the tertiary intensive care unit of a university hospital over 10.5 years. The outcome measure was "severe adverse outcome" defined as death before discharge or severe neurological injury on cranial ultrasound. The adjusted odd ratio (aOR and 95% confidence interval (95% CI of severe adverse outcome for hypoproteinemia (total plasma protein level <40 g/L was calculated by univariate and multivariate analyses. Calibration (Hosmer-Lemeshow goodness-of-fit was performed and the predictive ability for severe adverse outcome was assessed for total plasma protein and compared with CRIB, CRIB-II, SNAP-II and SNAPPE-II, by calculating receiver operating characteristic (ROC curves and their associated area under the curve (AUC. RESULTS: 761 infants were studied: 14.4% died and 4.1% survived with severe cerebral ultrasound findings. The aOR of severe adverse outcome for hypoproteinemia was 6.1 (95% CI 3.8-9.9. The rank order for variables, as assessed by AUCs and 95% CIs, in predicting outcome was: total plasma protein [0.849 (0.821-0.873], SNAPPE-II [0.822 (0.792-0.848], CRIB [0.821 (0.792-0.848], SNAP-II [0.810 (0.780-0.837] and CRIB-II [0.803 (0.772-0.830]. Total plasma protein predicted severe adverse outcome significantly better than CRIB-II and SNAP-II (both p<0.05. Calibration for total plasma protein was very good. CONCLUSIONS: Early hypoproteinemia has prognostic value for severe adverse outcome in very preterm, sick infants. Total plasma protein has a predictive performance comparable with CRIB and SNAPPE-II and greater than

  18. Prognostic value of plasma C-reactive protein in the evaluation of paraquat poisoning patients简

    Institute of Scientific and Technical Information of China (English)

    Zong; Ning; Yu-Long; Bai; Hua; Lu; Kang-Lin; Mo

    2015-01-01

    Objective: To investigate the prognostic value of plasma C-reactive protein(CRP) level in patients with paraquat poisoning.Methods: This study included 162 patients with paraquat poisoning. The data of plasma paraquat, CRP level and arterial blood gas were analyzed. Cox regression analysis was applied to evaluate the risk factors of prognosis. Receiver operating characteristics curve analysis and area under curve were used to calculate the predictive power of significant variable. Differences in patient survival were determined using the Kaplan–Meier method and a log-rank test.Results: Plasma CRP level was significantly increased in non-survival patients compared with survival patients(P < 0.05), and positively correlated with plasma paraquat level(P < 0.05). Cox regression analysis revealed that plasma CRP level was an independent prognostic marker of mortality within 30 days. The receiver operating characteristics curve analysis indicated that area under curve of plasma CRP level was0.867(95% CI: 0.81–0.93), and the cut-off value was 18 mg/L, and patients with CRP level over this value had a poor survival time compared with those with less than this value.Conclusions: These results suggest that plasma CRP level is distinct increased in patients with paraquat poisoning, and the plasma CRP level may be useful for the prediction of prognosis in paraquat poisoning.

  19. Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Directory of Open Access Journals (Sweden)

    Schlarman Maggie S

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development. Methods The P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR and green fluorescent protein (GFP-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined. Results The PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites. Conclusions PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

  20. Identification of differentially expressed serum proteins in gastric adenocarcinoma☆

    Science.gov (United States)

    Subbannayya, Yashwanth; Mir, Sartaj Ahmad; Renuse, Santosh; Manda, Srikanth S.; Pinto, Sneha M.; Puttamallesh, Vinuth N.; Solanki, Hitendra Singh; Manju, H.C.; Syed, Nazia; Sharma, Rakesh; Christopher, Rita; Vijayakumar, M.; Kumar, K.V. Veerendra; Prasad, T.S. Keshava; Ramaswamy, Girija; Kumar, Rekha V.; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu–Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. Biological significance Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well

  1. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Guo, Yan; Cuin, Tracey A.

    2007-01-01

    that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM Hþ-ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM Hþ-ATPase AHA2 at a novel......Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report...

  2. Adsorption of proteins from plasma at polyester non-wovens

    NARCIS (Netherlands)

    Klomp, A.J.A.; Engbers, G.H.M.; Mol, J.; Terlingen, J.G.A.; Feijen, J.

    1999-01-01

    Polyester non-wovens in filters for the removal of leukocytes from platelet concentrates (PCs) must be platelet compatible. In PC filtration, the adsorption of proteins at the plasma–non-woven interface can be of great importance with respect to the yield of platelets. Unmodified and radio frequency

  3. Plasma Membrane Protein Ubiquitylation and Degradation as Determinants of Positional Growth in Plants

    Institute of Scientific and Technical Information of China (English)

    Barbara Korbei; Christian Luschnig

    2013-01-01

    Being sessile organisms, plants evolved an unparalleled plasticity in their post-embryonic development, allowing them to adapt and fine-tune their vital parameters to an ever-changing environment. Cross-talk between plants and their environment requires tight regulation of information exchange at the plasma membrane (PM). Plasma membrane proteins mediate such communication, by sensing variations in nutrient availability, external cues as well as by controlled solute transport across the membrane border. Localiza-tion and steady-state levels are essential for PM protein function and ongoing research identified cis- and trans-acting determinants, involved in control of plant PM protein localization and turnover. In this overview, we summarize recent progress in our understanding of plant PM protein sorting and degradation via ubiquitylation, a post-translational and reversible modification of proteins. We highlight characterized components of the machinery involved in sorting of ubiquitylated PM proteins and discuss consequences of protein ubiquitylation on fate of selected PM proteins. Specifically, we focus on the role of ubiquitylation and PM protein degradation in the regulation of polar auxin transport (PAT). We combine this regulatory circuit with further aspects of PM protein sorting control, to address the interplay of events that might control PAT and polarized growth in higher plants.

  4. Improved detection specificity for plasma proteins by targeting cysteine-containing peptides with photo-SRM.

    Science.gov (United States)

    Enjalbert, Quentin; Girod, Marion; Simon, Romain; Jeudy, Jérémy; Chirot, Fabien; Salvador, Arnaud; Antoine, Rodolphe; Dugourd, Philippe; Lemoine, Jérôme

    2013-03-01

    Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as an alternative to immunoassays for protein quantification owing to faster development time and higher multiplexing capability. However, the SRM strategy is faced with the high complexity of peptide mixtures after trypsin digestion of whole plasma or the cellular proteome that most of the time causes contamination, irremediably, by interfering compounds in the transition channels monitored. This problem becomes increasingly acute when the targeted protein is present at a low concentration. In this work, the merit of laser-induced photo-dissociation in the visible region at 473 nm implemented in an hybrid quadrupole linear ion-trap mass spectrometer (photo-SRM) was evaluated for detection specificity of cysteine-containing peptides in a group of plasma proteins after tagging with a dabcyl chromophore. Compared with conventional SRM, photo-SRM chromatograms have improved detection specificity for most of peptides monitored. Comparison of the signals obtained for the best proteotypic peptides in SRM mode and those recorded by photo-SRM of cysteine-containing peptides for the same proteins reveals either increased (up to 10-fold) or similar signal to photo-SRM detection. Finally, photo-SRM has extended response linearity across a calibration plot obtained by diluting human plasma in rat plasma, down to the lowest concentrations. Hence, photo-SRM may advantageously complement conventional SRM in assay of proteins in complex biological matrices.

  5. Plasma protein fractions in healthy blood donors quantitated by an automated multicapillary electrophoresis system.

    Science.gov (United States)

    Larsson, Anders; Hansson, Lars-Olof

    2006-09-01

    During the last decade, capillary electrophoresis (CE) has emerged as an important alternative to traditional analysis of serum and plasma proteins by agarose or celluloseacetate electrophoresis. CE analysis of plasma proteins can now be fully automated and also includes bar-code identification of samples, preseparation steps, and direct post-separation quantitation of individual peaks, which permits short assay times and high throughput. For laboratory work, it is important to have reference values from healthy individuals. Therefore, plasma samples from 156 healthy blood donors (79 females and 77 males) have been analyzed with the Capillarys instrument and the new high resolution buffer, which yields higher resolution than the beta1-beta2+ buffer. Albumin concentrations in samples are measured using nephelometry in order to assign protein concentrations to each peak. The 2.5 and 97.5 percentiles for both the percentages of different peaks and the protein concentrations in the peaks are calculated according to the recommendations of the International Federation of Clinical Chemistry on the statistical treatment of reference values. The Capillarys instrument is a reliable system for plasma protein analysis, combining advantages of full automation with high analytical performances and throughput.

  6. Atmospheric pressure plasma polymers for tuned QCM detection of protein adhesion.

    Science.gov (United States)

    Rusu, G B; Asandulesa, M; Topala, I; Pohoata, V; Dumitrascu, N; Barboiu, M

    2014-03-15

    Our efforts have been concentrated in preparing plasma polymeric thin layers at atmospheric pressure grown on Quartz Crystal Microbalance-QCM electrodes for which the non-specific absorption of proteins can be efficiently modulated, tuned and used for QCM biosensing and quantification. Plasma polymerization reaction at atmospheric pressure has been used as a simple and viable method for the preparation of QCM bioactive surfaces, featuring variable protein binding properties. Polyethyleneglycol (ppEG), polystyrene (ppST) and poly(ethyleneglycol-styrene) (ppST-EG) thin-layers have been grown on QCM electrodes. These layers were characterized by Atomic Force Microscopy (AFM), Contact angle measurements, Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). The plasma ppST QCM electrodes present a higher adsorption of Concanavalin A (ConA) and Bovine Serum Albumin (BSA) proteins when compared with the commercial coated polystyrene (ppST) ones. The minimum adsorption was found for ppEG, surface, known by their protein anti-fouling properties. The amount of adsorbed proteins can be tuned by the introduction of PEG precursors in the plasma discharge during the preparation of ppST polymers. © 2013 Elsevier B.V. All rights reserved.

  7. Study of Plasma Malondialdehyde, Troponin I and C - Reactive protein in Acute Coronary Syndromes Patients

    Directory of Open Access Journals (Sweden)

    S. Shams

    2006-04-01

    Full Text Available Introduction & Objective: Ischemic injury of endothelium is associated with prostaglandin synthesis and platelet adhesion and aggregation, which may be associated with the release of aldehydes such as malondialdehyde (MDA. C-reactive protein and cardiac troponin I have been proposed as diagnostic markers of acute coronary syndromes. In this study, we compared the usefulness of plasma MDA as a marker of acute coronary syndromes with that of C-reactive protein and troponin I.Material & Methods: The study population contained 50 patients with unstable angina and 50 patients with acute myocardial infarction admitted to the hearth department of the Ekbatan Hospital of Hamadan. The subjects were matched according to age and sex. Total cholesterol, LDL and HDL cholesterol, triglycerides, plasma MDA, troponin I and C-reactive protein levels were determined in all patients. Results: Results showed that the plasma MDA levels were significantly higher in patients with acute myocardial infarction than in individuals with unstable angina (P<0.001 and were associated with increased levels of troponin I and C-reactive protein (P<0.001.Conclusion: The combination of the plasma MDA levels, which reflect endothelial injury, and troponin I and C-reactive protein levels may allow better discrimination in acute coronary syndromes patients.

  8. Maternal Low Quality Protein Diet Alters Plasma Amino Acid Concentrations of Weaning Rats

    Directory of Open Access Journals (Sweden)

    Arzu Kabasakal Cetin

    2015-12-01

    Full Text Available Several studies have indicated the influence of a maternal low protein diet on the fetus. However, the effect of a maternal low quality protein diet on fetal growth and development is largely unknown. Wistar rats (11 weeks old were mated and maintained on either a chow diet with 20% casein (n = 6 as the control group (C, or a low quality protein diet with 20% wheat gluten (n = 7 as the experimental group (WG through gestation and lactation. Maternal body weights were similar in both groups throughout the study. Birth weights were not influenced by maternal diet and offspring body weights during lactation were similar between the groups. Offspring’s plasma amino acid profiles showed that plasma methionine, glutamine and lysine were significantly lower and aspartic acid, ornithine and glycine-proline were significantly higher in the WG. Plant based protein comprises an important part of protein intake in developing countries. It is well-known that these diets can be inadequate in terms of essential amino acids. The current study shows differential effects of a maternal low quality protein diet on the offspring’s plasma amino acids. Future studies will examine further aspects of the influence of maternal low quality protein diets on fetal growth and development.

  9. Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins.

    Directory of Open Access Journals (Sweden)

    Ayasa Ochiai

    Full Text Available Elevated plasma low-density lipoprotein (LDL cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD-15 cells, which lack insulin-induced gene-1 (Insig-1 and Insig-2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity.

  10. Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins.

    Science.gov (United States)

    Ochiai, Ayasa; Miyata, Shingo; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2015-01-01

    Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD-15 cells, which lack insulin-induced gene-1 (Insig-1) and Insig-2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity.

  11. Expression of P53 protein after exposure to ionizing radiation

    Science.gov (United States)

    Salazar, A. M.; Salvador, C.; Ruiz-Trejo, C.; Ostrosky, P.; Brandan, M. E.

    2001-10-01

    One of the most important tumor suppressor genes is p53 gene, which is involved in apoptotic cell death, cell differentiation and cell cycle arrest. The expression of p53 gene can be evaluated by determining the presence of P53 protein in cells using Western Blot assay with a chemiluminescent method. This technique has shown variabilities that are due to biological factors. Film developing process can influence the quality of the p53 bands obtained. We irradiated tumor cell lines and human peripheral lymphocytes with 137Cs and 60Co gamma rays to standardize irradiation conditions, to compare ionizing radiation with actinomycin D and to reduce the observed variability of P53 protein induction levels. We found that increasing radiation doses increase P53 protein induction while it decreases viability. We also conclude that ionizing radiation could serve as a positive control for Western Blot analysis of protein P53. In addition, our results show that the developing process may play an important role in the quality of P53 protein bands and data interpretation.

  12. A molecular clock regulates angiopoietin-like protein 2 expression.

    Science.gov (United States)

    Kadomatsu, Tsuyoshi; Uragami, Shota; Akashi, Makoto; Tsuchiya, Yoshiki; Nakajima, Hiroo; Nakashima, Yukiko; Endo, Motoyoshi; Miyata, Keishi; Terada, Kazutoyo; Todo, Takeshi; Node, Koichi; Oike, Yuichi

    2013-01-01

    Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

  13. A molecular clock regulates angiopoietin-like protein 2 expression.

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    Tsuyoshi Kadomatsu

    Full Text Available Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2 contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

  14. Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane

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    Golan, Yonatan; Sherman, Eilon

    2017-06-01

    The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.

  15. Monocyte and plasma expression of TAM ligand and receptor in renal failure: Links to unregulated immunity and chronic inflammation.

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    Lee, Iris J; Hilliard, Brendan A; Ulas, Mehriban; Yu, Daohai; Vangala, Chandan; Rao, Swati; Lee, Jean; Gadegbeku, Crystal A; Cohen, Philip L

    2015-06-01

    Chronic inflammation is increased in patients with chronic kidney disease (CKD) and contributes to cardiovascular morbidity and mortality. Specific immune mechanisms and pathways that drive and maintain chronic inflammation in CKD are not well described. The TAM ligands (Gas6 and protein S) and receptors (Axl and Mer) have been recently recognized as playing a prominent role in immune regulation. The receptors exist in both soluble and cell-bound forms; the soluble receptors (sAxl and sMer) are believed to compete with the bound receptors and thus inhibit their function. In this study, we determined the expression of cell-bound and soluble TAM proteins in patients with CKD. CKD patients had significantly lower expression of Mer in monocytes, yet increased expression of soluble TAM receptors sAxl and sMer in plasma compared to controls. The metalloproteinase ADAM 17, responsible for cleavage of Mer to its soluble form, was increased in patient monocytes. Elevated levels of soluble TAM receptors were more evident in patients with progressive renal failure. These observations suggest that functional deficiency of TAM receptor-mediated regulation of inflammation may contribute to chronic inflammation in patients with CKD.

  16. Interaction of Globular Plasma Proteins with Water-Soluble CdSe Quantum Dots.

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    Pathak, Jyotsana; Rawat, Kamla; Sanwlani, Shilpa; Bohidar, H B

    2015-06-08

    The interactions between water-soluble semiconductor quantum dots [hydrophilic 3-mercaptopropionic acid (MPA)-coated CdSe] and three globular plasma proteins, namely, bovine serum albumin (BSA), β-lactoglobulin (β-Lg) and human serum albumin (HSA), are investigated. Acidic residues of protein molecules form electrostatic interactions with these quantum dots (QDs). To determine the stoichiometry of proteins bound to QDs, we used dynamic light scattering (DLS) and zeta potential techniques. Fluorescence resonance energy transfer (FRET) experiments revealed energy transfer from tryptophan residues in the proteins to the QD particles. Quenching of the intrinsic fluorescence of protein molecules was noticed during this binding process (hierarchy HSA<β-Lg protein molecules). Upon binding with QD particles, the protein molecules underwent substantial conformational changes at the secondary-structure level (50 % helicity lost), due to loss in hydration.

  17. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

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    Alberto Miranda

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  18. Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

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    Matti A Kjellberg

    Full Text Available Members of the glycolipid transfer protein superfamily (GLTP are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface.

  19. Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast.

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    Grossmann, Guido; Opekarová, Miroslava; Malinsky, Jan; Weig-Meckl, Ina; Tanner, Widmar

    2007-01-10

    The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

  20. Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

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    Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

    2013-03-01